EFFECTS OF RHO-KINASE INHIBITION ON ESTABLISHED

CHRONIC HYPOXIC PULMONARY HYPERTENSION IN THE

NEONATAL RAT

By

Emily Zhi Xu, H.B.Sc.

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Physiology

University of Toronto

© Copyright Emily Zhi Xu (2010)

ii

ABSTRACT

Effects of Rho-kinase inhibition on established chronic hypoxic pulmonary hypertension

in the neonatal rat.

Emily Zhi Xu, M.Sc, 2010

Department of Physiology, School of Graduate Studies

University of Toronto, Canada

Rationale: Vascular remodeling and right-ventricular (RV) dysfunction are features of

refractory pulmonary hypertension (PHT) in human neonates. These features are replicated in

rats chronically exposed to hypoxia (13% O2), in which increased pulmonary vascular resistance

(PVR) was acutely normalized by Y-27632, a Rho-kinase (ROCK) inhibitor, but not by inhaled

nitric oxide.

Objective: To examine the reversing effects of sustained ROCK inhibition on haemodynamic

(RV dysfunction and increased PVR) and structural (RV hypertrophy and arterial wall

remodeling) changes of chronic hypoxic PHT.

Methods: Rat pups were exposed to air or hypoxia from birth for up to 21 days and received Y-

27632 (15 mg/kg/b.i.d.) or vehicle from day 14.

Results: Y-27632 normalised RV dysfunction and reversed remodeling secondary to chronic

hypoxia. These changes were accompanied by increased apoptosis of smooth muscle and

attenuated endothelin-1 expression in pulmonary arteries.

Conclusion: ROCK inhibitors hold promise as a rescue therapy for refractory PHT in neonates.

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ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Robert Jankov, for his wonderful guidance and support

throughout my project. His knowledge of neonatal medicine and beyond has been of

monumental importance, and I am truly grateful for his mentorship.

I would like to thank Dr. Patrick McNamara for sharing his expertise in 2D

echocardiography and Doppler ultrasound, and for his much appreciated participation as a

member of my supervisory committee. Also, I would like to thank Drs. Keith Tanswell and

Jacques Belik for providing invaluable insights into my project as members of my supervisory

committee.

To my laboratory colleagues, Julijana Damchevska and Crystal Kantores, my sincerest

gratitude, for making this experience even more enjoyable and rewarding. I would also like to

thank Ms. Rosetta Belcastro, Dr. Azhar Masood, Dr. Linda Li, Ms. Mandy Lau, Ms. Doreen

Engelberts, and Mr. Todd Van Vliet for their help and support.

Lastly, and importantly, I would like to thank my mother, Jingbai Nie, father, Huimin

Xu, brother, Richard Xu, and my friends for their love and support throughout this journey.

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TABLE OF CONTENTS

ABSTRACT ………………………………………………………………………… ii

ACKNOWLEDGEMENTS ………………………………………………………......... iii

TABLE OF CONTENTS …………………………………………………………........ iv

LIST OF FIGURES……….…………………………………………………………... vii

ABBREVIATIONS…….……………………………………………………………... ix

CHAPTER 1……………………………………………………………………......... 1

1. INTRODUCTION ………………………………………………………………… 2

1.1. GENERAL DESCRIPTION …………………..……………….............................. 2

1.2. FETAL TO NEWBORN VASCULAR TRANSITION …………………………...……. 4

1.3. DISEASE ETIOLOGY …………………………………………...…………….... 6

1.4. MOLECULAR TRIGGERS IN PHT DEVELOPMENT…………………….………… 7

1.4.1. RHOA/RHO-KINASE PATHWAY …………………………………...…... 10

1.4.2. NO-CGMP PATHWAY …………………………….…………………. 15

1.5. ROCK INHIBITORS AS A NOVEL TREATMENT FOR PHT ………………….…… 17

1.6. OTHER EXPERIMENTAL TREATMENTS FOR PHT ……………………………… 20

1.6.1. ET-1 RECEPTOR ANTAGONISTS………………………….……………. 20

1.6.2. TYPE V PHOSPHODIESTERASE (PDE-V) INHIBITORS………………...... 20

1.6.3. PLATELET-DERIVED GROWTH FACTOR (PDGF) RECEPTOR INHIBITORS.. 21

1.7. CHRONIC HYPOXIA MODEL OF NEONATAL PHT …………………...…………. 21

1.8. RATIONALE, GLOBAL AIMS, AND SPECIFIC HYPOTHESES ……………………... 23

v

CHAPTER 2…………………………………………..…………………………….. 25

2. MATERIALS AND METHODS ……………..……………………………………… 26

2.1. MATERIALS …………………………………………………………………... 26

2.2. INSTITUTIONAL REVIEW …………………………………………………….... 27

2.3. HYPOXIA EXPOSURE SYSTEM ……………………………………………….... 28

2.4. INTERVENTIONAL STUDIES …………………………………………………… 28

2.5. TWO-DIMENSIONAL ECHOCARDIOGRAPHY & DOPPLER ULTRASOUND………... 29

2.5.1. PULMONARY VASCULAR RESISTANCE ……………………………….. 29

2.5.2. PULMONARY ARTERY SYSTOLIC PRESSURE ………………………….. 32

2.5.3. RIGHT VENTRICULAR OUTPUT …………………………...………… 32

2.6. EXAMINATION OF RIGHT VENTRICULAR HYPERTROPHY …………………….... 32

2.7. PERCENT MEDIAL WALL THICKNESS ………………………………………...... 33

2.8. IMMUNOHISTOCHEMISTRY ………………………………………………...…. 33

2.9. HART’S ELASTIN STAINING ………………………………………………….... 34

2.10. WESTERN BLOT ANALYSES ………………………………………………….... 35

2.11. PULMONARY ARTERY SMOOTH MUSCLE CELL (PASMC)-ENRICHED CULTURE. 36

2.12. TERMINAL DUTP NICK-END LABELLING (TUNEL) ASSAY …………………… 37

2.13. APOPTOSIS-DETECTION ELISA …………………………………………...….. 38

2.14. CELL VIABILITY ASSAY ………..…………………………………………..…. 39

2.15. DATA PRESENTATION AND ANALYSIS ……………………………………….... 39

vi

CHAPTER 3………………………...……………………………………………….. 40

3. RESULTS ………………………………………………………………….……. 41

3.1. BODY WEIGHT ………………………………………………………….…….. 41

3.2. PULMONARY VASCULAR RESISTANCE ………………………………………... 41

3.3. PULMONARY ARTERY SYSTOLIC PRESSURE …………………………………... 41

3.4. RIGHT VENTRICULAR PERFORMANCE …………………………………..…….. 45

3.5. RIGHT VENTRICULAR HYPERTROPHY ……………………………………... 45

3.6. ARTERIAL WALL REMODELLING …………………………………...…………. 45

3.7. PREPRO-ET-1 IMMUNOHISTOCHEMISTRY ……………………………..……… 50

3.8. PREPRO-ET-1 CONTENT ………..……………………………………..……… 50

3.9. ROCK-I AND ROCK-II CONTENT ….………………………………………… 50

3.10. MLC-2 PHOSPHORYLATION ……………………………………………..…… 50

3.11. PASMC PROLIFERATION …………………………………………………...… 56

3.12. KI-67 IMMUNOHISTOCHEMISTRY ………………………………………...…... 56

3.13. IN VITRO TUNEL AND ELISA ASSAY FOR APOPTOSIS ………………………... 56

3.14. IN VIVO TUNEL ASSAY ……………………………………………….……… 56

CHAPTER 4………………..……………………………………………………….. 62

4. DISCUSSION ……………………………………………………………..……... 63

4.1. LIMITATIONS AND FUTURE RESEARCH …………………………………...…… 66

5. REFERENCE LIST .…...………………………………………….……………... 69

vii

LIST OF FIGURES

CHAPTER 1

Figure 1 Fetal circulation ………………………………………………………….... 5

Figure 2 Progression from healthy to remodelled pulmonary vessels ……………… 7

Figure 3 Vasoconstriction pathway …………………………………………………. 8

Figure 4 The role of ROCK in calcium sensitisation, which results in smooth

muscle contraction ………………………………………………………… 10

Figure 5 Several mechanisms of ROCK activation and inactivation ……………….. 12

Figure 6 Smooth muscle contraction through activation of MAPK

by ROCK ………………………………………………………………….. 13

Figure 7 Smooth muscle contraction through activation of LIMK

by ROCK ………………………………………………………………….. 14

Figure 8 Smooth muscle contraction through activation of CPI-17 by PKC………... 15

Figure 9 Endogenous nitric oxide (NO) pathway …………………………………... 16

Figure 10 Molecular structures of Y-27632 and Fasudil …………………………….. 17

Figure 11 Actin immunostaining of pulmonary vessels ……………………………… 23

CHAPTER 2

Figure 12 Colour flow Doppler image of the pulmonary artery outflow tract ……... 30

Figure 13A Representative Doppler trace with pulmonary haemodynamic

measurements……………………………………………………..………... 30

Figure 13B Doppler trace of pulmonary artery outflow………………………….…….. 31

viii

CHAPTER 3

Figure 14 Body weights ……………………………………………………………… 42

Figure 15 Pulmonary vascular resistance (PVR) …………………………………….. 43

Figure 16 Pulmonary artery systolic pressure (PASP) …..…………………………… 44

Figure 17 Right ventricular performance ….…………………………………………. 46

Figure 18 Right ventricular hypertrophy (RVH) ……………………………………... 47

Figure 19 Percent medial wall thickness (% MWT) …………………………………. 48

Figure 20 Hart’s elastin stain ………………………………………………………….49

Figure 21 Prepro-ET-1 immunostaining ……………………………………………... 51

Figure 22 Prepro-ET-1 content ………..………………………………………………52

Figure 23 ROCK-I content ……….…………………………………………………... 53

Figure 24 ROCK-II content ….………………………………………………………. 54

Figure 25 Rho-kinase activity ………………………………………………………... 55

Figure 26 Proliferation index ………………………………………………………… 57

Figure 27 Immunohistochemical staining for Ki-67, a marker for DNA synthesis…... 58

