Investigations of Hepatic Hemodynamics and Alterations in ...

Investigations of Hepatic Hemodynamics and Alterations in the

NO-cGMP Pathway in an Animal Model of Liver Fibrosis / Cirrhosis Suggest PDE5 Inhibitors as Promising Adjunct in Portal Hypertension Therapy

INAUGURALDISSERTATION

zur Erlangung des Doktorgrades (Dr. rer. nat.)

der Fakultät für Chemie und Pharmazie

der Albert-Ludwigs-Universität Freiburg im Breisgau

vorgelegt im Jahr 2018

von

Denise Schaffner

geboren in Breisach am Rhein

Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber

Dekan: Prof. Dr. Manfred Jung

Referentin: Prof. Dr. Irmgard Merfort

Korreferent: Prof. Dr. Wolfgang Kreisel

Drittprüfer: Prof. Dr. Andreas Bechthold

Datum der mündlichen Prüfung: 29. Juni 2018

“With man this is impossible,

but with God all things are possible”

- Matthew 19:26 -

Scientific Activities

Publications D. Schaffner, D. von Elverfeldt, P. Deibert, A. Lazaro, I. Merfort, L. Lutz, J. Neubauer, M.W. Baumstark, W. Kreisel, W. Reichardt:

“Phase-contrast MR flow imaging: A tool to determine hepatic hemodynamics in rats with a healthy, fibrotic, or cirrhotic liver” J Magn Reson Imaging, 46(5), 1526-1534 (2017)

D. Schaffner, A. Lazaro, P. Stoll, P. Deibert, I. Merfort, A. Schmitt-Gräff, M.W. Baumstark, L. Vauth, P. Hasselblatt, W. Kreisel:

„Analysis of the NO-cGMP pathway in experimental liver cirrhosis suggests phosphodiesterase 5 as potential target in portal hypertension therapy” Manuscript in preparation

Short Oral Presentations D. Schaffner, D. von Elverfeldt, P. Deibert, A. Lazaro, I. Merfort, L. Lutz, J. Neubauer, M.W. Baumstark, W. Kreisel, W. Reichardt:

“Effect of Chronic Thioacetamide Treatment on Hepatic Hemodynamic Parameters in Rats: Evaluation by Magnetic Resonance Imaging” United European Gastroenterology (UEG) week 2016, Vienna, Austria

D. Schaffner, A. Lazaro, P. Deibert, I. Merfort, A. Schmitt-Gräff, P. Hasselblatt, W. Kreisel:

„Störungen des NO-cGMP-Systems im Tiermodell einer Leberzirrhose: Implikationen für die Therapie der portalen Hypertonie beim Menschen“ Annual Meeting of the German Society of Gastroenterology, Digestive and Metabolic Diseases (Deutsche Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten, DGVS) 2017, Dresden, Germany

Poster Presentations D. Schaffner, D. von Elverfeldt, P. Deibert, A. Lazaro, I. Merfort, L. Lutz, J. Neubauer, M.W. Baumstark, W. Kreisel, W. Reichardt:

“Phase-contrast Magnetic Resonance Flow Imaging: A Tool to Determine Hepatic Hemodynamics in Rats with a Healthy, Fibrotic, or Cirrhotic Liver” Annual Meeting of the International Society for Magnetic Resonance in Medicine (ISMRM) 2017, Honolulu, Hawaii, US

D. Schaffner, A. Lazaro, P. Deibert, M.W. Baumstark, I. Merfort, W. Kreisel:

“Investigation on Hepatic Hemodynamics in Animal Model of Liver Cirrhosis” Day of Research 2017, Faculty of Chemistry and Pharmacy, University Freiburg, Germany


“NO-cGMP Pathway Alterations may contribute to Portal Hypertension: Results of a Study in Rats with Liver Fibrosis/Cirrhosis” Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Liver Meeting 2017, Washington D.C., US


“Alterations of the NO-cGMP pathway in thioacetamide-induced liver fibrosis/cirrhosis in rats” United European Gastroenterology (UEG) week 2017, Barcelona, Spain


“The NO – cGMP Pathway in Experimental Liver Cirrhosis – Implications for Portal Hypertension Therapy” Day of Research 2017, Faculty of Medicine, University Hospital Freiburg, Germany

D. Schaffner, A. Lazaro, P. Hasselblatt, A. Schmitt-Gräff, M. Grosse-Perdekamp, I. Merfort, P. Deibert, W. Kreisel:

“Overexpression of Phosphodiesterase-5 in Liver Cirrhosis: A Rationale for Novel Therapy in Portal Hypertension” Digestive Disease Week® (DDW) 2018, Washington, D.C., US

Index 1. Summary .................................................................................................................... 1

2. Introduction ............................................................................................................... 5

2.1 The Liver - A Multifunctional Organ ........................................................................................................ 5

2.2 Hepatic Circulatory System ..................................................................................................................... 6

2.3 Regulatory Mechanisms of Hepatic Hemodynamics ........................................................................... 7

2.4 Regulatory Mechanisms of Hepatic Blood Flow ................................................................................... 7

2.5 Liver Cirrhosis ............................................................................................................................................ 9 2.5.1 Definition and Complications ........................................................................................................... 9 2.5.2 Epidemiology and Etiology ............................................................................................................ 10 2.5.3 Pathophysiology of Liver Fibrosis / Cirrhosis .............................................................................. 11 2.5.4 Pathophysiology of Portal Hypertension (PH) ............................................................................ 13

2.5.4.1 Cellular and Molecular Changes .......................................................................................... 14 2.5.5 Symptoms of Liver Cirrhosis and PH ........................................................................................... 17 2.5.6 Diagnosis and Classification of Liver Cirrhosis and PH ............................................................ 17 2.5.7 Therapy of Liver Cirrhosis and PH ............................................................................................... 21

2.5.7.1 NO – A Multifunctional Molecule .......................................................................................... 24 2.5.7.2 NO – Generation and Function ............................................................................................. 24 2.5.7.3 NO and NOS in the Pathophysiology of PH ....................................................................... 28 2.5.7.4 Strategies to Increase NO Availability and NO-cGMP Signaling ..................................... 29

2.5.8 PDE5 and PDE5 inhibitors ............................................................................................................ 31

2.6 Experimental Models of Liver Fibrosis / Cirrhosis .............................................................................. 34 2.6.1 Thioacetamide ................................................................................................................................. 35

2.7 Aims and Objectives ............................................................................................................................... 36

3. Results ..................................................................................................................... 38

3.1 Evaluation of the TAA Model ................................................................................................................. 38 3.1.1 General Remarks ............................................................................................................................ 38 3.1.2 Histological Assessment of the Degree of Liver Fibrosis .......................................................... 39 3.1.3 Mortality ............................................................................................................................................ 40

3.2 Noninvasive Hemodynamic Measurements ........................................................................................ 40 3.2.1 MR Assessment of the Degree of Liver Fibrosis ........................................................................ 42 3.2.2 Flow Velocity Patterns and Flow Curves ..................................................................................... 42 3.2.3 Hemodynamic Parameters ............................................................................................................ 43

3.3 Invasive Hemodynamic Measurements ............................................................................................... 46 3.3.1 Portal Flow Volume Rate ............................................................................................................... 47 3.3.2 Effect of Sildenafil on Hemodynamics ......................................................................................... 49 3.3.3 Effect of MAP on PVP ................................................................................................................... 55

3.4 Biochemical Investigations .................................................................................................................... 58 3.4.1 Serum Parameters (Clinical Chemistry) ...................................................................................... 60

3.4.1.1 Effect of TAA-induced Liver Disease ................................................................................... 60 3.4.1.2 Influence of Hemodynamic Measurements......................................................................... 61

3.4.2 Gene Expression and Serum cGMP Concentrations ................................................................ 64 3.4.2.1 Effect of TAA-induced Liver Disease ................................................................................... 64 3.4.2.2 Influence of Hemodynamic Measurements......................................................................... 64 3.4.2.3 Effect of Sildenafil on Serum cGMP Concentrations ......................................................... 65

3.4.3 Immunohistochemical Staining (PDE5) ....................................................................................... 70

4. Discussion ............................................................................................................... 72

4.1 Evaluation of the TAA Model ................................................................................................................. 72

4.2 Noninvasive Hemodynamic Measurements ........................................................................................ 74

4.3 Invasive Hemodynamic Measurements ............................................................................................... 78 4.3.1 Portal Flow Volume Rate ............................................................................................................... 78 4.3.2 Effect of Sildenafil on Hemodynamics ......................................................................................... 79 4.3.3 Effect of MAP on PVP .................................................................................................................... 83

4.4 Biochemical Investigations .................................................................................................................... 85

4.5 Concluding Remarks .............................................................................................................................. 90

5. Materials and Methods ........................................................................................... 93

5.1 Materials ................................................................................................................................................... 93 5.1.1 Chemicals, Reagents and Other Matters .................................................................................... 93 5.1.2 Anaesthetics and Drugs ................................................................................................................. 94 5.1.3 Antibodies, Kits, Primer, and Probes ........................................................................................... 94 5.1.4 Consumables ................................................................................................................................... 95 5.1.5 Apparatus ......................................................................................................................................... 97 5.1.6 Software ......................................................................................................................................... 100 5.1.7 Animals ........................................................................................................................................... 100

5.2 Methods .................................................................................................................................................. 101 5.2.1 Laboratory Animals ....................................................................................................................... 101 5.2.2 Induction of Liver Disease with TAA .......................................................................................... 101 5.2.3 Noninvasive Hemodynamic Measurements ............................................................................. 102

5.2.3.1 MR Scanning ......................................................................................................................... 102 5.2.3.2 Data Acquisition / Postprocessing ...................................................................................... 105 5.2.3.3 MR Assessment of the Degree of Liver Fibrosis .............................................................. 106

5.2.4 Invasive Hemodynamic Measurements..................................................................................... 107 5.2.4.1 Operative Procedure ............................................................................................................ 107

5.2.5 Serum Analyses ............................................................................................................................ 111 5.2.5.1 Serum Parameters ................................................................................................................ 111 5.2.5.2 Competitive cGMP Enzyme-linked Immunosorbent Assay (ELISA) ............................. 111

5.2.6 Two-step Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) ......................... 112 5.2.7 Histology ......................................................................................................................................... 117

5.2.7.1 Assessment of the Degree of Liver Fibrosis ..................................................................... 117 5.2.7.2 Immunohistochemical (IHC) PDE5 Staining ..................................................................... 117

5.2.8 Statistics ......................................................................................................................................... 120

6. References .............................................................................................................. 123

7. Attachments ........................................................................................................... 145

7.1 Score sheet to document the body condition of the rats (in German)........................................... 145

7.2 Photo series of the operative procedure............................................................................................ 147

8. Abbreviations ......................................................................................................... 151

9. Content of Figures ................................................................................................. 153

10. Content of Tables ................................................................................................ 155

11. Acknowledgments ............................................................................................... 157

12. Curriculum Vitae .................................................................................................. 159

Summary

1

1. Summary During the last 30 years phosphodiesterase 5 (PDE5) inhibitors had been

successfully integrated in the therapy of diseases with an underlying vascular

impairment, such as erectile dysfunction and pulmonary hypertension. Hence, the

use of PDE5 inhibitors is also considered as promising adjunct in the therapy of

portal hypertension (PH), one of the most crucial complications of liver cirrhosis, a

leading cause of death worldwide.

PH is associated with nitric oxide (NO) deficiency in the intrahepatic vasculature,

resulting in increased sinusoidal intrahepatic resistance. The latter is caused by a

mechanical and a functional component. However, up to now no drugs have been

approved to target the mechanical component, which occurs e.g. in the form of

fibrous connective tissue or regenerative nodules, responsible for around 70% of

increased intrahepatic resistance. The residual 30% is explained by the functional

component, which is determined by sinusoidal vasoreactivity. Impaired sinusoidal

vasoreactivty, in turn, can be caused by alterations in the key parameters of the nitric

nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway, a regulator of

vascular tone. PDE5 is one of these key parameters involved in the NO-cGMP

pathway, initiating cGMP inactivation and thus leads to vasoconstriction. Hence,

pharmaceutical inhibition of PDE5 is a promising option to counteract sinusoidal

vasoconstriction and increased intrahepatic resistance. Initial preclinical and clinical

hemodynamic studies however, showed variable results considering the effect of

PDE5 inhibitors. Therefore, in this thesis the potential of PDE5 inhibitors in PH

therapy was further elucidated based on hemodynamic measurements and

biochemical investigations.

A rat model of thioacetamide (TAA)-induced liver fibrosis/cirrhosis was established

and noninvasive magnetic resonance (MR) measurements of hepatic and systemic

hemodynamics in rats with healthy, fibrotic or cirrhotic livers were performed. Liver

disease-induced changes in hemodynamic parameters, emphasizing on portal flow

volume rate, were determined. A significant decrease in portal flow volume rate was

observed in diseased rats, which was validated by subsequent invasive

hemodynamic measurements with a flow probe.

Summary

2

Moreover, dose-dependent effects of the PDE5 inhibitor sildenafil on hepatic and

systemic hemodynamics were investigated using pressure transducers. Acute effects

of administration of either sodium chloride, sildenafil 0.1 mg/kg or sildenafil 1.0 mg/kg

were compared. After high-dosage sildenafil administration (1.0 mg/kg), a trend

towards decreased portal venous pressure (PVP), a significant decrease in heart rate

(HR), and a nonsignificant decrease in mean arterial pressure (MAP) were found in

rats with cirrhotic livers. Hemodynamic data also revealed a significant effect of MAP

on PVP among all subgroups regardless of intervention, suggesting that changes in

systemic blood pressure may lead to changes in hepatic blood pressure.

Additionally, biochemical analyses of the key parameters in the NO-cGMP pathway

were conducted. Hepatic gene expression of the enzymes endothelial and inducible

NO synthase (eNOS, iNOS), soluble guanylyl cyclase subunit a1 and b1 (sGCa1,

sGCb1) and phosphodiesterase 5 (PDE5) was analyzed by qRT-PCR. An up-

regulation of iNOS and a significant overexpression of PDE5 in diseased rats were

observed. Enhanced levels of PDE5 protein expression were confirmed

immunhistochemically. Furthermore, serum cGMP concentrations from carotid

arterial blood samples were determined by ELISA. In diseased rats a slight decrease

was observed, whereas sildenafil administration (1.0 mg/kg) nearly renormalized

serum cGMP concentrations. Finally, studies were performed to evaluate whether the

hemodynamic measurement and the associated operative procedure affected gene

expression or serum cGMP concentrations. A significant decrease in eNOS gene

expression was detected.

In summary, the results of this study contribute to the general understanding of the

pathophysiology of PH and highlight the valuable potential of PDE5 inhibitors as

promising adjunct in PH therapy.

Summary

3

1. ZUSAMMENFASSUNG In den letzten 30 Jahren wurden Phosphodiesterase 5 (PDE5)-Inhibitoren erfolgreich

in die Therapie von Erkrankungen mit einer zugrundeliegenden vaskulären

Beeinträchtigung, wie z.B. erektile Dysfunktion und pulmonale Hypertonie, integriert.

Daher wird der Einsatz von PDE5-Inhibitoren auch in der Therapie der portalen

Hypertension (PH) als vielversprechender Zusatz angesehen. PH ist eine der

wesentlichsten Komplikationen der Leberzirrhose, eine der weltweit führenden

Todesursachen.

PH ist mit einem Stickstoffmonoxid (NO)-Mangel im intrahepatischen Gefäßsystem

assoziiert, was zu einem erhöhten sinusoidalen intrahepatischen Widerstand führt.

Letzteres wird durch eine mechanische und eine funktionelle Komponente

verursacht. Bis jetzt wurden jedoch keine Arzneimittel zugelassen, die auf die

mechanische Komponente abzielen, welche z.B. in Form von fibrösem Bindegewebe

oder regenerativen Knötchen auftritt und für etwa 70% des erhöhten intrahepatischen

Widerstandes verantwortlich ist. Die restlichen 30% erklären sich durch die

funktionelle Komponente, die durch sinusoidale Vasoreaktivität bestimmt wird. Eine

gestörte sinusoidale Vasoreaktivität kann wiederum durch Veränderungen in den

Schlüsselparametern der Stickstoffmonoxid-cyclisches Guanosinmonophosphat (NO-

cGMP)-Signalkaskade, einem Regulator des vaskulären Tonus, verursacht werden.

PDE5 ist einer dieser Schlüsselparameter in der NO-cGMP-Signalkaskade, der für

die Inaktivierung von cGMP verantwortlich ist und somit zur Vasokonstriktion führt.

Aus diesem Grund stellt die pharmazeutische Inhibierung von PDE5 eine

vielversprechende Option dar, um der sinusoidalen Vasokonstriktion und dem

erhöhten intrahepatischen Widerstand entgegenzuwirken. Erste präklinische und

klinische hämodynamische Studien zeigten hinsichtlich der Wirkung von PDE5-

Inhibitoren jedoch unterschiedliche Ergebnisse. Daher wurde in der vorliegenden

Arbeit das Potenzial von PDE5-Inhibitoren in der Therapie der PH auf der Grundlage

von hämodynamischen Messungen sowie biochemischen Analysen untersucht.

Es wurde ein Ratten-Modell der Thioacetamid (TAA)-induzierten Leberfibrose/-

zirrhose etabliert und nichtinvasive Magnetresonanz (MR)-Messungen der

hepatischen und systemischen Hämodynamik in Ratten mit gesunden, fibrotischen

oder zirrhotischen Lebern durchgeführt. Dadurch sollten die durch

Lebererkrankungen induzierten Veränderungen der hämodynamischen Parameter,

Summary

4

unter besonderer Berücksichtigung der portalen Volumenflussrate, bestimmt werden.

Bei erkrankten Ratten konnte eine signifikante Abnahme der portalen

Volumenflussrate, welche durch die nachfolgenden invasiven hämodynamischen

Messungen mit einer Strömungssonde bestätigt wurde, beobachtet werden.

Zudem wurden dosisabhängige Effekte des PDE5-Inhibitors Sildenafil auf die

hepatische und systemische Hämodynamik mittels Drucksensoren untersucht. Die

Effekte einer Verabreichung von entweder Natriumchlorid, Sildenafil 0,1 mg/kg oder

Sildenafil 1,0 mg/kg wurden verglichen. Nach Verabreichung von hoch-dosiertem

Sildenafil (1,0 mg/kg) wurde bei Ratten mit zirrhotischen Lebern ein Trend zu

verringertem Pfortaderdruck (PVP), eine signifikante Abnahme der Herzfrequenz

(HR) und eine nicht signifikante Abnahme des mittleren arteriellen Blutdrucks (MAP)

beobachtet. Zudem wurde anhand der hämodynamischen Daten bei allen

Untergruppen, unabhängig von der Intervention, ein signifikanter Effekt des MAP auf

den PVP ermittelt. Dies deutet darauf hin, dass Veränderungen des systemischen

Blutdrucks zu Veränderungen des hepatischen Blutdrucks führen können.

Darüber hinaus wurden biochemische Analysen der Schlüsselparameter der NO-

cGMP-Signalkaskade durchgeführt. Die hepatische Genexpression der Enzyme

endotheliale und induzierbare NO-Synthase (eNOS, iNOS), lösliche Guanylyl-

Cyclase-Untereinheit a1 und b1 (sGCa1, sGCb1) und Phosphodiesterase 5 (PDE5)

wurde mittels qRT-PCR analysiert. Dabei zeigte sich eine Hochregulation von iNOS

und eine signifikante Überexpression von PDE5 bei erkrankten Ratten. Letzteres

wurde durch immunhistochemische Untersuchungen der PDE5-Proteinexpression

validiert. Außerdem wurden Serum-cGMP-Konzentrationen aus Blutproben der

Halsschlagader mittels ELISA bestimmt. In erkrankten Ratten wurde eine leichte

Abnahme beobachtet. Die Verabreichung von Sildenafil (1,0 mg/kg) führte dagegen

fast zu einer Renormierung der Serum-cGMP-Konzentrationen. Abschließend wurde

untersucht, ob die hämodynamische Messung und der damit verbundene operative

Eingriff die Genexpression oder Serum-cGMP-Konzentrationen beeinflussten.

Hierbei wurde eine signifikante Abnahme der eNOS-Genexpression nachgewiesen.

Zusammenfassend tragen die Ergebnisse dieser Studie zum allgemeinen

Verständnis der Pathophysiologie der PH bei und verdeutlichen das Potenzial von

PDE5-Inhibitoren als vielversprechenden Zusatz in der Therapie der PH.

Introduction

5

2. Introduction 2.1 The Liver - A Multifunctional Organ The liver is the largest gland in the human organism, and the second largest organ

after the skin 1. It is segmented into lobes, reddish-brown in color, and has a soft

consistency. Its central location in the upper-right portion of the abdomen, beneath

the diaphragm and to the right of the stomach, points out its importance for life.

The basic architectural unit of the liver is the hepatic lobule 2, where multiple

essential metabolic, detoxifying, and synthesizing processes take place:

• breaking down nutrients and turning them into energy

• storing glycogen, vitamins, iron and other essential chemicals

• controlling blood composition, i.e. levels of lipids, amino acids and glucose

• detoxifying potentially harmful substances, e.g. drugs and alcohol

• clearing the blood of particles and infections, e.g. toxins and bacteria

• converting ammonia to urea

• synthesizing immunologically active cells, plasma proteins and numerous

hormones

• synthesizing bile to digest lipids

• controlling blood clotting and repair of damaged tissues

To fulfill these tasks, the liver, together with its circulatory system and the associated

biliary duct, has evolved many structural and physiological features that underpin the

broad spectrum of critical functions. One major feature is functional liver tissue, which

encompasses at least seven different cell types. Among those, hepatocytes are the

major parenchymal cells, whereas sinusoidal endothelial cells (SECs),

cholanigocytes, as well as immunologically active cells such as hepatic stellate cells

(HSCs), Kupffer cells (KCs), natural killer cells (NKs) and lymphocytes of different

phenotypes are non-parenchymal cells 3. The most numerous cells are hepatocytes,

comprising 70-85% of the liver tissue 1,4. Other unique features of the liver are its

capacity for self-regeneration and its complex dual circulatory system 2.

Introduction

6

2.2 Hepatic Circulatory System The circulatory system of the liver is supplied by two distinct circulatory routes: the

hepatic artery and the portal vein 5. Each route provides blood of differing

compositions: the hepatic artery delivers well-oxygenated blood, accounting for 25%

of hepatic blood, whereas the residual 75% are supplied by the portal vein, which

delivers deoxygenated, but nutrient-rich blood 6–8. Both routes enter the liver via the

portal tracts, which are components of the hepatic lobules, and finally merge in the

sinusoids (Figure 1). The latter are a specialized network of intrahepatic blood

vessels, representing the hepatic microcirculation system and resembling systemic

capillaries 4.

Sinusoids, which are considered to be the functional vascular unit of the liver, are

composed of SECs, KCs, and HSCs 9,10. SECs form a loose physical barrier between

the blood circulating within the sinusoids and hepatocytes lining the sinusoids 10.

SECs and hepatocytes are in turn separated by the so-called “space of Disse”, where

HSCs are located 4. KCs are mainly located in the sinusoidal lumen, but they can

also make direct contact with the hepatocytes 11.

Figure 1: Schematic diagram of a portal tract (left) and a hepatic sinusoid (right) Original source: Y .Iwakiri et al. 2014: “Vascular pathobiology in chronic liver disease and cirrhosis –

Current status and future dicrections” (https://doi.org/10.1016/j.jhep.2014.05.047)

This article was published under the terms of the Creative Commons Attribution-NonCommercial-No

Derivatives License (CC BY NC ND).

SECs are highly specialized endothelial cells unique to their location 12. In contrast to

other endothelial cells, SECs lack a continuous endothelial lining and exhibit a

fenestration, which makes them the most permeable endothelial cells of the

mammalian organism 12. This “sinusoidal gap” most likely serves to facilitate the

https://doi.org/10.1016/j.jhep.2014.05.047

http://creativecommons.org/licenses/by-nc-nd/4.0/


Introduction

7

transport of macromolecules from the blood passing the sinusoidal lumen to the

abluminal located hepatocytes 13. Thus, an efficient exchange of, e.g. oxygen,

nutrients, hormones and inflammatory factors with the hepatocytes can be ensured

before the blood returns to the systemic circulation via the central venules, which

drain into hepatic veins, which in turn ultimately merge in the inferior vena cava 14.

In summary, the liver is a highly vascular organ and has the most complex circulation

of any organ in the body 7. The intrahepatic microvascular system is made up of

several discrete units, including portal venules, hepatic arterioles, sinusoids, and

central venules 13,15. All these vascular trees, as well as the sinusoids, the hepatic

microcirculation, have their own morphological and functional features, which

together determine hepatic hemodynamics 16.

2.3 Regulatory Mechanisms of Hepatic Hemodynamics The term “hemodynamics” refers to the study of the physiological aspects comprising

the blood circulation. The ultimate aim of an adequate blood circulation is to provide

sufficient blood flow to the different tissues of the body in order to sustain optimal

organ and tissue function 17. In the liver, hemodynamic homeostasis ensures

nutrients and hormone fluxes, hepatic clearance and elimination, adequate

oxygenation, as well as cardiovascular stability 5–7. Not only in the liver, but also in

any other organ, regulation of hemodynamics depends on a static component, which

is based on Ohm's Law, a physical principle that can be applied for the flow of any

fluid:

Flow (Q) = pressure gradient (ΔP) / resistance (R) 17.

This static component is superimposed by the dynamic component, which is based

on locally acting regulatory mechanisms and regulatory mechanisms that adjust the

current hemodynamic status to the demands of the organism as a whole 17.

2.4 Regulatory Mechanisms of Hepatic Blood Flow As already mentioned, 75% of the hepatic blood is supplied by the portal vein. But

portal blood flow is in fact simply the sum of outflows of splanchnic organs, which

means that the liver is not capable of regulating portal blood flow directly 7,5.

Introduction

8

However, to counteract acute or chronic changes in portal blood flow, the liver

evolved several interrelated regulatory mechanisms, which primarily influence blood

flow to extrahepatic splanchnic organs 5. As a result, a constant hepatic blood flow-

to- liver mass ratio can be ensured under physiological conditions. The regulatory

mechanisms have been elucidated by Lautt 5 and are summarized in the following:

The first mechanism is vascular compliance, which is based on the physical principle

of a volume-pressure relationship. In general, vascular compliance describes the

extent to which the volume of the vessel passively changes with changes in

pressure. The vessel volume itself is controlled by vasodilation or vasoconstriction.

Thus, a decreased portal flow is followed by a passive decrease in intrahepatic

pressure and furthermore a passive blood extrusion from the huge hepatic blood

reservoir into the central venous circulatory system. Thereby, cardiac output is

increased, which leads to an elevation of blood flow in the splanchnic arteries that

feed the portal venous system. As a consequence the initial flow deficit is, at least

partially, buffered.

Another well-described regulatory mechanism of the liver is the hepatic arterial buffer

response (HABR) 7,18. The key player in the HABR is adenosine, a potent vasodilator

of the hepatic artery. Adenosine is constantly secreted into the space of Mall that

surrounds the terminal branches of the hepatic artery and the portal vein before they

finally merge in the liver sinusoids. If portal flow is decreased, adenosine

accumulates, resulting in dilation of hepatic artery being stimulated. The induced

increase in hepatic arterial flow into the portal vein buffers changes in portal flow on

total hepatic flow. In former publications Lautt (et al) described the HABR to be

capable of compensating a 25% to 60% decrease in portal blood flow 19,20, however,

in a more current publication, he stated that the hepatic arterial buffer capacity is

challenging to quantify 5. Interestingly, the HABR only works unidirectionally, since a

decrease of hepatic arterial flow does not induce an elevation of portal flow 21,7.

Accumulation of adenosine also indirectly mediates the activation of the hepatorenal

reflex via hepatic afferent nerves. This reflex induces a decrease in renal output and

fluid retention, thereby leading to an increase in blood volume, venous return, cardiac

output, and ulitmately splanchnic blood flow.

Introduction

9

Moreover, the liver has a unique way of counteracting severe vasoconstriction.

