Chronic Intrauterine Pulmonary Hypertension Increases Endothelial Cell Rho-

Kinase Activity and Impairs Angiogenesis in vitro.

Jason Gien MD 1

Gregory J Seedorf, BS 2

Vivek Balasubramaniam MD 2

Nancy Tseng, BS, MS

Neil Markham, BS 2

Steven H Abman MD 2

From the Pediatric Heart Lung Center, Sections of Neonatology1 and Pulmonary Medicine2, Department of Pediatrics, University of Colorado School of Medicine, Denver, CO

Correspondence:Dr Jason Gien P18-4402K Mail Stop 831712800 East 19TH Ave.PO Box 6511Aurora, CO 80045 Phone: 303 724 4065Fax: 303 724 4072Email: jason.gien@uchsc.edu

Page 1 of 41Articles in PresS. Am J Physiol Lung Cell Mol Physiol (July 11, 2008). doi:10.1152/ajplung.00516.2007

Copyright © 2008 by the American Physiological Society.

Abstract

Persistent pulmonary hypertension of the newborn (PPHN) is characterized

by endothelial dysfunction and decreased vascular growth. The role of rho-kinase

activity in modulating endothelial function and regulating angiogenesis during

normal lung development and in PPHN are unknown. We hypothesized that PPHN

increases rho-kinase activity in fetal pulmonary artery endothelial cells (PAECs)

and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal

sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-

matched controls. Rho-kinase activity, was measured by rhoA, RhoGTP and P-

MYPT-1 protein content. The effects of rho-kinase activity on angiogenesis, eNOS

protein expression and NO production were determined in normal and PPHN

PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-

27632, (rho-kinase inhibitor), and calpeptin, (rho-kinase activator), in

presence/absence of L-NA (NOS inhibitor). RhoA, rho-GTP and P-MYPT-1 protein

were increased in PPHN PAECs. Tube formation was reduced by 29% in PPHN

PAECs (p<0.001) and increased with Y-27632 treatment in normal and PPHN

PAECs with PPHN PAECs achieving similar values to normal PAECs. L-NA

inhibited the Y-27632-induced increase in tube formation in normal but not PPHN

PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS

expression, was reduced by 42% in PPHN PAECs (p<0.01). Y-27632 increased

eNOS protein and NO production in normal and PPHN PAECs. Calpeptin

decreased eNOS protein only in normal PAECs, but reduced NO production in

Page 2 of 41

normal and PPHN PAECs. We conclude that rho-kinase activity is increased in

PPHN PAECs, which down-regulates eNOS protein and NO production and

impairs angiogenesis in vitro.

Key terms: Persistent pulmonary hypertension of the newborn, pulmonary

hypertension, angiogenesis, vasculogenesis, rho-kinase, nitric oxide, endothelial

nitric oxide synthase, endothelial cells, lung vascular development.

Page 3 of 41

Introduction

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical

syndrome characterized by elevated pulmonary vascular resistance (PVR) that

persists after birth, leading to extrapulmonary right to left shunting and profound

hypoxemia. Mechanisms responsible for elevated PVR in PPHN include increased

vascular tone, hypertensive remodeling and in the most severe cases, impaired

angiogenesis or vascular growth (12). Impaired angiogenesis is usually seen in the

setting of PPHN with lung hypoplasia, such as with congenital diaphragmatic hernia

(12,14). In the presence of lung hypoplasia, decreased arterial number plays an

especially prominent role in maintaining high PVR, resulting in disease that is often

refractory to vasodilator therapies, such as inhaled nitric oxide (14). In this setting,

novel strategies that can stimulate vascular growth and increase arterial number

may improve outcomes of neonates with severe PPHN and lung hypoplasia.

However, mechanisms that impair angiogenesis and enhance lung vascular growth

in severe PPHN are poorly understood.