Figure 28 Apoptosis in primary cultures of PASMC ………………………………… 59

Figure 29 Apoptosis index …………………………………………………………… 60

Figure 30 Apoptosis marker in frozen tissue sections ………………………………... 61

ix

ABBREVIATIONS

ANOVA Analysis of variance

Ca2+

Calcium ion

CaM Calmodulin

cGMP Cyclic guanosine monophosphate

CPI-17 Protein kinase C-potentiated myosin phosphatase inhibitor 17

ECMO Extracorporeal membrane oxygenation

ELISA Enzyme-linked immunosorbent assay

eNOS Endothelial nitric oxide synthase

ET-1 Endothelin-1

GAP Guanosine tri-phosphatase activating protein

GDI Guanine nucleotide dissociation inhibitors

GDP Guanine diphosphate

GEF Guanine nucleotide exchange factors

GTP Guanine triphosphate

IHC Immunohistochemistry

iNO Inhaled nitric oxide

LIMK Lin-II, Is1-1, and Mec-3 kinase

MAPK Mitogen-activated protein kinase

MLC-2 Myosin light chain

MLCK Myosin light chain kinase

MLCP Myosin light chain phosphatase

% MWT Percent medial wall thickness

x

NO Nitric oxide

NSAID Non-steroidal anti-inflammatory drug

O2•-

Superoxide radical

ONOO-

Peroxynitrite anion

PA Pulmonary artery

PAAT Pulmonary artery acceleration time

PASMC Pulmonary artery smooth muscle cell

PASP Pulmonary artery systolic pressure

PAVTI Pulmonary arterial velocity time integral

PDE-V Type 5 phosphodiesterase

PHT Pulmonary hypertension

PKA Protein kinase A

PKG Protein kinase G

PPHN Persistent pulmonary hypertension of the newborn

PVR Pulmonary vascular resistance

ROCK Rho-kinase

RV Right ventricle

RVET Right ventricular ejection time

RVH Right ventricular hypertrophy

RVI Right ventricular index

sGC Soluble guanylate cyclase

SMC Smooth muscle cell

TUNEL Terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labelling

1

Chapter 1

Introduction

2

1.1. General description

Pulmonary hypertension (PHT) is a debilitating disease characterised by an increase in

pulmonary arterial pressure and resistance. In early life, persistent pulmonary hypertension of

the newborn (PPHN) occurs in approximately 1 per 1000 term or near-term live births in North

America (Walsh-Sukys et al., 2000). PPHN usually presents within one to two days of birth,

and frequently results from clinical syndromes associated with persistent hypoxaemia, including

meconium aspiration and perinatal asphyxia (Evans & Archer, 1991; Walsh-Sukys, 1993;

Walsh-Sukys et al., 2000). Other conditions that may lead to PHT during the neonatal period

and infancy include congenital diaphragmatic hernia (Khemani et al., 2007; Mohseni-Bod &

Bohn, 2007; Soll, 2009) and severe cases of chronic lung disease of prematurity (Haworth,

1988; Rabinovitch, 1989; Thibeault et al., 2003; Parker et al., 2006). Persistent pulmonary

hypertension of the newborn has an associated mortality of 10-20% (Walsh-Sukys, 1993;

Walsh-Sukys et al., 2000) and survivors have an high morbidity (40-50%) in the form of

cognitive delays, cerebral palsy (Tanaka et al., 2007), hearing loss (Eriksen et al., 2009) and

frequent need for re-hospitalisation (Lipkin et al., 2002). The incidence of severe adverse

outcomes from other forms of neonatal PHT is likely higher.

An increased propensity to PHT in the newborn period, compared to other stages of life,

is predominantly related to two factors: (1) failure of the normal fall in pulmonary vascular

resistance (PVR) required for a successful transition to postnatal life and (2) the rapid

development and persistence of anatomical changes in the heart and pulmonary vasculature,

known collectively as “vascular remodelling”. Structural and functional consequences of

remodelling include narrowing of the vessel lumen, exaggerated responses to vasoconstrictors,

3

decreased relaxation and reduced responsiveness to vasodilators, all of which contribute to a

“fixed” increase in PVR. Increased PVR affects the right heart by increasing right ventricular

(RV) afterload, leading to right ventricular hypertrophy (RVH). Clinically, neonatal PHT

presents with an elevated FiO2 requirement and mechanical ventilation may be insufficient to

restore an adequate PaO2. If left untreated, right ventricular performance deteriorates, leading to

cor pulmonale and eventual death.

The current gold-standard treatment for neonatal PHT is inhaled nitric oxide (iNO),

which is approved for use in neonates above 35 weeks’ gestational age by Health Canada and

the United States Food and Drug Administration, and was the first therapy approved for

treatment of hypoxaemic respiratory failure following large clinical trials (NINOS, 1997;

Group, 1999; Clark et al., 2000; Clark et al., 2003; Li et al., 2009).

Though iNO is used extensively in NICUs across developed nations, there are a number

of serious drawbacks to its use, including non-responsiveness in up to 40% of patients. For

those patients who responded initially, rebound PHT (upon attempted weaning from iNO) or

non-sustained response is frequently observed. Rebound PHT is thought to be caused by a

down-regulation of endogenous NO production (Lee et al., 2008) and/or upregulated type 5

phosphodiesterase (PDE-V) expression following iNO treatment (Namachivayam et al., 2006).

Though most patients respond to iNO therapy in terms of improved systemic oxygenation and a

decreased need for more invasive interventions, such as extracorporeal membrane oxygenation

(ECMO), its use has not been associated with improvements in long term outcomes such as

cerebral palsy (NINOS, 2000; Clark et al., 2003), or in overall mortality rates (NINOS, 1997).

Furthermore, due to the high cost and the sophisticated equipment required for administration,

iNO is largely unavailable in developing nations. Currently, the usual second-line option for

4

neonates who do not respond to iNO is ECMO, which has been shown to decrease mortality

(Fakioglu et al., 2005), though it is associated with substantial risks of intracranial haemorrhage,

further contributing to neurocognitive impairment later in life (Short, 2005). Thus, the

investigation of novel treatments for PPHN patients who are nonresponsive to current

conventional therapies remains a high priority, and is the focus of this thesis.

1.2. Fetal-to-newborn vascular transition

All newborns must undergo a dramatic shift in circulation patterns in order to

successfully transition from an environment within the womb to the outside world. During fetal

life, less than 10% of the combined ventricular output passes through the lungs; the rest of the

outflow from the right ventricle passes through the patent foramen ovale or ductus arteriosus to

the systemic circulation (Fig. 1). These pathways are maintained because of the high resistance

in pulmonary arteries (PAs). The high resistance, in turn, is due to constriction of the relatively

abundant, muscular-walled, PAs. Transition from fetal to newborn circulation involves

elimination of the placental circulation, transfer of the function of gas exchange from the

placenta to the lungs, and closure of the fetal pulmonary-systemic shunts. A marked fall in

pulmonary vascular resistance (PVR), resulting in a dramatically increased pulmonary blood

flow, is central to this transition. The low-impedance placenta is lost at birth, and as a result,

high systemic and low PVR characterises the neonatal circulation. The fall in PVR occurs in

two phases. The first phase, commencing immediately after birth, is marked by a dramatic

decrease in pulmonary arterial tone, and therefore a dilatation of pulmonary vessels. Factors that

contribute to this phenomenon include expansion of the lungs with air after birth and the higher

oxygen tension of ambient air. The second, and more gradual, phase is associated with a

5

decrease in the muscularisation of arterial walls and a resultant relative increase in the number

of non-muscularised pulmonary arteries. A failure of the normal postnatal decrease in PVR

characterises PPHN, which presents as persistent cyanosis and hypoxaemia with or without

cardiac dysfunction.

Figure 1. Fetal circulation. The placenta oxygenates blood for the growing fetus, to which it

flows from the umbilical vein through the ductus venosus to the inferior vena cava, the right

atrium, the right ventricle, and is then pumped into the pulmonary artery. Due to high

pulmonary vascular resistance and an open ductus arteriosus, the majority of blood flow is from

the pulmonary artery through the ductus arteriosus and into the aorta; blood also flows across

the foramen ovale from the right to the left atria. Reproduced from the website of Lucile

Packard Children’s Hospital in Stanford:

http://www.lpch.org/DiseaseHealthInfo/HealthLibrary/hrnewborn/0181-pop.html.

6

The second phase, largely complete by two months of postnatal age, is dependent upon

the maintenance of low PVR. A reduction in the weight of the right ventricle relative to the left,

and a decrease in wall thickness of the major pulmonary arteries accompany this change in the

pulmonary arteries. Right ventricular weight is closely related to pulmonary arterial muscle

mass in vessels of varying sizes, making the presence of a relative hypertrophy of the right

ventricle a useful marker of PHT (Rabinovitch et al., 1979).

1.3. Disease etiology

Pulmonary hypertension presenting in early life results from one or more of the

following pathophysiological mechanisms: underdevelopment, maldevelopment, or

maladaptation of the pulmonary vasculature.

In cases of underdeveloped pulmonary vasculature, raised PVR is relatively permanent,

and is found in a variety of conditions, including congenital diaphragmatic hernia, cystic

adenomatoid malformation of the lung, and pulmonary hypoplasia syndromes. Because of the

relative permanence of PHT due to underdevelopment, patients who possess these conditions

tend to be the most difficult to treat and have the worst outcomes (Levin, 1978).

Maldevelopment of the pulmonary vasculature results from abnormalities involving

bronchiolar branching and alveolar differentiation, the number of pulmonary vessels in the

lungs, and/or an increase in the muscularisation of the pulmonary vessels, especially into the

typically non-muscularised intra-acinar vessels of the pulmonary system (Murphy et al., 1981),

leading to failure of normal pulmonary arterial relaxation after birth. Additionally, it has been

shown that some drugs taken by the mother during gestation may induce maldevelopment of the

pulmonary vasculature in newborns, resulting in an increased incidence of PPHN (Belik, 2008).

7

These drugs include non-steroidal anti-inflammatory drugs (NSAIDs) (eg: indomethacin,

ibuprofen) and the anti-depressant drugs of the serotonin reuptake-inhibitor class (SRIs) (eg:

Prozac, Zoloft, Paxil), which have been shown to increase the incidence of PHT, evidenced by

remodelling of the pulmonary vasculature (Alano et al., 2001; Chambers et al., 2006; Belik,

2008).

Lastly, PHT may result from maladaptation of a fetal pulmonary vascular bed that is ill-

equipped to adapt to extrauterine life. Maladaptation is known to result from such conditions as

perinatal asphyxia, pulmonary parenchymal diseases (meconium aspiration, congenital

pneumonia), and congenital sepsis (Curtis et al., 2003; Dakshinamurti, 2005).

1.4. Molecular triggers in PHT development

Increased PVR is related to two phenomena: (1) sustained vasoconstriction and (2)

vascular remodelling, the latter being the pathological hallmark of PHT in neonates and children

(Fig. 2).

Figure 2. Progression from a healthy to a remodelled pulmonary vessel. Reproduced with the

permission of Dr. A. Keith Tanswell.