Looking at the hepatic artery, vasoconstriction leads to decreased hepatic arterial

flow. The portal vein in comparison, responds to local intrahepatic vasoconstriction

by an increase in portal venous pressure (PVP) with no alterations in portal flow,

since portal flow is controlled by the outflow of the splanchnic organs. In addition to

adenosine, nitric oxide (NO) is also a potent vasodilator and antagonist to

vasoconstrictors. NO-induced vasodilation of the portal vein as well as the hepatic

artery occurs when intrahepatic vasoconstriction enhances shear stress. In contrast,

adenosine-induced vasodilation occurs only when vasoconstriction is more systemic

and causes a decrease in portal flow. Its vasodilatory effects, however, seem to be

limited to the hepatic artery.

If all these compensatory mechanisms are not sufficient to maintain hepatic blood

flow homeostasis, in the last resort liver mass is adapted to match the blood demand.

Therefore, hepatocyte proliferation is induced when portal flow is elevated, whereas

hepatocyte apoptosis is induced when portal flow is reduced.

Considering this massive compensatory machinery, it becomes obvious how

significant an adequate hepatic blood flow is to sustain liver function. Nevertheless,

the occurrence of hemodynamic disturbances and vascular insult, e.g. in association

with liver cirrhosis, cannot be excluded 6.

2.5 Liver Cirrhosis 2.5.1 Definition and Complications Liver cirrhosis is a serious chronic liver disease. Its pathogenesis describes a

prolonged and creeping progress characterized by fibrosis development, or scarring,

and structural modifications of the liver architecture 22–24. Secondary to liver cirrhosis,

the occurrence of impaired liver function as well as a characteristic vascular disorder,

namely portal hypertension (PH), is likely 25,26. Impaired liver function leads to

increased blood values of bilirubin and ammonia, and decreased blood values of

albumin, cholinesterase and clotting factors. Along with PH, further complications

emerge, such as ascites, esophageal or gastric varices, variceal bleeding,

spontaneous bacterial peritonitis, or dysfunction of other organs 27–29. The latter can

appear in form of the hepatorenal syndrome, hepatopulmonal syndrome,

Introduction

10

portopulmonal hypertension, cirrhotic cardiomyopathy or hepatic encephalopathy 28,29. Hence, in advanced stages of liver cirrhosis not only is there the risk of needing

a liver transplantation, but the risk of morbidity and mortality also increases

immensely 30,28,23,31.

2.5.2 Epidemiology and Etiology Liver cirrhosis is one of the most frequent chronic liver diseases worldwide that

appears in rich as well as in poor nations 32,33,24. With more than one million deaths

per year (data from 2010), it is the 14th most common cause of death worldwide 34,35.

In central Europe, it is in fact the fourth most common cause of death with around

170 000 deaths per year (data from 2002) 36,37. That makes up approximately 2% of

all deaths worldwide, and also 2% of deaths in Europe 35,32,34. However, it seems

likely that there is a high number of unreported and / or undetected cases as the

initial stage of liver cirrhosis is asymptomatic or the disorder remains undiagnosed 38,39.

Liver cirrhosis can arise as a consequence of a range of chronic stimuli including

toxic, viral, autoimmune, vascular, cholestatic or metabolic diseases (Table 1) 22,40,41,31. Among those, alcoholic and, increasingly non-alcoholic liver diseases

(NAFLD), as well as hepatitis B or C infections, are the most common risk factors 42,43,38,40,36,44,45.

Table 1: Causes of liver cirrhosis

Stimuli Examples

toxic

infectious - viral - others

autoimmune

vascular

cholestatic

metabolic

alcohol-induced steatohepatitis, medications and chemicals

hepatitis B, C, D schistosomiasis and toxoplasmosis

autoimmune hepatitis, primary sclerosing cholangitis and primary biliary cholangitis

right-heart failure, Budd-Chiari syndrome and, Osler disease

bile duct stenosisa and recurrent bacterial cholangitis

non-alcoholic steatohepatitis (NAFLD), hemochromatosis and Wilson’s disease

Introduction

11

2.5.3 Pathophysiology of Liver Fibrosis / Cirrhosis

Liver fibrosis, or scarring, describes a complex wound healing process in response to

acute or chronic liver inflammation and damage. Hence, when hepatocytes undergo

necrosis or apoptosis, the cascade starts and inflammatory signaling by cytokines

and chemokines, recruitment of immunologically active cells, and activation of HCS

are initiated 31. As a consequence, progressive extracellular matrix (ECM) generation

and accumulation in the liver tissue, and simultaneous inhibition of ECM remodeling

and degradation proceed 22,24.

The mechanisms involved in the pathophysiology of liver fibrosis / cirrhosis are

complex and still not completely understood 21,46. However, within a vast integrated

network of cellular and molecular components, the activation of HSCs is described to

be the main boosting factor 47,48,22. The transdifferentiation of quiescent HSCs into

activated HSC, so-called myofibroblasts, is regulated by their interaction with various

cellular and molecular components involved in the wound healing response 47,49.

Myofibroblasts present a cell type that is absent in healthy livers, but accumulates in

diseased livers. They are located in the space of Disse, between the hepatocytes

and the SECs, where they encircle the sinusoids 46,50. The origin of these hepatic

myofibroblasts is still a matter of debate, but it has been described that there are at

least two sources: HSC-derived and portal mesenchymal cell-derived myofibroblasts,

whereby the latter likely occur mainly in biliary disease 50–53.

HSC activation can be divided into the initiation and the perpetuation phase 54,55.

The initiation phase comprises the release of intracellular contents, such as growth

factors, DNA and ROS by stressed or damaged liver cells (hepatocytes, KCs, and

SECs), or infiltrating immunologically active cells 49. These stimuli activate KCs, the

liver-resident macrophages, to secrete cytokines and chemokines. This in turn leads

to the recruitment of bone marrow-derived monocytes into the liver 56. Once they

have reached the liver, the infiltrated monocytes differentiate into macrophages with

an inflammatory, profibrogenic phenotype (Ly6Chi) 57. This provides a rapid and

transient way to expand the macrophage pool in the liver. By secreting cytokines,

chemokines and growth factors, these macrophages promote inflammatory

responses, HSC activation, and hence fibrosis progression. While the bone-marrow

serves as a major source of Ly6Chi monocytes, the spleen serves as a reservoir for

Introduction

12

Ly6Clo monocytes 56, another subset of macrophages with an antifibrogenic or

“patrolling” phenotype (Ly6Clo). Ly6Clo macrophages trigger HSC deactivation,

including apoptosis, senescence and reversion to quiescent HSCs, and are thus

essential for fibrosis regression 56,47,57.

Due to these dual roles, recruited macrophages are major regulators of liver fibrosis

progression and resolution 46,57. However, KCs as well as recruited macrophages can

adopt their phenotype, depending on signals from the hepatic microenvironment,

making the role of the immune system in reversibility of hepatic fibrosis even more

complex 56,57.

The perpetuation phase starts once the HSCs are activated and aim to maintain their

activated phenotype, which is characterized by various changes in cell behavior and

properties. Whereas quiescent HSCs primarily serve as vitamin A reservoirs, the

activated phenotype shows acquisition of ECM-generating, contractile, proliferative,

migratory, immunomodulatory and phagocytic properties and simultaneously a loss

of vitamin A storage capacity 47,50,54.

The activated phenotype of HCSs, the myofibroblasts, are the principle source of

ECM constituents, including collagen. Moreover, myofibroblasts synthesize tissue

inhibitors of matrix metalloproteinases (TIMPs), which are secreted into the

extracellular environment to inhibit matrix metalloproteinases (MMPs), a family of

ECM-degrading enzymes 42. Being released from infiltrating macrophages and KCs,

MMPs are present in the liver even during progressive fibrogenesis, demonstrating

that ECM accumulation by far exceeds its degradation by MMPs 26.

Initially the encapsulation of inflamed or damaged liver tissue by ECM indeed

represents a beneficial mechanism in the wound healing process and ensures liver

repair; however, when the stimulus for wound healing remains sustained persistently,

fibrogenesis escalates. At first fibrosis develops around either portal tracts or central

veins, ultimately forming bridging fibrosis with nodule formation surrounded by thick

bands of fibrous connective tissue 48,26,24. As a consequence of ongoing distortion of

liver architecture, the transition from liver fibrosis into cirrhosis takes place.

Introduction

13

2.5.4 Pathophysiology of Portal Hypertension (PH) Secondary to liver cirrhosis one of the earliest and most crucial complication is PH,

which is characterized by an abnormally increased PVP 58,24,59. Defined clinically, the

term “PH” describes an increase of the hepatic venous pressure gradient (HVPG)

between the portal vein and the inferior vena cava above normal values (≥ 5 mmHg) 60. It is accompanied by distinct alterations not only in the intra-, but also in the

extrahepatic circulation, and underlies most of the clinically significant complications

of liver cirrhosis 61. The intrahepatic, sinusoidal PH (see 2.5.6), the most common

form occurring secondary to liver cirrhosis, will be focused on in the following 62,63.

When considering the pathophysiology of PH the first concept that needs to be

readdressed is the hemodynamic application of Ohm's Law 60:

Flow (Q) = pressure gradient (ΔP) / resistance (R) 17.

By transposing this equation, the pressure gradient (ΔP) is defined as the product of

amount of flow (Q) and resistance (R). Applied to the portal vein that means the

hepatic venous pressure gradient (equivalent to PVP) (ΔP) is directly proportional to

the amount of portal blood inflow (Q) and the intrahepatic resistance opposing this

inflow (R):

Pressure gradient (ΔP) = flow (Q) * resistance (R) 62,60,13.

Hence, from a theoretical point of view, an elevation in PVP can occur secondary to

either an increase in intrahepatic resistance, an increase in portal blood inflow, or

both 60.

After a paradigm shift, initiated in the 1970s, it has meanwhile been widely accepted

that the mechanisms involved in the pathophysiology of PH encompass two main

aspects 64,60,65,66:

1. Increased intrahepatic resistance due to a mechanical and functional component

2. Increased blood inflow into the portal vein due to splanchnic vasodilation

In the initial stage of liver cirrhosis, an elevated PVP occurs as a result of increased

intrahepatic resistance to portal blood flow, which is caused by a mechanical and a

functional component 60,67. The mechanical (or structural) modifications occur in the

form of fibrous connective tissue, regenerative nodules, angiogenesis and vascular

Introduction

14

occlusion, which explain around 70% of the increased intrahepatic resistance,

whereas the functional (or dynamic) change explains at least 30%. Since the latter is

determined by the vasoreactivity of sinusoids, it could as well be spoken of

“sinusoidal vascular tone” or “sinusoidal vascular resistance”.

Along with the progression of liver cirrhosis, splanchnic arterial vasodilatation occurs,

which leads to an elevated flow into the gut and into the portal venous system 68.

Vasodilation furthermore induces an activation of neurohumoral and vasoconstrictive

systems, sodium and water retention, and consequently an increase in blood volume,

cardiac output and heartrate 68. These factors in combination with a decreased

systemic vascular resistance ultimately cause a lowering of systemic blood pressure.

This so-called hyperdynamic circulatory state develops in advanced stages of

cirrhosis and further increases portal blood inflow 69. Concomitant or subsequent to

splanchnic vasodilation the generation of portal-systemic collaterals that are formed

by the opening of pre-existing vessels or angiogenesis, occurs to decompress the

portal system 65,70,71. The extrahepatic collateral formation results in a partial

rerouting of blood flow away from the liver through these collateral vessels into low-

pressure systemic veins, which finally significantly increases the risk of esophageal

or gastric variceal bleeding and systemic circulatory disturbances 72,73.

Taken together, the extrahepatic changes in the splanchnic and systemic circulatory

system do not compensate PH, but rather contribute to its maintenance or even

worsening, and evoke additional complications 74,75. It has therefore been of great

relevance to get deeper insights into the mechanisms causing these alterations in

intra- and extrahepatic circulation. Fortunately, the knowledge of the pathological

mechanisms in PH has become enlarged immensely during the past few decades,

showing that these hemodynamic alterations are caused by a vast integrated network

comprising several components, and which is still not completely understood.

2.5.4.1 Cellular and Molecular Changes Considering the intrahepatic vasculature, a diminished capability to adjust sinusoidal

vascular tone, which defines intrahepatic resistance, occurs that finally leads to

increased intrahepatic resistance. The latter is associated with a number of cellular

changes in vascular smooth muscle cells surrounding branches of the portal vein and

Introduction

15

in sinusoidal cells 76. And although even hepatocytes have been described to

undergo some changes, e.g. loss of microvilli, the most dominant changes are

manifested in sinusoidal cells, i.e. SECs and HSCs 60. Both SECs and HSCs change

on a structural as well as on a functional level, which has been referred to as

pathological sinusoidal remodeling 77. SECs lose their fenestration, resulting in

impaired liver function and alterations in their phenotype 13. The latter causes

endothelial dysfunction, which leads to reduced production of vasodilators and

triggers HSC activation 65. Once HSCs are activated they also alter their phenotype

and show marked contractile and ECM-generating properties, and increased

responsiveness to vasoconstrictors 63.

Under physiological conditions, the adjustment of intrahepatic vascular tone to

prevailing conditions requires a subtle paracrine and autocrine interplay between

SECs and HSCs 12,13. In PH however, massive chronic disorders in this cellular

interplay lead to a constriction of myofibroblasts, resulting in an elevation of vascular

tone and resistance 61,62. (Figure 2)

Considering the extrahepatic vasculature, the adjustment of the vascular tone

depends on the interplay between ECs of the vascular endothelium and vascular

smooth muscle cells. In the context of PH, an extensive vasodilation in splanchnic

and systemic arteries occurs due to functional changes in ECs and vascular smooth

muscle cells 13. Vasodilation is caused by the overproduction of vasodilators in the

ECs and / or the hypocontractility of vascular smooth muscle cells, describing a

decreased vascular responsiveness to vasoconstrictors 65. Moreover, structural

alterations of arteries, the so-called “thinning” of arterial walls, may also contribute to

extrahepatic vasodilation, but this needs further investigation 65.

Introduction

16

Figure 2: Changes in the hepatic sinusoid in response to liver cirrhosis

Original source: Y .Iwakiri et al. 2014: “Vascular pathobiology in chronic liver disease and cirrhosis –

Current status and future dicrections” (https://doi.org/10.1016/j.jhep.2014.05.047)

This article is published under the terms of the Creative Commons Attribution-Non-Commercial-No

Derivatives License (CC BY NC ND).

These dynamic cellular changes are associated with molecular changes, but whether

the latter is the cause or the consequence of cellular changes, has not yet been

clarified. On the molecular level, it has been well described that increased

intrahepatic resistance is associated with massive imbalances in vasoactive

molecules (Table 2). Whereas in the intrahepatic vasculature vasodilators are

decreased and vasoconstrictors are increased, in the extrahepatic vasculature

vasodilators are increased and vasoconstrictors are decreased 64,65. Regarding this

opposing regulation of vascular tone, as a potent vasodilator, NO plays a key role

among the vasoactive molecules involved. Details will be described later (see

2.5.7.3). Moreover, there is evidence that imbalances in growth factor pathways,

involving cytokines such as transforming growth factor b (TGF-b), vascular

endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are

involved in the pathophysiology of PH as well, particularly in HSC activation and

pathological intra- and extrahepatic angiogenesis 64,13,61.

https://doi.org/10.1016/j.jhep.2014.05.047



Introduction

17

Table 2: Vasoactive molecules

Vasodilators Vasoconstrictors

Nitric oxide (NO)

Adenosine

Carbon monoxide (CO)

Glucagon

Endocannabinoid

Prostaglandin

Hydrogen sulfide (H2S)

Endothelin

Angiotensin II

Norepinephrine

Vasopressin

2.5.5 Symptoms of Liver Cirrhosis and PH Since it can take years or even decades until liver cirrhosis causes any obvious signs

or symptoms, it is not surprising that in many affected people the disease remains

undiagnosed. The interval of progression from liver damage to liver cirrhosis seems

to be highly individual despite the same etiology; in some cases, the process can

take 40 years (slow fibrosers), whereas in others it can take less than 15 years (rapid

fibrosers). Accordingly, incidental liver screening tests, i.e. laboratory tests or

examinations using imaging modalities often lead to the diagnosis of liver cirrhosis in

an early stage than the disease itself. In a fairly advanced stage, it is much more

likely that the disease is diagnosed as a consequence of the occurrence of PH and

other clinically significant complications. 78,38,31,39

2.5.6 Diagnosis and Classification of Liver Cirrhosis and PH Once there are symptoms or indications of liver cirrhosis, defining the underlying

etiology and the stage of the disease is essential for the choice of therapy and the

prediction of the prognosis. Liver fibrosis per se can occur as a consequence of any

chronic liver disease regardless of etiology 42,47. However, the predominant

profibrogenic mechanisms, as well as the patterns of parenchymal damage indeed

vary with the etiology of the underlying liver disease 79. The etiology can be identified

Introduction

18

by the patient’s history combined with laboratory tests and histological examinations 39. Histological examinations are furthermore considered to be the reference standard

for the assessment of the degree of liver fibrosis, although this involves the invasive

procedure of a biopsy 80,30. In addition, laboratory tests and imaging modalities, e.g.

ultrasound (US) or magnetic resonance (MR) imaging can be used for the

assessment of the degree of liver fibrosis. In the following, only the reference

standard will be focused on.

Liver biopsy can be carried out from a percutaneous or a transjugular route under

local anesthesia 80. After having taken the liver tissue samples, cross-sections are

performed before liver tissue sections are evaluated histologically. For the evaluation

of the grade, measuring of necro-inflammatory activity, and the stage, measuring

fibrosis and architectural changes, several histological scores exist 81. One example

is the Desmet score (DS) with DS=0: no fibrosis, DS=1: mild fibrosis, DS=2:

moderate fibrosis, DS=3: severe fibrosis, and DS=4: cirrhosis 82. But regardless of

the scoring system used, from a histopathological perspective the diagnosis of

cirrhosis is established once liver fibrosis has reached its terminal stage and the

process is considered “end-stage” 59. Moreover, for many years only liver fibrosis was

regarded as a dynamic, and potentially reversible process, whereas liver cirrhosis

was described as a static and irreversible terminal disease 31,83. However, nowadays

the concept of a dynamic, and at least partly reversible multi-stage process for liver

cirrhosis is being increasingly accepted 38,61,59.

The course of liver cirrhosis can initially be classified into two major stages: a

compensated, or asymptomatic phase, followed by a rapidly progressive

decompensated stage 23. The decompensated stage is defined by the presence of

clinical complication events secondary to PH, such as variceal bleeding, ascites or

hepatic encephalopathy 23,38,59. PH can be categorized according to anatomical

location into either pre-, intra- or posthepatic, with the intrahepatic, sinusoidal PH

being the most common form secondary to liver cirrhosis, regardless of etiology

(Table 3) 62,63. Since prognosis and predictors of death differ between these two

major stages of compensated and decompensated cirrhosis, each of them should be

regarded as separate entities 58. In fact, much effort has been put into the clarification

of the predominant pathogenic mechanisms of PH in each stage, which finally led to

the discovery of further substages of cirrhosis. Referring to recent publications of

Introduction

19

Abrades et al 58, D’Amico et al 84, and Garcia-Tsao et al 68, five prognostic stages

with a significant increase in the risk of death can be proposed (Table 4).

Table 3: Classification of PH according to anatomical location

Classification Subclassification

prehepatic

intrahepatic

posthepatic

- congential portal atresia

- intraluminal obstruction (thrombus, neoplasia)

- extraluminal vascular compression

- presinusoidal

- sinusoidal

- postsinusoidal

- luminal vascular obstruction

- extraluminal vascular compression

Table 4: Stages of liver cirrhosis

Stage Definition

1

2a

2b

3

4

5

compensated cirrhosis with mild PH

compensated cirrhosis with clinically significant PH, no varices

compensated cirrhosis with clinically significant PH, and varices

(no bleeding)

bleeding without other disease complications

first non-bleeding decompensating even

any second decompensating event

Introduction

20

As the majority of complications are caused by PH, the diagnosis of liver cirrhosis

often implicates the necessity to evaluate PVP. In a clinical setting the reference

standard to assess PVP is the hepatic venous pressure gradient (HVPG), which

represents an indirect measurement of PVP. Also non-invasive imaging modalities,

e.g. ultrasound (US) or magnetic resonance (MR) imaging can be used to assess

portal hemodynamics, but not PVP. However, the use of these imaging techniques is

still a matter of debate.

The assessment of the HVPG has essential prognostic relevance that even might

exceed that of histological examinations 80,61. The determination of the HVPG

requires the measurement of two pressure values: the wedged (or occluded) hepatic

venous pressure (WHVP) and the free hepatic venous pressure (FHVP). To measure

WHVP, a balloon catheter is inserted under local anesthesia through the jugular,

femoral or cubital vein into the hepatic vein. Through inflation of the balloon, the

hepatic venous outflow is blocked. After 1 to 2 minutes of blockade, the pressure at

the tip of the catheter finally reflects that of the hepatic sinusoidal pressure. On the

other hand, to measure FHVP, the balloon is deflated at 2 to 3 cm from the hepatic

vein ostium, so that the pressure at the tip of the catheter usually reflects the

pressure in the inferior vena cava.80

HVPG is finally calculated as the difference between WHVP and FHVP and hence

represents the pressure gradient between the portal vein and the intraabdominal

portion of inferior vena cava:

HVPG = WHVP – FHVP. 80,85,86

Since the WHVP, and accordingly the HVPG, is a measure of sinusoidal pressure, it

is important to mention that this measurement does not deliver reliable data with

respect of prehepatic or presinusoidal PH 68. In intrahepatic, sinusoidal PH however,

the HVPG is a reliable diagnostic tool which gives an accurate estimation of PVP. It

can be interpreted as follows: A HVPG < 5 mmHg is considered to be normal,

whereas PH is defined as an HVPG > 5 mmHg, a HVPG > 5 but < 10mmHg being

defined as mild PH, and a HVPG ≥ 10 mmHg as clinically significant PH. Above this

threshold of 10 mmHg, all complications induced by PH are more likely to occur. 24,62,68

Introduction

21

An accurate evaluation of hepatic, but also systemic hemodynamic status in chronic

liver diseases is thus essential for prevention or therapy of PH and its complications.

2.5.7 Therapy of Liver Cirrhosis and PH The main aim in PH therapy is to reduce HVPG to less than 12 mmHg or at least

20% of baseline to reduce the risk of variceal bleeding or rebleeding 38,87,27. To attain

this goal, PH management ideally involves addressing the underlying etiology,

inhibiting fibrosis development and regression, diminishing intrahepatic resistance

and / or splanchnic vasodilation, and treating complications 27,67. According to the

Baveno guidelines, PH management can involve pharmaceutical, endoscopic and

mechanical therapies 88.

As a first step in PH management, correct identification and extinguishing of the

origin of the evil is essential, since clearance or control of the underlying etiology of

liver damage is always the most effective therapy 31,42,89. However, in many affected

people the primary event or relevant mediators cannot be eliminated. In addition,

since affected people commonly only appear at an advanced stage of the disease,

reversal may not be rapid enough to prevent complications 89.

In suspected variceal bleeding, pharmaceutical therapy with vasoactive substances

should be started as soon as possible, before endoscopic therapies, such as band

ligation or sclerosing, are applied 88. In acute esophageal variceal bleeding events

however, a combined pharmaceutical and endoscopic therapy is recommended 88.

When endoscopic therapies are applied, it should be considered that they indeed

help to stop the bleeding, but simultaneously lead to an enhancement of PVP,

thereby worsening PH.

In a very advanced stage, when initial pharmaceutical and endoscopic therapy show

no effect or are likely to show no effect, transjugular intrahepatic portosystemic stent

shunting (TIPS) presents another therapy option 90. This mechanical, minimal

invasive therapy lowers PVP, but at the same time increases the risk of serious side

effects, such as the development of hepatic encephalopathy. In the event that all

these therapies fail, radical treatment by liver transplantation is the only remaining

option to increase survival odds 28,31. Since donors for liver transplantations are rare,

and the current therapy options are far from satisfying, new approaches are urgently

needed 91,92. In the following, the focus will be on pharmaceutical therapies.

Introduction

22

Current pharmaceutical therapy is mainly stratified according to the presence and

characterization of esophageal varices, meaning that a complication rather than the

disease itself is treated 88,90,91. Hence, research and also pharmaceutical companies

have been working intensively on the development of novel drugs to improve PH

therapy. Some aimed at developing antifibrotic drugs to reverse or at least inhibit

fibrogenesis, but no drug has yet been approved for use in humans 42,48,31.

Consequently, treating PH it is still challenging, since up to now the mechanical

component of increased intrahepatic resistance remains mostly irreversible. The

good news is that the functional component of PH can indeed be targeted

pharmaceutically and might potentially improve the future management of PH 42,93.

The functional component of increased intrahepatic resistance can be influenced

positively, either by a decrease in intrahepatic vascular tone, a decrease in

splanchnic vasodilation, or ideally both (Table 5) 67.

The current reference standard in pharmaceutical therapy, i.e. nonselective beta

blockers (NSBBs, beta-adrenergic receptor antagonists), vasopressin derivatives or

intestinal hormones, mainly counteract splanchnic vasodilation. Oral administration of

NSBBs is recommended to prevent bleeding, whereas in acute variceal bleeding

events, vasopressin derivatives or intestinal hormones should be administered

intravenously to stop bleeding 91. However, since these vasoactive substances not

only affect intrahepatic, but also extrahepatic circulation and hence can cause

massive contrary effects, their use has always been a matter of debate 91,94. Looking

for better alternatives, modulating NO availability and / or NO downstream signaling

seem to be promising options, since NO plays a vital role in the pathophysiology of

PH.

Introduction

23

Table 5: Reference standards and potentially novel drugs for PH therapy

Mode of action Drug group and names

reduced splanchnic vasoconstriction

reduced intrahepatic resistance (intrahepatic vascular tone ↓)

- nonselective ß-blockers (NSBBs)

propaponol, nadolol and carvedilol only in acute variceal bleeding events: - vasopressin derivatives

terlipressin

- intestinal hormones

somatostatin and octreotide - organic nitrates

isosorbide mononitrate - ACE-inhibitors / AT1-receptor-blockers

benazepril and captopril / losartan and valsartan - statins (HMG-CoA-reductase-inhibitors)

simvastatin and atorvastatin

- PDE5 inhibitors

sildenafil, udenafil and vardenafil - endothelin-receptor-antagonists

ambrisentan, bosentan and macitentan

Introduction

24

2.5.7.1 NO – A Multifunctional Molecule NO is an unstable free radical with a short biological half-life 95. First of all, NO is

known to be a potent endothelium-derived vasodilator, but it is likewise involved in

various other physiological processes in the cardiovascular, immune, gastrointestinal,

genitourinary, respiratory and nervous systems 96–99.

After NO is generated, it quickly diffuses into surrounding cells, where it can interact

with different reactants, such as transition metals and free radicals, and affect

proteins, nucleic acids, as well as fatty acids 96,100,101. What kind of interactions are

finally favored depends on several factors like the cellular environment, the available

concentration of NO and reactants and the reaction rates 96,98,102,103. Its physiological

effects are caused either directly or indirectly by its reactive and radical nature 96,104,100. Its unique chemistry, specifically the unpaired electron, but also the fact of

nitrogen being able to reach various oxidation states to generate different reactive

nitrogen species (RNS), vastly raises the potential NO effects 101,98. Thus, it is still

challenging to specify its physiological effects in specific cell types or complex

neuronal assembles 105. However, a well-studied and recognized NO target is the

soluble guanylyl cyclase (sGC) 102,106,99, a key cytosolic enzyme in the NO-cGMP

signaling pathway. The activation of this pathway implicates vasodilation and is

therefore essential for vasoregulation, including vascular tone and resistance.