Past studies have shown that partial ligation of the ductus arteriosus (DA) in

late gestation fetal sheep provides a useful animal model for studying the

pathogenesis and treatment of PPHN (3,29,54). In this model, partial DA ligation

increases pulmonary artery pressure without causing sustained elevations of

pulmonary blood flow or hypoxemia (1). At delivery, PVR remains elevated and

causes hypoxemia due to extrapulmonary shunting despite mechanical ventilation

with supplemental oxygen (1). Physiologically, this model of PPHN is characterized

Page 4 of 41

by marked endothelial dysfunction, as reflected by the early loss of endothelium-

dependent vasodilation, with down-regulation of lung endothelial NO synthase

(eNOS) expression, impaired NO production, increased superoxide generation and

increased ET-1 expression (16,17,22,41,48,49). Overall, these and other findings

suggest that disruption of normal endothelial function in the fetal lung increases

pulmonary vasoconstriction and causes abnormal vasoreactivity in PPHN. In

addition to its role in the regulation of vascular tone, the endothelial cell also

modulates vascular structure and growth. Recent studies in this experimental

model have shown that chronic intrauterine pulmonary hypertension impairs lung

angiogenesis and cause lung hypoplasia (11). We recently demonstrated that

endothelial cells from PPHN fetal sheep maintain an abnormal phenotype in vitro,

which is characterized by decreased growth and impaired tube formation (9).

However, how hemodynamic stress induced by hypertension alters endothelial cell

function and impairs vascular growth in PPHN is unknown.

Rho-kinase signaling is a complex pathway responsible for cellular

proliferation, migration, differentiation and gene expression in diverse vascular beds

(26). Rho-kinase activity has been shown to regulate smooth muscle cell

contraction and vascular tone in systemic and pulmonary circulations (4,5,32,53).

During lung development, rho-kinase activity maintains high PVR in the fetal lung

(35) and may contribute to increased vascular tone in neonatal pulmonary

hypertension. In adult animal models of pulmonary hypertension, rho-kinase

activity is increased (21,26,32,52), however these studies have largely focused on

the effects of rho-kinase activation on smooth muscle cell function, demonstrating

Page 5 of 41

that increased rho-kinase activity elevates vascular tone, mediates calcium

sensitization and contributes to hypertensive remodeling (5,32,42,51,53). In adult

models of pulmonary hypertension, acute treatment with rho-kinase inhibitors

causes potent pulmonary vasodilation and chronic therapy prevents vascular

remodeling and improves survival (20,21,26,30,34).

In addition to its effects in smooth muscle cells, rho-kinase activity also

modulates endothelial cell function. RhoGTPases are key regulators of endothelial

permeability (2,50,55) and rhoA activation increases vascular permeability

(10,39,47). Whether rho-kinase activity regulates angiogenesis, especially in the

lung circulation, is controversial. In adult models of pulmonary hypertension due to

chronic hypoxia, inhibition of rho-kinase activity prevents pulmonary hypertension

and inhibits angiogenesis (13). Unlike these findings in the adult lung, experimental

pulmonary hypertension in fetal sheep is associated with reduced vascular growth

and impaired angiogenesis (11). Whether increased rho-kinase activity impairs

endothelial cell function and reduces vascular growth in severe pulmonary

hypertension, especially in the developing lung or in neonatal pulmonary

hypertension, remains unknown.

Since eNOS protein expression is decreased in PPHN PAECs (9) and rho-

kinase activity regulates eNOS protein expression and NO production (27,33,36),

we proposed to test whether reduced eNOS protein expression and tube formation

in PPHN PAECS is due to increased rho-kinase activity. Specifically, we

hypothesized that chronic intrauterine pulmonary hypertension would increase rho-

kinase activity in pulmonary artery endothelial cells, resulting in endothelial cell

Page 6 of 41

dysfunction, decreased eNOS expression and impaired angiogenesis. We further

hypothesized that increased rho-kinase activity would impair angiogenesis in fetal

and hypertensive PAECs due to reduced NO production. In this study, we report

increased rho-kinase activity in PAECs from PPHN lambs, and that rho-kinase

inhibition increases eNOS expression and NO production and enhances tube

formation in vitro. Overall, these findings support the hypothesis that rho-kinase

activation contributes to endothelial cell dysfunction and impaired angiogenesis in

PPHN.

Page 7 of 41

Methods

Isolation and culture of fetal ovine pulmonary arterial endothelial cells.

