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Am J Physiol Lung Cell Mol Physiol 294: L205-L213, 2008. First published November 21, 2007; doi:10.1152/ajplung.00234.2007
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Acute vasodilator effects of Rho-kinase inhibitors in neonatal rats with pulmonary hypertension unresponsive to nitric oxide

Patrick J. McNamara,3,5,* Prashanth Murthy,1,* Crystal Kantores,2 Lilian Teixeira,3 Doreen Engelberts,3 Todd van Vliet,1 Brian P. Kavanagh,3,4,6 and Robert P. Jankov1,2,5,6

1Newborn and Developmental Paediatrics, Sunnybrook Health Sciences Centre; 2Clinical Integrative Biology, Sunnybrook Research Institute; 3Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute; and the Departments of 4Anaesthesia, 5Paediatrics, and 6Physiology, University of Toronto, Toronto, Ontario, Canada

Submitted 13 June 2007 ; accepted in final form 20 November 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary hypertension (PHT) in neonates is often refractory to the current best therapy, inhaled nitric oxide (NO). The utility of a new class of pulmonary vasodilators, Rho-kinase (ROCK) inhibitors, has not been examined in neonatal animals. Our objective was to examine the activity and expression of RhoA/ROCK in normal and injured pulmonary arteries and to determine the short-term pulmonary hemodynamic (assessed by pulse wave Doppler) effects of ROCK inhibitors (15 mg/kg ip Y-27632 or 30 mg/kg ip fasudil) in two neonatal rat models of chronic PHT with pulmonary vascular remodeling (chronic hypoxia, 0.13 FIO2, or 1 mg·kg–1·day–1 ip chronic bleomycin for 14 days from birth). Activity of the RhoA/ROCK pathway and ROCK expression were increased in hypoxia- and bleomycin-induced PHT. In both models, severe PHT [characterized by raised pulmonary vascular resistance (PVR) and impaired right ventricular (RV) performance] did not respond acutely to inhaled NO (20 ppm for 15 min) or to a single bolus of a NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1; 2 µg/kg ip). In contrast, a single intraperitoneal bolus of either ROCK inhibitor (Y-27632 or fasudil) completely normalized PVR but had no acute effect on RV performance. ROCK-mediated vasoconstriction appears to play a key role in chronic PHT in our two neonatal rat models. Inhibitors of ROCK have potential as a testable therapy in neonates with PHT that is refractory to NO.

Y-27632; fasudil; two-dimensional echocardiography; pulse wave Doppler; SIN-1

PULMONARY HYPERTENSION (PHT) complicates the early neonatal course of 1–2 per 1,000 term and near-term live-born infants in North America (68) and contributes to poor outcomes related to chronic lung disease of prematurity (54), congenital diaphragmatic hernia (47), and some forms of congenital heart disease (23, 56). Clinical syndromes causing PHT in the newborn period (known as persistent PHT of the newborn; PPHN) include meconium aspiration, perinatal asphyxia, congenital sepsis, and respiratory distress syndrome (67, 68), all of which are characterized by persistent hypoxemia with or without pulmonary inflammation (8, 63). PPHN carries an associated mortality of up to 20% (67, 68), and survivors have high morbidity in the form of cognitive delay, hearing loss, chronic lung disease, and need for frequent hospitalization (44). An increased propensity to PHT in the newborn period, compared with other stages of life, is predominantly related to two factors: failure of the physiological fall in pulmonary vascular resistance (PVR) required for a successful transition to postnatal life and the persistence and/or rapid augmentation of anatomical changes in the heart and pulmonary vasculature, known collectively as vascular remodeling. Structural and functional consequences of remodeling include narrowing of the vessel lumen, exaggerated responses to constrictors, decreased relaxation, and reduced compliance, all of which are believed to contribute to a chronic form of PHT that is progressive and potentially irreversible (6, 13, 21, 36).