8

Vascular remodelling is characterised by the muscularisation of normally non-

muscularised pulmonary vessels, such as intra-acinar PAs, and the hyperplasia and hypertrophy

of medial wall smooth muscle cells (SMCs) in arteries that are normally muscularised (Kantores

et al., 2006). Once established, remodelling has traditionally been believed to be non-reversible,

contributing to poor responses to current therapies and to an increased potential for a

progressive worsening of the PHT and eventual death (Guilluy et al., 2009). Thus, it is

important to understand how this process occurs, and the mechanisms underlying the

development and persistence of sustained vasoconstriction and remodelling of the pulmonary

vasculature during the neonatal period.

A major cellular mechanism of pulmonary vasoconstriction involves the activation of G-

proteins, which are located at the plasma membranes of smooth muscle cells surrounding

pulmonary arterioles (Fig. 3).

Figure 3. Vasoconstriction pathway. Ca2+

= calcium ions, CaM= calmodulin, MLC-2= myosin

regulatory light chain, MLCK= myosin light chain kinase, MLCP= myosin light chain

phosphatase, P= phosphate molecule.

9

G-proteins are activated by hypoxia and binding of G-protein-coupled receptor (GPCR)

ligands, such as endothelin-1 (ET-1) and thromboxane A2. Activated G-proteins open calcium

ion (Ca2+

) channels at the plasma membrane and at the sarcoplasmic reticulum membranes

within the cell, allowing the entry of Ca2+

into the cell, and the release of sequestered Ca2+

from

stores in the endoplasmic reticulum, respectively. Calcium then binds to calmodulin, creating

the Ca2+

-calmodulin complex (Ca2+

-CaM). Ca2+

-CaM activates myosin light chain kinase

(MLCK), which in turn, phosphorylates myosin light chain (MLC-2, also known as MLC, LC-

20, or MRSLC), resulting in increased smooth muscle contraction. Conversely, the enzyme

myosin light chain phosphatase (MLCP) is involved in the dephosphorylation of MLC-2,

leading to smooth muscle relaxation.

10

1.4. 1. RhoA/Rho-kinase pathway

Another mechanism whereby sustained vasoconstriction may occur is through a pathway

involving a phenomenon known as “calcium sensitisation” (Fig. 4).

Figure 4. The role of ROCK in calcium sensitisation, which contributes to sustained smooth

muscle contraction. GDP= guanosine diphosphate, GTP= guanosine triphosphate, MLC-2=

myosin light chain, MLCK= myosin light chain kinase, MLCP= myosin light chain phosphatase,

RhoA= Rho GTP family of proteins, member A, ROCK= rho-kinase.

This phenomenon generally results when an increase in smooth muscle cell contraction occurs

without a change in Ca2+

concentration, and is thought to be mainly due to the phosphorylation

and deactivation of the enzyme MLCP. Phosphorylated MLCP is not able to dephosphorylate

MLC-2, and muscle contraction persists. This activity is mediated through the actions of a

small protein in the Rho GTP family of proteins, RhoA, and its effector protein, Rho-kinase

(ROCK) (Somlyo et al., 1999). ROCK has two known isoforms, ROCK-I (ROCK-β) and

11

ROCK-II (ROCK-α) and both are found ubiquitously throughout the vasculature (Nakagawa et

al., 1996).

Rho GTP binding proteins consist of a large number of Rho, Rac, and Cdc42 subfamilies

that are involved in many cellular processes such as gene transcription, differentiation,

proliferation, hypertrophy, apoptosis, phagocytosis, adhesion, migration, and contraction

(Riento & Ridley, 2003; Jaffe & Hall, 2005). Moreover, there are many molecules involved in

both the activation and deactivation of ROCK, such as guanine nucleotide exchange factors,

guanine nucleotide disassociation inhibitors, and GTPase-activating proteins, among others.

Cellular mechanisms leading to the activation of ROCK are believed to begin with

activation of G-proteins at the smooth muscle cell membrane. This activates GEFs, leading to

replacement of GDP by GTP on the RhoA GTPase (Fig. 5). RhoA-GTPase translocates to the

cell membrane, activating a number of downstream effectors, including ROCK. ROCK can

become deactivated by a series of negative regulators such as: GDIs that inhibit the exchange of

GDP by GTP; GAPs that convert RhoA-GTP to RhoA-GDP; statins that inhibit the

translocation of RhoA-GTP to the cell membrane; and protein kinases A (PKA) and G (PKG)

which also prevent translocation of RhoA-GTP to the cell membrane (Sawada et al., 2001;

Murthy, 2006).

12

Figure 5. Several mechanisms of ROCK activation and inactivation. GAP= GTPase activating

proteins, GDI= guanine nucleotide dissociation inhibitors, GDP= guanylate diphosphate,

GEF= guanine nucleotide exchange factors, GTP= guanylate triphosphate, PKA= protein

kinase A, PKG= protein kinase G.

The role of ROCK in the development of PHT has recently become an issue of

considerable interest, as it is now believed that ROCK may play a central role in the regulation

of vasoconstriction. This belief has evolved from an increased appreciation of the influence of a

number of physiological (eg: hypoxia) or biochemical (eg: serotonin; mitogen-activated protein

kinase- MAPK; Lin-II, Is1-1, and Mec-3 kinases- LIMK; nitric oxide, reactive oxygen species,

and endothelin-1) factors on ROCK, many of which are believed to contribute to the

pathogenesis of PHT (McMurtry et al., 2003).

Recently, RhoA was demonstrated to respond to the transport of serotonin (5-HT)

through the cell membrane of pulmonary artery smooth muscle cells (PASMCs) via serotonin

transporters (5-HTT) (Guilluy et al., 2009). The transport of 5-HT from outside of the cell into

the cell was demonstrated to result in RhoA serotonylation, which in turn resulted in increased

ROCK activity in the lungs, platelets, and PASMCs of patients with PHT (Guilluy et al., 2009).

13

ROCK is also known to regulate the expression of mitogen-activated protein kinase (p38

MAPK), Lin-II, Is1-1, and Mec-3 kinases (LIMK), and protein-kinase C-potentiated myosin

phosphatase inhibitor (CPI-17), all of which are known effectors of SMC contraction.

MAPK regulates smooth muscle contraction and, when activated by ROCK (Fig. 6),

phosphorylates heat shock protein 27 (HSP27) (Lopez-Ilasaca, 1998; Nwariaku et al., 2003;

Galaria et al., 2004). This activates HSP27, leading to increased polyermized actin stabilisation,

which leads to increased muscle contraction (Yamboliev et al., 2000).

Figure 6. Smooth muscle contraction through activation of MAPK by ROCK. HSP27= heat

shock protein 27, MAPK= mitogen-activated protein kinase, ROCK= Rho-kinase.

ROCK also affects the enzyme LIMK (Fig. 7). ROCK phosphorylates LIMK, which

then phosphorylates an actin-binding protein called cofilin, leading to its inactivation (Maekawa

et al., 1999). Inactivation of cofilin contributes to actin polymerization and thereby enhanced

potential for vasoconstriction (Sakamoto et al., 2003). Sakamoto et al. (2003) and Somlyo and

Somlyo (2003) have demonstrated that Y-27632 inhibited LIMK phosphorylation, which led to

a decrease in vaso-reactivity as well as a disruption in the actin cytoskeleton, which eventually

resulted in an attenuation of LIMK-related vasoconstriction.

14

Figure 7. Smooth muscle contraction through activation of LIMK by ROCK. LIMK= Lin-II,

Is1-1, and Mec-3 kinase, ROCK= Rho-kinase.

It is believed that CPI-17 is also affected indirectly by ROCK (Fig. 8). CPI-17 is

phosphorylated by protein kinase C (PKC), and when phosphorylated, CPI-17 inhibits the

activity of MLCP, leading to persistent vasoconstriction (Sakai, 2004). It has been shown that

ROCK inhibition has resulted in decreased CPI-17 phosphorylation, and thus attenuation of

SMC contraction (Sakai, 2004; Pourmahram et al., 2008).

15

Figure 8. Smooth muscle contraction through activation of CPI-17 by PKC. CPI-17= protein-

kinase C-potentiated myosin phosphatase inhibitor, MLC-2= myosin light chain, MLCK=

myosin light chain kinase, MLCP= myosin light chain phosphatase, PKC= protein kinase C,

ROCK= Rho-kinase.

1.4. 2. NO-cGMP pathway

Another major regulator of pulmonary vascular tone is the nitric oxide-cyclic guanosine

monophosphate (NO-cGMP) pathway. Nitric oxide is produced from L-arginine in endothelial

cells through the activity of the enzyme endothelial nitric oxide synthase (eNOS) (Fig. 9).

Nitric oxide diffuses into smooth muscle and acts upon its intracellular receptor, soluble

guanylate cyclase (sGC). Activation of sGC leads to increased production of cyclic guanosine

monophosphate (cGMP), which causes Ca2+

desensitisation and relaxation of smooth muscle via

increased activity of PKG.

16

Figure 9. Endogenous nitric oxide (NO) pathway. 5’-GMP= 5’- guanylate monophosphate,

cGMP= cyclic guanylate monophosphate, eNOS= endothelial NO synthase, GTP=guanine

triphosphate, NO= nitric oxide, PDE V= type 5 phosphodiesterase, PKG= protein kinase G,

sGC= soluble guanylate cyclase.

The bioavailability of NO may be directly affected by the oxygen radical superoxide

(O2•-), through the formation of NO into the peroxynitrite anion (ONOO

-), which may itself

contribute to the pathogenesis of PHT (Jankov et al., 2000; Belik et al., 2004; Abman, 2007).

Furthermore, low concentrations of L-arginine, a NO precursor, oxidisation of sGC and an

increase in activity of PDE-V (a cGMP-degrading enzyme), are also likely to further contribute

to PHT (Pearson et al., 2001; Andersen et al., 2008). The likelihood of reciprocal interactions

between the RhoA/ROCK and NO-cGMP pathways is evidenced by the following: reports of

the inhibitory effects of PKG on RhoA activation (Sawada et al., 2001; Murthy, 2006); the

decreased expression of endothelin-1 (ET-1), a major upstream effector of ROCK, in response

to stimulation of cGMP-mediated pathways (Kourembanas et al., 1993); and the down-

regulation of eNOS expression resulting from increased ROCK activity (Abe et al., 2006).

Therefore, the enhanced activity of one pathway appears to lead to the diminished activity of the

other.

17

1.5. ROCK inhibitors as a novel treatment for PHT

Two pharmacological Rho-kinase inhibitors, Y-27632 (Ishizaki et al., 2000) and Fasudil

(HA-1077) (Sasaki et al., 2002) (Fig. 10), though structurally dissimilar, have both been shown

to possess high specificity toward the two known ROCK isoforms (ROCK-I and ROCK-II)

when compared to a broad range of other kinases (Davies et al., 2000). This specificity has led

to the recognition that the activation of ROCK may be a central convergence point for multiple

pathways that can induce both sustained vasoconstriction and remodelling of pulmonary

resistance arteries.