Isolation and culture of fetal ovine pulmonary arterial endothelial cells. All

procedures and protocols were reviewed and approved by the Animal Care and

Use Committee at the University of Colorado Health Sciences Center, Denver, CO.

The left and right pulmonary arteries were isolated from late-gestation normal fetal

sheep (mixed-breed Columbia-Rambouillet pregnant ewes at 135 days gestation

(n=4), term = 147 days) and from fetal sheep that had undergone partial ligation of

the ductus arteriosus in utero 7-10 days prior to euthanasia (PPHN)(n=4)(as

previously described, (3,29,54). Proximal PAECs were isolated as previously

described (9,22) and endothelial cell phenotype confirmed by positive

immunostaining for von Willebrands Factor (vWF), eNOS, vascular endothelial

(VE)-cadherin. VEGF-R2 (KDR), positive uptake of ac-LDL and negative staining

for desmin. Cells from passage 4 and 5 were used for each of the study

experiments and cells from each animal were kept separate throughout all

passages and for all experiments.

ELISA: ELISA was performed using the G-LISA RhoA Activation Assay

(Cytoskeleton Inc, Denver, CO #BK124) and the assay was performed according

to the manufacturers instructions. Briefly PAECs from control and PPHN lambs

were grown to 50-70% confluence in 150mm dishes and cell lysate collected by

Page 8 of 41

scraping the dishes. Lysates were snap frozen in liquid nitrogen and stored at -

80°C. After thawing protein concentrations were determined and samples were

prepared with identical protein concentrations. The rhoA activation assay was

performed in triplicate and rho GTP signal was determined by measuring

absorbance at 490nm using a microplate spectrophotometer. Differences in

absorbance between normal and PPHN PAECs were measured and quantified.

Membrane-cytosolic separation: Membrane fraction separation was

performed using the ProteoExtract Native Membrane Protein Extraction Kit

(Calbiochem, Cat#444810 San Diego, CA). Briefly, PAECs from normal and PPHN

fetal sheep were grown to 95% confluence in 150mm dishes and cells were

detached from the dishes using 0.25% trypsin. Membrane protein was extracted

from whole cell lysates per manufacturer instructions.

The cytosolic fraction was extracted using the Mem-PER Eukaryotic

membrane extraction kit (Pierce Biotechnology Inc (catalog # 89826) Rockford, IL).

PAECs from normal and PPHN fetal sheep were grown to 95% confluence in

150mm dishes and cells were detached from the dishes using 0.25% trypsin.

Cytosolic protein was extracted from whole cell lysates per manufacturer

instructions.

Protein content in the membrane and cytosolic samples was determined

by the Bicinchoninic acid assay (BCA) (Pierce Biotechnology Inc (catalog #

23225) Rockford, IL), using bovine serum albumin as the standard. Twenty µg of

protein sample per lane was resolved by SDS polyacrylamide gel

Page 9 of 41

electrophoresis. Proteins from the gel were transferred to nitrocellulose

membrane and Rho A protein was detected by western blot analysis (See

protocol below).

Tube Formation Assay: The ability of fetal PAECs to form vascular

structures in vitro was assayed by plating PAEC’s on type 1 collagen. Collagen

was pipetted into 24 well tissue culture dishes (250 µl/well) and allowed to

polymerize at 37°C for one hour. PAEC from normal and PPHN fetal sheep were

seeded at a density of 5 x 104 cells/well in serum free DMEM supplemented with

and without Y-27632 (1µM; rho-kinase inhibitor) ,calpeptin (100µg/ml; rho-kinase

activator) and Y-27632 (1µM) with nitro-L-arginine (LNA; a NOS inhibitor; 4mM).

Doses for each drug were determined by preliminary experiments and published

studies (40). The lowest dose for which an effect was seen was used for all drugs.

PAECs were incubated in 3% oxygen conditions in order to simulate the low

oxygen environment in the normal fetus (9). After 6 hours, branch point counting

was performed in blinded fashion under 10X magnification from each of 4 wells, as

previously described (38).