The current gold standard vasodilator therapy for neonatal PHT is inhaled nitric oxide (NO), which acutely improves oxygenation in many patients (2, 11) but has been demonstrated to decrease neither mortality (2, 17) nor adverse pulmonary or neurological outcomes (1, 7). Unfortunately, ~40–50% of neonates with hypoxemic respiratory failure secondary to PPHN either do not respond to inhaled NO or respond only transiently (34), indicating a need for new therapies. To explore new therapies for PHT, we have developed two neonatal rat models of chronic PHT: secondary to exposure to chronic hypoxia (0.13 FIO2; Ref. 41) or to daily intraperitoneal bleomycin for 14 days from birth. Both models are characterized by increased PVR, right ventricular (RV) hypertrophy (RVH) and dysfunction, and remodeling of pulmonary resistance arteries. Moreover, as reported herein, neither model of PHT responds acutely to NO.

Recent studies have implicated the small GTPase, RhoA, and its effector protein, Rho-kinase (ROCK; Refs. 28, 61, 62) with two known isoforms (ROCK-I and ROCK-II) that are highly expressed in vascular tissues (16, 46), as a key pathway regulating pulmonary vascular tone. The availability of two pharmacological kinase inhibitors, Y-27632 (29) and fasudil (58), that possess high specificity toward both ROCK isoforms compared with a broad range of other kinases (10) has led to the appreciation that activation of ROCK may be central to sustained pulmonary vasoconstriction induced by hypoxia (14, 51, 57) and by mediators that are critical to the development of chronic PHT, including ligands of endothelin and thromboxane receptors (4, 19, 35, 65, 70, 72). Furthermore, inhibition of ROCK decreased PVR in fetal sheep, suggesting a role for activation of ROCK in maintenance of the normally high fetal pulmonary vasomotor tone and for its abnormally persistent activation in failure of the physiological circulatory transition after birth (55). Since there have been no previously published reports on the role of the RhoA/ROCK pathway in neonatal PHT, our aims were to determine whether pulmonary arterial ROCK is upregulated and activated by hypoxic and inflammatory (bleomycin-induced) insults and, if so, to determine whether acute inhibition of ROCK would have beneficial effects on abnormal pulmonary hemodynamics.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Y-27632 was purchased from Biomol International (Plymouth Meeting, PA), fasudil (HA-1077 dihydrochloride) was from LC Laboratories (Woburn, MA), and nitric oxide gas (400 ppm, balance N2) was from Praxair (Mississauga, Ontario, Canada). Plexiglas animal exposure chambers and automated O2 controllers (OxyCycler A84XOV) were purchased from BioSpherix (Redfield, NY). Acids, alcohols, organic solvents, paraformaldehyde, Permount, and Superfrost Plus microscope slides were from Fisher Scientific (Whitby, Ontario, Canada). Rabbit polyclonal antibodies against RhoA, ROCK-I, ROCK-II, and a goat anti-rabbit IgG-biotin secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against myosin phosphatase target (MYPT)-1 and Thr850-phosphorylated MYPT-1 were from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibodies against CD68 (ED-1) and {alpha}-smooth muscle actin were from Serotec (Raleigh, NC) and Neomarkers (Fremont, CA), respectively. A goat anti-rabbit IgG-peroxidase antibody was from Cell Signaling Technology (Beverly, MA). Avidin-biotin-peroxidase complex immunohistochemistry kits, diaminobenzidine staining kits, and normal goat serum were from Vector Laboratories (Burlingame, CA). Tris-glycine 4–20% gradient gels were from Pierce Biotechnology (Rockford, IL), and polyvinylidene difluoride (PVDF) membranes were from VWR (Mississauga, Ontario, Canada). A rabbit polyclonal antibody against {alpha}-catenin, 3-morpholinosydnonimine hydrochloride (SIN-1), and all other drugs, chemicals, and reagents were from Sigma (Oakville, Ontario, Canada).

Models. All procedures involving animals were performed in accordance with the standards of the Canadian Council on Animal Care and were approved by the Animal Care Committees of the Sunnybrook and Hospital for Sick Children Research Institutes. 1) Acute hypoxic PHT: day 14 Sprague-Dawley rat pups were exposed to 13% O2 for 30 min. 2) Chronic hypoxic PHT: litters (maintained at n = 10–12 pups to control for nutritional effects) were nursed in either hypoxia (13% O2) or 21% O2 (normoxia control) from birth for 14 days as previously described in detail (41). 3) Chronic bleomycin-induced PHT: commencing on the day of birth, pups received daily 1 mg/kg ip bleomycin sulfate in 0.9% saline (5 µl/g body wt by 30-gauge needle in the right iliac fossa) or 0.9% saline (vehicle control) daily intraperitoneally for 14 days.