Figure 10. Molecular structures of Y-27632 and Fasudil (HA-1077).

An increased appreciation of the importance of vascular remodelling in the pathogenesis

of chronic PHT has led to a shift in focus away from drugs that act solely as pulmonary

vasodilators, towards treatments that target and attenuate PASMC hyperplasia through pro-

apoptotic and/or anti-proliferative effects. Although patients with PHT have increased vascular

muscularisation, along with persistent pulmonary vasoconstriction, current treatments

(particularly iNO) target their role as vasodilators. However, once vascular remodelling is

18

established, such therapies are less likely to be efficacious (Guilluy et al., 2009). Drugs that

dilate pulmonary vessels and have pro-apoptotic/anti-proliferative effects on vascular wall

SMC’s, would be more likely to reverse chronic PHT. Rho-kinase inhibitors, such as Y-27632

and Fasudil, are unique in having been shown to both dilate the remodelled pulmonary

vasculature and to promote apoptosis in various cell types, thus providing a strong rationale for

the potential use of ROCK inhibitors as a treatment for chronic PHT.

Y-27632, the ROCK inhibitor used in experiments contributing to this thesis has been

shown to have a 50% lower IC50 on ROCK activity than Fasudil, and has been used in adult

animal models of PHT with beneficial attenuating affects on structural changes of PHT

(Doggrell, 2005; Rattan & Patel, 2008). Y-27632 (Fig.10) is a serine-threonine ROCK inhibitor

that competitively binds to ATP binding sites on the ROCK enzyme (Doggrell, 2005), and has

been shown to have vasodilatory effects in many different studies. ROCK inhibitors have been

reported to inhibit pulmonary artery myogenic responses in hypoxia-exposed adult rats

(Broughton et al., 2008) and fetal sheep (Tourneux et al., 2008) and to reverse sustained

pulmonary vasoconstriction in response to hypoxia (Fagan et al., 2004; McNamara et al., 2008),

bleomycin (McNamara et al., 2008) or the infusion of vasoconstrictors, such as endothelin-1

(Weigand et al., 2006). When delivered directly to the lung, Y-27632 was found to acutely

decrease PVR in adult rats with PHT induced by hypoxia (Nagaoka et al., 2005).

In cultured PASMCs, treatment with Y-27632 resulted in decreased formation of stress

fibres and attenuated Ca2+

sensitisation (Uehata et al., 1997). Chen reported attenuating effects

of ROCK inhibitors on PASMC proliferation induced by 5-HT (Chen et al., 2009). Such results

may help explain the effects of chronic treatment with ROCK inhibitors in preventing the

structural changes of PHT in adult rodent models. Systemic administration of ROCK inhibitors,

19

commenced at the onset of injury, has been reported to prevent vascular remodelling induced

either by hypoxia (Fagan et al., 2004) or monocrotaline (Abe et al., 2004). Y-27632 also

enhanced eNOS expression and resulted in the attenuation of chronic hypoxia-induced

angiogenesis (Fagan et al., 2004; Hyvelin et al., 2005). Furthermore, in monocrotaline-exposed

adult rats, a ROCK inhibitor was reported to prevent progression of disease leading to mortality

and to induce regression of vascular remodelling when commenced after the injury was already

well established (Abe et al., 2004).

Several pilot studies examining the effects of a single bolus dose of ROCK inhibitor

(Fasudil) have been published, demonstrating that intravenous Fasudil administered to adult

humans with idiopathic chronic PHT (Nagaoka et al., 2005; Ishikura et al., 2006) and children

with congenital heart disease-associated PHT (Li et al., 2009) lowered PA pressure and PVR.

Subsequent studies have shown the RhoA and ROCK pathway to be activated in pulmonary

vessels of adult humans with PHT (Do e et al., 2009; Guilluy et al., 2009).

Though there were significant benefits to the use of ROCK inhibitors for treatment of

PHT in both experimental models and humans, no improvements in right ventricular

performance have yet been described. This is an important factor to consider, as decreased right

ventricular performance is an important prognostic factor for PHT: its presence being highly

predictive of adverse outcome (McNamara et al., 2008; Bogaard et al., 2009).

20

1.6. Other experimental treatments for PHT

1.6.1. ET-1 receptor antagonists

ET-1 is an important vasoconstrictor that affects many pathways leading to PHT. ET-1

has two receptors, ETA and ETB; ETA is known to increase vasoconstriction and proliferation of

vascular SMCs and ETB is thought to be involved in ET-1 degradation, and therefore

contributing to clearance of circulating ET-1. The activation of ETB has been shown to result in

vasodilatation and NO release (Badesch et al., 2004). Bosentan (Tracleer®), an antagonist of

both ETA and ETB receptors, received Health Canada approval in 2001 for the treatment of

chronic PHT in adults (Michelakis et al., 2002). Clinical experience with Bosentan in newborns

is limited, partly related to potential concerns regarding hepatic toxicity (Zhao et al., 2001;

Michelakis et al., 2002). Expression of prepro-ET-1, a precursor to ET-1, was examined in this

thesis.

1.6.2. Type V phosphodiesterase (PDE-V) inhibitors

PDE-V is involved in the endogenous NO pathway (Fig. 9), by degrading cGMP,

resulting in smooth muscle cell contraction. PDE-V inhibitors, such as Sildenafil, have been

shown to improve PHT pathology by decreasing the rate of degradation of cGMP, resulting in

smooth muscle cell relaxation (Zhao et al., 2001; Lee et al., 2008). Based on the literature,

PDE-V inhibitors show promise as an adjunct treatment for neonatal PHT as well as a treatment

administered following NO weaning to decrease risks of rebound PHT (Galie et al., 2005;

Namachivayam et al., 2006; Lee et al., 2008), though experience with PHT in newborns is

currently limited to clinical trials.

21

1.6.3. Platelet-derived growth factor (PDGF) receptor inhibitors

PDGFs act as mitogens affecting the growth and development as well as the migration of

SMCs and fibroblast cells (Ostman et al., 1992; Haniu et al., 1993; Haniu et al., 1994). PDGFs

exist in five dimeric isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD

(Haniu et al., 1994). PDGF isoforms can bind to two different receptors, PDGF-α receptor

(PDGF-αR) and PDGF-β receptor (PDGF-βR) (Dalla-Favera et al., 1982). These receptors have

been found to be upregulated in lambs with chronic intrauterine pulmonary hypertension when

compared with control animals (Balasubramaniam et al., 2003). Furthermore, it was

demonstrated that human cases of severe pulmonary arterial hypertension exhibited an

overexpression of PDGF-AA (Humbert et al., 1998). Given the increasing evidence that one or

more PDGFs may play a role in the pathogenesis of vascular remodelling, there has been

interest in the use of drugs which antagonize this pathway. Tyrosine kinase inhibitors such as

imatinib mesylate, have been shown to reverse remodelling in adult models, have shown

therapeutic promise in experimental models of chronic PHT in which vascular SMC apoptosis

was increased (Schermuly et al., 2005; Chhina et al., 2010).

1.7. Chronic hypoxia model of neonatal PHT

Rodents are highly suitable as models for neonatal lung injury, given that alveolar and

acinar (distal airway-associated) vascular development are postnatal events (Burri, 1974) that

share many similarities with human neonatal PHT. Rats are the preferred species for modeling

PHT, as injury develops much more readily in rats than in mice (Rabinovitch et al., 1981; Chen

et al., 2006). Experimental studies in the immature rat have demonstrated a unique

susceptibility, when compared to adults, to the development of a rapidly progressive form of

22

pulmonary vasculopathy, characterised by severe vascular remodelling and cardiac dysfunction

(Rabinovitch et al., 1981; Kantores et al., 2006; McNamara et al., 2008), ultimately leading to

death. In humans with neonatal PHT, abnormalities in pulmonary vascular function have been

described to persist into adulthood (Sartori et al., 1999). Similarly, recovery from pulmonary

vascular injury in the immature rat is likely to be delayed and incomplete (Rabinovitch et al.,

1981; King et al., 1995; Keith et al., 2000), contributing to impaired lung growth (Meyrick &

Reid, 1981, 1982; Fabris et al., 2001) and a high potential for recurrence of PHT in later life

(Hampl & Herget, 1990; Caslin et al., 1991; King et al., 1995).

With the goal of testing novel approaches to therapy, our laboratory has developed a

neonatal Sprague Dawley (SD) rat model of chronic hypoxic PHT secondary to exposure to

13% O2 from birth until 14 days of life (Kantores et al., 2006). Pathological features of PHT in

this model include structural (RV hypertrophy and arterial medial wall thickening; Fig. 11) and

functional (increased PVR and RV dysfunction) abnormalities. Furthermore, once chronic PHT

is established (by 14 days), this injury model is unresponsive to iNO (McNamara et al., 2008),

replicating the “fixed” and often-fatal form of human neonatal PHT.

23

Figure 11. Actin immunostaining of pulmonary vessels. Note the increased expression of actin

(dark brown), a marker for smooth muscle, in lung sections from a chronic hypoxia-exposed

pup, compared to sections from a normoxia-exposed pup; Hematoxylin (light blue) stains all cell

nuclei. Arrows point to pulmonary vessels.

1.8. Rationale, global aims and specific hypotheses

We have previously shown that the RhoA/ROCK pathway is activated in the pulmonary

vasculature of SD rat pups with chronic PHT induced by exposure to 13% O2 from birth to 14

days (McNamara et al., 2008). In acute studies using this model, a single bolus of the ROCK

inhibitor, Y-27632, had far superior vasodilatory effects upon the hypertensive pulmonary

circulation when compared to the current “gold standard” therapy, iNO (McNamara et al.,

2008). Furthermore, we have shown that treatment with Y-27632 from birth, concurrent with

hypoxia exposure, prevented vascular remodelling through inhibitory effects on arterial wall

smooth muscle cell proliferation (Ziino et al., 2010). These studies indicate that ROCK is

critical to sustained vasoconstriction and mediates smooth muscle proliferation and arterial wall

remodelling in the chronic hypoxia-exposed neonatal rat.

24

Based upon these findings, the global aim of the studies upon which this thesis is based

was to determine whether late sustained (or “rescue”) treatment with Y-27632 (from day 14-21

of life), commenced after chronic PHT was already established, would reverse the structural and

functional changes of chronic hypoxic PHT. Reversal of the pathological changes of chronic

PHT is an important therapeutic goal, as the continued presence of these changes may increase

the likelihood of progression toward fatal PHT in later life (Hampl & Herget, 1990; Caslin et

al., 1991).