2.5.7.2 NO – Generation and Function The activation of the NO-cGMP pathway takes place once NO is generated and

diffuses into the cytoplasm of surrounding cells, where it binds to the enzyme soluble

guanylyl cyclase (sGC). The interaction of NO with sGC causes a conformational

change, which results in the catalytic conversation of guanosine-5’-triphosphate

(GTP) to cyclic guanosine-3’,5’-monophosphate (cGMP). cGMP, an intracellular

second messenger, triggers various downstream signaling effects, which induce

vasodilatation. (Figure 3)

Indeed, the NO-cGMP pathway is much more complex. The three main enzymatic

steps NO generation, cGMP generation and degradation will be described in more

detail.

Introduction

25

NO generation occurs in a broad number of different cell types; however, to regulate

vascular tone, its synthesis in ECs of the vascular endothelium, and in case of the

corpus cavernosum also in neurons, is particularly important. Both, biomechanical

and biochemical stimuli, such as shear stress, VEGF and bradykinin can precipitate

NO generation 100,107,108,63,109. The synthesis itself can occur in two different ways:

either non-enzymatically from the transformation or degradation of inorganic nitrogen

chemicals in the organism and diet, or enzymatically from the oxidation of L-Arginine

to NO and L-citrulline 95,110. In mammals, the enzymatic redox reaction can be

catalyzed by three different isoforms of the enzyme nitric oxide synthase (NOS),

which were named according to the cell type or condition first described: endothelial

NOS (eNOS), inducible or inflammatory NOS (iNOS) and neuronal NOS (nNOS) 98,102.

All NOSs differ slightly in expression profile and in physiological function: eNOS and

nNOS, are both expressed progressively and generate continuous, but moderate

amounts of NO. eNOS is primarily expressed in ECs and primarily regulates vascular

tone. In addition, it induces vasoprotective and anti-atherosclerotic effects 104. nNOS

is primarily expressed in neurons and skeletal muscle and is responsible for synaptic

plasticity in the central nervous system, central regulation of blood pressure, smooth

muscle relaxation and vasodilation via peripheral nitrergic nerves 98. These nerves

are involved in the relaxation of corpus cavernosum and penile erection 104. iNOS

expression was originally identified in macrophages. Later, however, it was

demonstrated in almost all cell types as a defense mechanism against infections

from invading bacteria, viruses and fungi or against inflammation 97,111,112. Since

iNOS up-regulation is usually a consequence of pathological conditions, induction of

iNOS expression generates huge amounts of NO. The cell-specific roles of iNOS-

derived NO, however, need further investigation. Under physiological conditions,

iNOS expression is minimal or even absent 113,114.

Regarding the liver, eNOS and iNOS are the major players, whereas only little is

known about the role of nNOS in this organ 100, eNOS being primarily expressed in

SECs and in ECs of the portal vein, hepatic artery, central vein, and lymphatic

vessels 100, whereas iNOS can potentially be expressed in almost all hepatic cells 100,115.

Introduction

26

Figure 3: Schematic diagram of the NO-cGMP pathway

eNOS: endothelial nitric oxide synthase, iNOS: inducible nitric oxide synthase, NO: nitrix oxide,

PDE5: phosphodiesterase 5, sGC: soluble guanylyl cyclase, GTP: guanosine-5’-triphosphate;

cGMP: cyclic guanosine-monophosphate (cGMP); GMP: guanosine-monophosphate,

PKG: protein kinase G, (S)EC: (sinusoidal) endothelial cell; HSC: hepatic stellate cell

NOSs are generated as inactive monomers. For activation monomers must dimerize

and bind different cofactors. Tetra-hydrobiopterin (BH4), haem, flavin adenine

dinucleotide (FAD) and flavin mononucleotide (FMN) are cofactors of all three

isoforms 104,98. On binding calmodulin, a calcium-binding protein, the active enzyme

catalyzes the oxidation of L-arginine to NO and L-citrulline. For eNOS and nNOS

calmodulin binding, and hence also enzyme activity, is highly calcium-dependent,

whereas in iNOS calmodulin is bound constitutively 98,101. Moreover, post-

translational modifications and protein-protein-interactions can also regulate NOS

activity 97,98. Finally, NO generation requires molecular oxygen and nicotinamide

adenine dinucleotide phosphate (NADPH) as co-substrates for the oxidation of L-

arginine.

cGMP generation requires direct interaction between NO and the enzyme soluble

guanylyl cyclase (sGC). sGC is a heterodimeric hemoprotein composed of an a- and

b-subunit, which are both required for enzyme activity 116. Two isoforms of each

Introduction

27

subunit exist: a1/a2 for the a-subunit, as well as b1/b2 for the b-subunit, but only

a1/b1 and a2/b1 are active heterodimers. The a1/b1 heterodimer is regarded as the

major sGC isoform, since it is expressed in most mammalian tissues, including liver

tissue 117–119. Essential for sCG activation is an interaction between the heme-binding

domain, located on the b-subunit, and a heme moiety. The heme moiety is a large

heterocyclic ring with a transition metal, building the metal center of sGC.

Once NO is generated and diffused into vascular smooth muscle cells, or in the liver

into HSCs, it induces a conformational change of the sGC heterodimer by binding

avidly to its transition metal (ferrous heme iron), thereby activating sCG, which, in

turn, catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine

monophosphate (cGMP), an intracellular second messenger 106. Increased cGMP

concentrations exert downstream signaling effects by directly modulating various

effector proteins, i.e. cGMP-dependent protein kinases (PKGs), cGMP-hydrolzying

phosphodiesterases (PDEs), as well as cGMP-gated ion channels 120,121.

Two PKG families (PKGI and PKGII) and PDE classes (class I and class II) exist,

whereby PDE class I includes all known mammalian PDEs, which comprise 11

families (PDE1-11) 122,120,123. Only the roles of the cGMP-dependent PKGI family and

the cGMP-selective PDE5 family will be focused on, since their members are key

players in the NO-cGMP pathway 120,124.

An increase of cGMP concentrations induces a decrease in intracellular free calcium

concentrations through multiple mechanisms. First, cGMP is capable of inhibiting

calcium release from intracellular stores; second, it triggers removal and

sequestration of intracellular calcium through calcium pumps; and third, it induces

direct, as well as indirect inhibition of the influx of extracellular calcium through

voltage-gated ion channels 125. As a result, the indirect inhibition of calcium influx is

mediated by PKGI. Upon cGMP binding to allosteric sites in the regulatory domain,

PKGI undergoes a conformational change. This conformational change leads to the

release of the N-terminus inhibition of the kinase domain, and hence to an increase

in phosphotransferase activity of the dimeric enzyme 126,120,125. Thus, PKGI

stimulation results in phosphorylation of several proteins, which results in two

primary effects: first, a decrease in intracellular calcium levels and second, calcium

desensitization of the actin-myosin contractile elements 127.

As a consequence of these cGMP-induced downstream signaling effects, vascular

dilation is initiated, eventually leading to a reduction in vascular tone.

Introduction

28

Moreover, cGMP binds to the high-affinity GAF-A domain of PDE5, thereby

increasing the hydrolytic activity of the dimeric holoenzyme. The hydrolytic activity

can be further promoted through stabilization of the cGMP binding by

phosphorylation at a separate N-terminal site by PKGI 128–130. Once PDE5 is

activated, it initiates the hydrolysis of cGMP into inactive guanosine-5′-

monophosphate (GMP). Hence, rising intracellular cGMP concentrations are

associated with activation of PDE5 as a negative feedback mechanism mediating

cGMP degradation 131. All these feedback mechanisms happen within seconds and

are pivotal in lowering cGMP concentrations to basal levels in the short term after NO

stimulation 120. Prolonged NO exposure and increased cGMP concentrations,

however, seem to induce more persistent modifications at several steps in the NO-

cGMP pathway, including down-regulation of PKGI and up-regulation of PDE5 120.

Knowing about the essential role of NO in terms of vasoregulation, the role of NO

and NOS in the pathophysiology of PH has been investigated extensively.

2.5.7.3 NO and NOS in the Pathophysiology of PH According to currently available data, NO is described as an important molecular

factor involved in the pathophysiology of PH secondary to liver cirrhosis. The

paradoxically controlled intra- and extrahepatic vascular tone is characterized by NO

deficiency in the intrahepatic vasculature and, on the other hand, NO excess in the

extrahepatic vasculature.

Considering the intrahepatic vasculature, a down-regulation of eNOS activity in SECs

is described to be primarily responsible for intrahepatic NO deficiency, whereas data

considering eNOS expression are inhomogeneous described 63,76,132–139. Underlying

causes of the down-regulated eNOS activity can involve different factors, such as

oxidative stress or decreased BH4 synthesis and activity 63,139,140. Furthermore, an

up-regulation of iNOS expression is associated with liver cirrhosis, which can be

stimulated by endotoxins, cytokines and bacterial infections 63,141–144. iNOS

expression can take place in all hepatic cell types, but little can be revealed about

iNOS activity in the different cell types. However, it is known, that iNOS activity is

dependent on several factors such as availability of its substrate arginine and BH4 115. Interestingly, eNOS-derived NO is described to maintain liver homeostasis and

Introduction

29

counteract pathological conditions within the liver 100, while iNOS-derived NO is in

general pathological, and does not seem to cause vasodilation 100. The paradox of

decreased eNOS-derived NO and increased iNOS-derived NO, finally resulting in

increased intrahepatic resistance, might point out that the source of NO and the

surrounding microenvironment strongly influence the NO-induced effects 100. Its

inducible nature as well as the observation that iNOS can act as regulator of other

effectors, e.g. eNOS, increases the potential impact of iNOS massively and needs

further investigation 63,111.

Regarding the extrahepatic vasculature, an up-regulation of primarily eNOS activity,

but also eNOS expression in ECs of the vascular endothelium is assumed to cause

NO excess 64,63,145. The up-regulation of eNOS can be triggered by several stimuli,

e.g. shear stress, VEGF, or increased BH4 synthesis and activity 64,145–147. For iNOS,

however, data are inconsitstent. Some studies have implicated that enhanced iNOS

expression and activity is involved in the vasodilation of the extraheptic vasulature 141,148, whereas others found evidence against the involvement of iNOS 149. It seems

like eNOS- rather than iNOS-derived NO contributes to NO excess in the

extrahepatic vasculature, but equivalent to the intrahepatic vasculature the role of

iNOS-derived NO needs to be clarified further 74,150–152 .

Moreover, NO contributes to angiogenesis and as a result to collateral formation,

which again promotes the progression of PH 63.

Bearing that paradox in mind, the ideal concept for PH therapy should specifically

target intrahepatic vasculature and on site enhance NO availability and / or NO-

cGMP downstream signaling to counteract intrahepatic NO deficiency 60.

2.5.7.4 Strategies to Increase NO Availability and NO-cGMP Signaling To increase NO availability and / or NO-cGMP downstream signaling, different

pharmaceutical strategies can be used (Table 6). However, it must always be kept in

mind that drugs acting within the liver may have extrahepatic effects as well 94.

Hence, testing those, intra- and extrahepatic effects should be subject of scrutiny 91.

In this experimental study, we investigated the effect of PDE5 inhibitors, which act as

indirect stimulators of NO downstream signaling; thus, they will be focused on in the

following.

Introduction

30

Table 6: Reference standards and potentially novel strategies to increase NO

downstream signaling 96

Mode of action Drug group and name

1. NO delivery / generation a) NO-delivering compounds b) NO-releasing compounds c) enhancing endogenous NO generation by promoting eNOS activity d) other drugs that enhance endogenous NO generation by promoting eNOS activity and expression 2. Prevention of NO scavenging by other radicals or transition metals 3. Stimulating NO downstream signaling

- organic nitrates

isosorbide mononitrate (unspecific) - UDCA derivatives

NCX-1000 (UDCA derivative, liver-specific)

- eNOS substrates, cofactors, or the like

L-arginine, L-citrulline and BH4

- statins

Simvastatin and atorvastatin

- ACE inhibitors / AT1 receptor-blockers

Benazepril and captopril / losartan and valsartan

- PDE5 inhibitors

sildenafil, vardenafil, tadalafil and udenafil

- sGC stimulators

riociguat

- sGC activators

cinaciguat and vericiguat

Introduction

31

2.5.8 PDE5 and PDE5 inhibitors The PDE5 family is part of a PDE superfamily. PDEs are in general enzymes that

regulate the concentrations of the intracellular cyclic nucleotides, such as cyclic

adenosine-3’,5’-monophosphate (cAMP) and cyclic guanosine 3’,5’-monophosphate

(cGMP). Cyclic nucleotides act as second messengers and initiate a broad range of

downstream effects. By hydrolyzing cAMP into adenosine-5′-monophosphate (AMP)

and / or cGMP into guanosine-5′-monophosphate (GMP), respectively, PDEs

terminate the cyclic nucleotides’ downstream signaling (see 2.5.7.2).

PDEs can be classified into class I and class II, but since class I contains all known

mammalian PDEs 123, the focus will be on this class. The class I PDE superfamily

comprises 11 families (PDE1-11) involving more than 100 PDE isoforms 153,123. Their

differing substrate specificity allows a further division into three subgroups: some

PDEs are highly cAMP-specific; some are highly cGMP-specific, whereas some are

cAMP- and cGMP-specific (Table 7). Furthermore, they differ in primary structure,

catalytic properties, responses to specific inhibitors, and in cellular as well as

subcellular distribution, although all PDE families are structurally related 153.

Their regulatory key role in combination with the fact that PDEs exist ubiquitously, but

with distinct cellular and subcellular distribution, has made them a favorable target for

pharmaceutical therapies 153,154. The PDE5 inhibitor sildenafil citrate (sildenafil),

better known as Viagra®, is probably the most popular example for a successful

market launch of a PDE targeting drug (Figure 4).

Introduction

32

Table 7: Substrate specificity and distribution of PDE families 155,156

Family Specificity Tissue / Cellular distribution

PDE1 PDE2 PDE3 PDE4 PDE5 PDE6 PDE7 PDE8 PDE9 PDE10 PDE11

cAMP and cGMP cAMP and cGMP cAMP and cGMP cAMP cGMP cGMP cAMP cAMP cGMP cAMP and cGMP cAMP and cGMP

heart, brain, lung and smooth muscle adrenal gland, heart, lung, liver, platelets and endothelial cells heart, smooth muscle, lung, liver, platelets, adipocytes and immunologically active cells brain, Sertolli cells, kidney, liver, heart, smooth muscle, lung, endothelial cells and immunologically active cells smooth muscle, lung, platelets, heart, endothelial cells and brain photoreceptors, pineal gland and lung skeletal muscle, heart, kidney, brain, pancreas and T lymphocytes testes, eye, liver, skeletal muscle, heart, kidney, ovary, brain, T lymphocytes and thyroid kidney, liver, lung and brain testes, brain and thyroid skeletal muscle, prostate, pituitary gland, liver and heart

Introduction

33

Figure 4: Comparison of the structures of cGMP (native molecule) and sildenafil

Figure reprinted with permission of Springer Nature.

Original source: H .Ghofrani et al. 2006: “Sildenafil: from angina to erectile dysfunction to pulmonary

hypertension and beyond”

PDE5 is mainly expressed in smooth muscle cells and is encoded by one gene

PDE5A with three isoforms: PDE5A1, PDE5A2 and PDE5A3 (all 95-100 kDa) 155,157.

Due to its specific hydrolysis of cGMP and its presence in vascular smooth muscle

cells and in platelets, PDE5 was selected 30 years ago to undergo further research in

the context of vasoregulation. In some primilinary preclinical studies, the assumption

that PDE5 inhibitors mediate vasodilating effects could be confirmed 156. The

subsequent approach to use PDE5 inhibitors in diseases caused by vascular

dysfunction was obvious and eventually sildenafil was brought into the clinic as a

potential therapy for angina pectoris 154. The results of the first clinical trial proved the

mode of action of sildenafil by inducing moderate vasodilating effects in patients with

angina pectoris. However, the results of a later second study in healthy persons

showed that co-administration of sildenafil enhanced the vasodilating effects and the

hyperdynamic circulatory state induced by organic nitrates, the standard reference

therapy for angina pectoris at that time 156. Hence, it seemed to be critical to use

sildenafil in patients taking organic nitrates. Furthermore, sildenafil provided no

additional therapeutic effect compared to the available organic nitrates 154. But since

many patients reported penile erection as a side effect of sildenafil treatment, it came

to pass that sildenafil evolved from a potential anti-angina pectoris drug to a therapy

for erectile dysfunction, and more recently also for pulmonary hypertension 156,158.

Introduction

34

Consequently, the use of sildenafil in the context of cirrhotic PH therapy also seems

obvious and was the topic of this experimental study. Therefore, first of all, an

appropriate experimental model had to be established.

2.6 Experimental Models of Liver Fibrosis / Cirrhosis For experimental liver disease research primarily rodents are used as laboratory

animals 159. To induce liver fibrosis / cirrhosis in laboratory animals, the currently

most popular models are treatment with hepatotoxic agents like thioacetamide (TAA)

or carbon tetrachloride (CCl4) and surgical bile duct ligation (BDL).

Considering the toxic models of TAA and CCl4 administration, the available protocols

widely vary in application, frequency and dosage 159,160. Toxic models usually need

more time for the process of liver disease development compared to BDL. The BDL

presents a reliable cholestatic model 159,161, but requires a surgical procedure,

implying potential complications 162, as well as burdensome wound control and

aftercare.

After intensive literature research the model of TAA-induced liver fibrosis / cirrhosis

was used in this study. Thus, focus is placed on this agent in the following.

TAA causes liver disease which resembles human fibrosis and cirrhosis 160,163 and

leads to the development of PH 159. Application can be conducted by intraperitoneal

injection or oral treatment via drinking water. The advantages of oral application are

the ease of administration, noninvasiveness of the procedure 160,163,164, and the

decrease of extrahepatic toxicity due to the first-past effect, enabling a more

consistent intoxication 160. In this study, the model of oral and weight-adapted TAA

treatment according to the protocol previously described by Li et al. 163 was chosen.

Trusting their results, using this model induced a well-developed macronodular

cirrhosis in 90% of the rats after 12 weeks of TAA administration. They additionally

pointed out that the rats’ individual response to TAA intake could be easily evaluated

based on their weekly body weight change. Therefore, adaption of the TAA dosage

referring to the weekly body weight change was recommended to increase the

incidence and a more homogenous production of liver cirrhosis compared to the

administration of a constant TAA dosage. Furthermore, by adapting the TAA dosage,

mortality could be reduced to zero. All these aspects led to the decision to use this

particular TAA model in this study.

Introduction

35

2.6.1 Thioacetamide Thioacetamide (TAA) is a small, polar organosulfur compound, which is soluble in

water and alcohol 165,166. It was originally used to preserve citrus fruits, especially

oranges, due to its fungicidal activity 165,167. Nowadays it is known to be a potent

selective hepatotoxin and carcinogen, which has been used extensively in preclinical

research to develop animal models of acute and chronic liver disease 165,167.

However, although TAA is characterized by high liver-specificity 168, it has also been

described that it can harm other organs, such as the lungs, kidneys, spleen, thymus

and pancreas 165,167,169–171.

A short time after administration TAA accumulates in the liver 172. Provoking TAA

toxicity within the liver, however, requires a metabolic two-step bioactivation. This

bioactivation progress is triggered by hepatic cytochrome P450 enzymes, particularly

cytochrome P4502E1, and flavin-containing monooxygenases (FMO) 165,173,174. As a

result TAA is converted into its initial reactive metabolite TAA-sulfoxide (TAASO),

and subsequently into its ultimate reactive metabolite TAA-sulfdioxide (TAASO2).

Both metabolites induce noxious effects in hepatocytes, resulting in apoptosis,

necrosis and in the development of cholangiocellular carcinoma (CCCs) and

hepatocellular carcinoma (HCCs) 160,165,175. Several studies indicate that the

hepatotoxic effect induced by these metabolites is characterized by inflammation,

HSC activation, enlargement of nucleoli, mitochondrial and cytochrome P450

dysfunction, covalent binding to cellular macromolecules and / or production of

oxidative stress 161,165,168,172,174,176–178. The detailed underlying molecular mechanisms

causing TAA-induced intrahepatic organic and functional damage, however, are

complex and still not completely understood 159,160,168.

Introduction

36

2.7 Aims and Objectives The first aim of this experimental study was to establish and evaluate an animal

model of liver fibrosis / cirrhosis. To induce liver disease the model of oral and

weight-adapted thioacetamide (TAA) application was chosen, in which rats should be

used as laboratory animals. Relative portions of rats with fibrotic and cirrhotic livers,

the presence of cholangiocellular carcinoma (CCCs), and mortality induced by TAA

administration should be quantified.

The second aim was to noninvasively evaluate hepatic and systemic hemodynamic

changes induced by liver fibrosis / cirrhosis in rats with healthy, fibrotic or cirrhotic

livers by magnetic resonance (MR) measurements. Alterations in the parameters

portal cross-sectional area, portal flow velocity, portal flow volume rate, and aortic

flow volume rate should be determined. Moreover, MR data should be used to

investigate whether the degree of liver fibrosis can be assessed using a self-

established MR score. Therefore, a scanning protocol had to be established.

The third aim was to invasively evaluate hepatic and systemic hemodynamic

changes induced by liver fibrosis / cirrhosis. Alterations in the parameters portal flow

volume rate and mean arterial pressure (MAP) should be determined. Portal flow

volume rate should be detected with a flow probe, whereas MAP should be

determined using a pressure transducer. Moreover, results for the portal flow volume

rate between noninvasive and invasive measurements should be compared.

Additional invasive hemodynamic measurements aimed to evaluate hepatic and

systemic hemodynamic changes which are induced by the administration of the

PDE5 inhibitor sildenafil. Acute effects of administration of either sodium chloride,

sildenafil 0.1 mg/kg or sildenafil 1.0 mg/kg on the parameters portal venous pressure

(PVP), mean arterial pressure (MAP), microvascular flow (MF), and heart rate (HR)

over 50 minutes should be examined. PVP, MAP and HR should be measured using

pressure transducers, whereas MF should be determined with a microvascular flow

probe. Moreover, hemodynamic data should be used to investigate the effect of MAP

on PVP over the first 30 minutes. Therefore, a work protocol for the operative

procedure had to be established.

Introduction

37

The fourth aim was to evaluate changes biochemically in the key parameters of the

nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway induced by liver

fibrosis / cirrhosis. Alterations in hepatic gene expression of the enzymes endothelial

and inducible NO synthase (eNOS, iNOS), soluble guanylyl cyclase subunit a1 and

b1 (sGCa1, sGCb1) and phosphodiesterase 5 (PDE5) should be analyzed by qRT-

PCR, whereas changes in serum cGMP concentrations from carotid arterial blood

samples should be determined using ELISA. In this context it should also be

evaluated whether the hemodynamic measurement and in particular the associated

operative procedure affected gene expression or serum cGMP concentrations.

Moreover, the effect of sildenafil administration (1.0 mg/kg) on serum cGMP

concentrations should be determined. The main finding(s) of gene expression

analyses should finally be verified by immunohistochemical staining.

Results

38

3. Results 3.1 Evaluation of the TAA Model The model of TAA-induced liver fibrosis / cirrhosis by oral and weight-adapted TAA

administration is one of the commonly used models in preclinical liver research.

However, since recommendations for appropriate TAA exposure time vary, the first

step in the current study was to evaluate this model. Thereby, Dr. Lisa Lutz (Institute of

Clinical Pathology, Medical Center, University of Freiburg) conducted the histological

evaluation of the rats’ liver tissue samples.

3.1.1 General Remarks The model of TAA-induced liver fibrosis / cirrhosis by oral and weight-adapted TAA

administration was easy and safe to perform.

Rats receiving TAA (41 Sprague Dawley and 101 Wistar rats) showed symptoms

which were mostly induced by liver fibrosis / cirrhosis (Table 8). Their level of

discomfort was classified as minor.

Table 8: Symptoms observed in rats during TAA exposure time

on a regular basis: less frequently:

- slightly reduced intake of drinking water

- reduced body weight increase

- drier and light-colored feces

- bloody scabs on the nose

- markedly visible blood vessels in the ears

- yellowness of the skin

- ragged fur

- sensitivity to touch

- frightened behavior

- aggressive behavior

- unsteady gait

- mild ascites

Results

39

3.1.2 Histological Assessment of the Degree of Liver Fibrosis

Table 9: Number of rats sorted by strain and their histological degree of liver fibrosis

with corresponding TAA exposure time

a) Strain: Sprague Dawley

Desmet score 0 1 2 3 4 n total

TAA exposure: 12 weeks 1 15 2 (1) 2 1 (1) 21

TAA exposure: 15 weeks 1 1 (1) 2

TAA exposure: 17 weeks 1 1





TAA exposure: 23 weeks 1 (1) 1

TAA exposure: 24 weeks 1 (1) 1

Group classification CON FIB CIR 40 b) Strain: Wistar


TAA exposure: 12 weeks 10 7 1 18

TAA exposure: 16 weeks 4 8 18 (1) 17 35 (17) 82

Group classification CON FIB CIR 100

The degree of liver fibrosis was assessed according to the histological Desmet score (DS) 82 with

DS=0: no fibrosis, DS=1: mild fibrosis, DS=2: moderate fibrosis, DS=3: severe fibrosis and DS=4:

cirrhosis. The number of rats developing cholangiocellular carcinoma (CCCs) simultaneously is given

in subscript brackets.

Results

40

Regarding Sprague Dawley rats (Table 9a), all untreated rats and 5% (1/21) of the

rats treated with TAA for 12 weeks had no fibrosis. 5% (1/21) of the rats treated with

TAA for 12 weeks developed cirrhosis. A prolonged TAA exposure time from 15 up

to 24 weeks increased the incidence of cirrhosis to 68% (13/19). Among the Sprague

Dawley rats with a cirrhotic liver, all had CCCs simultaneously. For this reason a

second rat strain was tested subsequently.

Regarding Wistar rats (Table 9b), all untreated rats and 5% (4/82) of the rats treated

with TAA for 16 weeks had no fibrosis. None (0/18) of the rats treated with TAA for

12 weeks, but 43% (35/82) of the rats treated with TAA for 16 weeks developed

cirrhosis. Among the Wistar rats with a cirrhotic liver, 49% (17/35) had CCCs

simultaneously.

In both strains, a TAA exposure time of 12 weeks mainly induced mild fibrosis, but

not cirrhosis. 3% (1/40) of the Sprague Dawley and 4% (4/100) of the Wistar rats

developed no fibrosis or cirrhosis irrespective of TAA exposure time.

3.1.3 Mortality The mortality caused by TAA administration was 1-2%. 2% (1/41) of the Sprague

Dawley and 1% (1/101) of the Wistar rats died within the first week of TAA

administration. In comparison, no mortality (0/133) occurred in the untreated rats.

3.2 Noninvasive Hemodynamic Measurements In order to better characterize the TAA model and to address the lack of a

noninvasive and repeatable assessment of hemodynamics in the preclinical setting,

an MR scanning protocol was established to determine hepatic and systemic

hemodynamic alterations induced by liver fibrosis / cirrhosis. Thereby, MR

measurements were performed in cooperation with the working group of PD Dr.

Dominik von Elverfeldt (Department of Radiology – Medical Physics, Medical Center, University of

Freiburg). Their group member Dr. Wilfried Reichardt conducted the subsequent

postprocessing of MR data. He and Dr. Jakob Neubauer furthermore evaluated the

MR rat liver images with the MR score.

Results

41

Fifty-four Wistar rats were included in this study and sorted by their histological

degree of liver fibrosis (Table 10). However, due to the development of CCCs or low

imaging quality, the data sets of some rats were excluded. The actual number of data

sets of rats evaluated for the assessment of the degree of liver fibrosis by histological

and MR scoring, as well as for MR hemodynamic measurements is listed (Table 11).

Table 10: Rats sorted by their histological degree of liver fibrosis with corresponding

TAA exposure time


TAA exposure: 0 weeks 15 0 0 0 0 15


TAA exposure: 16 weeks 0 4 6 1 13 (4) 24

Group classification CON FIB CIR


DS=0: no fibrosis, DS=1: mild fibrosis, DS=2: moderate fibrosis, DS=3: severe fibrosis, and DS=4:


in subscript brackets. The histological results served as a basis for the group classification of the rats:

CON=control, FIB=fibrosis (any degree), and CIR=cirrhosis for the MR hemodynamic measurements.