Interventions. Pups received continuous inhaled NO (20 ppm) or a single bolus (5 µl/g body wt) of Y-27632 (15 mg/kg ip in 0.9% NaCl), fasudil (30 mg/kg ip in 0.9% NaCl), SIN-1 (2 µg/kg ip in 0.9% NaCl), or vehicle alone 15–30 min before assessing response. The intraperitoneal dose of SIN-1 used was similar to that previously shown to be effective at attenuating allergic encephalomyelitis in adult rats (71). Delivered NO concentration was constantly monitored using a PulmoNOx mini dosing device (Namu, Messer Griesheim, Austria). At the end of each experiment, pups were killed either by pentobarbital overdose or by exsanguination after anesthesia.

Two-dimensional echocardiography and Doppler ultrasound. Echocardiographic evaluation of pulmonary hemodynamics was performed using the Vivid 7 Advantage cardiovascular ultrasound system (GE Medical Systems, Milwaukee, WI) with a small high frequency linear probe (I13L) as previously described in detail (41). Immediately following induction of anesthesia with intraperitoneal ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (6 mg/kg), the animal was laid supine and, while spontaneously breathing 13% O2 (acute hypoxic PHT group) or room air (other groups), underwent a baseline echocardiographic study, followed by the intervention (continuous inhaled NO with 0.21 FIO2 or bolus intraperitoneal dose of vehicle, Y-27632, fasudil, or a NO donor, SIN-1) and a second study 15–30 min later. All pups chronically exposed to hypoxia or bleomycin that were included in the study had a baseline pulmonary arterial acceleration time (PAAT) of ≤18 ms, indicative of severe PHT (41, 43). For measurements of pulmonary arterial hemodynamics, a short axis view at the level of the aortic valve was obtained, and the pulmonary artery was identified using color flow Doppler. The PAAT and RV ejection time (RVET) of the pulmonary Doppler profile were then measured. 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 completion of systolic pulmonary flow. A surrogate of PVR was calculated according to the formula 1/(PAAT/RVET). We (41) have found that PAAT-to-RVET ratio inversely correlates with the severity of vascular remodeling, and others have shown a close inverse correlation with direct measurement of pulmonary arterial pressure (40). RV stroke volume (RVSV) was also calculated as a measure of RV performance using the formula (pulmonary artery diameter/2)2 x 3.14 x pulmonary artery velocity time integral. Left ventricular (LV) systolic performance was estimated from the parasternal short axis view by calculating the shortening fraction according to the formula (LV end diastolic dimension – LV end systolic dimension/LV end diastolic dimension) x 100.

Arterial blood gas measurement. Pups were anesthetized with intraperitoneal ketamine/xylazine, and the neck was dissected to expose the external carotid artery. The carotid artery was transected, and blood was immediately collected into a heparinized capillary tube and analyzed (ABL500; Radiometer, Copenhagen, Denmark).

RV hypertrophy. RVH was quantified by measuring the RV-to-LV-and-septum (LV+S) dry weight ratios as previously described (32).

Histological and morphometric studies. Four animals from each group (2 from each of 2 separate litters) were anesthetized with ketamine (80 mg/kg ip) and xylazine (12 mg/kg ip). After opening of the thoracic cavity and cannulation of the trachea, the pulmonary veins were divided. The pulmonary circulation was flushed with 1x PBS containing 1 U/ml heparin via a needle inserted in the RV to clear the lungs of blood while the lungs were inflated at a constant pressure of 20 cmH2O. The lungs were then perfusion-fixed for 5 min at 100 cmH2O pressure with ice-cold 4% (wt/vol) paraformaldehyde in 1x PBS, excised en bloc, and then dehydrated, cleared in xylene, and embedded in paraffin. Sections (5 µm) were stained for elastin or immunostained as previously described (30–32, 41). Concentrations of the primary antisera were 1/100 (2 µg/ml) for both ROCK-I and -II, 1/200 (1.25 µg/ml) for ED-1, and 1/1,000 (0.25 µg/ml) for {alpha}-smooth muscle actin. Negative controls were generated by omission of the primary antibody. For measurements of medial wall thickness (MWT), intraacinar pulmonary arteries (associated with respiratory bronchioles, alveolar ducts, and alveolar walls), where the complete circumference of the vessel was visible (>20 per animal), were identified on Hart's elastin-stained sections and digitally photographed by an observer blinded to group identity. Percentage MWT was calculated using the formula (distance between internal and elastic laminae x 2)/(mean external diameter) x 100 as previously described (41). Mean external diameter was calculated from measurements in two perpendicular planes to account for any irregularities in vessel shape. Results are shown as mean values from three to four animals per group.