Specifically, my hypotheses were that late-sustained treatment with the ROCK inhibitor,

Y-27632, in neonatal rats with established chronic hypoxic PHT would:

1. normalise raised PVR and RV dysfunction

2. reverse structural abnormalities, including RVH and arterial medial wall thickening, and

lead to inhibited proliferation and augmented apoptosis of pulmonary arterial smooth

muscle cells

25

Chapter 2

Materials and Methods

26

2.1. Materials

The Rho-kinase inhibitor, Y-27632, was purchased from Alexis Biochemicals (San

Diego, CA, U.S.A.). Oxygen exposure chambers and automated controllers (OxyCycler model

A84XOV) were from Biospherix (Redfield, NY, U.S.A.). Phos-tag acrylamide was purchased

from NARD Institute (Amagasaki City, Hyogo, Japan). Total protein assay kits, tris-glycine

gels and poly-vinylidene difluoride (PVDF) membranes were from BioRad (San Diego, CA,

U.S.A.). Protease inhibitors, Cell Death Detection ELISAPLUS

and terminal deoxyuridine

triphosphate (dUTP) nick-end labelling (TUNEL) assay kits were purchased from Roche (Laval,

QC, Canada). Dulbecco’s modified Eagle’s medium (DMEM), antibiotic-antimycotic solution,

trypsin and ethylenediamine tetra-acetic acid (EDTA) were from Gibco BRL (Burlington, ON,

Canada). Heat-inactivated fetal bovine serum (FBS) and normal goat serum (NGS) were from

Flow Laboratories (McLean, VA, U.S.A.); collagenase and DNAse were from Worthington

(Freehold, NJ, U.S.A.). Acids, alcohols, chromatography-grade organic solvents, H2O2,

Permount and Superfrost/Plus microscope slides were purchased from Fisher Scientific

(Markham, ON, Canada). All other chemicals and reagents were purchased from Sigma

Chemical Co. (Oakville, ON, Canada).

Goat anti-rabbit and anti-mouse immunoglobulin (IgG)-peroxidase antibodies were from

Cell Signaling Technology (Beverly, MA). Goat anti-rabbit, goat anti-mouse and donkey anti-

goat IgG-biotin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). 6-

diamino-2´-phenylindole (DAPI) mounting medium, and 3, 3´ diaminobenzidine (DAB)

staining kits were from Vector Laboratories (Burlingame, CA, U.S.A.). Aqueous mounting

medium was from DAKO Cytomation (Carpenteria, CA, U.S.A.). Wst-8 Cell Proliferation kits

27

were from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). Primary antibody sources and

concentrations used for Western blot (WB) and/or immunohistochemistry (IHC) on paraffin-

embedded tissue are as follows:

Antibody (Source) WB IHC Catalogue #

α-Smooth muscle actin 1:5000 1:1000 MS-113-PI

(Neomarkers, Fremont, CA, U.S.A.)

Glyceraldehyde-3-phosphate dehydrogenase 1:5000 ---------- SC-25778

(Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.)

Myosin Regulatory Light Chain 1:2000 ---------- SC-15370

(Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.)

Rho-kinase I (Santa Cruz, CA, U.S.A.) 1:2000 ---------- SC-17794

Rho-kinase II (Santa Cruz, CA, U.S.A.) 1:200 ---------- SC-5561

Ki67 antigen (Dako, Carpenteria, CA, U.S.A.) ---------- 1:25 M-7248

Prepro-endothelin-1 ---------- 1:200 T-4306.0050

(Cedarlane, Burlington, ON, Canada)

Prepro-endothelin-1 1:3000 ---------- MA3-005

(Thermo Scientific, Rockford, IL, U.S.A.)

2.2. Institutional review

All procedures involving animals were conducted according to criteria established by the

Canadian Council on Animal Care. Approval for studies was obtained from the Sunnybrook

Research Institute Animal Care Committee.

28

2.3. Hypoxia exposure system

Pathogen-free timed pregnant SD rats were obtained from Taconic (Germantown, NY,

U.S.A.). Experiments were conducted as paired exposures with one litter receiving 13% O2

(hypoxia) and another litter receiving 21% O2 (air) from day one of life for up to 17 or 21 days,

depending upon the endpoint of interest. O2 and CO2 concentrations, temperature and humidity

were continuously monitored, recorded, and regulated by computer using customised software

(AnaWin2 Run-Time, version 2.2.18, Watlow-Anafaze, St. Louis, MO, U.S.A.). Gas delivery

was automatically adjusted to maintain an O2 concentration within 0.1% of the set point. O2

sensors were calibrated weekly. On the anticipated day of delivery, each dam was placed in an

100 × 80 × 60 cm Plexiglass chamber with 12 h/12 h light-dark cycles, and with temperature

maintained at 25 ± 1°C, humidity ≈ 50%, and CO2 concentration <0.5%. Equal litter sizes (10-

12 pups) and sex distribution were maintained throughout the exposure period. Food and water

were available ad libitum. Dams were exchanged daily between paired air and hypoxia

chambers to prevent any maternal toxicity and consequent nutritional effects on the pups. At

the end of each exposure period, pups were either killed by exsanguination after anaesthesia or

by pentobarbital sodium overdose.

2.4. Interventional studies

Injections (15 mg/kg body weight) were given subcutaneously (s.c.) via a 30-gauge

needle twice per day (bis in die or b.i.d.) for a daily dose of 30 mg/kg body weight) starting on

postnatal day 14.

29

2.5. Two-dimensional echocardiography and Doppler ultrasound

Two-dimensional echocardiography and Doppler ultrasound, performed by Dr.

McNamara, was employed as a non-invasive method for assessment of pulmonary

haemodynamics using a Vivid 7 Advantage cardiovascular ultrasound system (GE Medical

Systems, Milwaukee, WI) with a small high frequency linear probe (I13L).

An ultra-high frame

rate provided high image quality, which was found to be particularly suitable for small animal

imaging, in which the heart rate is very rapid. Immediately following administration of

anesthesia with intraperitoneal (i.p.) ketamine hydrochloride (40 mg/kg) and xylazine

hydrochloride (6 mg/kg), the animal was laid supine and the probe was gently applied to the

chest while the animal was spontaneously breathing room air. For measurements of pulmonary

arterial haemodynamics, a short axis view at the level of the aortic valve was obtained, and the

pulmonary artery was identified using colour flow Doppler (Fig. 12). Analysis was undertaken

in a blinded fashion.

2.5.1. Pulmonary vascular resistance

Pulmonary vascular resistance is widely used as a marker for severity of pulmonary

hypertension (Ananthakrishnan et al., 2009; Corte et al., 2009; Rame, 2009). The pulsed

Doppler gate was placed proximal to the pulmonary valve leaflets and aligned, with an angle of

insonation < 20°, to maximize laminar flow. The pulmonary artery acceleration time (PAAT)

and right ventricular ejection time (RVET) were then estimated using the pulmonary Doppler

profile (Figs. 13A and 13B). PAAT was measured as the time from the onset of systolic flow to

peak pulmonary outflow velocity, and RVET was measured as the time from onset to

30

completion of systolic pulmonary flow. A surrogate measure of PVR was calculated according

to the formula: PVR= 1/ (PAAT: RVET) (Jones et al., 2002).

Figure 12. Colour flow Doppler image of the pulmonary artery outflow tract, used to derive

measurements of pulmonary vascular resistance and right ventricular output. Blue colour

represents blood flow away from the echo probe.

Figure 13A. Representative Doppler trace used to obtain measurements of pulmonary

haemodynamic. PAAT= pulmonary artery acceleration tract, RVET=right ventricular ejection

time, and PAVTI (area under the curve)= pulmonary arterial velocity time integral. Arrow

highlights mid-systolic “notching” pattern often seen in severe PHT.

31

Figure 13B. Doppler trace of pulmonary artery outflow. Note differences in shape of traces in

the untreated hypoxia control (top) and the Y-27632-treated hypoxia animals (bottom). Used

with permission from the American Physiological Society (McNamara et al., 2008).

32

2.5.2. Pulmonary artery systolic pressure

An estimation of pulmonary artery systolic pressure (PASP) was derived from the

PAAT, using the formula PASP=137.2 -3.3 x PAAT (Jones et al., 2002).

2.5.3. Right ventricular output

Right ventricular output was used as an index of RV performance, which is decreased in

severe pulmonary hypertension (Hoeper et al., 2001; Liu et al., 2009). The diameter of the

pulmonary artery (PAD) was measured by colour flow Doppler at the hinge-point of the

pulmonary valve leaflets. From the same Doppler interrogation of the pulmonary artery used to

measure PAAT and RVET, right ventricular stroke volume was estimated using the formula:

(PAD/2)2 x 3.14 x pulmonary artery velocity time integral (PAVTI). PAVTI was measured by

tracing the leading edge of the velocity time graph from the onset to completion of systolic

pulmonary flow. Right ventricular stroke volume was corrected for heart rate and body weight

to derive a right ventricular index (RVI). (Hoeper et al., 2001).

2.6. Examination of right ventricular hypertrophy

Right ventricular hypertrophy is a well-established index of PHT (Fulton and

Hutchinson, 1952), and has been shown to closely correlate with medial thickening of distal

pulmonary arteries in hypoxia-exposed rats (Rabinovitch et al., 1979; Rabinovitch et al., 1981).

At sacrifice, the thoracic contents were removed en bloc. The heart was then separated from the

lungs, the atria removed just inferior to the atrio-ventricular valves and discarded and the right

ventricle was dissected free from the left ventricle and septum. Each component was freeze-

33

dried and weighed separately and RVH was measured using the right ventricle/ (left ventricle +

septum) dry weight ratio as a marker (Kantores et al., 2006).

2.7. Percent medial wall thickness

Percentage arterial medial wall thickness (% MWT) was used as a marker of pulmonary

vascular remodelling (Ambalavanan et al., 2008; Xue et al., 2008; Mercier et al., 2009; Zhai et

al., 2009). Pulmonary arteries, identified by the presence of both inner and outer elastic lamina

using Hart’s elastin stain (detailed below). Images were digitally captured (Pixera Penguin

600CL, San Jose, CA) and analysis was undertaken by an observer blinded to group identity.

Vessel sizes of less than 20 µm or larger than 80 µm in external diameter were excluded, and at

least 20 vessels per animal were analysed. Mean external diameter was calculated from

measurements of round and ovoid vessels in two perpendicular planes (to account for any

irregularities in vessel shape), excluding vessels which were cut tangentially (more than three-

fold difference in MWT between perpendicular planes). Percent medial wall thickness was

measured using the formula: medial wall thickness x 2/mean external diameter x 100

(Rabinovitch et al., 1979).