Table 11: Number of data sets of rats evaluated for the assessment of the degree of

liver fibrosis by histological (Desmet score) and MR scoring (MR score), as well as

MR hemodynamic measurements.

CON FIB CIR n total

Histological scoring 15 26 13 54

MR scoring 12 23 13 48

MR hemodynamics 13 25 8 46

From the total of 54 rats, six were excluded from MR scoring due to low imaging quality. Eight were

excluded from MR hemodynamic measurements: Four due to low imaging quality and a further four

(from group CIR) since they had CCCs.

Results

42

3.2.1 MR Assessment of the Degree of Liver Fibrosis MR scoring of the rat livers (n=48) by two readers showed an interobserver

agreement of 64% or referring to weighted kappa analysis a substantial agreement

(ƙw=0.76). If the histological score is taken as standard, the self-established MR

score has a sensitivity of 89% (32/36) to identify diseased livers and a specificity of

100% (12/12) to identify healthy livers (Figure 5). The accurate assessment of the

degree of liver fibrosis in diseased rats, as well as the detection of CCCs, was not

possible by MR rat liver image evaluation.

Figure 5: Dot plot illustrating the assessment of the degree of liver fibrosis using

histological (Desmet score) and MR scoring (MR score)

3.2.2 Flow Velocity Patterns and Flow Curves Detected flow velocity patterns and flow curves indicate a constant portal and a

pulsatile aortic flow.

A flow velocity pattern and a flow curve (Figure 6a and 6b) for the portal vein and

abdominal aorta of an exemplary rat is shown for selected time points of a cardiac

cycle (16.5 - 115.5 ms). Flow velocities (m/min) in the flow velocity pattern (Figure

6a) are color-coded and depicted with positive values flowing towards the heart

(caudo cranially) and negative values flowing away from the heart (cranio caudally).

Flow volume rates (ml/min) in the flow curve (Figure 6b) are depicted likewise.

Results

43

Figure 6: Color-coded image displaying the flow velocities (m/min) (a) and a diagram

of flow volume rates (ml/min) (b) in the portal vein (red, yellow) and abdominal aorta

(blue) at selected time points of a cardiac cycle

3.2.3 Hemodynamic Parameters For the statistical analysis of the hemodynamic parameters, the remaining 46 rats

were classified into three groups depending on their histologically assessed degree

of liver fibrosis: CON (control, n=13), FIB (fibrosis, n=25), and CIR (cirrhosis, n=8)

(Table 11).

Rats in FIB were tested in advance to determine if TAA exposure time (12 vs 16

weeks) has any influence on the results, but for all parameters investigated only

minimal and nonsignificant differences occurred. Hence, these data were pooled in

one group.

Since the most diseased rats in CIR showed a significant reduction of 8% in body

weight compared to FIB (p=0.006) (Table 12), it was assessed if there is a correlation

between body weight and other parameters of interest. In the total group (n=46), a

poor but significant correlation between body weight and aortic flow volume rate was

found (rs=0.408, p=0.005). The correlations between body weight and portal

hemodynamic parameters (i.e. cross-sectional area, flow velocity and volume rate)

were nonsignificant.

The portal cross-sectional area (=ROI) showed no differences among groups

(p=0.622) (Table 12, Figure 7a). For the mean portal flow velocity, a significant

lowering of 21% in FIB (p=0.006) and a nonsignificant lowering of 17% in CIR

(p=0.105) was found in comparison to CON (Table 12, Figure 7b).

Results

44

Comparing FIB and CIR, the differences were nonsignificant (p=1.000). A correlation

in the total group (n=46) including portal cross-sectional area and mean portal flow

velocity revealed a significant negative correlation (rs=-0.546, p=0.001).

Portal cross-sectional area (=ROI) and the mean portal flow velocity had already

been determined for each rat’s portal vein and abdominal aorta (Table 3). The

corresponding flow volume rates were calculated according to the physical principle

‘Volume rates = Area * Velocity’. Results showed a significant reduction in mean

portal flow volume rate of 20% in FIB (p=0.009) and of 25% in CIR (p=0.024) when

compared to CON (Table 12, Figure 7c). Comparing FIB and CIR, the differences

were nonsignificant (p=1.000). Moreover, there was a trend towards a lower mean

aortic flow volume rate in CIR compared to CON and FIB, but differences among

groups were nonsignificant (p=0.101) (Table 12, Figure 7d).

To determine if portal and aortic flow volume rate are related to one another, these

two parameters were correlated in the total group. A poor but significant correlation

was found (rs=0.353, p=0.016). If healthy rats in CON were exclusively considered,

reflecting a healthy physical state, the correlation coefficient was higher than in the

total group; however, the correlation was nonsignificant (rs=0.407, p=0.168).

Results

45

Table 12: Median ± interquartile range (IQR) of body weight and hemodynamic

parameters of the groups

CON

(n=13) Median ± IQR

FIB (n=25)

Median ± IQR

CIR (n=8)

Median ± IQR

Body weight [g] 367 ± 34 375 ± 46 345 ± 27 Δ

Portal cross-sect. area [mm²] 3.1 ± 1.0 3.1 ± 0.6 2.7 ± 1.4

Portal flow velocity [m/min] 7.0 ± 1.7 5.5 ± 1.9 * 5.8 ± 2.1

Portal flow volume rate [ml/min] 20 ± 7 16 ± 3 * 15 ± 5 *

Aortic cross-sect. area [mm²] 4.6 ± 0.8 4.6 ± 1.1 4.5 ± 1.4

Aortic flow velocity [m/min] -17.1 ± 2.4 -16.7 ± 3.3 -16.2 ± 7.5

Aortic flow volume rate [ml/min] -79 ± 12 -78 ± 20 -68 ± 16

Pairwise comparisons between groups are determined according to Dunn 179. Significant differences

(p<0.05) between FIB and CON or CIR and CON are marked by an *, whereas significant differences

between CIR und FIB are marked by a Δ.

Results

46

Figure 7: Boxplots showing the distributions of portal cross-sectional area [mm2] (a),

portal flow velocity [m/min] (b), portal and aortic flow volume rate [ml/min] (c and d) in

the groups. Significant differences among groups are corrected for multiple

comparisons and denoted by *p<0.05

3.3 Invasive Hemodynamic Measurements In addition to the noninvasive MR measurements, hepatic and systemic

hemodynamic alterations induced by liver fibrosis / cirrhosis were determined by

invasive hemodynamic measurements. Since portal flow volume rate was measured

in both cases, it was possible to compare the results between invasive and

noninvasive measurements. In the same context the potential of PDE5 inhibitors in

PH management was further elucidated. PDE5 inhibitors are considered as

promising option to treat PH, initial preclinical and clinical hemodynamic studies,

however, showed different results. Therefore, additional invasive hemodynamic

measurements were performed to evaluate hepatic and systemic hemodynamic

changes, induced by acute administration of the PDE5 inhibitor sildenafil. Moreover,

based on these hemodynamic data, the effect of systemic blood pressure on portal

Results

47

blood pressure was determined. Corresponding regression analyses for the latter

were conducted by Thomas Heister (Institute of Medical Biometry and Statistics, Medical

Center, University of Freiburg).

3.3.1 Portal Flow Volume Rate Eighty-three Wistar rats were included in this study and sorted by their histological

degree of liver fibrosis (Table 13). However, due to the development of CCCs, the

data sets of 13 rats in CIR were excluded.


TAA exposure time



TAA exposure: 16 weeks 0 16 17 10 25(13) 68





in subscript brackets.The histological results served as a basis for the group classification of the rats:

CON=control, FIB=fibrosis (any degree),andCIR=cirrhosis for the invasive portal flow volume rate

measurements. However, due to the development of CCCs, the data sets of 13 rats in CIR had to be

excluded.

For the statistical analysis of the hemodynamic parameters, the remaining 70 rats

were classified into three groups depending on their histologically assessed degree

of liver fibrosis: CON (control, n=15), FIB (fibrosis, n=43), and CIR (cirrhosis, n=12).

Since the most diseased rats in CIR showed a significant reduction of 9% in body

weight compared to CON (p=0.006) and a significant reduction of 6% compared to

FIB (p=0.027) (Table 14), it was assessed if there is a correlation between body

weight and other parameters of interest. In the total group (n=70), a poor but

significant correlation between body weight and mean portal flow volume rate

https://www.imbi.uni-freiburg.de/

Results

48

(rs=0.472, p=0.001), as well as between body weight and MAP was found (rs=0.241,

p=0.044).

Results showed a significant reduction in mean portal flow volume rate of 38% in FIB

(p=0.003) and of 50% in CIR (p=0.003) when compared to CON (Table 14, Figure

8a). Comparing FIB and CIR, a significant decrease of 20% in CIR (p=0.066) was

detected. Moreover, in CIR a significant decrease in MAP of 26% compared to CON

(p=0.003) and of 19% compared to FIB (p=0.033) was found (Table 14, Figure 8b).

To determine if portal flow volume rate and MAP are related to one another, these

two parameters were correlated in the total group. A poor but significant correlation

was found (rs=0.546, p=0.001). If healthy rats in CON were exclusively considered,

reflecting a healthy physical state, the correlation coefficient was lower than in the

total group and the correlation was nonsignificant (rs=0.116, p=0.680).



CON


FIB (n=43)

Median ± IQR

CIR (n=12)

Median ± IQR

Body weight [g] 374 ± 42 363 ± 34 342 ± 28 * Δ

Portal flow volume rate [ml/min] 16 ± 4 10 ± 4 * 8 ± 2 * Δ

MAP [mmHg] 103 ± 18 97 ± 31 78 ± 42 * Δ

Pairwise comparisons between groups are determined according to Dunn 179. Significant differences

(p<0.05) between FIB and CON or CIR and CON are marked by an *, whereas significant differences

between CIR und FIB are marked by a Δ.

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49

Figure 8: Boxplots showing the distributions of portal flow volume rate [ml/min] (a)

and MAP (b) in the groups. Significant differences among groups are corrected for

multiple comparisons and denoted by *p<0.05

3.3.2 Effect of Sildenafil on Hemodynamics One hundred and ten rats were included in this study and sorted by their histological

degree of liver fibrosis (Table 15), the healthy rats in CON being Sprague Dawley

and the diseased rats in FIB and CIR Wistar rats. However, the data sets of the 2

rats having no fibrosis after 16 weeks of TAA exposure, were excluded. The data

sets of the 12 rats in CIR developing CCCs, were included to maintain a sufficiently

large group size.


TAA exposure time



TAA exposure: 16 weeks 2 7 15 7 24(12) 55






CON=control, FIB=fibrosis (any degree), and CIR=cirrhosis for the invasive hemodynamic

measurements.

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50

For the statistical analysis of the hemodynamic parameters and HR, the remaining

108 rats were classified into three groups depending on their histologically assessed

degree of liver fibrosis: CON (control, n=55), FIB (fibrosis, n=29), and CIR (cirrhosis,

n=24). Each of those had 3 subgroups which are categorized based on intervention

in the form of sodium chloride (NaCl), sildenafil 0.1 mg/kg (Sil 0.1 mg/kg) or sildenafil

1 mg/kg (Sil 1 mg/kg), which was applied in a standardized volume of 600 µl.

All parameters of interest were normalized (PVPrel, MAPrel, MFrel and HRrel) to

compensate differences in absolute values (Table 16) between healthy and diseased

rats, the time point “10 min” being taken as baseline and set to 100% since the

administration of 600 µl liquid volume into the right atrium caused parameter

variations for the next few minutes before they reached a new steady state.

Regarding the course during the measurement interval, a decrease in parameter

values was observed for all subgroups regardless of intervention. The decrease in

PVPrel (%) compared to the decrease in MAPrel (%) becoming more pronounced with

sildenafil and increasing dosage (Figure 9a). CVPrel, respiration raterel and oxygen

saturationrel remained unchanged in all subgroups (data not shown).

The effect of sildenafil was evaluated by comparing the change in parameter values

at time point “60 min” to baseline (“10 min”) (Table 17, Figure 9a and 9b).

In CON intragroup comparisons showed nonsignificant effects of sildenafil on the

parameters PVPrel (p= 0.399), MAPrel (p=0.867), MFrel (p=0.770) and HRrel (p=0.664).

In FIB as well, intragroup comparisons revealed nonsignificant effects of sildenafil on

the parameters PVPrel (p= 0.320), MAPrel (p=0.272), MFrel (p=0.133) and HRrel

(p=0.311). In CIR in contrast, sildenafil caused a trend towards a lower PVPrel, but

intragroup comparisons were nonsignificant (p= 0.088). Moreover, nonsignificant

effects on MAPrel (p=0.915) and MFrel (p=0.974) were determined, whereas for HRrel

a significant decrease of 8% (RMD) in Sil 1mg/kg was found when compared to NaCl

(p=0.024).

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51

Figure 9a: Changes of relative median (%) in PVPrel (blue) and MAPrel (red) ± 95% CI

in the subgroups. Parameters were normalized with time point “10 min” being set to

100%. Intervention in the form of sodium chloride (NaCl) or sildenafil (Sil) was

applied in a standardized volume of 600 µl.

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52

Figure 9b: Changes of relative median (%) in MFrel (green) and HRrel (orange) ± 95%

CI in the subgroups. Parameters were normalized with time point “10 min” being set

to 100%. Intervention in the form of sodium chloride (NaCl) or sildenafil (Sil) was


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53

Table 16: Median ± interquartile range (IQR) of body weight, hemodynamic parameters and HR of the subgroups at time points 0, 10,

30 and 60 min

CON FIB CIR

NaCl


Sil 0.1 mg/kg (n=18)

Median ± IQR


Median ± IQR

NaCl (n=7)

Median ± IQR


Median ± IQR

Sil 1.0 mg/kg (n=7)

Median ± IQR

NaCl (n=7)

Median ± IQR

Sil 0.1 mg/kg (n=7)

Median ± IQR


Median ± IQR

Body weight [g] 375 ± 15 370 ± 28 380 ± 27 359 ± 35 363 ± 28 361 ± 24 336 ± 40 350 ± 46 337 ± 15

PVP_0 [mmHG] 6.4 ± 0.7 6.6 ± 0.4 6.3 ± 0.7 6.0 ± 0.9 6.2 ± 1.3 6.2 ± 0.9 6.8 ± 1.6 7.5 ± 1.7 7.7 ± 1.1

PVP_10 [mmHG] 6.0 ± 0.9 6.6 ± 0.6 6.6 ± 0.9 5.7 ± 1.2 5.5 ± 1.3 5.1 ± 1.4 6.8 ± 1.4 7.1 ± 1.6 7.1 ± 2.2

PVP_30 [mmHG] 5.9 ± 0.7 6.3 ± 0.7 6.4 ± 0.8 5.3 ± 1.4 5.4 ± 1.3 4.8 ± 1.4 6.5 ± 1.2 6.6 ± 1.0 6.3 ± 1.3

PVP_60 [mmHG] 5.9 ± 0.7 6.3 ± 0.8 6.4 ± 1.1 5.1 ± 1.2 5.2 ± 1.2 4.8 ± 1.5 6.6 ± 2.0 6.3 ± 1.0 5.8 ± 1.2

MAP_0 [mmHG] 89 ± 12 106 ± 14 101 ± 28 65 ± 30 59 ± 21 58 ± 28 49 ± 19 47 ± 15 60 ± 19

MAP_10 [mmHG] 83 ± 20 94 ± 14 85 ± 27 55 ± 25 45 ± 10 42 ± 8 42 ± 11 41 ± 5 38 ± 16

MAP_30 [mmHG] 79 ± 21 88 ± 17 77 ± 21 50 ± 14 43 ± 12 37 ± 6 41 ± 12 35 ± 8 36 ± 8

MAP_60 [mmHG] 76 ± 21 82 ± 23 74 ± 28 44 ± 11 40 ± 8 37 ± 7 38 ± 3 34 ± 5 34 ± 7

MF_0 [flux] 208 ± 68 209 ± 39 206 ± 38 108 ± 46 111 ± 38 112 ± 24 117 ± 44 115 ± 21 107 ± 34

MF_10 [flux] 196 ± 65 215 ± 65 192 ± 44 109 ± 34 102 ± 43 104 ± 32 114 ± 33 106 ± 28 90 ± 22

MF_30 [flux] 195 ± 64 194 ± 57 184 ± 40 96 ± 33 103 ± 44 92 ± 19 110 ± 46 101 ± 19 89 ± 24

MF_60 [flux] 190 ± 56 190 ± 53 181 ± 45 94 ± 17 100 ± 50 85 ± 18 106 ± 52 103 ± 22 80 ± 34

HR_0 [bpm] 373 ± 40 377 ± 47 368 ± 31 320 ± 30 335 ± 30 314 ± 22 307 ± 38 308 ± 76 365 ± 28

HR_10 [bpm] 362 ± 55 383 ± 44 380 ± 50 313 ± 23 339 ± 41 351 ± 29 286 ± 33 316 ± 71 374 ± 22

HR_30 [bpm] 356 ± 57 366 ± 52 373 ± 41 291 ± 20 310 ± 43 310 ± 21 268 ± 40 313 ± 57 350 ± 36

HR_60 [bpm] 347 ± 39 359 ± 43 362 ± 49 284 ± 26 304 ± 41 295 ± 33 254 ± 36 302 ± 47 324 ± 30

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54

Table 17: Relative median of differences (RMD) (%) ± interquartile range (IQR) of hemodynamic parameters and HR in the subgroups.

Parameters were normalized with time point “10 min” being set to 100%. RMD was calculated for the 50 min interval (“60 min”

compared to baseline (“10 min”))

CON FIB CIR

NaCl

(n=19) RMD [%] ± IQR


RMD [%] ± IQR


RMD [%] ± IQR

NaCl (n=7)

RMD [%] ± IQR


RMD [%] ± IQR

Sil 1.0 mg/kg (n=7)

RMD [%] ± IQR

NaCl (n=7)

RMD [%] ± IQR

Sil 0.1 mg/kg (n=7)

RMD [%] ± IQR


RMD [%] ± IQR

PVPrel - 3 ± 7 - 6 ± 10 - 3 ± 6 - 9 ± 11 - 8 ± 8 - 7 ± 6 - 3 ± 7 - 13 ± 7 - 19 ± 26

MAPrel - 10 ± 17 - 8 ± 16 - 7 ± 19 - 21 ± 24 - 14 ± 21 - 10 ± 10 - 17 ± 16 - 14 ± 11 - 17 ± 23

MFrel - 6 ± 14 - 8 ± 16 - 5 ± 8 - 16 ± 16 - 8 ± 19 - 15 ± 10 - 8 ± 10 - 10 ± 8 - 11 ± 26

HRrel - 4 ± 6 - 4 ± 5 - 4 ± 5 - 8 ± 5 - 8 ± 14 - 12 ± 13 - 6 ± 6 - 8 ± 11 - 14 ± 10*

Significant differences (p<0.05) between Sil 0.1mg/kg and CON or Sil 1.0mg/kg and CON are marked by an *. No significant differences between Sil 0.1mg/kg

and Sil 1.0mg/kg were observed.

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55

3.3.3 Effect of MAP on PVP 1 Regarding the course of MAP and PVP of the individual rats during the measurement

interval (Figure 10), a change in MAP led to a slightly delayed change in PVP in the

same direction (decrease / increase). Hence, hemodynamic data were also used to

evaluate the effect of MAP on PVP over the first 30 minutes.

For the statistical analysis of the correlation between MAP and PVP, the 108 rats

(Table 15) were classified into nine subgroups as before (see 3.3.2).

All parameters of interest were normalized (PVPrel, MAPrel) to compensate

differences in absolute values (Table 16) between healthy and diseased rats. Since

the correlation of the two parameters was visible, particularly in the first few minutes

in which administration of 600 µl liquid volume into the right atrium caused parameter

variations, time point “0 min” was taken as the baseline and set to 100%.

Figure 10: Course of MAP (black) and PVP (blue) of an exemplary rat after sodium

chloride (NaCl) administration

In CON a significant effect of MAPrel on PVPrel (p=0.001) was found in all subgroups

(Table 18). For every 1% change in MAPrel in the NaCl subgroup, PVPrel varies by

0.35%. 40% of the variation in PVPrel within one rat can be explained by MAP. For

every 1% change in MAPrel in the subgroups Sil 0.1 mg/kg and Sil 1.0 mg/kg, PVPrel

varies by 0.37% and 0.53%, respectively. 46% and 43% of the variation in PVPrel

within one rat can be explained by MAP.

1 Data of this analysis were previously published in the medical dissertation by Adhara Lazaro: “Correlation between mean arterial pressure (MAP) and portal venous pressure (PVP) in rats” (2018).

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56

In FIB a significant effect of MAPrel on PVPrel (p=0.001) was found in all subgroups

(Table 18). For every 1% change in MAPrel in the NaCl subgroup, PVPrel varies by

0.52%. 42% of the variation in PVPrel within one rat can be explained by MAP. For

every 1% change in MAPrel in the subgroups Sil 0.1 mg/kg and Sil 1.0 mg/kg, PVPrel

varies by 0.48% and 0.61%, respectively. 46% and 43% of the variation in PVPrel

within one rat can be explained by MAP.

In CIR as well a significant effect of MAPrel on PVPrel (p=0.001) was found in all

subgroups (Table 18). For every 1% change in MAPrel in the NaCl subgroup, PVPrel

varies by 0.32%. 61% of the variation in PVPrel within one rat can be explained by

MAP. For every 1% change in MAPrel in the subgroups Sil 0.1 mg/kg and Sil 1.0

mg/kg, PVPrel varies by 0.32% and 0.39%, respectively. 40% and 23% of the

variation in PVPrel within one rat can be explained by MAP.

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57

Table 18: Regression analysis between MAPrel and PVPrel in the subgroups. Parameters were normalized with time point “0 min” being

set to 100%.

The change in PVPrel for every 1% change in MAPrel is described by the regression coefficient (s). Significant effects of MAP on PVP (p<0.05) are marked

by an *. The explained variation (%) within one rat is described by r-squared (r²).

CON FIB CIR

NaCl

(n=19) PVPrel

Sil 0.1 mg/kg (n=18) PVPrel


NaCl (n=7) PVPrel



NaCl (n=7) PVPrel



MAPrel s 0.345* 0.367* 0.532* 0.512* 0.479* 0.612* 0.319* 0.318* 0.388*

MAPrel r2 0.404 0.461 0.431 0.424 0.463 0.431 0.613 0.406 0.234

Results

58

3.4 Biochemical Investigations In order to determine alterations in serum parameters and in the key parameters of

the NO-cGMP pathway induced by liver fibrosis / cirrhosis different biochemical

analyses were conducted. The NO-cGMP pathway is a key regulator of vascular tone

and thus plays an important role in sinusoidal vasoreactivity, which is impaired in PH.

Further expertiments were performed to evaluate whether the hemodynamic

measurement and the associated procedure and / or administration of the PDE5

inhibiitor sildenafil affect those.

One component of the biochemical investigations were the immunohistochemical

stainings, which were conducted in cooperation with Birgit Hockenjos (Department of

Medicine II, Gastroenterology, Hepatology, Endocrinology, and Infectious Diseases, Medical Center,

University of Freiburg), whereas Prof. Dr. Annette Schmitt-Graeff (Institute of Clinical

Pathology, Medical Center, University of Freiburg) contributed to the diagnosing of the

stainings.

Sixty-eight Wistar rats were included in this study and sorted by their histological

degree of liver fibrosis and whether or not they underwent the hemodynamic

measurements (and the associated operative procedure), which included

administration of sodium chloride (NaCl) or sildenafil 1 mg/kg (Sil 1 mg/kg) (Table

19a-c). However, the data sets of the 6 rats having no fibrosis after 16 weeks of TAA

exposure were excluded. The data sets of the 13 rats in CIR developing CCCs were

included to maintain a sufficiently large group size.

Table 19a-c: Rats sorted by their histological degree of liver fibrosis and group

classification

a) Rats used for biochemical investigations only




Group classification CON 1 FIB 1 CIR 1

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59

b) Rats used for hemodynamic measurements (intervention: NaCl) and

biochemical investigations



Group classification -- FIB 2 CIR 2

c) Rats used for hemodynamic measurements (intervention: Sil 1mg/kg) and

biochemical investigations (cGMP only)


TAA exposure: 0 weeks 12 -- -- -- 0 12

TAA exposure: 16 weeks 2 -- -- -- 10 (6) 12

Group classification CON 3 -- CIR 3





CON=control, FIB=fibrosis (any degree), and CIR=cirrhosis for biochemical investigations

For the statistical analysis of serum parameters, hepatic gene expression of eNOS,

iNOS, PDE5, sGCa1 and sGCb1, and serum cGMP concentrations, the remaining 61

rats were classified depending on their histologically assessed degree of liver

fibrosis: CON (control), FIB (fibrosis), and CIR (cirrhosis). Depending on whether or

not they underwent the hemodynamic measurement and the respective intervention,

they were subsequently divided into seven subgroups (Table 20). In CON 3 and CIR

3, serum cGMP concentrations were investigated exclusively, while immunohisto-

chemical staining (PDE5) was performed on liver tissue samples of CON 1, CIR 1,

and CIR 2 only.

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60

Table 20: Overview subgroups

CON 1 (n=11)

FIB 1 (n=6)

CIR 1 (n=8)

FIB 2 (n=7)

CIR 2 (n=7)

CON 3 (n=12)

CIR 3 (n=10)

TAA [weeks] 0 16 16 16 16 0 16

hemodynamic measurement

no no no yes yes yes yes

intervention -- -- -- NaCl NaCl Sil 1mg/kg Sil 1mg/kg

3.4.1 Serum Parameters (Clinical Chemistry)

3.4.1.1 Effect of TAA-induced Liver Disease To investigate whether the development of liver fibrosis or cirrhosis affects serum

parameters from carotid arterial blood samples, differences among rats with healthy

(CON 1), fibrotic (FIB 1) and cirrhotic livers (CIR 1) were evaluated. Those rats have

not undergone the hemodynamic measurement and the associated operative

procedure; they were exclusively used for biochemical investigations.

In FIB 1 analysis of serum parameters showed a significant decrease of 37% for Glc

(p=0.012), and of 27% (p=0.003) for ALT, as well as a significant increase of 275%

(p=0.003) for Bil compared to CON 1.

In CIR 1 a significant decrease of 42% (p=0.003) for Glc, and of 13% (p=0.003) for

Alb, as well as a significant increase of 400% (p=0.003) for Bil was determined

compared to CON 1.

If exclusively diseased rats in FIB 1 and CIR 1 were considered, no significant

differences between subgroups were observed.

(Table 21 and 22)

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61

3.4.1.2 Influence of Hemodynamic Measurements To investigate whether the hemodynamic measurement and the associated operative

procedure leads to changes in serum parameters, differences between diseased rats

which have undergone the hemodynamic measurement (FIB 2 and CIR 2) and those

which have not undergone any (FIB 1 and CIR 1) were ascertained.

In FIB 2 analysis of serum parameters showed a significant increase of 2% (p=0.002)

for Na compared to FIB 1.

In CIR 2 a significant increase of 1% (p=0.020) for Na, and of 12% (p=0.014) for K,

as well as of 139% (p=0.012) for Crea, and a significant decrease of 36% (p=0.032)

for AP was determined when compared to CIR 1.