Western blot analyses. RhoA activation (measured by increased membrane localization of RhoA as previously described in Ref. 72) increased ROCK activity (quantified by increased ratio of phospho- to total MYPT-1), and arterial ROCK-I and -II expression were assessed by Western blot analyses on third to fourth generation intrapulmonary arteries dissected from three to four litters per group (the pooled vessels of 4 animals were used from each litter). For total protein, tissue was lysed in RIPA buffer with protease inhibitors, leupeptin, aprotinin (1 µg/ml each), and 1 mM PMSF. For phospho-MYPT-1, tissue was lysed in NETF buffer (0.1 M NaCl, 2 mM EDTA, 50 mM Tris·HCl, 50 mM NaF, pH 7.4) containing protease inhibitors (as above) and phosphatase inhibitor cocktail (Sigma). For isolation of membrane protein (particulate fraction), commercially available extraction and purification kits were used (Pierce Biotechnology). Total or membrane protein samples (50 µg per lane) were boiled for 5 min in Laemmli buffer, fractionated by SDS-PAGE, transferred to PVDF membranes, and blotted as previously described (30). Dilutions of primary antisera were 1:4,000 (0.05 µg/ml) for {alpha}-catenin (150 kDa), 1:2,000 (0.1 µg/ml) for phospho- and total MYPT-1 (130 kDa), 1:1,000 (0.2 µg/ml) for RhoA (20 kDa), and 1:200 (1 µg/ml) for ROCK-I (150 kDa) and -II (160 kDa). The protein bands of interest were identified using enhanced chemiluminescence reagent and exposure on blue film. Bands were quantified by digital densitometry of nonsaturated radiographs with the background density removed (ImageJ version 1.30; National Institutes of Health, Bethesda, MD). After blotting for phospho-MYPT-1, membranes were stripped and reblotted for total MYPT-1. For ROCK-I and -II, differences in protein loading were accounted for by reblotting for {alpha}-catenin, the expression of which we determined in preliminary experiments to be unaffected by injury in pulmonary arteries.

Data presentation and analysis. Values are expressed as means ± SE. All analyses were performed using SigmaStat version 3.0.1 (Systat Software, San Jose, CA). Where three or more groups were compared, statistical significance (P < 0.05) was determined by two-way ANOVA followed by pairwise multiple comparisons using the Tukey test. Where two groups were compared, statistical significance (P < 0.05) was determined using the unpaired t-test.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline parameters. Body weight and blood gas values at 14 days are shown in Table 1. Pups chronically exposed to hypoxia or treated with daily intraperitoneal bleomycin had significantly (P < 0.01) lower body weight than controls, with the most severe growth restriction being evident in bleomycin-treated pups (P < 0.05 vs. chronic hypoxia group). There were no differences in the severity of growth restriction between NO- and ROCK inhibitor-treated groups (P > 0.05; combined body weight data are shown in Table 1). Pups chronically exposed to hypoxia from birth had significantly lower pH and PO2 values (while anesthetized and breathing room air) than controls (P < 0.05) and a lower baseline heart rate (P < 0.05) than bleomycin-exposed pups. There was a trend toward higher PCO2 values in both chronic hypoxia- and bleomycin-exposed pups, but these differences did not reach statistical significance (P > 0.05). In pups chronically exposed to hypoxia, a return to 21% O2 (normoxia) or, in both models, exposure to hyperoxia (50% O2) for >30 min did not significantly affect baseline PAAT-to-RVET ratio as measured by pulse wave Doppler (P > 0.05 by t-test compared with baseline; n = 6 animals per group, data not shown); therefore, all subsequent studies were performed under normoxic conditions.