Western Blot Analysis: PAECs from normal and PPHN animals were

grown on 150mm cloning dishes in DMEM supplemented with 5% serum. At 70%

confluence, PAECs were treated with Y-27632 (1µM) for 24 hours and calpeptin

(100µg/ml) were for 30 minutes per the manufacturer’s recommendations. Cells

were washed with ice cold PBS x 2 and lysed in radioimmunoprecipitation (RIPA)

buffer (PBS, 1% Nonidet P-40, 0.5%, sodium deoxycholate, 0.1% SDS, PMSF

Page 10 of 41

[10mg/ml], aprotinin [16 µl/ml], and sodium orthovanadate [1mM]). Cell lysates

were scraped off the dishes, sonicated, and centrifuged at 10,000 x g for 30 min

at 4°C. The supernatant was removed and protein content in the supernatant

was determined by the BCA assay (Pierce Biotechnology Inc (catalog # 23225)

Rockford, IL), using bovine serum albumin as the standard. 20 µg of protein

sample per lane was resolved by SDS polyacrylamide gel electrophoresis, and

proteins from the gel were transferred to nitrocellulose membrane.

Rho A: Blots were blocked for 30 minutes in 5% nonfat dry milk dissolved in

buffer 1 (10mM tris-hcl, 150mM NaCl, 0.05% tween-20, PH 8.0) Blots were

incubated for 2 hours at room temperature with anti ROCK-II/ROK∝ (BD610624

BD Biosciences, San Jose, CA) (1:500) diluted in 5% nonfat dry milk in buffer 1.

After washing, blots were incubated for 1 hour at room temperature with goat

anti-mouse HRP conjugated secondary (Chemicon, Billerica, MA)(1:10000).

Bands of interest were visualized by enhanced chemiluminenscence (ECL+ kit;

Amersham Pharmacia Biotech, Buckinghamshire, UK), identified by molecular

weight as identified by the manufacturer for the protein of interest.

Phospho-MYPT-1: Blots were blocked for 30 minutes with 5% nonfat dry

milk dissolved in buffer 1 (10mM tris-hcl, 150mM NaCl, 0.05% tween-20, PH 8.0).

Blots were then incubated overnight with Phospho-MYPT1 (Thr853) Antibody

(#4563 Cell Signaling, Danvers, MA) (1:500). After washing, blots were incubated

for 1 hour at room temperature with goat anti-rabbit HRP conjugated secondary

Page 11 of 41

(Santa Cruz Biotech, SC2054). Bands of interest were visualized by enhanced

chemiluminenscence (ECL+ kit; Amersham Pharmacia Biotech,

Buckinghamshire, UK), identified by molecular weight as identified by the

manufacturer for the protein of interest.

eNOS: Blots were blocked for 30 minutes in 2% ECL advance

(Amersham Pharmacia Biotech, Buckinghamshire, UK) dissolved in PBS with

0.05% Tween 20 after which time the blots were incubated for 1 hour with

BD610297 (eNOS/NOS III)(1:1000) diluted in 2% ECL advance. After washing,

blots were incubated for 1 hour at room temperature with goat anti-mouse HRP

conjugated secondary (Chemicon)(1:20000). Bands of interest were visualized

using the ECL advance kit, identified by molecular weight as identified by the

manufacturer for the protein of interest.

All blots were then stripped and reprobed with an antibody to β-actin

(Sigma,St. Louis, A5316). Densitometry was performed using NIH Image (v1.61).

Changes in protein expression were analyzed after normalizing for β-actin

expression.

Nitric Oxide Assay: NO production was determined with the DAF-FM

Nitric Oxide indicator (Molecular Probes, Eugene, OR #D-23844). 5 x 103 normal

and PPHN PAECs were plated in 96 well plates in DMEM with 5% FBS under

3% oxygen conditions. Cells were allowed to adhere overnight after which normal

and PPHN PAECs were incubated with DAF-FM with and without Y-27632 (1µM)

and calpeptin (100µg/ml) in PBS for 1 hour. PBS containing DAF-FM fluorescent

Page 12 of 41

probe was transferred to a black 96 well plate and NO production measured in

response rho-kinase activation and inhibition using a microplate reader with

fluorescence excitation and emission maxima of 495 and 515 nm, respectively.