(Table 21 and 22)

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62

Table 21: Median ± interquartile range (IQR) of body weight and serum parameters in

subgroups

CON 1 (n=11)

Median ± IQR

FIB 1 (n=6)

Median ± IQR

CIR 1 (n=8)

Median ± IQR

FIB 2 (n=7)

Median ± IQR

CIR 2 (n=7)

Median ± IQR

body weight [g]

364 ± 22 368 ± 45 340 ± 26 359 ± 35 336 ± 40

Glc [mg/dl]

130 ± 9.0 82 ± 31 75 ± 35 76 ± 19 72 ± 34

Na [mmol/l]

144 ± 4.0 143 ± 2.5 143 ± 4.8 146 ± 4.0 145 ± 4.0

K [mmol/l]

5.4 ± 0.3 5.0 ± 0.4 5.0 ± 0.9 5.4 ± 0.2 5.6 ± 0.4

Bil [mg/dl]

0.08 ± 0.02 0.30 ± 0.20 0.40 ± 0.55 0.20 ± 0.00 0.20 ± 0.10

Crea [mg/dl]

0.32 ± 0.10 0.45 ± 0.09 0.44 ± 0.19 0.98 ± 0.51 1.05 ± 0.20

Alb [g/dl]

3.7 ± 0.2 3.3 ± 0.4 3.2 ± 0.3 3.2 ± 0.5 2.8 ± 0.4

AST [U/l]

161 ± 40 137 ± 51 136 ± 70 148 ± 263 188 ± 56

ALT [U/l]

63 ± 15 46 ± 13 53 ± 13 51 ± 61 55 ± 28

AP [U/l]

189 ± 50 152 ± 61 167 ± 67 86 ± 39 106 ± 31

Body weight and the following serum parameters were determined: glucose (Glc), sodium (Na),

potassium (K), total bilirubin (Bil), creatinine (Crea), albumin (Alb), aspartate aminotransferase (AST),

alanine aminotransferase (ALT), and alkaline phosphatase (AP).

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63

Table 22: Pairwise comparisons between subgroups are determined according to Dunn 179. Adjusted comparisons are corrected

according to Bonferroni separately for the two hypotheses, which are marked by a double line. Significant differences (p<0.05) are

marked by an *.

Glc

Na K Bil Crea Alb AST ALT AP

CON 1 vs. FIB 1

sig. (pairwise) 0.004* 0.019* 0.062 0.001* 0.068 0.039* -- 0.001* 0.190

sig. (adjusted) 0.012* 0.057 0.186 0.003* 0.204 0.117 > 0.050 0.003* 0.570

CON 1 vs. CIR 1

sig. (pairwise) 0.001* 0.058 0.223 0.001* 0.087 0.001* -- 0.072 0.468

sig. (adjusted) 0.003* 0.174 0.669* 0.003* 0.261 0.003* > 0.050 0.216 1.000

FIB 1 vs. CIR 1

sig. (pairwise) 0.496 0.564 0.480 0.605 0.807 0.270 -- 0.105 0.545

sig. (adjusted) 1.000 1.000 1.000 1.000 1.000 0.810 > 0.050 0.315 1.000

FIB 1 vs. FIB 2

sig. (pairwise) 0.853 0.001* 0.072 0.126 0.034* 0.371 -- 0.056 0.039

sig. (adjusted) 1.000 0.002* 0.144 0.252 0.068 0.742 > 0.050 0.112 0.078

CIR 1 vs. CIR 2

sig. (pairwise) 0.905 0.010* 0.007* 0.138 0.006* 0.476 -- 0.396 0.016*

sig. (adjusted) 1.000 0.020* 0.014* 0.276 0.012* 0.952 > 0.050 0.792 0.032*

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3.4.2 Gene Expression and Serum cGMP Concentrations

3.4.2.1 Effect of TAA-induced Liver Disease To investigate whether the development of liver fibrosis or cirrhosis affects hepatic

gene expression and / or serum cGMP concentrations from carotid arterial blood

samples, differences among rats with healthy (CON 1), fibrotic (FIB 1) and cirrhotic

livers (CIR 1) were determined. Those rats have not undergone the hemodynamic

measurement and the associated operative procedure; they were exclusively used

for biochemical investigations.

Gene expression became the higher the more the rats became diseased (CON < FIB

1 < CIR 1). iNOS expression was detected in diseased rats only. Analysis of serum

cGMP concentration showed no such pattern.

In FIB 1 gene expression analysis revealed significantly increased expression of

PDE5 (7.7-fold; p=0.006), and sGCb1 (2.1-fold; p=0.018) compared to CON 1. For

serum cGMP concentrations, a nonsignificant lowering of 34% (p=0.453) was found.

In CIR 1 a significantly increased expression of eNOS (2.2-fold; p=0.003), PDE5 (11-

fold; p=0.003), sGCa1 (1.7-fold; p=0.003) and sGCb1 (3-fold; p=0.003) was

measured when compared to CON 1. Moreover, there was a trend towards a

decrease in cGMP concentrations of 40% (p=0.054).

If exclusively diseased rats in FIB 1 and CIR 1 were considered, no significant

differences between subgroups were assessed.

(Table 23, 24 and 25, Figure 11a-f)

3.4.2.2 Influence of Hemodynamic Measurements To investigate whether the hemodynamic measurement and the associated operative

procedure leads to changes in hepatic gene expression and / or serum cGMP

concentrations from carotid arterial blood samples, differences between diseased

rats which have undergone the hemodynamic measurement (FIB 2 and CIR 2) and

those which have not undergone any (FIB 1 and CIR 1) were determined.

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65

In FIB 2 gene expression analysis showed a significant decrease in expression of

eNOS when compared to FIB 1 (p=0.036). For serum cGMP concentrations, a

nonsignificant lowering of 32% (p=1.000) was found.

In CIR 2 as well a significantly decreased expression of eNOS was exposed

compared to CIR 1 (p=0.004), whereas for serum cGMP concentrations a

nonsignificant elevation of 12% (p=0.192) was found.

(Table 23, 24 and 25, Figure 11a-f)

3.4.2.3 Effect of Sildenafil on Serum cGMP Concentrations To investigate whether sildenafil affects serum cGMP concentrations, differences

between rats which received no intervention (CON 1 and CIR 1) or sodium chloride

(NaCl) (CIR 2), and those which received sildenafil 1mg/kg (Sil 1.0 mg/kg) (CON 3

and CIR 3) were determined.

In healthy rats, comparing CON 3 to CON 1, sildenafil led to a significant increase of

64% (p=0.036) in serum cGMP concentrations. A significant increase of 85%

(p=0.027) could also be found in diseased rats when CIR 3 is compared to CIR 1.

Furthermore, in diseased rats, the effect of sildenafil and NaCl on serum cGMP

concentrations was analyzed. By comparing CIR 3 and CIR 2, sildenafil induced a

nonsignificant increase of 65% (p=1.000).

(Table 24 and 25, Figure 11f)

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66

Figure 11: Dotplots showing the distributions of hepatic gene expression of the

enzymes eNOS (a), iNOS (b), PDE5 (c), sGCa1(d) and sGCb1(e), as well as

distributions of serum cGMP concentrations [pmol/ml] (f) in the subgroups. Significant

differences among subgroups are corrected for multiple comparisons separately for

the three hypothesis and denoted by *p<0.05. Gene expression levels are given as

fold expression compared to CON 1. Since iNOS expression in CON 1 was below the

detection limit, it was set to “1.0”.

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67

Table 23: Mean ± standard deviation (SD) of hepatic enzyme gene expression in the

subgroups. Fold expression is referring to CON 1. qRT-PCRs were performed in

duplicates; thus, values are based on mean values of duplicates.

CON 1 (n=11)

Mean ± SD

FIB 1 (n=6)

Mean ± SD

CIR 1 (n=8)

Mean ± SD

FIB 2 (n=7)

Mean ± SD

CIR 2 (n=7)

Mean ± SD

eNOS [fold exp.] 1.0 ± 0.4 1.5 ± 0.3 2.2 ± 1.0 0.9 ± 0.3 1.0 ± 0.4

iNOS [fold exp.] n.d.1 4.6 ± 3.0 5.3 ± 2.3 10.0 ± 3.0 15.6 ± 8.7

PDE5 [fold exp.] 1.0 ± 1.0 7.7 ± 0.9 11.0 ± 3.1 7.6 ± 2.6 6.1 ± 2.8

sGCa1 [fold exp.] 1.0 ± 0.3 1.4 ± 0.3 1.7 ± 0.4 1.1 ± 0.3 1.4 ± 0.4

sGCb1 [fold exp.] 1.0 ± 0.5 2.1 ± 0.3 3.0 ± 1.3 1.1 ± 0.3 1.4 ± 0.5

1 Since iNOS expression in CON 1 was below the detection limit, it was set to “1.0”

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68

Table 24: Median ± interquartile range (IQR) of serum cGMP concentrations in the

subgroups. ELISAs were performed in duplicates; thus, values are based on mean

values of duplicates.

CON 1 (n=11)

Median ±

IQR

FIB 1 (n=6)

Median ±

IQR

CIR 1 (n=8)

Median ±

IQR

FIB 2 (n=7)

Median ±

IQR

CIR 2 (n=7)

Median ±

IQR

CON 3 (n=12)

Median ±

IQR

CIR3 (n=10)

Median ±

IQR

cGMP [pmol/ml]

152 ±

86

100 ±

68

91 ± 22

68 ±

105

102 ±

134

249 ±

153

168 ± 52

Results

69

Table 25: Pairwise comparisons between subgroups are determined according to

Dunn 179. Adjusted comparisons are corrected according to Bonferroni separately for

the three hypotheses, which are marked by a double line. Significant differences

(p<0.05) are marked by an *.

eNOS

iNOS PDE5 sGCa1 sGCb1 cGMP

CON 1 vs. FIB 1

sig. (pairwise) 0.024* -- 0.002* 0.065 0.006* 0.151

sig. (adjusted) 0.072 -- 0.006* 0.195 0.018* 0.453

CON 1 vs. CIR 1

sig. (pairwise) 0.001* -- 0.001* 0.001* 0.001* 0.018*

sig. (adjusted) 0.003* -- 0.003* 0.003* 0.003* 0.054

FIB 1 vs. CIR 1

sig. (pairwise) 0.280 0.732 0.201 0.194 0.267 0.495

sig. (adjusted) 0.840 1.000 0.603 0.582 0.801 1.000

FIB 1 vs. FIB 2

sig. (pairwise) 0.018* 0.461 0.982 0.327 0.119 0.748

sig. (adjusted) 0.036* 0.992 1.000 0.654 0.238 1.000

CIR 1 vs. CIR 2

sig. (pairwise) 0.002* 0.079 0.050 0.171 0.146 0.096

sig. (adjusted) 0.004* 0.158 0.100 0.342 0.292 0.192

CON 1 vs. CON 3

sig. (pairwise) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 0.012*

sig. (adjusted) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 0.036*

CIR 1 vs. CIR 3

sig. (pairwise) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 0.009*

sig. (adjusted) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 0.027*

CIR 2 vs. CIR 3

sig. (pairwise) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 0.446

sig. (adjusted) n.m.2 n.m.2 n.m.2 n.m.2 n.m.2 1.000

2 not measured

Results

70

3.4.3 Immunohistochemical Staining (PDE5)

To investigate if the PDE5 overexpression in diseased rats found on the gene

expression level can be verified on the protein expression level, liver tissue samples

of some rats, which have been included in the previous gene expression analyses,

were stained immunohistochemically. Therefore, 4 tissue samples of rats with

healthy (CON1) and cirrhotic livers (CIR 1 and CIR 2) were randomly chosen for

PDE5 staining and cell counts.

Immunohistochemical staining revealed a markedly increased PDE5 protein

expression (brown) in diseased rats in CIR 1 und CIR 2 compared to healthy rats in

CON 1. In contrast, no differences were distinguished between diseased rats which

have undergone the hemodynamic measurement and the associated operative

procedure (CIR 2) and those which have not undergone any (CIR 1).

Considering the distribution in healthy rats in CON 1, PDE5 protein was

predominantly expressed by perivenular hepatocytes around the central vein (zone

3) and to a lesser extent by perisinusodial cells in the parenchyma. In contrast, in

diseased rats in CIR 1 and CIR 2 hepatic zoning got lost, whereas bands of fibrous

connective tissue (septa) were formed. PDE5 protein was expressed nonzonally by

perisinusoidal cells in the parenchyma, but was also present in fibrous connective

tissue (Figure 12).

Subsequent microscopic quantification revealed 3 stained cells per HPF in healthy

rats in CON1, and in diseased rats 23 stained cells per high power field (HPF) for

CIR 1, and 22 stained cells per HPF for CIR 2. This corresponds to a 7.7-fold

increase in CIR 1 and a 7.3-fold increase in CIR 2. Exclusively stained cells in the

parenchyma were included in cell counts, whereas PDE5 staining around the central

vein (CON 1) and in fibrous connective tissue (CIR 1 and CIR 2) was not considered.

Results

71

CON 1 CIR 1 CIR 2

Figure 12: Immunohistochemical PDE5 staining (brown) of liver tissue samples of

rats with healthy (CON 1) and cirrhotic livers (CIR 1 and CIR 2). Rats in CON 1 and

CIR 1 were used for biochemical investigations only, whereas the rats in CIR 2

underwent the hemodynamic measurement and the associated operative procedure.

PC: perisinusoidal cell; HC: hepatocyte, CV: central vein.

Discussion

72

4. Discussion 4.1 Evaluation of the TAA Model The aim of this part of the study was to establish and evaluate the animal model of

TAA-induced liver fibrosis / cirrhosis by oral and weight-adapted TAA administration

described previously by Li et al 163. Relative portions of rats with fibrotic and cirrhotic

livers, the presence of cholangiocellular carcinoma (CCCs), and mortality induced by

TAA administration were quantified.

Two outbred rat strains were investigated: Sprague Dawley and Wistar. In contrast to

inbred strains, which are characterized by a minimal genetic variation, outbred strains

show a broad genetic variation. Outbred strains are used to imitate the genetic

variation, which is a natural and unavoidable occurrence in any population. In

general, outbred strains are considered to be multipurpose models, but their main

fields of applications are pharmaceutical and toxicological studies.

Independent of the strain rats showed various symptoms during TAA exposure time,

mostly associated with liver fibrosis / cirrhosis. However, their level of discomfort was

classified as minor.

A frequently observed symptom was reduced body weight increase, which is in good

agreement with data from previous studies using TAA-treated rodents 160,167.

Moreover, reduced water intake was oberserved once TAA was added into the

drinking water. Three rats shared a cage and a water bottle, making it impossible to

determine each rats’ individual water intake. Regarding the amount of water in the

bottles, it was, however, obvious that the rats noticed the TAA additive and drank

less than untreated rats. As a consequence, 2.5 ml liquid sweetener per 750 ml

drinking water was added to mask the bitter taste of TAA. Assuming an normal

average daily water intake of 30 ml per rat 180 and a TAA dosage of 0.03%, each rat

consumed an average of 9 mg TAA and 0.1 ml liquid sweetener per day. The liquid

sweetener comprised steviol glycosides which, according to the European Food

Safety Authority (EFSA), represented no toxicity or carcinogenicity potential 181. By

adding liquid sweetener, the rats’ water intake increased once again, but was still

slightly lower than the water intake of untreated rats. Furthermore, previous

preclinical studies reported that after an initial peak of serum transaminases (AST

Discussion

73

and ALT) within the first four weeks of oral TAA administration, serum transaminase

levels returned to baseline values regardless of TAA exposure time and degree of

liver fibrosis 160,178. Also in the current study serum parameters analysis revealed the

absence of an increase in transaminase levels after 16 weeks of TAA treatment

regardless of the rats’ degree of liver fibrosis (see 3.4.1).

Considering genetic susceptibility to liver disease, strain differences have been

reported for different models, but for TAA-induced liver disease no systematic

analysis on strain-specific susceptibility has been conducted so far 160,182–187. In the

current study the TAA model was initially established in Sprague Dawley rats. On the

basis of the histological assessment of their degree of liver fibrosis, it has been

shown that a TAA exposure time of 12 weeks, as recommended by Li et al 163, was

insufficient to induce severe liver disease. However, it is a delicate matter to prolong

TAA exposure time since it has been known that TAA is a carcinogen and can lead to

the development of cholongiocellular carcinoma (CCCs) and hepatocellular

carcinoma (HCCs) after chronic administration 167,188. In the current study, the

incidence of Sprague Dawley rats with cirrhotic livers increased with prolonged TAA

exposure time (15 to 24 weeks), but all of them had CCCs simultaneously (HCCs

were not detectable with the staining used). These findings contradict the results of

Yeh et al 167, who also used the model of oral TAA administration to induce liver

disease in Sprague Dawleys, but applied a constant dosage of 0.03%. According to

them, development of CCCs started after 16 weeks, whereas the development of

HCCs occurred earliest after 20 weeks of TAA administration.

Having found that in Sprague Dawley rats the development of liver cirrhosis is

principally associated with the emergence of CCCs, a second rat strain was tested.

Therefore, Wistar rats, the rat strain originally used by Li et al 163, was chosen. This

time TAA exposure time was fixed at 12 and 16 weeks. However, histological

assessment of the degree of liver fibrosis showed that in Wistar rats a TAA exposure

time of 12 weeks is also insufficient to induce severe liver disease. Barely half of the

rats treated with TAA for 16 weeks developed cirrhosis. Thereof half had CCCs

simultaneously. These findings are in good accordance with the experiences made

by Laleman et al 189. According to their results in Wistar rats, TAA exposure time

should be prolonged to 18 weeks to obtain a homogenous and reproducible model of

Discussion

74

liver cirrhosis when following the protocol described by Li et al 163. Moreover, they

observed the development of CCCs and HCCs after 18 weeks of TAA treatment.

Considering mortality in the current study, there was a minor loss of 2 rats out of 142

that died within the first week of TAA administration, probably due to TAA-induced

acute liver failure 76,165,190.

In terms of limitations in the study design, the use of the model of oral TAA

application via the drinking water implicates an unstandardized intake of TAA since

the rats´ water intake varies and can not be controlled. This form of application could

also lead to gastrointestinal tract irritation 159 and thus influence splanchnic

circulation. Moreover, the model should ideally resemble human alcoholic or

hepatitis-associated liver fibrosis / cirrhosis, but this was not evaluated in the current

study.

In conclusion, the model of TAA-induced liver disease by oral and weight-adapted

TAA administration previously described by Li et al 163 caused tolerable physical

burden and minor mortality. A prolonged TAA exposure time of 16 to 18 weeks is

recommendable to attain a high incidence of rats developing a cirrhotic liver.

However, since the degree of liver fibrosis / cirrhosis varies even when TAA

exposure time is standardized, it is reasonable to make a histological evaluation. For

the simultaneous development of CCCs seems to be more common among Sprague

Dawley rats. The frequent presence of CCCs should be considered in biochemical,

hemodynamic and all other studies using this model.

4.2 Noninvasive Hemodynamic Measurements The aim of this part of the study was to noninvasively evaluate hepatic and systemic

hemodynamic changes induced by liver fibrosis / cirrhosis in rats with healthy, fibrotic

or cirrhotic livers by MR measurements. Alterations in the parameters portal cross-

sectional area, portal flow velocity, portal flow volume rate, and aortic flow volume

rate were determined. Moreover, MR data were used to test whether the degree of

liver fibrosis can be assessed using a self-established MR score.

Discussion

75

The idea for this study was born, since at present, the reference standard for

hemodynamic evaluation in clinical practice is the assessment of portal blood

pressure by measuring the HVPG 191,192. However, this technique is an invasive

procedure and involves some degree of inconvenience and risk for the patients.

Moreover chronic liver diseases, as well as the induced hemodynamic alterations

(mainly PH) are heterogeneous and dynamic conditions 38,81. Consequently, a

noninvasive and repeatable assessment of hemodynamics is warranted.

To date, there are few noninvasive modalities that quantify blood flow in clinical

practice. Most of them are based on ultrasound (US) and MR. Both of these

modalities have been investigated whether measurements of portal parameters (e.g.

flow velocity or volume rate) may correlate with the degree of PH with variable results 193–195. Even if technical modalities have substantially advanced over the last

decades, low reproducibility and lack of standardized examination protocols are still

mentioned as limitations of noninvasive techniques 196,197. Thus, preclinical studies

are warranted in order to evaluate and further optimize recently developed clinical

imaging techniques, and to aid in biomedical and pharmaceutical research.

Multi-dimensional phase-contrast MR (PC-MR) imaging is a preferred technique to

determine blood flow in preclinical and clinical studies. Some preclinical

hemodynamic studies in small laboratory animals using multi-dimensional PC-MR

imaging have already been performed, mostly investigating cardiovascular

hemodynamics 198–201. Only a few focus on portal hemodynamic changes induced by

liver cirrhosis 202–204. To address this lack of portal hemodynamic data, two-

dimensional PC-MR (2D PC-MR) imaging, a technique being well-established,

validated and in use for clinical practice and research 205, was chosen to determine

different hemodynamic parameters in this study. Thereby, hemodynamic changes in

the portal vein and the abdominal aorta, as well as morphological changes of the liver

tissue were focused on.

For the MR scoring of the rat livers, an MR approach and a self-established MR

score were used. Therefore, histological criteria 82 were adapted for MR imaging to

determine morphological alterations induced by liver inflammation, fibrosis, or

cirrhosis including liver tissue density, nodules and liver surface 206. From the

diseased rats 11% (4/36) were scored false negative, but all of the healthy rats were

identified as such. The accurate assessment of the degree of liver fibrosis in

Discussion

76

diseased rats, as well as the detection of CCCs was not possible by MR rat liver

image evaluation. Histological evaluation is required instead. Thus, even if histology

remains to be the reference standard, the evaluation of MR rat liver images can be

helpful to noninvasively discriminate between a healthy and a diseased liver,

consequently reducing the unnecessary use of laboratory animals.

Looking at the detected flow velocity patterns for the portal vein and the abdominal

aorta during a cardiac cycle, these reflect the physiological conditions as one would

expect in a venous or arterial vessel: the flow velocity pattern in the portal vein

appears constant, whereas the observed flow velocity pattern in the abdominal aorta

reveals a pulsatile structure. These flow velocity patterns are consistent with those

previously demonstrated by Wang et al 207 for a phantom and preclinical in vivo study

in rats using PC-MR. However, compared to Wang et al, substantially higher field

strength was used in this study (9.4 T vs. 1.5 T); thus, it can be assumed that at least

the same or, even more likely, a better signal-to-noise ratio for the detection of PC-

MR data, and therefore more robust hemodynamic parameters were achieved.

Considering the hemodynamic alterations induced by the chronic treatment of the

hepatotoxic agent TAA, the most distinct alteration in diseased rats in comparison

with healthy rats was the marked reduction of portal flow velocity and volume rate.

Results indicate that in the model of TAA-induced liver disease, the development of

fibrosis is sufficient to cause a significant decrease in portal flow velocity and volume

rate. In contrast, the development of cirrhosis caused no further significant decrease

in portal flow velocity and volume rate.

However, from these results it cannot reliably be concluded that the total liver

perfusion in the diseased rats is diminished. A reduction of portal perfusion can at

least partially be compensated (25%-60%) by an increase of arterial hepatic

perfusion – the so-called hepatic buffer response 7. Parameters of the hepatic artery

were not obtained in this study. Since aortic flow volume rate in diseased rats

remained constant, the reduction of portal flow is not a consequence of a reduced

aortic flow, but most likely an indicator of increased intrahepatic resistance.

The findings of a reduced portal flow velocity and volume rate in diseased rats are

contrary to the results given by Wang et al 207. In their preclinical study, differences in

portal flow volume rate (portal flow velocity data were not shown) between CON and

Discussion

77

FIB, as well as between CON and CIR were nonsignificant. They also investigated

Wistar rats, but used the model of CCl4-induced liver disease.

According to clinical studies, portal perfusion in humans is altered depending on the

degree of liver fibrosis and with advanced PH the portal blood flow may even become

reversed 107,208,209. Furthermore, the portal cross-sectional area may increase with

rising PVP, but the determination of the portal cross-sectional area does not seem to

be a reliable diagnostic indicator for PH 210–212.

In the present study, the portal cross-sectional area of the rats remained constant

even in severely diseased rats. It could be likely that the increase in PVP in the

tested animal model is not pronounced enough to cause a dilation of the portal vein.

In further experiments, it should be clarified to what extent PVP in diseased rats is

indeed enhanced. Moreover, an investigation should be made in which morphological

or biochemical modifications are responsible for the unexpected finding of the strong

portal hemodynamic effect even in rats with fibrotic livers.

In terms of limitations in the study design, the anesthesia, which is unavoidable,

could have added more variability and uncertainty to the measurements 213. The

physical states of the rats during the MR measurement were not perfectly equal for

all of them even if every possible precaution was taken to keep the conditions and

their physical state stable. The procedure itself - the preparation of the rats and the

MR measurement - lasted about 1 h per rat and was performed at different times of

the day.

The main technical causes of errors in the 2D PC-MR technique were possibly the

2D plane application and positioning in the complex liver vasculature, as well as the

Venc setting 205,214.

In conclusion, in rats with fibrotic or cirrhotic livers, markedly reduced portal flow

velocity and volume rate were found compared with rats with healthy livers.

Moreover, the evaluation of the MR rat liver images of the livers enables

differentiation between healthy and diseased livers.

Discussion

78

4.3 Invasive Hemodynamic Measurements 4.3.1 Portal Flow Volume Rate The aim of this part of the study was to invasively evaluate hepatic and systemic

hemodynamic changes induced by liver fibrosis / cirrhosis in rats with healthy, fibrotic

or cirrhotic livers. Alterations in the parameters portal flow volume rate and mean

arterial pressure (MAP) were determined. Portal flow volume rate was measured with

a flow probe, whereas MAP was measured by a pressure transducer. Moreover,

results for the portal flow volume rate between noninvasive and invasive

measurements were compared.

Considering the hemodynamic alterations induced by the chronic treatment of the

hepatotoxic agent TAA, the most distinct alteration in diseased rats in comparison

with healthy rats was the marked reduction of portal flow volume rate and MAP.

Equivalent to the results of the noninvasive measurements, the current findings also

indicate that in the model of TAA-induced liver disease, the development of fibrosis is

sufficient to cause a significant decrease in portal flow volume rate.

However, in contrast to the noninvasive measurements, in which no significant

differences between diseased rats in FIB and CIR were observed, a significant

reduced portal flow volume rate in CIR was detected by invasive measurements

when compared to FIB. Moreover, absolute values for the portal flow volume rate

measured in the groups were around 20 to 47% higher in the noninvasive

measurements. A comparison of methods was not performed due to several reasons:

First, at least partially different rats were used for the noninvasive and invasive

measurements, as well as a different kind of anesthesia. Second, even if rats were

investigated twice, noninvasive and invasive measurements were not performed on

the same day, but with a 2- or 3-day recovery period in between. Third, it is beneficial

to have an arterial reference parameter since changes in the arterial circulatory

system could lead to changes in the venous circulatory system but, whereas for the

noninvasive measurement the abdominal aorta flow volume rate was referred to,

MAP was used as an arterial reference parameter for the invasive measurement

since the anatomical closeness to the vena cava does not allow the positioning of a

flow probe at the abdominal aorta. Hence, the arterial reference parameters differed

between noninvasive and invasive measurements and were not comparable.

Discussion

79

In terms of limitations in the study design, the anesthesia as well as the operative

conditions, which are unavoidable, could have added more variability and uncertainty

to the measurements 213. The physical states of the rats during the MR measurement

were not perfectly equal for all of them even if every possible precaution was taken to

keep the conditions and their physical state stable. The procedure itself – the

preparation of the rats and the portal flow volume rate measurement - lasted about 1

to 1.5 h per rat and was performed at the same time of the day.

The main technical causes of errors in the measurement technique were the size and

positioning of the ultrasonic transit time flow probe, and the loss of the applied

ultrasound gel due to body fluids.

In conclusion, in rats with fibrotic or cirrhotic livers, markedly reduced portal flow

volume rate and MAP were found compared with rats with a healthy liver.

Comparing the results for the portal flow volume rate between noninvasive and

invasive hemodynamic measurements, the same pattern of a liver disease-induced

decrease was found, but absolute values were not equivalent.

4.3.2 Effect of Sildenafil on Hemodynamics The aim of this part of the study was to evaluate hepatic and systemic hemodynamic

changes induced by the administration of the PDE5 inhibitor sildenafil in rats with

healthy, fibrotic or cirrhotic livers. Therefore, additional invasive hemodynamic

measurements were performed to determine the acute effects of administration of

either sodium chloride (NaCl) or sildenafil (Sil 0.1 mg/kg or Sil 1.0 mg/kg) on the

parameters portal venous pressure (PVP), mean arterial pressure (MAP),

microvascular flow (MF), and heart rate (HR) over 50 minutes. PVP, MAP and HR

were measured using pressure transducers, whereas MF was determined with a

microvascular flow probe.