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  		Table 1. Baseline parameters at day 14

 
Chronic treatment with bleomycin leads to pulmonary vascular remodeling and macrophage influx. Chronic exposure to hypoxia (13% O2) for 14 days from birth causes elevation of PVR, RV dysfunction, severe RVH, and structural remodeling of pulmonary resistance arteries (41). Pups treated daily for 14 days from birth with bleomycin also develop significant RVH and vascular remodeling, characterized in major part by medial arterial wall smooth muscle thickening (Fig. 1A), demonstrated by {alpha}-smooth muscle actin and elastin staining (Fig. 1B, top and middle). Furthermore, in contrast to the lungs of chronic hypoxia-exposed pups in which few macrophages were evident (Fig. 1B, bottom, Hypoxia), the lungs of bleomycin-treated pups had greatly increased numbers of alveolar and interstitial macrophages (ED-1 immunoreactive macrophages are highlighted by arrows; Bleomycin), which have been shown to be a critical component of bleomycin-induced pulmonary injury (33, 48).


Figure 1
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  		Fig. 1. Chronic treatment with bleomycin leads to pulmonary vascular remodeling and inflammation. A: increased right ventricle (RV)-to-left ventricle-and-septum (LV+S) dry weight ratios as a marker of RV hypertrophy (RVH; n = 10 animals per group) and increased percentage arterial medial wall thickening (MWT; n = 3–4 animals per group) as evidence of pulmonary vascular remodeling in pups exposed to 1 mg·kg–1·day–1 ip bleomycin sulfate from birth until 14 days. *P < 0.01, by ANOVA, compared with vehicle-treated controls. B, top: representative low-power images of {alpha}-smooth muscle actin immunostaining ({alpha}-Actin; arrows highlight pulmonary arterial smooth muscle shown as brown stain) demonstrating the thickened medial smooth muscle layer of pulmonary arteries in pups exposed to bleomycin. Middle: high-power images of Hart's elastin staining (Elastin) further demonstrating the thickened medial smooth muscle layer, delineated on either side by the internal and external elastic laminae in the pulmonary artery (pa) of a pup exposed to bleomycin. Bottom: ED-1 (macrophage CD68) immunostaining. Macrophage numbers were not increased by exposure to chronic hypoxia from birth for 14 days (Hypoxia). In contrast, exposure to bleomycin (Bleomycin) led to a striking increase in macrophage numbers in the interstitium and air spaces (some macrophages are highlighted by arrows). Bar lengths = 100 µm.

 
Increased PVR induced by chronic hypoxia or bleomycin is acutely unresponsive to inhaled NO or systemically administered NO donor, SIN-1. As shown in Fig. 2, A and B, pups chronically exposed to hypoxia or given daily intraperitoneal treatment with bleomycin had significantly elevated PVR (P < 0.05 vs. controls). Neither exposure to inhaled 20 ppm NO for >15 min (Fig. 2A) nor a systemically administered NO donor (2 µg/kg ip SIN-1; Fig. 2B) had any significant effect on PVR (P > 0.05 compared with baseline).


Figure 2
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  		Fig. 2. Increased pulmonary vascular resistance (PVR) induced by chronic hypoxia or bleomycin is acutely unresponsive to inhaled or systemic NO. PVR was raised by chronic exposure to 13% O2 (Chronic Hypoxia) or by 1 mg·kg–1·day–1 ip bleomycin sulfate (Bleomycin) for 14 days from birth as quantified by the inverse ratio of pulmonary arterial acceleration time (PAAT) to RV ejection time (RVET). *P < 0.05, by ANOVA, compared with all other groups (n = 6–8 animals per group representing 2 litters). This parameter was acutely responsive to inhaled nitric oxide (20 ppm; P > 0.05 compared with respective baseline group; A) or to systemic administration of a NO donor, SIN-1 (2 µg/kg ip; P > 0.05 compared with respective baseline group; B).