Comparisons were made between normal and PPHN cells with respect to NO

production.

Statistical analysis. Data are presented as means ± SEM. Statistical analysis

was performed with the Prism 4 software package (GraphPad Software, San Diego,

CA). Statistical comparisons were made using analysis of variance for tube formation

assays with Bonferroni post test analysis. Unpaired t test was used for western blot

and ELISA analysis and NO production studies. P < 0.05 was considered significant.

Page 13 of 41

Results

Increased Rho-kinase activity in fetal PAECS from PPHN Lambs. In

comparison with controls, PAECs from PPHN lambs exhibited increased rho-

kinase activity. Western blot analysis on whole cell lysates from normal and PPHN

PAECs demonstrated a 93% increase in total Rho A protein in PAECs from PPHN

lambs (p<0.05)(fig 1a). As determined by ELISA, rho-GTP, the active form of rho,

was increased by 53% in PPHN PAECs (p<0.001) (fig 1b.) In addition to increased

rhoA and rho-GTP protein, phosphorylation of MYPT-1, another measure of rho-

kinase activity, was increased 65% in PPHN PAECs (p<0.01) (fig 1c). When

separated into membrane and cytosolic protein fractions, cell lysates from PPHN

PAECs demonstrate increased rho A protein in both membrane and cystosolic

fractions when compared with normal controls. In comparison with control PAECs,

rho A membrane and cytosolic protein contents were increased by 34% and 52%,

respectively (p<0.01) (fig 2).

Activation of rho-kinase with calpeptin. Western blot analysis on whole cell

lystates from normal and PPHN PAECs demonstrated that rho-kinase activation

with calpeptin increased phosphorylation of MYPT-1 by 85% in normal PAECs

(p<0.01) (fig 3a). With calpeptin treatment there was no further increase in

phosphorylation of MYPT-1 in PPHN PAECs (p=NS)(fig3b.).

Effect of Rho-kinase inhibition and stimulation on tube formation in vitro.

Treatment with Y-27632, a rho-kinase inhibitor, increased tube formation in both

Page 14 of 41

normal and PPHN PAECs. Tube formation was increased by 13% (p<0.01) and

31% (p<0.001) in normal and PPHN PAECs, respectively (fig 4a). Rho-kinase

inhibition increased tube formation by PPHN PAECs to values achieved in normal

PAECs. Treatment with calpeptin, decreased tube formation in both normal and

PPHN PAECs by 29% (p<0.001) and 21% (p<0.01), respectively. (fig 4b). The

addition of S-nitroso-N-acetylpenicillamine (SNAP) as an NO donor did not prevent

the decrease in tube formation due to rho-kinase activation. Tube formation

remained decreased by 25% (p<0.001) in normal and by 17% (p<0.001) in PPHN

PAECs (fig not shown).

Effect of Rho-kinase inhibition and stimulation on eNOS expression. Rho-kinase

inhibition with Y-27632 increased eNOS protein content in both normal and PPHN

PAECs. eNOS protein expression was increased by 30% (p<0.01) and 58% (p<0.05)

in normal and PPHN PAECs, respectively (fig 5a). Rho-kinase activation with

calpeptin decreased eNOS protein expression by 28% (p<0.01) in normal PAECs,

however, calpeptin did not cause a further decrease in eNOS protein expression in

PPHN PAECs ((fig 5b), which was decreased by 42% (p<0.01) at baseline.

Effect of Rho-kinase inhibition and activation on nitric oxide (NO) production

in normal and PPHN PAECs. Rho-kinase inhibition with Y-27632 increased NO

production in both normal and PPHN PAECs (fig 6a.). With rho-kinase inhibition

NO production increased 63% (p<0.001) in normal and 64% (p<0.001) in PPHN

Page 15 of 41

PAECs. Rho-kinase activation decreased NO production by 31% (p<0.05) and

25% (p>0.05) in normal and PPHN PAECs respectively (fig 6b).