Considering the hemodynamic alterations induced by acute sildenafil administration,

a dose-dependent effect was observed. The most distinct alteration was observed

after high-dosage administration (1mg/kg) in rats with cirrhotic livers, which led to a

trend towards a decreased PVP and was associated with a significant reduction of

HR and a nonsignificant lowering of MAP.

Discussion

80

Looking at PVP, a decrease among all subgroups was determined. In rats with

healthy and fibrotic livers, the decrease was nonsignificant regardless of intervention.

However, in rats with cirrhotic livers sildenafil administration led to a trend towards a

decreased PVP, PVP decreasing more prominently after high-dosage administration.

But while healthy rats were considered to have a physiological PVP, it is hard to

make any statement about how pronounced the elevation in PVP in diseased rats

really was, particularly in association with their markedly lower baseline MAP values

in comparision with healthy rats. On the other hand, the latter could indicate that

those rats have been in a hyperdynamic circulatory state, which is typically a

consequence of PH 74.

For MAP and MF, a nonsignificant decrease was also observed among all subgroups

regardless of intervention. Changes in MAP were measured as administration of

PDE5 inhibitors has been reported to be associated with a decrease in MAP, which

could influence PVP. The decrease in MF, however, occured unexpectedly since

administration of PDE5 inhibitors should lead to sinusoidal vasodilation and hence

increased MF. It might be speculated that the effect of sildenafil on MF has been

covered by the decrease in the residual hemodynamic parameters. Moreover, a

nonsignificant decrease in HR was found among almost all subgroups regardless of

intervention, exclusively in rats with a cirrhotic liver, which received the high-dosage

sildenafil administration (1mg/kg), HR decreased significantly.

In a preclinical study, investigating the effects of PDE5 inhibitors in healthy rats, it

was shown that after acute administration of either sildenafil or vardenafil (1-100

µg/kg, intravenous) PVP remained unchanged or showed a trend towards a decrease 215. A dosage of 10 µg/kg, which was most effective, led to a significant increase in

MF, but at the same time to a significant reduction in MAP. HR remained unaltered

regardless of the dosage applied.

In contrast in the model of BDL-induced liver disease, acute administration of

sildenafil (0.01-10 mg/kg, intravenous or intramesenteric) led to a dose-dependent

increase in PVP and a significant decrease in MAP 216. Diseased rats also tended to

have lower baseline MAP values compared to sham-operated rats, which is

consistent with our observations. The same model was used in another study

considering the effect of a chronic one-week administration of sildenafil (0.25 mg/kg,

2 x daily, oral) 217. Whereas in sham-operated rats no effect was found, in diseased

Discussion

81

rats a nonsignificant decrease in PVP and portal perfusion pressure, and a significant

increase in MF were determined. These findings coincide with the results of a further

study, which also used the model of BDL-induced liver disease, showing that after

chronic administration of the PDE5 inhibitor udenafil (1, 5 or 25 mg/kg; 1 x daily,

oral) for 3 weeks PVP decreased by approximately 30% 218.

In a clinical trial on patients with liver cirrhosis and a significantly elevated HVPG,

acute administration of sildenafil (50 mg, oral) caused no changes in HVPG and HR,

whereas MAP decreased 219. These findings were confirmed by a subsequent study

with a similar study design in patients with compensated cirrhosis (Child A) 220. In a

further study investigating patients with compensated and decompensated liver

cirrhosis (Child A-C), acute administration of sildenafil (50 mg, oral) showed no effect

on PVP, MAP and HR, but induced a significant reduction in intrahepatic resistance 221. Moreover, the effect of an acute and chronic one-week administration of udenafil

(12.5 -100 mg, 1 x daily, oral) was tested in patients with decompensated liver

cirrhosis (Child A-B), a dosage of 75 mg or 100 mg being found to be most effective 222. After one hour HVPG was lowered by 25% (75 mg) or 17% (100 mg)

respectively, whereas after one week HVPG was reduced by 14% (75 mg) or 17%

(100 mg) respectively. By combining the results of these two dosages a significant

reduction in HVPG of 19% in the acute setting and of 16% in the chronic setting was

found, while HR remained unchanged. However, reduced HVPG was associated with

a significant lowering of MAP of 4% in the acute setting and of 6% in the chronic

setting which, according to the authors, was well tolerated by the patients.

In a further pilot study on patients with compensated liver cirrhosis (Child A), acute

administration of vardenafil (10 mg, oral) caused a decrease in HVPG and

intrahepatic resistance in four out of five patients, whereas HR remained constant 223.

Moreover, a recent case-report about a female patient with compensated liver

cirrhosis (Child A) revealed promising results for the chronic use of PDE5 inhibitors 224. In the acute setting, administration of vardenafil (5 mg, 1 x daily, oral) led to a

reduction of HVPG by 13%. For the maintenance medication over the following eight

years with tadalafil (5 mg, 1 x daily, oral), similar effects on HVPG were described.

MAP also slightly decreased in the acute as well as in the chronic treatment phase,

but changes were reported to be clinically irrelevant.

Discussion

82

Taken together, data from preclinical and clinical studies provided promising results

regarding the effect of PDE5 inhibitor administration on PVP (or HVPG), even though

results were partly variable. The associated decrease in MAP seemed to be

tolerable. Only in the current study there was a significant decrease in HR

determined after acute high-dosage sildenafil administration (1mg/kg), which

contradicts all other existing data and needs to be clarified.

Believing the hypothesis that the presence of high PDE5 expression in a particular

tissue should predict the effect of a PDE5 inhibitor 225, the current finding of hepatic

PDE5 overexpression in diseased rats (see 3.4) reveals the need for further

investigations in order to better evaluate drug- and dose-dependent effects of PDE5

inhibitors in the acute and chronic setting. It might be assumed that in particular a

prolonged chronic administration of PDE5 inhibitors could be beneficial to counteract

PDE5 overexpression. However, potential therapy effect heterogeneity should always

be considered since the degree of liver fibrosis / cirrhosis and PH could also

influence the effectiveness of a therapy 58.

In terms of limitations in the study design, administration of 600 µl liquid volume into

the right atrium most likely caused transient cardiac decompensation probably due to

volume overload, which was accompanied by variations in PVP, MAP, MF and HR

during the first minutes of the hemodynamic measurements. After approximately 10

minutes a new steady state was reached. Therefore, timepoint “10 min” was taken as

the baseline value for further calculations. However, that measure did not seem to be

ideal for the NaCl subgroup in FIB since relative median of differences for the

parameters seemed to be markedly lower compared to those in CON and CIR. This

might have influenced the results of intragroup comparisons in FIB. Moreover, the

decrease in parameter values during the measurement interval, which occurred

consistently among all subgroups even in the absence of a vasoactive drug (NaCl

subgroups), indicates that the anesthesia as well as the operative conditions, which

are unavoidable, could have added more variability and uncertainty to the

measurements 213,226. This disruptive factor occurred although pentobarbital was

used for anesthesia, which is described to be one of the best forms of anesthesia for

the performance of invasive blood pressure measurements in rats 227.

The physical states of the rats during the hemodynamic measurement were not

perfectly equal for all of them regardless of every possible precaution taken to keep

the conditions and their physical state stable. The procedure itself – the operative

Discussion

83

procedure of the rats and the hemodynamic measurement - lasted about 2.5 to 3 h

per rat and was performed at the same time of the day. The use of different rat

strains as well as the fact that some of the diseased rats had CCCs could have

influenced the results of the hemodynamic measurements.

The main technical causes of errors in the hemodynamic measurement technique

were the location and a potential clogging of the catheters.

In conclusion, acute high-dosage sildenafil administration (1mg/kg) led to a trend

towards decreased PVP in rats with cirrhotic livers, which were characterized by

hepatic PDE5 overexpression, and furthermore to a significant lowering of HR and a

nonsignificant reduction of MAP. Hence, PDE5 inhibitors might be a promising

adjunct in PH therapy and should be investigated further.

4.3.3 Effect of MAP on PVP The aim of this part of the study was to determine the influence of systemic blood

pressure on portal blood pressure. Therefore, the effect of MAP on PVP over the first

30 minutes was evaluated based on the available hemodynamic data (see 3.3.2).

Considering the course of MAP and PVP of the individual rats, a change in MAP led

to a slightly delayed change in PVP in the same direction (decrease / increase). This

was best visible within the first minutes of measurements, in which hemodynamic

parameters were “manipulated” unintentionally by the bolus injection of 600 µl liquid

volume into the right atrium.

The results showed that the effect of MAP on PVP was significant in all subgroups

regardless of intervention. For every 1% change in MAPrel ,PVPrel varies by 0.32% to

0.61%, which implies a distinct relationship between the change in MAP and the

change in PVP.

These findings are of particular importance regarding the current pharmaceutical

options in PH therapy. As mentioned before (see 2.5.7), the functional component of

increased intrahepatic resistance can be influenced positively, either by a decrease

in intrahepatic vascular tone, a decrease in splanchnic vasodilation, or ideally both 67.

In general, NSBBs, the current cornerstone in pharmaceutical PH therapy, block the

binding of catecholamines, such as norepinephrine and epinephrine, to beta1 and

Discussion

84

beta2 adrenergic receptors 33,228. To lower PVP (HVPG), NSBBs act in two different

ways: whereas a beta1 blockade reduces portal inflow by decreasing the heart rate

and cardiac output, a beta2 blockade leads to unopposed alpha1 activity resulting in

splanchnic vasoconstriction 90. The latter however, seems to be the essential mode

of action 68. In comparison, beta1-selective beta blockers, which only lower cardiac

output, show a less pronounced effect on PVP (HVPG) than NSBBs 229–233.

Unfortunately, not only NSBBs, but most of the drugs used in PH therapy are

vasoactive and do not only affect hepatic, but to some extent systemic

hemodynamics as well. Statins exclusively, best known for their cholesterol lowering

effects, are able to decrease intrahepatic resistance without affecting systemic

hemodynamics simultaneously 66. However, since PH per se is already associated

with splanchnic vasodilation and the development of a hyperdynamic circulatory

state, a further reduction of systemic blood pressure induced by vasoactive drugs is a

matter of concern, especially in advanced stages of the disease 234.

Even the use of NSBBs has been a matter of ongoing controversy due to potential

hemodynamic, but also nonhemodynamic adverse effects in patients with liver

cirrhosis 68,228,235–240. Those side effects led to treatment termination in approximately

15% of patients, whereas another 15% a priori had contraindications to the use of

NSBBs 241. Moreover, 30 to 40% of patients did not show a portal hemodynamic

response to NSBB administration 242,243. In addition, the therapeutic window

hypothesis resulting from a meta-analysis by Krag et al 33 limits their use as well. The

therapeutic window hypothesis states that in patients with liver cirrhosis NSBBs

improve survival only during a certain time window in the disease. This window

opens when medium to large esophageal varices occur 244–246, and closes when a

very advanced stage of liver cirrhosis is reached 87,229,247–249. Since outside this

therapeutic window NSBBs have been described to be potentially ineffective or even

detrimental, it is obvious that novel therapeutic strategies are needed.

Nevertheless, NSBBs might be an excellent example to investigate the correlation

between systemic and hepatic hemodynamics. In a recent review by Tripathi 250, the

changes in HVPG and the associated changes in MAP after acute and chronic

administration of propaponol and carvediol in patients with PH were presented.

These data also suggest a correlation between systemic and hepatic hemodynamics

since after acute or chronic administration of propranolol, a conventional NSBB,

Discussion

85

reduction of HVPG, but also MAP was less pronounced than after acute or chronic

administration of carvediol, a NSBB with an additional alpha1 blocking capacity.

However, whereas those results are based on measurements at selected time points

only, results of the current experimental study were based on continuous invasive

hemodynamic measurements over 30 min, and therefore more robust results were

achieved. Another advantage of this study was the unintentional “manipulation” of

hemodynamics within the first minutes, which best illustrated the correlation between

MAP and PVP. Moreover, even in the absence of a vasoactive drug (NaCl

subgroups) the course of hemodynamic parameters revealed a decrease not only in

MAP but also in PVP, suggesting a correlation between these two parameters, but on

the other hand, indicating that the sildenafil-induced decrease in PVP was also at

least partly a consequence of the lowering of MAP. However, although no intragroup

comparisons were performed, results showed that for a 1% change in MAP the

change in PVP was highest in the subgroups, in which the high dosage of sildenafil

(Sil 1 mg/kg) was applied, which might imply a partly liver-specific effect. Hence,

future studies evaluating the effect of vasoactive drugs on PVP (HVPG) should

distinguish between the portion induced by the decrease in MAP, and the portion that

indeed reflects a liver-specific mode of action. Ideally, the effect on PVP should be

markedly higher than the effect on MAP.

In terms of limitations, reference can be made to those listed above (see 4.3.2).

In conclusion, there is a distinct correlation between MAP and PVP, i.e. between

systemic blood pressure and portal blood pressure. This should be considered in all

other studies evaluating the effect of vasoactive drugs on PVP (HVPG).

4.4 Biochemical Investigations The aim of this part of the study was to evaluate changes biochemically in the key

parameters of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway

induced by liver fibrosis / cirrhosis in rats with healthy, fibrotic or cirrhotic livers. The

NO-cGMP pathway is a key regulator of vascular tone and thus plays an important

role in sinusoidal vasoreactivity, which is impaired in PH. Alterations in hepatic gene

expression of the enzymes endothelial and inducible NO synthase (eNOS, iNOS),

soluble guanylyl cyclase subunit a1 and b1 (sGCa1, sGCb1) and phospho-

Discussion

86

diesterase 5 (PDE5) were analyzed by qRT-PCR, whereas changes in serum cGMP

concentrations from carotid arterial blood samples were determined using ELISA.

Additionally, blood samples were used to determine serum parameters (clinical

chemistry) for the sake of completeness, but the results will not be discussed further.

In this context, it was also evaluated whether the hemodynamic measurement and in

particular the associated operative procedure affected gene expression or serum

cGMP concentrations. Moreover, the effect of sildenafil administration (1.0 mg/kg) on

serum cGMP concentrations was determined. The main finding of gene expression

analyses was finally confirmed by immunohistochemical staining.

Considering the alterations in the NO-cGMP pathway induced by the chronic

treatment of the hepatotoxic agent TAA, distinct alterations in diseased rats in

comparison with healthy rats were observed. In terms of hepatic gene expression, an

iNOS up-regulation and a marked PDE5 overexpression were detected. The less

pronounced increase in the expression of the residual genes, i.e. eNOS, sGCa1 and

sGCb1, might represent a compensatory mechanism to balance PDE5 over-

expression. The latter was confirmed by immunohistochemical investigation of PDE5

protein expression, which furthermore revealed a loss of hepatic zoning. Serum

cGMP concentrations were slightly decreased in diseased rats, but high-dosage

sildenafil administration (1mg/kg) nearly led to renormalization. Finally, a significant

decrease in eNOS gene expression was detected due to the hemodynamic

measurement and the associated operative procedure.

Regarding hepatic eNOS gene expression, a significant elevation in rats with cirrhotic

livers was found in the current study, whereas eNOS elevation in rats with fibrotic

livers was significant in nonadjusted pairwise comparisons only. As described above

(see 2.5.7.3), other preclinical and clinical studies investigating eNOS gene and / or

protein expression showed inhomogeneous results, including enhanced, unchanged,

or diminished eNOS expression, whereas a down-regulation of eNOS activity, in

particular in SECs, has been consistently described in the context liver cirrhosis 63,76,132–139. Moreover, results revealed that iNOS gene expression was absent in

healthy rats, but up-regulated in diseased rats, which is in good agreement with

findings from other preclinical and clinical studies 251–253. Since iNOS can be

expressed potentially by all hepatic cell types, its activity might vary in dependency

with its localization. The cell-specific role of iNOS-derived NO and its potential impact

Discussion

87

on vascular regulation needs to be clarified further (see 2.5.7.3). So far, up-regulated

iNOS protein expression has been described as not causing vasodilation 100, but

instead has been associated with intrahepatic microvascular dysfunction in some

other animal studies investigating endotoxemia and steatosis 112,254. These

observations make sense in that eNOS and iNOS compete for their common cofactor

BH4, meaning iNOS up-regulation could lead to reduced eNOS activity and hence

HSC activation 255,256.

Looking at sGC, the results showed a slightly increased gene expression in the

current model of TAA-induced liver cirrhosis, which coincides with former preclinical

studies reporting elevated sGC protein expression in the model of CCl4-induced 257

and BDL-induced liver cirrhosis 217. Furthermore, in both models a markedly elevated

expression of PDE5 protein was shown by western blot analyses 257,217, which was

validated by the current immunohistochemical PDE5 staining in the TAA model. In

contrast, sGC activity was found to be significantly decreased in the model of BDL-

induced liver cirrhosis 258, whereas data for PDE5 activity are lacking. Increased sGC

activity, however, occurred regardless of the amount of NO available in the liver,

indicating that with increasing intrahepatic NO deficiency the effect on vascular

regulation might be magnified by reduced cGMP generation 258.

In terms of protein distribution in healthy livers, eNOS has been described to show a

nonzonal distribution in human livers, eNOS being expressed predominantly by

hepatocytes, but also by ECs of hepatic arteries, terminal venules, sinusoids and

biliary epithelium 137, whereas iNOS is normally absent in healthy livers. In rat livers,

sGC showed a zonal distribution, whereby in the parenchyma almost all HSCs

expressed sGC, while the number of sGC-expressing HSCs decreased towards the

central vein 106. No sGC has been expressed in the innermost region around the

central vein, in which in the current study an accumulation of PDE5, expressed by

perivenular hepatocytes, was found.

The opposing enzyme zoning of sGC and PDE5 within a healthy hepatic lobule could

represent a regulatory mechanism to control sinusoidal cGMP concentrations.

Whereas cGMP generation in the parenchyma is maintained by high sGC expression

to ensure appropriate sinusoidal vasoreactivity, it is attenuated towards the central

vein due to reduced sGC expression. The latter, together with the increased PDE5

Discussion

88

expression around the central vein, might serve to inactivate excess cGMP in the

blood before it finally passes from the intrahepatic into the extrahepatic vasculature.

In the context of liver cirrhosis, loss of hepatic zoning and formation of bands

composed of fibrous connective tissue (septa) was observed, leading to altered

enzyme distribution. In diseased human livers, not only eNOS-, but also iNOS protein

showed nonzonal distribution within the hepatic lobule 137,252. Furthermore, Mc

Naughton et al 137 found a translocation of eNOS from hepatocytes to hepatocyte

nuclei. This phenomenon of translocation to cell nuclei has also been described

under other pathological conditions for enzymes and molecules, such as NOS (all

isoforms), sGC and cGMP 259. Data for sGC distribution are lacking, but for PDE5

results of the current study revealed that its expression in diseased rats is not only

markedly up-regulated, but postponed towards the parenchyma and fibrous

connective tissue (septa) (CIR 1 and CIR 2). In the parenchyma, PDE5 was

expressed by different perisinusoidal cells, predominantly HSCs (and / or

myofibroblasts), but also likely by macrophages and SECs. Which cell types in the

fibrous connective tissue express PDE5 has yet to be clarified.

In general, it seems like hepatic zoning is an underrated topic regarding the

pathophysiology of PH, although the concept of hepatic zoning itself and functional

differences between zones is nothing new as exemplified by hepatic enzymes

involved in different metabolic pathways 260,137. For hepatocytes it has also been

described that their functions and gene profiles depend on their location within the

hepatic lobule 261. Consequently, it is obvious that correct hepatic zoning is required

to ensure physiological liver functions, including adequate NO generation and / or

subsequent downstream signaling. Liver cirrhosis, however, is associated with loss of

enzymatic and metabolic zoning due to structural modifications of the liver

architecture. Resulting alterations in expression, activity and distribution of key

enzymes involved in the NO-cGMP pathway and their specific role in the

pathophysiology of PH have never been investigated systemically and should be

clarified further.

Referring to intrahepatic cGMP concentrations, it might be assumed that these

should be decreased in liver cirrhosis due to hepatic PDE5 overexpression, which

leads to an enhanced hydrolysis of cGMP into inactive GMP. In the current study, a

trend towards decreased cGMP concentrations in carotid arterial serum was indeed

Discussion

89

found, but most clinical studies detected increased cGMP concentrations in

association with liver cirrhosis in arterial as well as portal venous blood 262–264. These

opposing results need further clarification, but it might be that cGMP concentrations

depend on the location blood samples are taken from. In the current study, blood

samples were taken from the carotid artery since the blood volume from the portal

vein would not have been sufficient for all serum analyses. One could also speculate

that the rats developed pulmonary hypertension secondary to liver cirrhosis. In turn,

pulmonary hypertension leads to increased PDE5 expression in lung vascular

smooth muscle cells 265,225, which could have contributed to the reduction of cGMP

concentrations in the blood before it eventually reached the carotid artery. After acute

high dosage sildenafil administration (1mg/kg, intravenous) a significant increase in

serum cGMP concentrations in rats with healthy and cirrhotic livers was revealed by

current results. This is in good agreement with findings from a clinical study by Lee et

al 221, showing a significant increase in intraheptic NO and cGMP concentrations

after acute administration of sildenafil (50 mg/d, oral) in patients with liver cirrhosis.

These data were furthermore supplemented by a preclinical study by Lee et al 217 in

the model of BDL-induced liver cirrhosis, in which a one-week administration of

sildenafil (0.25 mg/kg, 2 x daily, oral) led to increased sGC and simultaneously

decreased PDE5 expression, accompanied by a reduction of PVP and portal

perfusion pressure, and a significant increase in MF.

Moreover, further experiments were performed in the current study to evaluate

whether the hemodynamic measurement and the associated operative procedure

affected gene expression or serum cGMP concentrations. A significant decrease in

eNOS gene expression was detected that might have influenced hepatic

hemodynamic parameters. This finding should be considered in all other invasive

hemodynamic studies.

In terms of limitations in the study design, qRT-PCR data does not allow any

conclusion about the enzymes’ activity. Moreover, cGMP concentration determined in

carotid arterial serum does not necessarily reflect cGMP concentrations in portal

venous serum or intrahepatic cellular cGMP concentrations. Carotid arterial serum

was taken to ensure an adequate amount of serum for serum parameter analyses

and ELISA.

Discussion

90

For the microscopic quantification of the PDE5, stained liver tissue samples only

stained cells in the parenchyma were counted (see 3.4.3).

In conclusion, this part of the study contributes to the understanding of the

pathophysiology of PH and particularly its functional component. Not only marked

alterations in the key parameters of the NO-cGMP pathway, a key regulator of

vascular tone, but also loss of hepatic zoning were found in association with liver

cirrhosis. These changes support the hypothesis that sinusoids remain in a

contractile state, thereby contributing to PH.

4.5 Concluding Remarks The ongoing interest in PDE research since their discovery coincides with the

development of their inhibitors 122. The fact that PDEs exist ubiquitously in every cell

in the body, but with distinct cellular and subcellular distribution of the 11 PDE

families, provided new opportunities for selective therapeutic targets 154. For diseases

with an underlying vascular impairment, notably PDE5 has been described to

represent a promising target due to its presence in vascular smooth muscle cells and

in platelets, and its specific hydrolysis of cGMP 266.

Regarding the historical development of PDE5 inhibitors, sildenafil synthesized by

Pfizer was the first potent und selective PDE5 inhibitor that was finally marketed for

the therapy of erectile dysfunction 156, but zaprinast was the first compound ever

described for selective inhibition of PDE5 267. In vitro studies investigating human

corpus cavernosum tissue showed a sildenafil effect, which was around 240-fold

more potent at inhibiting PDE5 than zaprinast 268. Later on, two further PDE5

inhibitors, vardenafil and tadalafil, were developed and also approved for treatment of

erectile dysfunction and pulmonary hypertension 117,269,270,154. But whereas PDE5

inhibitors had been successfully launched in the therapy of erectile dysfunction and

pulmonary hypertension, their use in the management of PH still needs approval.

For this reason, in the current study the potential of PDE5 inhibitors in PH therapy

was further elucidated in the animal model of TAA-induced liver fibrosis/ cirrhosis. As

a basis, liver disease-induced hemodynamic changes in this model were determined,

before additional hemodynamic measurements were conducted to evaluate the

changes induced by the administration of the PDE5 inhibitor sildenafil. Moreover,

Discussion

91

biochemical analyses of alterations in the key parameters of the NO-cGMP pathway

were performed to extend the general understanding of the pathophysiology of PH,

emphasizing on the functional component. In summary, current findings suggest that

administration of PDE5 inhibitors might at least partly correct the intrahepatic

dysregulation of the NO-cGMP pathway and the associated changes in hepatic

hemodynamics. Hence, PDE5 inhibitors could present a promising adjunct in PH

therapy.

As a future perspective, the use of PDE5 inhibitors as an antifibrotic drug should also

be taken into account since a preliminary preclinical study showed that chronic

administration of the PDE5 inhibitor udenafil over 3 weeks exhibited antifibrotic

effects, probably due to HSC deactivation 218. Equivalent to the effects induced by

chronic administration of PDE5 inhibitors, chronic administration of sGC activators

also led to a decrease in PVP and antfibrotic effects in some initial preclinical studies 91,271,272. Thus, a combined therapy of PDE5 inhibitors and sGC activators could also

represent a favorable adjunct in PH therapy. Another interesting approach for a

future use of PDE5 inhibitors is the sinusoidal pressure hypothesis, which states that

an elevation of sinusoidal pressure is the major upstream event that initiates fibrosis 21. This hypothesis contradicts the commonly accepted opinion that pressure

changes are exclusively a consequence of liver cirrhosis, but assuming it turns out to

be true, the potential of PDE5 inhibitors would be further extended.

When it comes to clinical use of PDE5 inhibitors however, differences in clinical

pharmacology should be considered. For sildenafil, vardenafil and tadalafil

differences regarding pharmacokinetics and pharmacodynamics has been well

described in a review by Mehrotra et al 273. In short, sildenafil and vardenafil are very

similar in terms of their chemical structure, while tadalafil has a markedly different

structure 274. These chemical similarities and differences are reflected in the clinical

pharmacokinetics and pharmacodynamics of these compounds, which lead to

substance-specific properties 273,274. Appreciation of the latter is needed to ensure a

rational dosage and compound selection based on the individual needs of the patient 275. Moreover, treatment with PDE5 inhibitors can implicate adverse events, such as

headache, flushing, dyspepsia, rhinitis, and visual disturbances 155,276,154,277. The

latter were reported in particular in association with sildenafil and vardenafil

administration, and are most likely caused by their nonselectivity towards PDE6, an

Discussion

92

enzyme located in the retina 278,279,154. Tadalafil, on the other hand, shows a clearly

higher selectivity towards PDE5 relative to PDE6, which might explain the lower

frequency of visual disturbances associated with tadalafil administration 273,277. In

general, however, the use of PDE5 inhibitors for the treatment of erectile dysfunction

or pulmonary hypertension has been reported to be safe, effective and well-tolerated 280,270,277,281. Should they become approved prospectively for the treatment of PH in

liver cirrhosis patients, it should be considered that drug safety in this particular

setting is a more delicate matter. Since marked changes in terms of drug disposition,

metabolism, excretion and elimination might occur as a consequence of liver

cirrhosis, it can be challenging to determine how best to prescribe drugs, including

PDE5 inhibitors, or to predict drug-drug interactions in these patients 282–284.