 
Pulmonary vascular RhoA is activated and ROCK activity is increased by chronic exposure to hypoxia or bleomycin. As shown in Fig. 3, RhoA was activated in the arteries of both hypoxia- and bleomycin-exposed animals, as demonstrated by its increased membrane localization (P < 0.05 compared with respective controls). In addition, ROCK activity was increased in both models, as shown by significantly increased phosphorylation of Thr850 MYPT-1 (Fig. 3; P < 0.05 compared with respective controls).


Figure 3
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  		Fig. 3. Pulmonary vascular RhoA is activated and Rho-kinase (ROCK) activity is increased by chronic exposure to hypoxia or bleomycin. Western blot analyses showing increased membrane (compared to total) RhoA, as a marker of activation (left), and increased phospho-MYPT-1 (expressed as a ratio of total MYPT-1), as a marker of increased ROCK activity (right), in intrapulmonary arteries derived from rat pups exposed to 13% O2 (Hypoxia) or daily intraperitoneal bleomycin sulfate (Bleomycin) for 14 days from birth, compared with their respective controls. *P < 0.05, by t-test, vs. respective control (n = 3–4 litters per group). Bottom: 2 representative protein bands per group are shown (corresponding to bars in graph above), each representing the total protein derived from pooled homogenized intralobar (3rd to 4th generation) pulmonary arteries dissected from 4 animals per group.

 
Pulmonary vascular ROCK expression is increased by chronic exposure to hypoxia or bleomycin. As shown by immunohistochemistry in Fig. 4, increased immunoreactive ROCK-I and -II is evident in the lungs of hypoxia- or bleomycin-exposed pups, particularly localized to the walls of pulmonary arteries and on the distal airway epithelium. Intrapulmonary arterial expression of both ROCK-I and -II was also examined by Western blot (Fig. 5), which confirmed upregulation of both ROCK isoforms in both chronic hypoxia- and bleomycin-exposed animals (P < 0.05 compared with respective controls).


Figure 4
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  		Fig. 4. Pulmonary ROCK expression is increased by chronic exposure to hypoxia or bleomycin. Representative immunohistochemistry (brown stain) for ROCK isoforms ROCK-I (left) and ROCK-II (right) of lung sections from rat exposed to normoxia (Air), chronic hypoxia (Hypoxia), daily intraperitoneal saline vehicle (Vehicle), or 1 mg·kg–1·day–1 ip bleomycin sulfate (Bleomycin) for 14 days from birth. Bar lengths = 100 µm. Immunoreactivity was largely restricted to proximal airway epithelium in control (Air or Vehicle) rat pups. Immunoreactivity of ROCK-I and ROCK-II was increased in pups with hypoxia- or bleomycin-induced pulmonary hypertension, particularly on the walls of pulmonary arteries and on distal airway epithelium. Insets: sections from hypoxia- or bleomycin-exposed animals in which the primary antibody was omitted, showing lack of staining.

 

Figure 5
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  		Fig. 5. Pulmonary arterial ROCK-I and -II expression is increased by chronic exposure to hypoxia or bleomycin. Western blot analyses showing increased ROCK-I (left) and ROCK-II (right) expression in intrapulmonary arteries dissected from rat pups exposed to 13% O2 (Hypoxia) or 1 mg·kg–1·day–1 ip bleomycin sulfate (Bleomycin) for 14 days from birth, compared with their respective controls. *P < 0.05, by t-test, vs. respective control (n = 3–4 litters per group). Bottom: 2 representative protein bands per group are shown (corresponding to bars in graph above), each representing the total protein derived from pooled homogenized intralobar (3rd to 4th generation) pulmonary arteries dissected from 4 animals per group.