Increase in tube formation with rho-kinase inhibition is nitric oxide dependent in

normal but not PPHN PAECs. The increase in tube formation seen with rho-kinase

inhibition was reversed with nitric oxide synthase inhibition using L-NA in normal

PAECs. Tube formation decreased by 30% (p<0.001) with the addition of L-NA to

Y-27632 (fig 7). Values achieved were 22% lower than that achieved by normal

controls. In PPHN PAECs, L-NA had no effect on the increase in tube formation

with rho-kinase inhibition (fig 7).

Page 16 of 41

Discussion

In addition to increased pulmonary vascular tone and hypertensive remodeling,

impaired angiogenesis also contributes to high PVR in severe PPHN, especially in

the setting of lung hypoplasia (12). Previous studies have shown that pulmonary

hypertension during late gestation impairs fetal lung vascular growth in vivo (11),

and causes abnormalities in endothelial cell phenotype that persist in vitro (9).

However, mechanisms through which sustained elevations of pulmonary arterial

pressure inhibit lung angiogenesis during development are unknown. Since rho-

kinase activity modulates eNOS protein expression and activity, we hypothesized

that increased rho-kinase activity may account for the change in endothelial cell

phenotype seen in PPHN. We found that rho kinase activity, as assessed by rhoA,

and rhoGTP protein expression and phosphorylation of MYPT-1, was increased in

PAECs harvested from PPHN lambs. We also found that treatment with Y27632, a

rho kinase inhibitor, increased eNOS protein expression and NO production and

rescued the abnormal in vitro phenotype, restoring tube formation by PPHN

PAECs to normal levels. In addition, treatment with calpeptin, a rho kinase

activator, increased phosphorylation of MYPT-1, decreased eNOS protein

expression and NO production, and decreased tube formation in vitro in normal

PAECs. These findings demonstrate that chronic intrauterine pulmonary

hypertension increases rho kinase activity in lung vascular endothelium, which

Page 17 of 41

contributes to impaired angiogenesis, reduced eNOS protein content and

decreased NO production in PPHN.

This is the first study of rho-kinase activity in fetal PAECs and these findings

demonstrate increased rho-kinase activity in PPHN PAECS, suggesting a role for

the rho-kinase pathway in regulating angiogenesis in the developing lung. Prior

studies in experimental pulmonary hypertension have demonstrated increased rho-

kinase activity in the adult, but these reports have primarily focused on rho-kinase

activity in the smooth muscle cell and its effect on vascular tone and hypertensive

remodeling in pulmonary hypertension. With exposure to acute hypoxia inhibition

of rho-kinase activity attenuates the constrictor response in adult rats (37,52), while

after chronic hypoxia, rho-kinase inhibition decreases mean pulmonary artery

pressure (30,32). Pulmonary hypertension induced by chronic hypoxia has

previously been attributed to structural changes in the pulmonary vasculature

including hypertensive remodeling which produces a fixed increase in resistance

(13). Sustained inhibition of rho-kinase throughout the period of hypoxic exposure

attenuates pulmonary hypertension and prevents vascular remodeling (20). Adult

rats when treated with a single dose of monocrotaline develop severe pulmonary

hypertension and vascular remodeling (8,46). Chronic rho-kinase inhibition in this

setting prevents pulmonary vascular remodeling, by suppressing vascular smooth

muscle cell proliferation and macrophage infiltration (19). These studies indicate

that rho-kinase–mediated pathways are substantially involved in the pathogenesis

of pulmonary hypertension contributing significantly to vascular tone and

hypertensive remodeling in PPHN.

Page 18 of 41

We recently reported that prolonged intrauterine pulmonary hypertension

impairs angiogenesis and decreases alveolarization and lung weight in fetal lambs

(11). Intrauterine pulmonary hypertension also directly alters endothelial cell

function and impairs growth and tube formation by isolated PAECs in vitro. (9).

Thus, pulmonary hypertension itself can impair endothelial cell function, reduce

vascular growth and cause lung hypoplasia. How pulmonary hypertension alters

endothelial cell function and contributes to impaired angiogenesis in PPHN

remains unknown, but our results implicate the rho-kinase signal transduction

pathway as contributing to endothelial cell dysfunction and impaired vascular

growth in PPHN.