2.8. Immunohistochemistry

Four randomly selected animals from each group were anaesthetised with intraperitoneal

(i.p.) ketamine (80 mg/kg) and xylazine (20 mg/kg) injections. After opening of the thoracic

cavity and cannulation of the trachea, the pulmonary veins were divided. The pulmonary

circulation was flushed with 1 × phosphate buffered saline (PBS) containing 1 U/ml heparin to

clear the lungs of blood, and perfusion fixed with 4% (w/v) neutral buffered paraformaldehyde

while inflated with air at a constant airway pressure of 20 cm H2O which was maintained via a

34

tracheal catheter. Paraffin-embedded tissues were cut into 5-µm sections and mounted on

Superfrost/Plus slides, allowed to air dry and baked overnight at 43°C. Sections were then

immersed in xylene (3 × 10 min) and rehydrated starting with 100% ethanol (3 × 5 min),

followed by 5 min each of 95, 90, 80, 70, and 50% ethanol. After washing (1 × PBS for 3 × 5

min), sections were boiled in 10 mM Na citrate for antigen retrieval. Slides were then immersed

for 30 min in 100% methanol with 0.3% (v/v) H2O2 to quench endogenous peroxidases. After

further washing, sections were blocked with 1% BSA (w/v)/5% NGS (v/v) for 1 h followed by

overnight incubation at 4°C with primary antibody or, where available, by pre-incubation with

the appropriate blocking peptide, according to the manufacturer’s instructions. Following

further washing, sections were incubated with biotin-conjugated secondary antisera diluted to

1:200 with blocking solution, at room temperature for 2 h. Sections were then washed and

incubated with avidin-biotin-peroxidase complex using the Vectastain ABC system, according

to the manufacturer’s instructions, at room temperature for 1-2 h. Finally, sections were stained

for 30 s to10 min with DAB. After further washing, sections were lightly counterstained with

Carazzi hematoxylin and rinsed in water for 15 min. Sections were dehydrated, cleared in

xylene and mounted using Permount. Images from stained sections were digitally captured

using a Zeiss Axioskop upright microscope (Carl Zeiss Inc., Oberkochen, Germany) equipped

with a Penguin 600CL camera and Viewfinder capture software (Pixera Corp., Los Gatos, CA).

2.9. Hart’s elastin staining

Paraffin-embedded tissue sections were de-waxed and rehydrated, followed by several

rinses with distilled water, and left overnight for 20 hours in Weigert’s resorcin-fuchsin stain

(Rowley Biochemical, Danvers, MA, U.S.A.). Slides were then washed in distilled water for 10

35

min, followed by counterstaining in tartrazine for 3 minutes. Slides were then dehydrated and

mounted by coverslip using a 50%/50% Permount/xylene solution. Images were digitally

captured as described above.

2.10. Western blot analyses

Whole (flash frozen) lung tissue or third-fourth generation PAs (dissected from 4 litters per

group; the pooled vessels of 2-3 animals from each litter representing 1 sample) were

homogenised and sonicated (40 W for 30 s) in RIPA cell lysis buffer (10 mM NaPO4, 0.3 M

NaCl, 0.1% (w/v) sodium dodecyl sulphate (SDS), 1% (v/v) Nonidet P-40, 1% (v/v) sodium

deoxycholate, 2 mM EDTA, pH 7.2) containing protease inhibitors (Protease Inhibitor Cocktail

Set I, EMD Chemicals, Gibbstown, NJ) and phosphatase inhibitor cocktail (Sigma). The

homogenate was left on ice for 10 min before centrifugation at 7,000 × g for 10 min. The

supernatant was then collected and protein concentration measured by a commercially-available

spectrophotometric assay using known concentrations of BSA as standards (Bradford, 1976).

Samples were then aliquoted and stored at -80°C until analysis. Tissue containing 50-100 µg of

protein was boiled for 5 min in SDS sample buffer (60 mM Tris-HCl, 10% (w/v) SDS, 5% (v/v)

glycerol, 2 mM β-mercaptoethanol, pH 6.8) and fractionated under reducing conditions by SDS

polyacrylamide gel electrophoresis for 2 h at 120 V. Following electrophoresis, proteins were

transferred to PVDF membranes. All membranes were blocked with 5% (v/v) Tween 20

overnight at 4°C and incubated with the appropriate primary antibodies for > 1 h at room

temperature. Blots were then washed in TBST and placed in secondary antibody for > 1 h at

room temperature.

36

Increased ROCK activity, characterised by an increased ratio of phosphorylated MLC-2:

unphosphorylated MLC-2, was assessed by using an acrylamide-pendant Phos-tagTM

ligand in

an SDS-PAGE system. This specialised variation on Western blot analysis incorporates a di-

nuclear metal complex, Mn2+

-Phos-tagTM

, into the gel matrix of a standard SDS-PAGE Western

blot. This allows for the elucidation of a ratio between phosphorylated to unphosphorylated

proteins of a particular sample on the same Western blot; in this case, the detection of

phosphorylated MLC-2 protein. A concentration of 5 mM of Mn2+

-Phos-tagTM

was incorporated

into a 12.5% acrylamide gel. Proteins were run under a constant current of 30mA, and then

transferred to PVDF membranes overnight at 4C under a constant voltage of 27 V. Following

electrophoresis and transfer, membranes were blotted with anti-MLC-2, yielding two bands (an

upper band representing phosphorylated and a lower band representing unphosphorylated MLC-

2). For each sample, density of the upper band was expressed as a ratio of the combined

densities of the upper and lower bands. After blotting, protein bands were imaged using an

enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.) and

exposed on Kodak X-OmatTM

Blue XB-1 film (Eastman Kodak, Rochester, NY, U.S.A.). Film

was electronically scanned and band density was quantified using Image J software (NIH,

Bethesda, MD, U.S.A.). Compensation for differences in protein loading was achieved by re-

blotting for GAPDH, and expressing results after normalisation to the GAPDH band.

2.11. Pulmonary artery smooth muscle cell (PASMC)-enriched culture

PASMCs were isolated using pulmonary artery explants from the pooled pulmonary

arteries of a litter (10-12 pups) of day 14 Sprague-Dawley rats, by a modification of a

previously described method (Jones & Rabinovitch, 1996). Pulmonary arteries were harvested,

37

opened with fine scissors, and the endothelium was scraped off by repeatedly rubbing the

luminal side with a scissor edge. The arteries were minced with scissors and washed in 1 × PBS

(pH 7.6) with antibiotic-antimycotic solution (10 U/ml penicillin G sodium, 10 µg/ml

streptomycin sulphate, 0.25 µg/ml amphotericin B, and 0.1 mg/ml gentamicin sulphate). Tissue

was placed in DMEM with 20% (v/v) FBS and antibiotic-antimycotic solution, and incubated

for 1-2 weeks at 37°C at a gas phase of 21% O2, 5% CO2, and 74% N2 until the cells had

attached. Cells were then passaged by trypsinisation using 0.05% (w/v) trypsin/EDTA and

centrifugation at 300 × g for 5 min, then reseeding. PASMCs were identified by their

characteristic hill-and-valley morphology and positive immunostaining for α-smooth muscle

actin. All experiments using PASMCs from the same litter were performed using cells from the

first passage. Equal numbers of cells (105 cells per well) were seeded in 96-well plates and

grown to semi-confluence with 10% (v/v) FBS and then serum-starved for 48 h. PASMCs were

incubated with DMEM alone (control) or DMEM with various concentrations of Y-27632, 10%

(v/v) FBS or Paclitaxel for 24 h. Values are means ± SEM for 6 wells per experiment. All

experiments were performed in duplicate, using cells derived from a different litter to ensure

reproducibility of results.

2.12. Terminal dUTP nick-end labelling (TUNEL) assay

PASMCs or frozen lung sections were examined for apoptotic cells using a

commercially-available TUNEL assay. For collection of frozen lung sections, two randomly

selected animals from each group were anaesthetised with i.p. ketamine (80 mg/kg) and

xylazine (20 mg/kg). After opening of the thoracic cavity and cannulation of the trachea, the

pulmonary veins were divided. The pulmonary circulation was flushed with 1 × PBS containing

38

1 U/ml heparin to clear the lungs of blood, as the lungs were inflated with frozen tissue matrix

(Tissue-Tek OCT®, Sakura Finetek, Torance, CA, U.S.A.), diluted 1:3 with 20% (w/v) sucrose

in PBS. The heart and lungs were then removed en bloc and covered with OCT® matrix using a

plastic mold placed on dry ice until the tissue and matrix were frozen, and stored at -80°C until

sectioning. Frozen lung tissue, cut by cryostat in to 15-20 µm-thick sections and placed on

SuperFrost/Plus slides or cultured PASMCs seeded on to Lab-Tek™ chamber slides (VWR,

Mississauga, ON, Canada) were stained with fluorescein, according to the manufacturer’s

instructions. Slides were then mounted with fluorescent mounting medium and digitally

photographed using an epifluorescent microscope (Zeiss Axioskop) with appropriate filter sets

(Fluorescein: excitation 495 nm and emission 520 nm; DAPI: excitation 350 nm and emission

470 nm).

2.13. Apoptosis-detection ELISA

Quantification of apoptosis in PASMCs was achieved using a commercially-available

ELISA designed to detect ssDNA (Cell Death Detection ELISAPLUS

, Roche). Cells were

seeded at a concentration of 2 105cells/ml, and incubated until sub-confluent, following which

they were trypsinised, washed and diluted with 20% FBS in DMEM culture medium to

neutralise trypsin. Y-27632 at concentrations of 0.1, 1, and 10 µM or a prototypic apoptosis-

inducer, Paclitaxel (10 µM; used as a positive control) in DMEM were added to the PASMCs,

and incubated at 37°C for 24 h. Following incubation, microplates were centrifuged, the cell

pellet combined with lysis buffer, and incubated at room temperature for 30 minutes. The lysate

was then centrifuged for 10 minutes, and the supernatant transferred on to a streptavidin-coated

microplate. Immunoreagent was added to each well, followed by incubation at room

39

temperature with gentle shaking. The wells were then emptied and washed with incubation

buffer, and 2, 2´-azino-bis-(3-benzthiazoline-6-sulfonic acid) (ABTS) solution was added. The

microplate was then incubated at room temperature with gentle shaking for 10-20 minutes, or

until colour development, followed by the addition of the ABTS Stop solution. Optical density

was measured by plate reader at a wavelength of 405 nm.