Materials and Methods

93

5. Materials and Methods 5.1 Materials 5.1.1 Chemicals, Reagents and Other Matters

Chemicals / Reagents Manufacturer

b-mercaptoethanol (98+%)

EDTA (ethylenediamine tetraacetic

acid disodium) (99+%)

Entellan® mounting medium

ethanol (100%)

formalin solution

(neutral buffered, 10%)

hematoxylin

Histoacyrl®

InvitrogenTM SYBR® Green

phosphate buffered saline (PBS)

stevia liquid sweetener

thioacetamide

tris (trishydroxymethylamio-

methane) (99.8+%)

Tween® 20 solution

Sigma-Aldrich, Schnelldorf, Germany

Serva Electrophoresis, Heidelberg, Germany

Merck Chemicals, Darmstadt, Germany

Honeywell, Morris Plains, New Jersey



B. Braun Melsungen, Melsungen, Germany

Thermo Fisher Scientific, Waltham,

Massachusetts

Oxoide, Hampshire, England

Borchers fine food, Oyten, Germany



PanReac AppliChem, Darmstadt, Germany


94

5.1.2 Anaesthetics and Drugs

Anaesthetics / Drugs Manufacturer

Forene®

Heparin sodium (25000 I.E./5 ml)

Jonosteril®

Pentobarbital sodium

Pancuronium bromide (2mg/ml)

Revatio® (0.8mg/ml)

sodium chloride (0,9%)

AbbVie, Wiesbaden, Germany

ratiopharm, Ulm, Germany

Fresenius Kabi, Bad Homburg, Germany

Fagron, Barsbüttel, Germany

Inresa Arzneimittel, Freiburg, Germany

Pfizer, Berlin, Germany


5.1.3 Antibodies, Kits, Primer, and Probes

Antibodies / Kits / Primer / Probes Manufacturer

anti-PDE5a-antibody (ab64179)

cDNA synthesis kit

cGMP ELISA kit (ab133052)

Dako EnVision® +, System-HRP

(DAB) kit

dNTP mix (10mM each)

primer

RNeasy® Plus Mini kit

Taq DNA polymerase kit

(Taq DNA polymerase: 500 units)

Abcam, Cambridge, UK


Massachusetts

Abcam, Cambridge, UK

Dako, Glostrup, Denmark


Massachusetts

Microsynth, Balgach, Switzerland

Qiagen, Hilden, Germany

InvitrogenTM, Thermo Fisher Scientific,

Waltham, Massachusetts


95

5.1.4 Consumables

Consumables Manufacturer

Bepanthen® eye and nose cream

catheter

(Tygon® R3607, ID 1.14 mm)

catheter PE-10

(PE, ID 0.28mm)

catheter PE-50

(Portex®, ID 0.58mm)

Cellstar® serological pipette

(5ml, 10ml, 50ml)

Cellstar® centrifuge tube

(15ml, 50ml)

cotton swab

culture dish (sterile, ID 60mm)

Discofix® C three-way tap

electrode gel

Eppendorf® reaction vessel

(1.5ml)

face mask

Feather® standard scalpel (sterile)

Foliodress® head cover

gauze compress (sterile)

Graseby® respiration sensor

gigasept® FF(new) disinfection

Bayer, Leverkusen, Germany

IDEX Health & Science, Wertheim, Germany

Becton Dickinson Primary Care Diagnostics,

Sparks, Maryland

Smiths medical International, Kent, UK

Greiner Bio-One, Frickenhausen, Germany


neoLab Migge, Heidelberg, Germany

Carl Roth, Karlsruhe, Germany


Gello Geltechnik, Ahaus, Germany

Eppendorf, Hamburg, Germany

3M Health Care, St. Paul, Minneapolis

pfmmedical, Osaka, Japan

Hartmann, Heidenheim, Germany

Fuhrmann, Much, Germany

Medicare Health & Living, Kilmacanogue,

Ireland

Schülke & Mayr, Norderstedt, Germany


96

Infuvalve® non-return valve

KendallTM neonatal ECG

electrodes H207PG

Kodan® tincture disinfection

(forte, colorless)

LightCycler® 480, 96 well plate

LightCycler® 480, cover sheeting

Omnifix® F Solo syringes (1ml)

Omnifix® Solo syringes

(5ml, 10ml)

Original-Perfusor® syringes (50ml)

Original-Perfusor® line (2m)

Mini-Spike® filter (green)

mirco tube (1.5ml)

MoliNea® operation pad

Microtouch® latex cloves

(powder-free)

Microtouch® Nitratex® nitrile

cloves (powder-free)

Parafilm M®

pipette tips (10µl)

pipette tips (diverse)

reaction vessel (1.5ml)

sample tube

(2ml, DNA-, DNase-, RNA-free)

silicone hose (ID 5mm)


Covidien-Medtronic, Minneapolis, Minnesota

Schülke & Mayr, Norderstedt, Germany

Roche, Basel, Switzerland







Sarstedt, Nümbrecht, Germany

Paul Hartmann, Heidenheim, Germany

Ansell, Brussels, Belgium

Ansell, Brussels, Belgium

Bemis, Neenah, Wisconsin

Biozym Scientific, Oldendorf, Germany

Mettler-Toledo Rainin, Oakland, California



Ketterer & Liebherr, Freiburg, Germany


97

Seraflex® (EP 1.5 / USP 4/0)

screw caps with sealing ring

(yellow, DNA-, DNase-, RNA-free)

screw caps (white)

Sterican® cannula (22G, 30G)

surgical instruments

ultrasound gel Caleo

Versatus® peripheral venous

catheter (26G)

QIAshredder

QIAxpert slide

Serag-Wiessner, Naila, Germany


Sarstedt, Nümbrecht, Germany


Aesculap, Tuttlingen, Germany

Caesar & Loretz, Hilden, Germany

Terumo, Eschborn, Germany



5.1.5 Apparatus

Apparatus Manufacturer

Data acquisition system

(HSE-USB-HAEMODYN)

DPC MicroMix 5 shaker

ECG and respiration monitoring

and gating system Model 1030

Eppendorf® table centrifuge

5417C

Eppendorf® table centrifuge

5424R

Eppendorf® Thermomixer

Compact

Eppendorf® Multipipette® plus

Hugo Sachs Elektronik - Havard Apparatus,

March-Hugstetten, Germany

DPC systems, Benbrook, Texas

SA instruments, Stony Brook, New York






98

Eppendorf® pipettes (diverse)

freezer (-20°C)

freezer (-80°C)

hair clipper / trimmer QC5115

Heraeus® Megafuge® 1.0

universal centrifuge

Ice-maker

IKA® magnetic mixer

(COMBIMAG RET)

isoflurane vapor 19.3

LightCycler® 480

laser doppler blood flow monitor

DRT4 with a Titanium tipped low

profile disc probe type DP8C

liquid nitrogen container (TR11)

microwave

MouseOx®Plus pulse oximeter

system

MR scanner BioSpec 94/21 URS

(preclinical, 9.4T)

operating light KL 1500 LCD

operation table rat type 872H with

homeothermic controller type 874

(230 vac)

oven 400 HY-E

Perfusor® fm syringe pump


Liebherr, Ochsenhausen, Germany

Heraeus, Hanau, Germany

Philips, Singapore, Singapore


Massachusetts

Hoshizaki Europe, Amsterdam, Netherlands

IKA-Werke, Staufen, Germany

Drägerwerk, Lübeck, Germany


Moor Instruments, Devon, UK

KGW-Isotherm, Karlsruhe, Germany

Siemens, Munich, Germany

Starr Life Sciences, Oakmont, Pennsylvania

Bruker, Ettlingen, Germany

Schott, Mainz, Germany



Bachofer, Reutlingen, Germany



99

Pipetboy pro (pipetting aid)

Pipetman® pipettes (diverse)

pressure infusion cuff (500ml)

pressure transducer ATP300

(arterial)

pressure transducer P75 type 379

(venous)

QIAxpert spectrophotometer

quadrature volume rat coil

BioSpin MRI (Item: RF RES 400 1

H 112/072 QUAD TR AD)

rodent ventilator type 7025

Rotiolabo® Economy magnetic

bars

TAM-A plugsys transducer

amplifier module type 705/1

Transonic® animal research

flowmeter T206 series with

perivascular flow probe type 2.5S

Vortex-Genie2

water recirculator

µQuantTM spectrophotometer

Zeiss Axioplan microscope

Integra Biosciences, Biebertal, Germany

Gilson, Middelton, Wisconsin

Droh, Mainz, Germany







Ugo Basile, Gemonio, Italy

Carl Roth, Karlsruhe, Germany



Transonic Systems, Ithaka, New York

Scientific Industries, Bohemia, New York

supplied from Bruker, Ettlingen, Germany

Bio Tek Instruments, Bad Friedrichshall,

Germany

Carl Zeiss Microscopy, Göttingen, Germany


100

5.1.6 Software

Software Manufacturer

HSE-Basic Data Acquisition

Software (BDAS) 1.5

KC4

LightCycler® 480 Software 1.5

MatLab® 14b

ParaVision 5.1

PC-sam 32

SPSS® software 23.0 / 24.0

STATA® software 14

QIAxpert Software 2.2.0.21



Bio Tek Instruments, Bad Friedrichshall,

Germany


MathWorks, Natick, Massachusetts


SA instruments, Stony Brook, New York

IBM, Armonk, New York

StataCorp LLC, Lakeway Drive, Texas


5.1.7 Animals

Animals Manufacturer

Sprague Dawley rats

Wistar rats

Charles River, Sulzfeld, Germany

Charles River, Sulzfeld, Germany


101

5.2 Methods

5.2.1 Laboratory Animals The laboratory animal research protocol was approved by the local institutional

animal care and use committee (Regierungspräsidium Freiburg, ref. no.: G-13/89)

Animal care was performed in accordance to the rules and regulations of the German

animal protection law and the animal care guidelines of the European community

(2010/63/EU). A total of 275 male rats, specifically 147 Sprague Dawley and 128

Wistar rats (Charles River) were studied (Table 26). All of them were clearly

recognizable from their permanent and unique identifiers using an ear punch code.

Rats were housed in individually ventilated cages in a laboratory animal facility and

received daily human care. Their body condition was documented at least three

times a week according to a self-established score sheet (see 7.1). All rats had free

access to food and water and were exposed to a 12:12-h light–dark cycle at an

ambient temperature of 22-25 °C.

Before starting any experiments, the rats were allowed to acclimatize to the ambient

conditions for at least one week.

5.2.2 Induction of Liver Disease with TAA 133 rats were left untreated, whereas 142 rats received thioacetamide (TAA) (Sigma-

Aldrich) to induce liver disease (Table 26). The protocol of liver fibrosis / cirrhosis

induction described previously by Li et al 163 was used. TAA was administered orally

via drinking water for 12 to 24 weeks. 2.5 ml liquid sweetener (stevia liquid

sweetener) was added per 750 ml drinking water to mask the bitter taste of TAA.

Starting with an initial dosage of 0.03% TAA (30 mg TAA / 100 ml) in the first week,

TAA administration was continued with an individual TAA dosage adjusted weekly

according to each rat’s body weight change. If a rat gained or lost more than 20 g

body weight per week, the dosage was increased or decreased by 0.015%

accordingly. An increase of the TAA dosage by 0.015% was done in rats with an

overall body weight increase of more than 60 g.


102

Table 26: Number of untreated and TAA-treated rats sorted by strain

Strain Untreated TAA-treated n total

Sprague Dawley 106 41 147

Wistar 27 101 128

n total 133 142 275

5.2.3 Noninvasive Hemodynamic Measurements 5.2.3.1 MR Scanning Before MR scanning was started rats were fasted for 1.5 h to avoid prandial effects

on portal flow parameters. Anesthesia was initiated in an animal induction chamber

using a mixture of 3% isoflurane (Forene®) and 97% oxygen. It was maintained with

an animal nose mask applied with a mixture of 1.5% isoflurane (Forene®) and 98.5%

oxygen at a flow rate of 0.6 l/min. Eye cream (Bepanthen® eye and nose cream) was

applied on the eyes of the rats to prevent desiccation. ECG electrode pads

(KendallTM neonatal ECG electrodes H207PGT) with applied conductive gel

(electrode gel) were fixed on the forepaws, and a respiration sensor (Graseby®) on

the abdomen. Both were connected with a monitoring and gating system (Model

1030). ECG and respiration rate (spontaneous breathing) were continuously

monitored by the corresponding software (PC-sam 32). The body temperature was

not determined, but a warm water recirculator (supplied from Bruker) was used to

keep it stable at 37 ± 0.5 °C during the measurement. Then rats were scanned using

a 9.4 T preclinical scanner (BioSpec 94/21 URS), a dedicated quadrature volume rat

coil with an inner diameter of 68 mm (BioSpin MRI, Item: RF RES 400 1 H 112/072

QUAD TR AD) and the corresponding software (ParaVision 5.1). (Figure 13)


103

Figure 13: Preparation of the rat for the MR measurements and insertion into

MR scanner

The following scanning protocol was tested for the evaluation of MR rat liver images

to assess the degree of liver fibrosis and to determine the cross-sectional areas and

mean flow velocities of the rats’ portal vein and abdominal aorta:

To get a morphological overview for the planning of the measurements, several

localizers were used in multiple orientations (Figure 1). Then an ECG- and

respiratory-gated T1-RARE axial sequence was acquired to determine the

morphological alterations induced by liver inflammation, fibrosis, or cirrhosis. These

included liver tissue density, nodules and liver surface. Parameters for the T1-RARE

were FoV: 4.5 x 6 cm, MTX: 256 x 336, TE/TR: 8.87 ms / 1555 ms, slice thickness: 1

mm and spatial resolution: 0.0176 x 0.0179 cm/pixel.

The T1-RARE was also used for the planning of the two flow-sensitive 2D PC-MR

sequences measuring the hemodynamic parameters in the portal vein and the

abdominal aorta in a single slice perpendicular to the respective vessels (Figure 14).


104

Figure 14: T1-RARE images displaying the positioning of the PC-MR slice

orthogonally to the portal vein (a and b) and the abdominal aorta (c and d) of a rat on

the coronal (a and d) and sagittal (b and c) reference scans

The 2D PC-MR technique uses the fact that spins dephase in the presence of a field

gradient to produce a contrast between stationary tissues and flowing blood (Figure

15). Initially a reference scan is performed, in which all spin phases are in the same

position (flow-compensated image). Subsequently, the spin phase is manipulated by

a bipolar gradient pulse, such that the phase shifts of stationary spins are

compensated and the phase shifts of moving spins are proportional to the flow

velocity (flow-encoded image) 285. The faster the spins are moving the greater is their

phase shift and thus their phase angle (ϕ). The sensitivity to slow or fast flows is

determined by the velocity encoding (VENC), a user-defined parameter which

describes the amplitude, duration, and spacing of the bipolar gradient. The phase-

contrast image is generated by subtraction of these two sets of phase information

(flow-encoded data - flow-compensated data). The remaining phase difference (Δϕ)

can then be used for voxel wise calculation of flow velocities 286. Hyperintense voxels

(bright, white) represent a high flow velocity in the positive direction, whereas

hypointense voxels (dark, black) represent a high flow velocity in the opposite

(negative) direction. Stationary spins in stationary tissue with no net spin phase and a

flow velocity of zero are illustrated as mean gray areas. Scattered areas represent

irregular flow or noise.


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Figure 15: Principle of 2D PC-MR Figure reprinted with permission of the Korean Society of Radiology.

Original source: H .Ha et al 2016: “Hemodynamic Measurement Using Four-Dimensional

Phase-Contrast MRI: Quantification of Hemodynamic Parameters and Clinical Applications”

Parameters of the axial flow-sensitive 2D PC-MR sequences were FoV: 4.5 x 6 cm,

MTX: 388 x 512, TE/TR/FA: 5 ms / 16.5 ms / 70°, slice thickness: 2.5 mm, spatial

resolution: 0.0116 x 0.0156 cm/pixel for the portal vein and FoV: 4.5 x 6 cm, MTX:

388 x 512, TE/TR/FA: 5 ms / 16.5 ms / 70°, slice thickness: 7.5 mm and spatial

resolution: 0.0116 x 0.0117 cm/pixel for the abdominal aorta.

Velocity encoding (Venc) settings were preset following literature values 82,202,

optimized in test measurements, and fixed at 18 m/min for the portal vein and 72

m/min for the abdominal aorta for the final experiment.

5.2.3.2 Data Acquisition / Postprocessing Data were taken directly from the software (ParaVision 5.1). The flow-sensitive 2D

PC-MR data were postprocessed by a blinded preclinical imaging expert (10 years of

experience) with a homebuilt analysis tool (MatLab® 14b), including noise filtering,

flow-compensated flow-encoded


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correction for eddy currents and Maxwell terms, as well as velocity antialiasing 287.

2D PC-MR slices which were positioned orthogonally to the portal vein and the

abdominal aorta enabled the selection of the region of interest (ROI) of the two

vessels. Since ROI is equivalent to the vessel’s cross-sectional area, it was multiplied

by the corresponding flow velocity to calculate the flow volume rate (‘Volume rate =

Area * Velocity’) of the portal vein and the abdominal aorta for a cardiac cycle.

5.2.3.3 MR Assessment of the Degree of Liver Fibrosis Rat livers were scored via T1-RARE cross-sectional images based on morphological

hallmarks such as increased nodularity and irregular tissue appearance. The self-

established MR score is derived from the semiquantitative histological five-level

Desmet score (see 5.2.7.1). Only a four-level scoring system was used for the MR

score, as the difference between the first two levels described in the Desmet score is

not detectable with an MR approach. A blinded preclinical imaging expert performed

the evaluation of the MR rat liver images with the MR score (Figure 16): MR score=0:

no visible irregularities, MR score=1: minor irregularities and small nodular structures,

MR score=2: more prominent irregularities, solitary medium sized to large nodular

structures, and MR score=3: severe irregularities and prominent nodular structures

throughout the liver.

An independent and blinded radiologist repeated the MR scoring of the rat livers to

assess interobserver variability.


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Figure 16: T1-RARE images showing morphological hallmarks of the MR score, such

as increased nodularity and irregular tissue appearance (arrows) with MR score=0:

no visible irregularities (a), MR score=1: minor irregularities and small nodules (b),

MR score=2: more prominent irregularities and medium-sized nodules (c), and MR

score=3: severe irregularities and prominent nodules (d)

5.2.4 Invasive Hemodynamic Measurements 5.2.4.1 Operative Procedure Before the invasive hemodynamic measurements were started rats were fasted for

1.5 h to avoid prandial effects on portal flow parameters. If rats have already passed

the MR measurements, they were again invasively measured two or three days after

scanning. Anesthesia was initiated in an animal induction chamber using a mixture of

3% isoflurane (Forene®) and 97% oxygen. It was maintained by an intraperitoneally

injected bolus of 0.3 - 0.4 ml pentobarbital [125 mg/ml] (pentobarbital sodium). After

having verified the depth of anesthesia, rats were shaved (hair clipper / trimmer

QC5115) and fixed on a homeothermic controlled operating table (Typ 872H), which

kept body temperature stable at 37 ± 0.5 °C. Vital parameters (i.e. heart and

respiration rates (HR), oxygen saturation) were determined by a pulse oximeter

(MouseOx®Plus) which was fixed on a hind paw.


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After thorough disinfection (Kodan®) of the front neck region, a tracheotomy was

performed and a tracheal cannula was inserted. Since rats were mechanically

ventilated [50 breaths/min] (Rodent Ventilator Typ 7025), a muscle relaxation was

induced by intraperitoneal injection of 0.5 ml pancuronium [0.4 mg/ml] (pancuronium

bromide) to prevent spontaneous breathing.

To monitor central venous pressure (CVP) the right external jugular vein was

exposed and cannulated with PE-10 tubing (Becton Dickinson), which was positioned

near the right atrium. A second PE-10 tubing was inserted and was used to

compensate evaporative losses during the surgical procedure by a continuous

infusion of isotone electrolyte solution [1 ml/h] (Jonosteril®). The electrolyte solution

was enriched with pentobarbital [15 mg/ml] to ensure continuous anesthesia. Both

tubings were fixed with a ligature. To monitor mean arterial pressure (MAP) the left

carotid artery was exposed and cannulated with PE-50 tubing (Portex®). This tubing

was fixed with a ligature and was also used for the blood withdrawal at the end of

measurements (Figure 17). The surgical site around the front neck region was then

covered with wet gauze compress to avoid drying.

After thorough disinfection (Kodan®) of the abdomen, a median laparotomy (Figure

17) was performed and the portal vein was exposed. To measure the portal flow

volume rate an ultrasonic transit time flow probe (Transonic Animal Research

Flowmeter T206 with perivascular flow probe type 2.5S) was placed at the portal

vein, loosely encircling the vessel, before ultrasound gel (Caelo) was applied. After a

stabilization period of 10 to 15 minutes portal flow volume rate was measured over

five minutes without any intervention given, but presupposing a stable MAP.

The flow probe consists of a probe body which houses two ultrasonic transducers

and a probe reflector. The latter is positioned on one side of the vessel of interest,

whereas the two transducers are positioned on the opposite side. The first transducer

emits an ultrasonic beam that traverse the full width of the vessel. This beam is then

reflected by the probe reflector and captured by the second transducer. The time the

beam needs to travel from the first to the second transducer is termed “transit time”.

Basically, the beams traveling back and forth alternately cross the flowing blood in

upstream and downstream direction. During the upstream measurement the beam

travels against flow, and the resulting transit time is increased by a flow-dependent

factor. In contrast, during the downstream measurement, the beam travels with flow,


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and the resulting transit time is reduced by the same flow dependent factor. The

subtraction of the upstream from the downstream integrated transit times provides an

accurate measure of the flow volume rate. Since the transit time is determined across

the cross section of the vessel, the flow volume rate is determined independently of

the vessel diameter 288.

To monitor portal venous pressure (PVP) the ultrasonic transit time flow probe was

removed and a peripheral venous catheter (Versatus®) was inserted into the portal

vein and fixed with a tissue adhesive (Histoacyrl®). In addition, a microvascular flow

probe (Laser Doppler blood flow monitor DRT4 with a Titanium tipped low profile disc

probe type DP8C) was set on the surface of the left liver lobe to determine

microvascular flow (MF). Thereby a laser beam (785 nm) emitted from the optic fiber

penetrates the liver tissue starting from the liver surface. The laser light emitted

interacts randomly with both, moving objects (primarily erythrocytes) and stationary

tissue. If the light hits a moving erythrocyte it is reflected in a different frequency and

magnitude of the wavelength (scatter) than it is emitted, i.e. a Doppler shift occurs. In

contrast, if the light is reflected from stationary tissue it remains unchanged. The light

changes induced by moving erythrocytes are detected by a photosensitive optic fiber

on the liver surface and returned to a photodetector and the signal processing

electronics. The Laser-Doppler system analyses the Doppler shift and calculates flux

values, a quantity proportional to the product of the average velocity of the

erythrocytes and their number concentration. Hence, erythrocyte motion in the

outmost layer of liver tissue was measured continuously. To provide consistent

measurements for all tissue types, the probes were calibrated with a motility standard

supplied with the monitoring system. The motility standard consists of a low

concentration of polystyrene microspheres in water undergoing thermal motion

(Brownian motion).


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Figure 17: Insertion of catheter for MAP measurements and laparotomy.

A more detailed photo series of the operative procedure has been attached (see 7.2).

As a second measure, in addition to the homeothermic controlled operating table, the

torso of the rats was covered with aluminum foil to prevent it from becoming

hypothermic. After a stabilization period of 10 to 15 minutes, basal values of all

parameters were obtained and the intervention was administered through the second

CVP-tubing. The intervention was either sodium chloride (B. Braun Melsungen,

Melsungen, Germany) or sildenafil (Revatio®, Pfizer, Berlin, Germany). Rats were

randomly allocated in one of three intervention groups: sodium chloride (NaCl),

sildenafil 0.1 mg/kg (Sil 0.1 mg/kg) or sildenafil 1 mg/kg (Sil 1mg/kg). To minimize

hemodynamic alterations due to plasma volume changes, the intervention was


To monitor arterial and venous pressures invasively, a solid column of liquid

connecting blood to the pressure transducer (ATP300 (arterial) or P75 type 379

(venous)) is required. Therefore tubings are be pre-filled with a heparinized isotone

electrolyte solution (Jonosteril®). The heparin sodium prevents occlusion of the

tubing due to thrombosis. The liquid within the tubing is in contact with a flexible

diaphragm which is located within the pressure transducer. The diaphragm moves in

response to the transmitted pressure waveform. The pressure transducer then

converts this movement into a proportional electrical signal (voltage, e.g.

5mV/V/mmHg), being enhanced by an amplifier module and send to the data

acquisition system (HSE-USB-HAEMODYN).


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The high calibration point of the pressure transducers was calibrated with a pressure

manometer at 100 mmHg. Zero point calibration was performed 1 cm above the

operating table (heart height) by opening the pressure transducer to atmospheric

pressure and electronically zeroing the system.

Using the data acquisition system (HSE-USB-HAEMODYN) and the corresponding

software (HSE-Basic Data Acquisition Software (BDAS) 1.5) all measured

parameters were monitored continuously. Portal flow volume rate was recorded over

5 minutes before the intervention was applied. All other parameters (i.e. heart and

respiration rate, oxygen saturation rate, CVP, MAP, PVP, microvascular flow) were

recorded over 60 minutes starting from time point “0min”. Right after determining

baseline values the intervention took place. Data were taken directly from the

software (BDAS).

5.2.5 Serum Analyses

5.2.5.1 Serum Parameters At the end of the invasive hemodynamic measurements blood samples were taken

via the left carotid artery. Serum was used for the analysis of the following serum

parameters by semi-automated clinical routine methods: glucose (Glc), sodium (Na),

potassium (K), total bilirubin (Bil), creatinine (Crea), albumin (Alb), aspartate amino-

transferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP).

5.2.5.2 Competitive cGMP Enzyme-linked Immunosorbent Assay (ELISA) A second serum sample was stored at −80 °C until used for the quantification of

cGMP concentrations. After defrosting, an in vitro competitive ELISA was performed

according to the manufacturers’ instructions of the cGMP ELISA kit (Abcam

ab133052). In total, two kits, including one 96 well plate each, were used. Each

sample was assayed in duplicates. In each set of experiments a standard curve was

assayed for calibration using standard serial dilution. In addition to the serum

samples, a negative control (blank) and two positive controls (two different standard

samples) were included. One out of these two positive controls was applied in six

replicates to determined inter- and intra-assay variability.


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A goat anti-rabbit IgG capture antibody (secondary antibody) has been precoated

onto the wells. 100 µl of standard or serum samples (undiluted, non-acetylated) were

incubated togheter with 50 µl of an alkaline phosphatase (AP)-conjugated cGMP

antigen and 50 µl of a polyclonal rabbit cGMP antibody (primary antibody) per well at

room temperature for two hours on a plate shaker at 500 rpm. Thereby the AP-

labeled antigens from the conjugate and the unlabeled antigens from the serum

sample compete for binding to the target specific antibody (primary antibody) which

in turn binds to the capture antibody (secondary antibody).

After incubation, the excess reagents (unbound antibodies and other biological

materials) were removed by three wash steps using each 400 µl wash buffer per well.

Then 200 µl of substrate solution were applied and incubated at room temperature

for one hour without shaking. As a phosphatase substrate p-Nitrophenylphosphate

(pNpp) is used, which turns yellow, when dephosphorylated by ALP. The intensity of

the color change is inversely proportional to the amount of cGMP in the well. To

quench the enzyme reaction, 50 µl stop solution were pipetted into each well.

Immediately after, optical density absorbance at 405 nm was read using a scanning

microplate spectrophotometer (µQuantTM) and the corresponding software (KC4).

The fluorescence data were taken directly from the software (KC4). Since each

sample was assayed in duplicates, mean values of cGMP concentration were

determined and used for further statistical calculations.

5.2.6 Two-step Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) Sample preparation, mRNA extraction, and cDNA synthesis At the end of the invasive hemodynamic measurements, the left lateral lobe of each

rat’s liver was excised, cut into pieces, snap frozen in liquid nitrogen, and stored at

−80 °C until used for qRT-PCR. qRT-PCR was used to quantified hepatic gene

expression of endothelial and inducible NO synthase (eNOS and iNOS),

phosphodiesterase 5 (PDE5), soluble guanylate cyclase subunits a1 and b1 (sGCa1

and sGCb1). Therefore appropriate primer pairs (Table 27) were designed in

advance using NCBI Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast), which

were then manufactured (Microsynth).


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Table 27: Nucleotide sequences of forward and reverse primers.