 
ROCK inhibitors acutely normalized increased PVR induced by chronic exposure to hypoxia or bleomycin. Treatment with 15 mg/kg Y-27632 completely normalized increased PVR induced by acute and chronic exposure to hypoxia (Fig. 6A; P < 0.05 compared with baseline) or by treatment with bleomycin (Fig. 6B; P < 0.05 compared with baseline). An alternative ROCK inhibitor (fasudil) that is structurally dissimilar to Y-27632 also had a significant (P < 0.05 compared with baseline) attenuating effect on raised PVR secondary to either acute or chronic exposure to hypoxia (Fig. 6C). Importantly, neither Y-27632 (Fig. 6A) nor fasudil (data not shown) had any effect on PVR in control animals nor were any significant (P > 0.05) changes observed in heart rate or LV systolic function observed with either treatment (data not shown), suggesting a lack of any major inhibitory effects on cardiac output and intrinsic myocardial performance.


Figure 6
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  		Fig. 6. ROCK inhibitors acutely normalize raised PVR induced by chronic exposure to hypoxia or bleomycin. PVR was raised by acute (13% O2 for 30 min at day 14; Acute Hypoxia) or chronic exposure to 13% O2 for 14 days from birth (Chronic Hypoxia) (A), or by 1 mg·kg–1·day–1 ip bleomycin sulfate for 14 days from birth (Bleomycin; C), as quantified by the inverse ratio of pulmonary arterial PAAT to RVET. Raised PVR was completely normalized by a single intraperitoneal bolus of ROCK inhibitor, Y-27632 (15 mg/kg; A) or fasudil (30 mg/kg; B) in acute- or chronic hypoxia-exposed pups and Y-27632 (15 mg/kg; C) in bleomycin-exposed pups. *P < 0.01, by ANOVA, compared with respective baseline (n = 6–7 animals per group representing 2 litters). D: representative Doppler traces showing mid-systolic "notching" (arrow) and rapid time to peak acceleration, characteristic of severe pulmonary hypertension, which is no longer present shortly after treatment with Y-27632.

 
Y-27632 does not improve impaired RV function induced by chronic exposure to hypoxia or bleomycin. As shown in Fig. 7, RVSV (as a marker of RV output) was greatly decreased (P < 0.05 compared with control) in animals with chronic PHT secondary to hypoxia or bleomycin. Neither inhaled nor systemic NO (data not shown) nor treatment with Y-27632 (Fig. 7) led to any improvements in this parameter (P > 0.05 compared with baseline).


Figure 7
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  		Fig. 7. Y-27632 does not improve impaired RV performance induced by chronic exposure to hypoxia or bleomycin. RV output, as quantified by the RV stroke volume (RVSV), was greatly decreased in pups chronically exposed to 13% O2 (Chronic Hypoxia) or to 1 mg·kg–1·day–1 ip bleomycin sulfate for 14 days from birth (Bleomycin). Treatment with a single intraperitoneal bolus of the ROCK inhibitor, Y-27632 (15 mg/kg), had no significant effects on this parameter. *P < 0.01, by ANOVA, compared with respective baseline and control groups (n = 6–7 animals per group representing 2 litters; data from air-exposed and vehicle-treated controls were combined; n = 12 animals).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study were: 1) PHT in neonatal rats chronically exposed to hypoxia or bleomycin did not acutely respond to inhaled NO or to systemic administration of a NO donor; 2) ROCK was upregulated in the pulmonary arteries in both models of chronic neonatal PHT, and its activity was increased, as demonstrated by increased phosphorylation of MYPT-1; 3) a single intraperitoneal bolus of either Y-27632 or fasudil, both selective ROCK inhibitors, completely normalized PVR in both chronic PHT models and in neonatal rats acutely exposed to hypoxia; and 4) none of the interventions tested had any significant short-term impact on impaired RV performance (as measured by decreased RVSV) in animals with chronic PHT. A disconnect between normalization of PVR by inhibition of ROCK and lack of acute improvement in RV performance, and therefore cardiac output, has also been described in adult rats with severe vaso-occlusive PHT (53). We interpret these findings as a reflection of the limited short-term impact of effective vasodilator treatment to be expected in chronic PHT, which leads to a dilated and poorly contracting RV (41). Further studies are required to determine whether "unloading" the right heart for a longer period improves its performance over time.