2.14. Cell viability assay

PASMC proliferation was quantified using the Wst-8 cell proliferation assay kit

(Cayman Chemicals). Cells were seeded in a 96-well plate at a concentration of 105 cells per

well, and treated with DMEM alone, DMEM with 20% (v/v) FBS, or DMEM with Y-27632 at

concentrations of 0.1, 1, and 10 µM. for 24 h. Reconstituted Wst-8 mixture was added to each

well, and the plate was put on a shaker at low speed for 1 minute, followed by an incubation

period of 2 hours at 37°C. Optical density was measured in a spectrophotometer using a

wavelength of 405 nm.

2.15. Data presentation and analysis

All values are expressed as the mean ± standard error of the mean (SEM). For in vitro

studies of cultured PASMCs, at least one replicate experiment using pooled explants from a

different litter of pups was performed to demonstrate reproducibility of results. Statistical

significance (p< 0.05) was determined using Sigma Stat 3.0 software (SPSS, Chicago, IL,

U.S.A.) using one-way analysis of variance (ANOVA) followed by pair-wise multiple

comparisons using the Tukey’s test when significant differences were found (Snedecor, 1968;

Glantz, 2002).

40

Chapter 3

Results

41

3.1. Body weight

Hypoxia-exposed animals, regardless of whether they received vehicle or Y-27632, had

significantly lower body weights when compared to air-exposed animals. Treatment with Y-

27632 had no impact on body weight in either group (Fig. 14).

3.2. Pulmonary vascular resistance

Hypoxia-exposed, vehicle-treated animals had significantly higher PVR values when

compared to air-exposed, vehicle-treated animals (Fig. 15). PVR was completely normalized in

animals exposed to hypoxia and treated with Y-27632. Animals exposed to air and treated with

Y-27632 had similar PVR values compared to vehicle-treated controls.

3.3. Pulmonary artery systolic pressure

Hypoxia-exposed, vehicle-treated animals had significantly higher PASP compared to

air-exposed, vehicle-treated animals (Fig. 16). Animals treated with Y-27632 and exposed to

hypoxia had significantly lower PASP compared to vehicle-treated animals. Animals exposed

to air and treated with Y-27632 had similar PASPs compared to vehicle-treated controls.

42

Figure 14. Body weights. Body weights of day-21 rat pups. *p<0.001, by ANOVA, compared

to respective air-exposed groups, n=12 animals; representing 2 litters/group.

43

Figure 15. Pulmonary vascular resistance. Doppler-derived inverse ratio of pulmonary artery

acceleration time (PAAT) and right ventricular ejection time (RVET) as a measure of PVR in

day-21 rat pups. *p<0.001, by ANOVA, compared to all other groups. n=7-8 animals

representing 2 litters/group.

44

Figure 16. Pulmonary artery systolic pressure (PASP). PASP was estimated in day-21 rat

pups.*p<0.05, by ANOVA, compared to all other groups. #p<0.05, by ANOVA, compared to

air-exposed Y-27632-treated group. n=7-8 animals representing 2 litters/group.

45

3.4. Right ventricular performance

Hypoxia-exposed, vehicle-treated animals had significantly lower RVIs compared to air-

exposed, vehicle-treated animals (Fig. 17). Animals treated with Y-27632 and exposed to

hypoxia had values comparable to air-exposed controls. Animals exposed to air and treated

with Y-27632 had RVIs comparable to air-exposed, vehicle-treated controls.

3.5. Right ventricular hypertrophy

Hypoxia-exposed, vehicle-treated animals developed significant RVH when compared to

air-exposed, vehicle-treated animals (Fig. 18). Animals treated with Y-27632 and exposed to

hypoxia had significantly attenuated RVH compared to vehicle-treated hypoxia-exposed

animals.

3.6. Arterial wall remodelling

Hypoxia-exposed, vehicle-treated animals had significantly higher %MWT values than

air-exposed, vehicle-treated animals (Fig. 19). Hypoxia-exposed animals treated with Y-27632

had completely normalised %MWT values. Animals exposed to air and treated with Y-27632

had significantly decreased %MWT values when compared to air-exposed vehicle-treated

animals. Representative Hart’s elastin stained sections are shown in Figure 20.

46

Figure 17. Right ventricular performance index. Right ventricular index, as a measure of right

ventricular output, in day-21 rat pups. *p<0.05, by ANOVA, compared to other groups, n=7-8

animals representing 2 litters/group.

47

Figure 18. Right ventricular hypertrophy (RVH). Right ventricle (RV)/left ventricle + septum

(LV+S) weight ratio as a marker of RVH in day-21 rat pups. *p<0.001, by ANOVA, compared

to all other groups, #p<0.01, by ANOVA, compared to air-exposed groups, n=7 animals

representing 2 litters/group.

48

Figure 19. Percent medial wall thickness (% MWT). % MWT was derived from day-21 lung

sections stained with Hart’s elastin stain. *p<0.001, by ANOVA, compared to all other groups,

and #p<0.05, by ANOVA, compared to air-exposed vehicle-treated group, n=4 animals

representing 2 litters/group.

49

Figure 20. Hart’s elastin stain. Hart’s elastin staining for elastin (dark brown) and tartrazine

counterstain (light brown) in paraffin-embedded lung tissue from day-21 rat pups.

50

3.7. Prepro-ET-1 immunohistochemistry

Animals exposed to hypoxia and treated with vehicle had increased vascular wall prepro-

ET-1 immunoreactivity compared to animals exposed to air and treated with vehicle (Fig. 21).

Animals treated with Y-27632, whether exposed to hypoxia or air, had comparable prepro-ET-1

immunoreactivity to air-exposed, vehicle-treated animals.

3.8. Prepro-ET-1 content

Consistent with findings on immunohistochemistry, Western blot analyses of pulmonary

arterial prepro-ET-1 showed increased prepro-ET-1 content in hypoxia-exposed vehicle-treated

animals when compared to air-exposed groups and hypoxia-exposed animals treated with Y-

27632 (Fig. 22).

3.9. ROCK-I and ROCK-II content

No changes in pulmonary arterial ROCK-I (Fig. 23) or ROCK-II (Fig. 24) content were

observed between groups.

3.10. MLC-2 phosphorylation

Hypoxia-exposed, vehicle-treated animals had significantly increased MLC-2

phosphorylation, as a marker of ROCK activity, compared to air-exposed, vehicle-treated

animals (Fig. 25). In hypoxia-exposed animals treated with Y-27632, MLC-2 phosphorylation

was completely normalised, indicating that Y-27632 completely attenuated the hypoxia-

mediated increase in ROCK activity. Animals exposed to air and treated with Y-27632 had no

change in MLC-2 phosphorylation compared to air-exposed controls.

51

Figure 21. Prepro-ET-1 immunostaining. Immunohistochemical staining for the ET-1

precursor, prepro-ET-1 (dark brown) and hemotoxylin (light blue) in paraffin-embedded lung

tissue of postnatal day-21 control and Y-27632-treated rat pups. Inset image. Hypoxia vehicle-

treated negative control.

52

Figure 22. Prepro-ET-1 content. Top. Prepro-ET-I content in pulmonary arterial tissue of

postnatal day-21 control and Y-27632-treated rat pups. Bottom. Representative Western blots.

Molecular weight of Prepro-ET-1 is 24.4 kDa. *p<0.01, by ANOVA, compared to all other

groups, n=4 samples/group.

53

Figure 23. ROCK-I content. Top. ROCK-I content in pulmonary arterial tissue of postnatal

day-21 control and Y-27632-treated rat pups. Bottom. Representative Western blots. Molecular

weight of ROCK-I is 160 kDa. n=4 samples/group.

54

Figure 24. ROCK-II content. Top. ROCK-II content in pulmonary arterial tissue of postnatal

day-21 control and Y-27632-treated rat pups. Bottom. Representative Western blots. Molecular

weight of ROCK-II is 160 kDa. n=4 samples/group.

55

Figure 25. Rho-kinase activity. Top. MLC-2 phosphorylation in pulmonary arterial tissue of

postnatal day-17 control and Y-27632-treated rat pups, as a marker of Rho-kinase activity.

Bottom. Representative immunoblots for MLC-2 using Phos-tag Western blot analysis.

*p<0.001, by ANOVA, compared to all other groups, n=4 samples/group.

56

3.11. PASMC proliferation

Compared to control (DMEM only)-treated PASMCs, cells treated with Y-27632 (1 and

10 µM) had significantly decreased proliferation when compared to positive controls (Fig. 26).

3.12. Ki-67 immunohistochemistry

Ki-67 immunostaining was used to identify cells undergoing DNA synthesis. Increased

numbers of proliferating medial arterial wall smooth muscle cells were observed in vessels of

animals exposed to hypoxia and treated with vehicle when compared to all other groups (Fig.

27).

3.13. In vitro TUNEL assay and ELISA for apoptosis

Treatment with Y-27632 led to greatly increased apoptosis in PASMCs when compared

to untreated controls (Fig. 28). This finding was confirmed using quantitative ELISA (Fig. 29).

3.14. In vivo TUNEL assay

Using fluorescent TUNEL on day-17 frozen lung tissue to identify cells undergoing

apoptosis increased apoptosis was observed in the arterial walls of both air- and hypoxia-

exposed animals treated with Y-27632 when compared to vehicle-treated animals (Fig. 30).

57

Figure 26. Proliferation index. Proliferation index of primary cultures of PASMCs using a

colorimetric assay. *p<0.05, by ANOVA, compared to Y-27632-free negative control group.

The positive controls were cells placed in 10% FBS for 24 hours.

58

Figure 27. Immunohistochemical staining for Ki67, a marker for DNA synthesis. Ki67-positive

staining (dark brown) and hemotoxylin (light blue) in paraffin-embedded lung tissue from

postnatal day-17 control and Y-27632-treated rat pups. Black arrows point to two Ki67-positive

cells in the medial arterial wall.

59

Figure 28. Apoptosis in primary cultures of PASMC. Fluorescent TUNEL stain showing

increased apoptotic nuclei (green) in primary cultured PASMCs treated with Y-27632. Positive

control cells were treated with DNAse I.

60

Figure 29. Apoptosis index. Apoptosis index of primary cultures of PASMCs measured by

colorimetric ELISA. *p<0.001 compared to all other groups, #p<0.001 compared to negative

control group. Positive control cells were treated with 10 µM Paclitaxel.

61

Figure 30. Apoptosis marker in frozen tissue sections. Fluorescent TUNEL assay to identify

apoptotic nuclei (green). Non-apoptotic nuclei are stained by DAPI (blue) in postnatal day-17

frozen lung tissue of control and Y-27632-treated air-exposed and hypoxia-exposed rat pups;

pa= pulmonary artery.

62

Chapter 4

Discussion

63

The aims of this project were to examine whether sustained treatment with a ROCK inhibitor,

Y-27632, would normalise PVR, augment RV performance, and reverse vascular remodelling in

a neonatal chronic hypoxic rat model of PHT. As hypothesised, treatment with Y-27632

reversed the structural (RVH and arterial wall remodelling, as reflected by %MWT) and

functional (raised PVR and attenuated RV performance) abnormalities of chronic hypoxic PHT.