Our studies demonstrate a role for the rho-kinase pathway in regulating

these important endothelial cell functions and contributing to normal blood vessel

formation in the developing lung as well as impaired angiogenesis in PPHN.

Whether decreased alveolarization in PPHN is mediated by rho-kinase is unknown.

Prior studies have implicated rho-kinase signaling in regulating alveolarization

during development (26,28), but mechanisms underlying these findings were not

explored. Past studies have suggested that inhibition of vascular growth impairs

alveolarization (18,44,45). We speculate that high rho-kinase activity in PPHN

PAECs decreases vascular growth and subsequent alveolarization. Fetal lung

explants incubated for 48 hours in 3% oxygen show increased branching as well

as membrane associated rhoA when compared with room air controls (7,26).

Page 19 of 41

Recent studies have demonstrated that under hypoxic condition rho-kinase is

activated (26,37,43,52), suggesting that the increase in lung branching seen under

hypoxic conditions may be mediated by rho-kinase. The fawn hooded rat (FHR) is

a genetic model of pulmonary hypertension and is characterized by increased rho-

kinase activity (31). FHRs when exposed to mild hypoxia seen at Denver’s altitude

develop alveolar simplification and pulmonary hypertension (23,24). In this model

chronic rho-kinase inhibition improves alveolarization and vascular growth. These

studies support our findings that while rho-kinase activity during fetal life may

regulate lung growth, inappropriate rho-kinase activation during fetal life or

persistence of rho-kinase activation after birth, may impair angiogenesis and lung

growth.

Prior studies have demonstrated that inhibition of rho-kinase upregulates

and activates eNOS, increasing the production of NO (27,33,36). Long-term

inhibition of rho-kinase activity is protective against pulmonary hypertension and

right ventricular hypertrophy in hypoxia exposed adult mice (20), but was less

effective in eNOS (-/-) mice (20), which suggests that eNOS activation after rho-

kinase inhibition is responsible for these protective effects. We report that rho-

kinase inhibition increases eNOS protein expression and activity in both normal

and PPHN PAECs, which supports the concept that eNOS activation and

increased NO production may be responsible for enhanced angiogenesis in vitro

by normal and PPHN PAECs. However, the effect of rho-kinase inhibition on

angiogenesis was lost in normal but not PPHN PAECS with NOS inhibition using

Page 20 of 41

L-NA. This finding suggests an NO independent effect of rho-kinase inhibition

and, raises the possibility that in conditions associated with dysfunctional eNOS,

and impaired NO production, rho-kinase inhibitors may have even greater

therapeutic benefit. Interestingly, Lohn et al demonstrated that in the models of

genetically reduced endothelial NO production (eNOS-/- mice and spontaneous

hypertensive rats) and in models of pharmacologically reduced endogenous NO

production (LNAME treatment), rho-kinase inhibition produced a strong

vasodilator response, which also suggests that inhibition of rho kinase has NO-

independent effects as well (25).

Potential limitations of this study include the use of fetal PAECs harvested

from relatively large vessels, and that differences may exist in the behavior of

these cells as compared to microvascular PAECs. Since microvascular PAECs

may primarily be involved in lung angiogenesis during development in vivo, future

studies are needed to compare and contrast rho-kinase activity in microvascular

PAECs. While calpeptin markedly increased phosphorylation of MYPT-1 in normal

PAECs, there was no further increase in phosphorylation of MYPT-1 in PPHN

PAECs. NO production, however, was significantly reduced with calpeptin

treatment in both normal and PPHN PAECs. While the effects of calpeptin

treatment may not be mediated through rho-kinase, the decrease in NO production

with calpeptin treatment may support the concept that the detrimental effects of

rho-kinase activation are the result of decreased eNOS activity. Another potential

limitation is the fact that angiogenesis was only measured in vitro, and whether

Page 21 of 41

rho-kinase inhibition enhances angiogenesis in vivo remains unknown.

In conclusion, we found that chronic intrauterine pulmonary hypertension

increases rho-kinase activity in PAECs harvested from PPHN lambs and that this

increase in rho-kinase activity directly contributes to downregulated eNOS,

decreased NO production and impaired angiogenesis in vitro. Rho-kinase inhibition

reversed the abnormal in vitro phenotype previously described in PPHN PAECs.