Gene Forward primer (5´-3´) Reverse primer (5´-3´) Product length (bp)

eNOS 5'-AAGTGGGCAGCATCACCTAC-3´

5´-GCCTGGGAACCACTCCTTTT-3´

211

iNOS 5´-CTCACTGGGACTGCACAGAA-3´

5´-TGTTGAAGGGTGTCGTGAAA-3´

128

PDE5 5´-GCGGAGGAAGAAACAAGGGA-3´

5´-ATCGGCAAAGAACCTCGTGT-3´

196

sGCa1 5´-GCCCCACGACATACAGGTTA-3´

5´-GCGGCTCACTAATCTACCCC-3´

229

sGCb1 5´-AATTACGGTCCCGAGGTGTG-3´

5´-ACCAGCATTGAGGTTGAGGAC-3´

147

18sRNA (reference)

5´-GTAACCCGTTGAACCCCATT-3´

5´-CCATCCAATCGGTAGTAGCG-3´

151

srsf4 (reference)

5´-GGTTCTGGACGCAGTGGATA-3´

5´-CTCCTTCGTTTTTGCGTCCC-3´

193

Hepatic total mRNA extraction was performed according the manufacturer’s

instructions of the mRNA extraction kit (RNeasy® Mini Kit).

For mRNA extraction RNase-free materials, water and ethanol were used, since the

thermostable and ubiquitous occurring RNase leads to a rapid mRNA degradation. In

addition a thorough disinfection of the work area using ethanol (70%) was essential.

Initially, a maximum of 20 µg liver tissue was placed in a tube filled with 350 µl buffer

(RLT), containing 1% b-mercaptoethanol. In there, the tissue was ground with a

thoroughly disinfected pestle. After tissue disruption, the lysate was pipetted directly

into an eliminator column (QIAshredder) and centrifuged (2 min / 15.000 rpm / 24 °C)

for homogenization. Then 350 µl ethanol (70%) were added to the cleared lysate and

mixed by pipetting. 700 µl of the sample were pipetted directly into a spin column

(RNeasy®) and centrifuged (10 sec / 10.000 rpm / 24 °C). The flow-through was

discarded. 700 μl buffer (RW1) were added to the spin column and centrifuged (15

sec / 10.000 rpm / 24 °C) to wash the spin column membrane. After centrifugation,

the spin column was removed carefully avoiding contact with the flow-through which

was then discarded. In the next step, 500 μl buffer (RPE), diluted with ethanol

(100%), were added to the spin column and centrifuged (15 sec / 10.000 rpm / 24

°C). Again the flow-through was discarded. This wash step was repeated with a

longer centrifugation (2 min / 10.000 rpm / 24 °C) to dry the spin column membrane.


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Hence, it was ensured that no ethanol was carried over during RNA elution, since

residual ethanol could interfere with downstream reactions. After centrifugation, the

spin column was removed carefully avoiding contact with the flow-through and placed

into a new collection tube. Finally 50 µl RNase-free water was added directly to the

spin column membrane and centrifuged (1.5 min / 10.000 rpm / 24 °C) to elute the

mRNA.

In the next step, the capillary channels of a disposable microfluidic slide (QIAxpert

slide) were loaded with 4 µl of each mRNA sample. mRNA concentration was

determined using a microfluidic spectrophotometer (QIAxpert reader) and the

corresponding software (QIAxpert Software 2.2.0.21). No additional mRNA

purification was needed.

The reverse transcription of mRNA into cDNA as well as the PCR amplification was

performed according to the manufacturer’s instructions of the RT-PCR kit (First

Strand cDNA Synthesis Kit).

For cDNA synthesis 1 µg hepatic mRNA and 1µl random hexamer primer were

pipetted into a reaction vessel before RNase-free water was added up to a final total

volume of 11 µl. The components were mixed by pipetting.

Then a master mix was prepared containing the following components (amounts are

given per well):

• 4 µl 5x reaction buffer

• 1 µl RiboLock RNase inhibitor (20U/µl)

• 2 µl 10 mM dNTP mix

• 2 µl M-MuLV reverse transcriptase (20U/µl)

Again the components were mixed by pipetting before 11 µl of the mRNA / primer

mixture were added to 9 µl of the master mix. Once again components were mixed

by pipetting. Afterwards samples were incubated for 5 min at 25 °C, 60 min at 37 °C

and for 5 min at 70 °C. After sample incubation, 20 µl of RNase-free water were

added and components were mixed by pipetting. The resulting final total volume of

40 µl of first strand cDNA synthesis reaction mixture was stored at -20 °C until used

for subsequent qRT-PCR runs.


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qRT-PCR For the qRT-PCR runs a second master mix was prepared containing the following

components (amounts are given per well):

• 2 µl PCR buffer

• 0.3 µl 10 mM dNTP mix

• 1.25 µl 50 mM MgCl2

• 2 µl DMSO

• 0.25 µl forward primer

• 0.25 µl reverse primer

• 0.2 µl Taq DNA polymerase

• 0.2 µl SYBER Green

• 12.55 µl RNase-free water

The components were mixed by pipetting before 1 µl of the defrosted first strand

cDNA synthesis reaction mixture was added to 19 µl of the master mix. Again

components were mixed by pipetting. Hence, PCR runs were carried out in a final

total volume of 20 μl. In total, five 96 well plates were used. Each sample was

assayed in duplicates. Furthermore, a negative control (water bidest blank) was

included in each set of experiments. The PCR runs were performed under defined

thermocycling conditions (Table 28) using a thermocycler (LightCyler® 480) and the

corresponding software (LightCycler® 480 Software 1.5).

Table 28: Thermocycling conditions for qRT-PCR runs

Step Temperature Time

initial denaturation for one cycle

denaturation

annealing for 40 cycles

elongation

94 °C

94 °C

60 °C

72 °C

3 min

30 sec

30 sec

40 sec

melt-curve gradient from 65 °C - 95 °C in 0.5 °C

increments (5 sec each)


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During the PCR run cDNA amplification is detected in real time by accumulation of an

emitted fluorescence signal. First, during the denaturation step, all double-stranded

DNA (dsDNA) is separated into single-stranded DNA (ssDNA) which prevents

occurrence of a fluorescence signal. During the annealing step the forward and

reverse primers hybridize to the template cDNA strands and form little segments of

dsDNA. The SYBER Green binding dye immediately intercalates into all dsDNA

present in the sample. During the elongation step the DNA polymerase starts to

elongate these new dsDNA segments and more SYBER Green can intercalate. At

the end of each elongation step, when the maximum amount of SYBER Green has

intercalated, the fluorescence signal is measured (excitation at 465 nm / detection at

510 nm). This fluorescence signal is proportional to the amount of dsDNA

synthesized. However, the presence of a perpetual background fluorescence signal

has to be taken into account for the quantification of cDNA amplification. Therefore it

is essential to assess the Ct (threshold cycle) which marks the beginning of the

exponential increase of the reaction curve and is defined as the number of cycles

being required for the fluorescence signal to cross the background fluorescence

signal (threshold). The Ct is inversely proportional to the amount of cDNA present in

the sample. This method, however, assumes that the PCR is operating at 100%

efficiency, meaning the amount of cDNA doubles with each amplification cycle.

Since SYBER Green intercalates nonspecifically into all dsDNA present, a final melt-

curve step was included post-PCR to ensure that there are no nonspecific

amplification products.

The fluorescence data were taken directly from the software (LightCycler® 480

Software 1.5). Levels of gene expression were quantified relatively with the

comparative Ct method 289. To use the comparative Ct method, a validation

experiment was run in advance to show that the efficiencies of the gene

amplifications of the target (gene of interest) and the reference (endogenous control)

are approximately equivalent. Thereby the need of a standard curve for calibration

was eliminated.

Since each sample was assayed in duplicates, mean Ct values for all target and

reference genes were calculated (Ct). The individual mRNA levels in the samples

were normalized for the two reference genes 18sRNA (a ribosomal subunit) and

srsf4 (a splicing factor) by calculating the difference between Ct target gene and Ct


117

reference gene (ΔCt). Both reference genes were expressed stably in all groups. The

result was finally calculated as “fold expression” of the samples in terms of the

control as 2-ΔΔCt which is defined as follows:

2-ΔΔCt = [(Ct target gene - Ct reference gene (ΔCt)) sample A

- [(Ct target gene - Ct reference gene) sample B (ΔCt))] 290.

5.2.7 Histology

5.2.7.1 Assessment of the Degree of Liver Fibrosis Instantly at the end of the invasive hemodynamic measurements, the median lobe of

each rat’s liver was excised. A cross-section of the median lobe was fixed in 10%

neutral buffered formalin (Sigma-Aldrich) and embedded in paraffin. Serial step

sections were semi-automatically stained for hematoxylin-eosin, sirius red, reticulin,

periodic acid-schiff diastase and iron. Fibrosis was evaluated semiquantitatively

according to the five-level score described previously by Desmet et al 82. A blinded

pathologist performed the cross-sections and the histological evaluation of the liver

tissue sections with the Desmet score (DS): DS=0: no fibrosis, DS=1: mild fibrosis,

DS=2: moderate fibrosis, DS=3: severe fibrosis, and DS=4: cirrhosis.

In addition, the development of cholangiocellular carcinoma (CCCs) was determined

based on histomorphological criteria (i.e. degree of cytological atypia and

desmoplastic stroma reaction).

5.2.7.2 Immunohistochemical (IHC) PDE5 Staining Paraffin-embedded liver tissue sections were furthermore used for PDE5 protein

detection and localization. Therefore, immunohistochemical staining was performed

with an polyclonal rabbit anti-PDE5A-antibody (Abcam ab64179) using the polymer

chain two-step indirect technique according to the manufacturers’ instructions of the

Dako EnVision® +, System-HRP (DAB) kit (Dako).

Deparaffinization / Rehydration

To enable staining the paraffin-embedded sections had to be deparaffinized and

rehydrated first. Therefore sections were heated overnight at 60 °C in an oven


118

(Bachofer). The next day sections were placed in a rack and the following wash steps

were performed by immersing the sections in a series of alcohols and water:

• xylene 1 for 10 min


• ethanol (100%) for 2 x 3 min

• ethanol (95%) for 1 min


• running cold water (demineralized) for 10 min

Antigen retrieval

In the next step, heat-mediated antigen retrieval was performed as formalin-fixed

tissues tend to form methylene bridges during fixation that cross-link proteins and

therefore mask antigenic sites. To expose the antigenic site in order to enable

antibody binding, tissue sections were immersed in an antigen retrieval buffer (Tris /

EDTA pH 9.0 (Sigma-Aldrich / Serva Electrophoresis)) and put into a microwave

(Siemens). Once the buffer came to boil, boiling was maintained for 10 min at 580 W.

Afterwards, sections were removed from the boiling buffer and immersed in ice water

for 10 min before they were washed in running cold water (demineralized) for the

next 10 min.

Staining

During the whole staining procedure sections were placed in a humid environment to

avoid drying. Furthermore, between the single steps of the staining protocol any

remaining liquid was removed by carefully wiping around the sections using gauze

compresses (Fuhrmann).

Initially sections were immersed in 3% hydrogen peroxide for 10 min at 24 °C to

block all endogenous peroxidase activity, before they were rinsed gently with wash

buffer (1x PBS + 0.1% Tween® 20). Then sections were placed in a rack and the

polyclonal rabbit anti-PDE5A-antibody (Abcam ab64179) (primary antibody) was

diluted in antibody diluent (1:500) and applied on each section. Sections were

covered with a sealing film (Parafilm M®) and incubated for 60 min at 24 °C. During

incubation the anti-PDE5A-antibody binds to its target antigen. Afterwards sections

https://www.cellsignal.com/product/productDetail.jsp?productId=8112


119

were rinsed gently with wash buffer (1x PBS (Oxoide) + 0.1% Tween® 20 (PancReac

AppliChem)) before 1 to 2 drops of a peroxidase labelled polymer, which was

conjugated to a goat anti-rabbit IgG capture antibody (secondary antibody), were

applied on each section. Sections were covered with a sealing film (Parafilm M®) and

incubated for 60 min at 24 °C. During incubation the capture antibody reacts with the

anti-PDE5A-antibody, which has already bound to its target antigen. The labelled

polymer does not contain avidin or biotin, thus any nonspecific staining as a

consequence of endogenous avidin-biotin activity in the liver is eliminated or

significantly reduced. Afterwards sections were rinsed gently with wash buffer (1x

PBS (Oxoide) + 0.1% Tween® 20 (PancReac AppliChem)). In the next step

substrate-chromogen solution (DAB) was applied on each section and incubated for

10 min at 24 °C. During incubation antibody binding is visualized on the basis of the

occurring brown color reaction. Once more sections were rinsed gently with wash

buffer (1x PBS + 0.1% Tween® 20). (Figure 18)

Figure 18: Principle of the polymer two-step indirect technique for immunhisto-

chemical staining.

Figure reprinted with permission of Sudhanshu Goyal, employee of BioGenex.

Original source: http://www.biogenex.com/us/detection-systems (July 24, 2017)

To provide contrast that helps the primary stain stand out, a hematoxylin nuclear

counterstaining step was performed in which sections were incubated in filtered

hematoxylin (Sigma-Aldrich) for 5 min at 24 °C.


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Dehydration

After the hematoxylin counterstaining sections were dehydrated. Therefore the

following wash steps were performed by immersing the sections in water and a

series of alcohols:

• running cold water (demineralized) for 5 min



• ethanol (100%) for 2 x 5 min



Finally, sections were mounted and coverslipped using a mounting medium

(Entellan®).

Microscopic analysis

For microscopic quantification (Zeiss Axioplan microscope) the number of stained

cells was counted in 20 random high power-fields (HPF) (400x magnification) for

each sample. Exclusively stained cells in the parenchyma were included in cell

counts, whereas PDE5 staining around the central vein (CON 1) and in fibrous

connective tissue (CIR 1 and CIR 2) was not considered.

5.2.8 Statistics Results were expressed as median ± interquartile range (IQR). Only results of the

qRT-PCR experiments were expressed as mean ± standard deviation (SD) to enable

the quantification of gene expression with the comparative Ct method.

To determine differences among groups or subgroups for the measured parameters,

the non-parametric Kruskal-Wallis test was used. Post-hoc pairwise comparisons

between groups or subgroups 179 were corrected for multiple comparisons according

to Bonferroni. For reasons of consistency the non-parametric Kruskal-Wallis test and

post-hoc pairwise comparisons with Bonferroni correction were also used for the

qRT-PCR experiments. A two-tailed p-value of < 0.05 was considered as statistically

significant.


121

For statistical analyses SPSS® software 23.0 (IBM Corp., Armonk, New York) was

used, only the regression analysis was calculated with STATA® software 14

(StataCorp LLC, Lakeway Drive, Texas).

Specific statistic information for each part of the study will be described in the

following.

Non-invasive hemodynamic measurements

To assess interobserver variability (2 readers) of the self-established MR score, a

weighted kappa analysis was performed. The kappa coefficient (ƙw) indicates the

strength of agreement and was categorized as follows: 0–0.20: slight; 0.21–0.40: fair;

0.41–0.60: moderate; 0.61–0.80: substantial; and 0.81–1.00: almost perfect 291.

Correlations were calculated using Spearman’s rank correlation coefficient (rs).

Invasive hemodynamic measurement

Invasive portal flow volume rate measurement

Correlations were calculated using Spearman’s rank correlation coefficient (rs).

Effect of sildenafil on hemodynamics

All parameters were normalized (PVPrel, MAPrel, MFrel and HRrel, CVPrel, respiration

raterel and oxygen saturationrel) to compensate differences in absolute values

between healthy and diseased rats. Thereby, time point “10 min” was taken as

baseline and set to 100% since the administration of 600 µl liquid volume into the

right atrium caused parameter variations for the next few minutes.

To evaluate the effect of sildenafil the change in parameter values at time point “60

min” compared to baseline (“10 min”) was determined by calculating relative median

of differences (RMD).

To illustrate the course of parameters during the measurement interval data of time

points 0, 1, 3, 5, 10, 20, 30, 45 and 60 min were chosen.


122

Effect of MAP on PVP

All parameters were normalized (PVPrel, MAPrel) to compensate differences in

absolute values between healthy and diseased rats. Thereby, time point “0 min” was

taken as baseline and set to 100%. The effect of MAP on PVP was evaluated by

linear regression analysis, including changes from baseline (“0 min”) for every

recorded time stamp (1 time stamp = 2 sec) over the first 30 minutes.

The change in PVPrel for every 1% change in MAPrel is described by the regression

coefficient (s), whereas the explained variation (%) within one rat is described by r-

squared (r²).

Biochemical investigations

Levels of gene expression were quantified relatively with the comparative Ct method 289. The individual mRNA levels in the samples were normalized for two reference

genes. Analyses of both, gene expressions and cGMP concentrations, were

performed in duplicates. Mean values of duplicates were used for further statistical

analyses.

For microscopic quantification of the immunohistochemical stainings, the number of

PDE5 stained cells was counted in 20 random high power fields (HPF) for each

tissue sample. Mean values of stained cells per HPF were used for further statistical

analyses.

123

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7. Attachments

7.1 Score sheet to document the body condition of the rats (in German)

147

7.2 Photo series of the operative procedure

a) Operation table

b) Tracheotomy

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c) Insertion of venous catheters

d) Insertion of arterial catheters

e) Laparotomy

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f) Exposure of the portal vein and positioning of the flow probe

g) Insertion portal vein catheter

h) Positioning microvascular flow probe

150

i) Apparatuses and monitoring system

j) Blood sample collection

151

8. Abbreviations Alb albumin

ALT alanine aminotransferase

AP alkaline phosphatase

AST aspartate aminotransferase

BDL bile duct ligation

BH4 tetra-hydrobiopterin

Bil bilirubin

Ca calcium

cAMP cyclic adenosine guanosine monophosphate

CCC cholangiocellular carcinoma

CCl4 carbon tetrachloride

cGMP cyclic guanosine monophosphate

Crea creatinine

EC endothelial cell

ECM extracellular matrix

ELISA enzyme-linked immunosorbent assay

eNOS endothelial nitric oxide synthase

FAD flavin adenine dinucleotide

FHVP free hepatic venous pressure

FMN flavin mononucleotide

FMO flavin-containing monooxygenase

Glc glucose

HABR hepatic arterial buffer response

HCC hepatocellular carcinoma

HPF high power field

HSC hepatic stellate cell

HR heart rate

HVPG hepatic venous pressure gradient

iNOS inducible nitric oxide synthase

K potassium

KC Kupffer cell

Ly6Chi macrophages with a profibrogenic phenotype

Ly6clo macrophages with an antifibrogenic phenotype

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MAP mean arterial pressure

MF microvascular flow

MMP matrix metalloproteinase

MR magnetic resonance

Na sodium

NaCl sodium chloride

n.d. not detectable

NK natural killer cell

n.m. not measured

NO nitric oxide

NO-cGMP nitric oxide-cyclic guanosine monophosphate

NOS nitric oxide synthase

NSBB nonselective beta-blocker, beta-adrenergic receptor antagonists

PDE phosphodiesterase

PDGF platelet derived growth factor

PH portal hypertension

PKG protein kinase G

PVP portal venous pressure

qRT-PCR quantitative real-time polymerase chain reaction

ROS reactive oxygen species

SEC sinusoidal endothelial cell

sGC soluble guanylyl cyclase

sGCa1 soluble guanylyl cyclase (alpha1 subunit)

sGCb1 soluble guanylyl cyclase (beta1 subunit)

Sil sildenafil

TAA thioacetamide

TAASO TAA-sulfoxide

TAASO2 TAA-sulfdioxide

TGF-b transforming growth factor beta

TIMP tissue inhibitors of matrix metalloproteinases

TIPS transjugular intrahepatic portosystemic stent shunting

US ultrasound

VEGF vascular endothelial growth factor

WHVP wedged hepatic venous pressure

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9. Content of Figures Figure 1: Schematic diagram of a portal tract and a hepatic sinusoid

Figure 2: Changes in the hepatic sinusoid in response to liver cirrhosis

Figure 3: Schematic diagram of the NO-cGMP pathway

Figure 4: Comparison of the structures of cGMP and sildenafil

Figure 5: Dot plot illustrating the assessment of the degree of liver fibrosis using

histological (Desmet score) and MR scoring (MR score)

Figure 6: Color-coded image displaying the flow velocities (m/min) (a) and a

diagram of flow volume rates (ml/min) (b) in the portal vein (red, yellow)

and abdominal aorta (blue) at selected time points of a cardiac cycle

Figure 7: Boxplots showing the distributions of portal cross-sectional area [mm2]

(a), portal flow velocity [m/min] (b), portal and aortic flow volume rate

[ml/min] (c and d) in the groups

Figure 8: Boxplots showing the distributions of portal flow volume rate [ml/min] (a)

and MAP (b) in the groups

Figure 9a: Changes of relative median (%) in PVPrel and MAPrel ± 95% CI in the

subgroups

Figure 9b: Changes of relative median (%) in MFrel and HRrel ± 95% CI in the

subgroups

Figure 10: Course of MAP (black) and PVP (blue) of an exemplary rat after sodium

chloride (NaCl) administration

Figure 11: Dotplots showing the distributions of hepatic gene expression of the

enzymes eNOS (a), iNOS (b), PDE5 (c), sGCa1(d) and sGCb1(e), as

well as distributions of serum cGMP concentrations [pmol/ml] (f) in the

subgroups

Figure 12: Immunohistochemical PDE5 staining (brown) of liver tissue samples of

rats with healthy (CON 1) and cirrhotic livers (CIR 1 and CIR 2)

Figure 13: Preparation of the rat for the MR measurements and insertion into

MR scanner

Figure 14: T1-RARE images displaying the positioning of the PC-MR slice

orthogonally to the portal vein (a and b) and the abdominal aorta (c and

d) of a rat on the coronal (a and d) and sagittal (b and c) reference

scans

Figure 15: Principle of 2D PC-MR

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Figure 16: T1-RARE images showing morphological hallmarks of the MR score

Figure 17: Insertion of catheter for MAP measurements and laparotomy

Figure 18: Prinicple of the polymer two-step indirect technique for

immunhistochemical staining

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10. Content of Tables Table 1: Causes of liver cirrhosis

Table 2: Vasoactive molecules

Table 3: Classification of PH according to anatomical location

Table 4: Stages of liver cirrhosis

Table 5: Reference standards and potentially novel drugs for PH therapy

Table 6: Reference standards and potentially novel strategies to increase

NO downstream signaling

Table 7: Substrate specificity and distribution of PDE families

Table 8: Symptoms observed in rats during TAA exposure time

Table 9: Number of rats sorted by strain and their histological degree of liver

fibrosis with corresponding TAA exposure time

Table 10: Rats sorted by their histological degree of liver fibrosis with

corresponding TAA exposure time

Table 11: Number of data sets of rats evaluated for the assessment of the degree

of liver fibrosis by histological (Desmet score) and MR scoring (MR

score), as well as MR hemodynamic measurements.









Table 16: Median ± interquartile range (IQR) of body weight, hemodynamic

parameters and HR of the subgroups at time points 0, 10, 30 and 60min

Table 17: Relative median of differences (RMD) (%) ± interquartile range (IQR) of

hemodynamic parameters and HR in the subgroups

Table 18: Regression analysis between MAPrel and PVPrel in the subgroups

Table 19: Rats sorted by their histological degree of liver fibrosis and group

classification

Table 20: Overview subgroups

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Table 21: Median ± interquartile range (IQR) of body weight and serum

parameters in subgroups

Table 22: Pairwise comparisons between subgroups

Table 23: Mean ± standard deviation (SD) of hepatic enzyme gene expression in

the subgroups

Table 24: Median ± interquartile range (IQR) of serum cGMP concentrations in the

subgroups

Table 25: Pairwise comparisons between subgroups

Table 26: Number of untreated and TAA-treated rats sorted by strain

Table 27: Nucleotide sequences of forward and reverse primers

Table 28: Thermocycling conditions for qRT-PCR runs

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11. Acknowledgments The present dissertation was performed in the Institute for Exercise- und Occupational

Medicine, Medical Center, University of Freiburg. So I would like to thank the medical director

of the institute, Prof. Dr. Peter Deibert, for his support and for providing the topic and

workplaces for my research.

In addition, I would like to express my sincere gratitude to my advisor Prof. Dr.

Wolfgang Kreisel (Department of Medicine II, Gastroenterology, Hepatology, Endocrinology, and

Infectious Diseases, Medical Center, University of Freiburg) for the continuous support of my

PhD study and related research, for his jokes, patience, motivation, and immense

knowledge. His guidance and our discussions helped me in all the time of research

and writing of this thesis. Although he has already been retired, he spent a lot of time

for this project and our small working group; therefore I will be always grateful to him.

Besides my advisor, I would like to thank the rest of my thesis committee, in

particular Prof. Dr. Irmgard Merfort (Department of Pharmaceutical Biology and Biotechnology,

University of Freiburg) for her support, her insightful comments and encouragement, but

also for her questions, which incented me to widen my research from various

perspectives.

Furthermore, I would like to thank all my lab colleagues: PD Dr. Manfred Baumstark,

Sabine Jotterand, Sabine Linser-Haar, Heidrun Zurmoehle, and Sabine Well-

Zimmermann (Institute for Exercise- und Occupational Medicine, Medical Center, University of

Freiburg). Hereby, my sincere thanks goes to PD Dr. Manfred Baumstark who advised

me with all statistical matters. I really admired his knowledge covering so many

different areas, so that in the end he was not only a colleague but also a mentor.

Moreover, I thank Sabine Jotterand who thought me how to perform ELISA

experiments and who together with her teammate, Sabine Linser-Haar, was always

willing to help with organizational matters in the lab. I also thank Heidrun Zurmoehle

and Sabine Well-Zimmermann who ordered all material I needed.

Very special thanks also goes to my former teammate and meanwhile friend, Dr.

Adhara Lazaro (previously: Institute for Exercise- und Occupational Medicine, Medical Center,

University of Freiburg) for the inspiring discussions on technical as well as private topics,

for the tough times we were working together before deadlines, and for all the fun we

have had in these 2.5 years. It has been a pleasure to work with her.

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I am also very thankful for the excellent and reliable cooperation with the animal care

attendants Claudia Bravo, Monika Kolterjahn and Lisa Zota (Center for Experimental

Models and Transgenic Service, Medical Center, University of Freiburg).

A special thanks also goes to Dr. Patrick Stoll (previously: Anesthesiology and Critical Care,

Medical Center, University of Freiburg) who taught me how to perform the anesthesia and

the operative procedure for the invasive hemodynamic measurements.

I also thank Dr. Lisa Lutz and Prof. Dr. Annette Schmitt-Graeff (Institute of Clinical

Pathology, Medical Center, University of Freiburg). Dr. Lisa Lutz conducted the histological

evaluation of the rats’ liver tissue samples, whereas Prof. Dr. Annette Schmitt-Graeff

contributed to the diagnosing of the immunhistochemical PDE5 staining.

In addition, a special mention goes to PD Dr. Dominik von Elverfeldt, Dr. Wilfried

Reichardt, Dr. Jakob Neubauer, Annette Merkle and Michaela Schaeper (Department of

Radiology – Medical Physics, Medical Center, University of Freiburg). In cooperation with this

team the MR measurements were performed. Dr. Wilfried Reichardt also conducted

the postprocessing of MR data. Furthermore, he and Dr. Jakob Neubauer evaluated

the MR rat liver images with the MR score.

A gratidude also goes out to Prof. Dr. Hasselblatt, Isabel Schulien, and Birgit

Hockenjos (Department of Medicine II, Gastroenterology, Hepatology, Endocrinology, and Infectious

Diseases, Medical Center, University of Freiburg) who provided me an opportunity to

temporarily join their team. They taught me how to perform and evaluated qRT-PCR

experiments (including the primer design) and assisted me with the immuno-

histochemical stainings.

I also thank Thomas Heister (Institute of Medical Biometry and Statistics, Medical Center,

University of Freiburg) for the conduct of the regression analyses to evaluate the effect of

MAP on PVP.

Moreover, I am grateful to my family members and friends who supported me

mentally throughout writing this dissertation and in general.

And last, but for sure not least, I thank God for the strength, health, perseverance,

and patience he gave me during these years which have been real challenging

sometimes.

https://www.imbi.uni-freiburg.de/

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12. Curriculum Vitae This page contains personal information and is therefore not part of the online

publication.

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