Our finding that Y-27632 led to quantitatively increased apoptosis and decreased proliferation

of PASMCs in vitro and qualitative differences in vivo provides a mechanistic explanation for

the reversal of established structural changes of vascular remodelling. Therefore, our results

point towards a structural as well as a functional attenuation of chronic hypoxic PHT, which

may be due to the effectiveness of Y-27632 both as a vasodilator and as a pro-apoptotic/anti-

proliferative agent. Moreover, normalisation of RV performance with Y-27632 has not, to our

knowledge, been observed in any previous studies examining treatments for established chronic

PHT in adult animals, including other ROCK inhibitors, such as Fasudil. Together, our findings

could have important therapeutic implications for chronic forms of PHT in infancy and

childhood, because severe vascular remodelling is a pathological hallmark of fatal PHT and RV

dysfunction carries a high risk of early death (Utsunomiya et al., 2009).

The observed vasodilatory effects of Y-27632 are likely to be caused by the inhibition of

the RhoA/ROCK-mediated pathway (Fig. 3) Ca2+

sensitisation by Y-27632, in which MLC-2

becomes phosphorylated (Hanazaki et al., 2008). If ROCK is activated, as it is in this chronic

hypoxic model, the inhibition of this pathway would thereby lead to the attenuation of sustained

vasoconstriction. Other possible contributing factors, not tested in this thesis, include attenuated

actin polymerization and/or augmented function of the NO-cGMP pathway. ROCK inhibition

may also contribute to a decrease in the number of vascular SMCs, as ROCK is involved in

64

many cellular processes, including mitogenesis (Liu et al., 2004). A decrease in the number of

vascular SMCs, or a cessation of the proliferation of such cells, would thus lead to an

attenuation of vascular remodelling. Previous research examining production of endothelin-1 by

pulmonary artery SMCs in response to thromboxane (TXA2) receptor stimulation with 8-

isoprostane, has demonstrated that treatment with Y-27632 resulted in a decrease in ET-1

production and release (Yi et al., 2006). Interestingly, the same tissues exhibiting increased ET-

1 expression also exhibited increased ROCK activity, which was attenuated through treatment

with the ROCK inhibitor Y-27632. The importance of this finding is the overwhelming

evidence that ET-1 plays an important role in PHT development both in newborn and adult

experimental models (Jankov et al., 2000; Porchia et al., 2008), such that ET-1 receptor

antagonists are now an established therapy for children and adults with chronic PHT (Rubin et

al., 2002; Barst et al., 2006). Thus, a relationship between ROCK and ET-1 could potentially

explain the effects of ROCK inhibition on promoting apoptosis of SMCs, as it is known that

along with having a vasoconstrictive role, ET-1 also promotes SMC growth and survival

through the inhibition of apoptosis (Wu-Wong et al., 1997; Jankov et al., 2000; Jankov et al.,

2006). Therefore, treatment with Y-27632 may induce apoptosis, at least in part, by attenuating

ET-1 expression. Other mechanisms for putative actions of ROCK as an anti-apoptotic

molecule have also been postulated, particularly in the fields of cancer and tumorigenesis.

Effects on the expression of p53 protein (Xue et al., 2007), the activation and release of

caspases through mitochondrial membrane depolarisation (Maeda et al., 2009), as well as the

inhibition of the proper adhesion of cells for continued growth (Rattan et al., 2006) have all

been proposed as possible mechanisms for the pro-apoptotic effects of Y-27632 that could also

apply to SMC apoptosis.

65

Concerning the mechanisms of Y-27632-induced attenuation of chronic hypoxia-induced

RV dysfunction, it could be inferred that a decrease in RV afterload helped significantly in

improving RV function, although other mechanisms may have contributed. It has been shown

by Sugiyama and colleagues (Sugiyama et al., 2002) that Y-27632 enhances heart function by

improving sinoatrial automaticity, increasing coronary blood flow and reducing myocardial O2

demand through altered myofibrillar Ca2+

sensitivity. Y-27632 has also been reported to

attenuate increased myocardial contractility (and thus O2 demand) caused by the administration

of ET-1, angiotensin-II, or prostaglandin F2α, all of which are powerful vasoconstrictors

(Nishimaru et al., 2003). This attenuation was believed to result from two mechanisms: the

inhibition of α-adrenoceptor-mediated increased Ca2+

sensitivity, and/or from the decrease of a

Na2+

/H+ exchanger that was found to be activated together with the RhoA/ROCK pathway

(Nishimaru et al., 2003). Therefore, it is possible that Y-27632 exerted direct effects on the

myocardium in addition to reducing afterload through attenuation of Ca2+

sensitisation.

In previous work, our laboratory has studied the acute haemodynamic effects of a single

bolus of Y-27632 on hypoxia- and bleomycin-induced chronic neonatal PHT models

(McNamara et al., 2008). Y-27632 resulted in completely normalised PVR but did not improve

RV output/function. Subsequently, the effects of Y-27632 when given from birth at the onset of

chronic hypoxic exposure were examined (Ziino et al., 2010). The results of this study showed

that there was also attenuation of PVR, RVH, and vascular remodelling (at least in part through

inhibitory effects of PDGF-β receptor signaling), indicating that ROCK is a critical initiating

factor in the pathogenesis of hypoxia-induced vascular remodelling. In addition, no adverse

effects on lung growth and development were observed in Y-27632-treated animals during a

period when the increase in alveolar density is maximal in the rat (Ziino et al., 2010). The study

66

outlined in this thesis represented a logical continuum of previous work, whereby the reversing

effects of Y-27632 were examined once chronic hypoxic PHT was already well-established.

Collectively, these studies strongly suggest that Y-27632, and possibly other inhibitors of

ROCK, have potential as a novel treatment for PHT in infancy and childhood that is non-

responsive to conventional therapies.

4.1. Limitations and future research

Much remains to be understood about the involvement of ROCK in chronic PHT in

human infants and children and the possible side-effects of ROCK inhibitors, prior to clinical

translation. For example, systemic administration of ROCK inhibitors from birth was found to

worsen somatic growth restriction in chronic hypoxia-exposed pups (Ziino et al., 2010), which

has not been reported in mature rodents receiving either Y-27632 or Fasudil. Evidence from

that study strongly suggested that growth restriction was related to systemic inhibition of

ROCK, rather than through an off-target effect of Y-27632 (Ziino et al., 2010). We believe that

systemic hypoperfusion, due to parallel dilating effects of ROCK inhibitors on the systemic

vasculature (Nagaoka et al., 2005), is likely to account for many adverse effects, including

growth restriction. Importantly, in the present study, treatment with Y-27632 caused no adverse

effects on somatic growth, which is likely explained by the different timing of treatments (days

14-21, rather than days 1-14 of life). In pilot dose-response studies carried out at 14 days of life,

we observed immediate ill effects consistent with hypotensive shock, including gray pallor,

weakness, decreased activity and (in many cases) death. These effects were more frequent and

severe the higher the dose (30 mg/kg>>25 mg/kg>>20 mg/kg), appeared to be worse in air-

exposed animals than in animals chronically exposed to hypoxia, and were not observed at 15

67

mg/kg, which was the dose ultimately used in the present study. We chose to perform twice

daily injections of Y-27632, given that studies in adult rodents suggest inhibitory effects on

ROCK activity (as measured by vasodilatory effects on the pulmonary circulation) last

approximately 4-6 hours after a single bolus injection (Nagaoka et al., 2004).

ROCK is known to be expressed in both vascular and non-vascular cell types in many

tissues; therefore, it is likely that ROCK mediates many processes in cell types other than

vascular SMCs, including cell motility, adhesion and proliferation. Therefore, the need for

more information concerning systemic effects of ROCK inhibition is evident. Furthermore,

given the apparent increased sensitivity of newborn animals to the systemic effects of ROCK

inhibitors and the narrow therapeutic-toxic dose range, our results suggest that systemic

treatment with this class of agents should be undertaken in neonates and infants with caution. A

potentially safer and equally efficacious approach may be through delivery of smaller doses

directly to the lung, either by intratracheal instillation (similar to exogenous surfactant) or by

nebulisation, which has been shown in adult animals to limit associated effects on the systemic

vasculature (Nagaoka et al., 2005).

Chronic PHT is a complex disease with multiple factors contributing to pathogenesis,

including hypoxia, inflammation, and the underdevelopment of the pulmonary vasculature.

Therefore, it would be important to confirm the present findings in models of neonatal PHT in

which inflammation is critical, including chronic exposure to hyperoxia or systemic treatment

with bleomycin (Yi et al., 2004; McNamara et al., 2008; Tourneux et al., 2008). Additionally,

longer-term studies are required to determine whether the beneficial effects of Y-27632 last

beyond the treatment period and whether combining Y-27632 with existing treatments, such as

iNO, would enhance its effects.

68

A complete understanding of the role in the RhoA/ROCK pathway in the initiation and

persistence of chronic PHT is lacking; therefore, examination of the effects of more possible

upstream and/or downstream effector molecules of ROCK is needed to pinpoint how and where

ROCK contributes to the pathogenesis of PHT. Some of these molecules include: GEF, GDI,

GAP, MAPK, LIMK, cofilin, CPI-17, and many other proteins and enzymes that were discussed

earlier. The examination of the role of ROCK can also be more precisely elucidated through the

use of siRNA-mediated knockdown or conditional knockout animal models. Currently, there

exist both ROCK-I and ROCK-II knockout mice. ROCK-I knockouts exhibit defects in eyelid

epithelia while the loss of ROCK-II expression results in placental dysfunction, leading to

intrauterine growth restriction (Thumkeo et al., 2005). Interestingly, in contrast to the effect of

ROCK inhibition in the lung, it was observed that in mouse models of systemic hypertension,

deletion of ROCK-I expression resulted in reduced cardiac fibrosis, and reduced cardiomyocyte

apoptosis, even though an increase of apoptosis was observed in the lung in hypertensive

models (Chang et al., 2006), suggesting differing effects of ROCK in various organ systems.

Thus, it would be useful in the future to use conditional knockouts or siRNA-induced models for

the study of PHT development, which might allow for the specific knockdown of ROCK in the

lungs only.

Though much remains to be understood before ROCK inhibitors may be introduced into

clinical practice, this project yielded many promising and important results that may drive future

research aimed at increasing our understanding about the effects of ROCK inhibitors in the

neonate. With an improved understanding of the optimal dose, mode of administration and

associated effects, ROCK inhibitors could have great potential as a testable therapy in patients

who are non-responsive to current treatments for chronic PHT.

69

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