This effect however was found to be NO independent. These findings suggest that

treatment strategies that down regulate or inhibit rho-kinase activation in PPHN

may enhance angiogenesis in vivo, and may be especially important in treating

pulmonary hypertension in the presence of endothelial dysfunction and lung

hypoplasia.

Page 22 of 41

Acknowledgements:

This work was supported in part by grants from iNO Therapeutics (Gien) and the

NIH R01 HL068702 (Abman).

Page 23 of 41

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Figure Legends

Figure 1. Increased Rho-kinase activity in PAECs from PPHN Sheep.

RhoA, rhoGTP and phosphorylated MYPT-1 protein expression was assessed in

fetal ovine PAECs from normal and PPHN lambs by western blot analysis and

ELISA. In comparison with normal PAECs total rhoA (fig 1a), rhoGTP (fig1b) and

phosphorylated MYPT-1( fig 1c.) protein was increased in PPHN PAECs.

Error bars represent SD from mean.

Figure 2. Increased Membrane and Cystosolic Rho A Protein Expression in

PAECs from PPHN Sheep. Whole cell lysates were separated into membrane

and cytosolic fractions and both membrane and cytosolic RhoA protein was

increased in PPHN PAECs when compared with normal controls.

Error bars represent SD from mean.

Figure 3. Effects of Calpeptin on Rho-Kinase Activation in Normal and PPHN

PAECs. Phosphorylated MYPT-1 protein expression was assessed in normal

and PPHN PAECs in response to calpeptin treatment (rho-kinase activator). In

response to calppeptin treatmet phosphorylation of MYPT-1 was increased in

normal (fig 3a.) but not PPHN PAECs (fig 3b).

Page 31 of 41

Figure 4. Effect of Rho-kinase Inhibition and Stimulation on Tube Formation in vitro.

Fetal ovine PAECs from normal and PPHN sheep were plated on collagen in serum

free media under 3% oxygen conditions with and without Y-27632 (1µM) (rho-kinase

inhibitor) and calpeptin (100µg/ml)(rho-kinase activator). Y27632 treatment increased

the number of branch points in PPHN and normal PAECs (fig 4a), increasing the

number of branch points by PPHN PAECs to similar values seen in normal PAECs.

Calpeptin decreased tube formation in both normal and PPHN PAECs (fig 4b). Error

bars represent SD from mean.

Figure 5. Effect of Rho-kinase Inhibition and Activation on eNOS Protein Expression.

Cell lysates were collected from normal and PPHN PAECs with and without Y-27632

(1µM) (rho-kinase inhibitor) and calpeptin (100µg/ml)(rho-kinase activator). Rho-kinase

inhibition increased eNOS protein expression in both normal and PPHN PAECs (fig

5a.). Rho-kinase activation decreased eNOS protein expression in normal but not

PPHN PAECs (fig 5b.). Error bars represent SD from mean.

Figure 6. Effect of Rho-kinase Inhibition and Activation on Nitric Oxide (NO)

production in normal and PPHN PAECs. Rho-kinase inhibition with Y-27632

increased NO production in both normal and PPHN PAECs (fig 6a). Rho-kinase

activation decreased NO production in both normal and PPHN PAECs (fig 6b).

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Figure 7. Increase in Tube Formation with Rho-kinase inhibition is Nitric Oxide

Dependent in Normal but not PPHN PAECs. Fetal ovine PAECs from normal and

PPHN sheep were plated on collagen in serum free media under 3% oxygen

conditions with and without Y-27632 (1µM) (rho-kinase inhibitor) in the presence and

absence of L-NA(4mM). The addition of L-NA to Y-27632 decreased tube formation in

normal but not PPHN PAECs (fig 7.). Error bars represent SD from mean.

Page 33 of 41

Figure 1a.

Figure 1b.

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Figure 1c.

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Figure 2.

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Figure 3a

Figure 3b

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Figure 4a

Figure 4b

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Figure 5a.

Figure 5b.

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Figure 6a

Figure 6b

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Figure 7.

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