In this study, we describe a new rat model of chronic neonatal PHT secondary to bleomycin toxicity. As hypoxia and/or inflammation are important initiating and perpetuating factors in human neonates with PHT (8, 63), our finding that pulmonary inflammation was not present to any extent in hypoxia-exposed neonatal rats led us to develop a model of PHT in which inflammatory cell (predominantly macrophage) influx is critical to pulmonary injury (33, 48). We believe that the strikingly similar findings in both models strengthen the argument for a putative central role for ROCK-mediated vasoconstriction in chronic neonatal PHT. Similar findings using ROCK inhibitors in adult rodent models of experimental chronic PHT (14, 51, 53) have led to the dogma being challenged that structural remodeling in hypoxic PHT is the main contributor to a fixed increase in PVR through luminal obstruction and a decreased potential for relaxation (64). Our current observations extend this challenge to neonatal animals, further suggesting that PHT unresponsive to conventional therapies may in many cases be caused by ROCK-mediated vasoconstriction (which appears not to be unmasked by short-term administration of NO) rather than by remodeling. However, augmented cGMP-dependent protein kinase G signaling, which is primarily responsible for NO-mediated vasodilation (37), has also been described to have inhibitory effects on the RhoA/ROCK pathway (20, 38, 49, 59). We therefore speculate that the lack of significant acute vasodilatory effect of NO, in contrast to the complete normalizing effect of ROCK inhibitors observed in our models, may be related to dysfunction of the intracellular NO receptor, soluble guanylate cyclase (12, 66), or to increased phosphodiesterase type 5 expression/activity and therefore accelerated degradation of cGMP (37). It is also plausible that a longer duration of exposure to NO may be required to attenuate RhoA/ROCK signaling. All of these possibilities warrant exploration in future studies.

An increasing number of studies implicate oxidant stress, due to increased generation of reactive oxygen species (ROS), as an important initiating and perpetuating factor in chronic PHT (5, 69). In the lung, increased ROS are implicated in hypoxia-induced vasoconstriction, endothelial dysfunction, and vascular remodeling (24, 25, 45). A role for increased numbers of ROS-producing inflammatory cells (especially macrophages) in chronic PHT is also likely (42, 52). Oxidant stress and the consequent upregulation of G protein-coupled receptor (60) ligands that are critical to the pathogenesis of chronic PHT, including endothelin-1 and thromboxane A2 (4, 19, 35, 65, 70, 72), are known to activate RhoA (39). We speculate that these ROS-induced mediators lead to activation of the RhoA/ROCK pathway in our rat models of chronic neonatal PHT, although the sources and specific ROS and ROS-induced mediators involved likely differ between them.

A key substrate of ROCK involved in sustained vasoconstriction and enhanced contractile potential of smooth muscle is myosin light chain phosphatase (MLCP; Ref. 15), which mediates smooth muscle relaxation when in its dephosphorylated form by leading to actin-myosin cross-bridge dissociation (9). Phosphorylation of the MYPT-1 regulatory subunit of MLCP by ROCK leads to repression of its activity (22) and dissociation from myosin (15). Activation of ROCK therefore leads to increased and sustained smooth muscle contraction at a given level of intracellular Ca2+, a phenomenon known as Ca2+ sensitization (61). As current efforts to treat PPHN are frequently hampered by nonresponsiveness to currently available vasodilators, our results suggest that ROCK inhibitors could represent a promising future first or second line therapy, potentially leading to improved survival and reduced morbidity from this difficult-to-treat condition. Furthermore, and similar to sildenafil (3), the oral availability of Y-27632 and fasudil make them an attractive treatment option in the developing world where inhaled NO is generally not available. However, although it has been suggested that the RhoA/ROCK pathway may contribute less to vasomotor tone in the systemic (compared to the pulmonary) circulation (26), a potential limitation to the use of oral or parenterally administered ROCK inhibitors in the clinic is the likelihood of parallel effects on systemic vascular resistance. Indeed, small (but significant) decreases in systemic arterial blood pressure have been documented following systemic administration of ROCK inhibitors in adult animals (50) and in pilot studies on adult humans with idiopathic PHT (27). An alternative treatment approach may be through direct airway delivery of ROCK inhibitors to the lung by inhalation, which, by allowing for smaller doses, has been shown to confer relative pulmonary selectivity in rat models (50).

There are a number of limitations to this study. First, although the pathology (18) and underlying pathophysiology (8) for pulmonary vascular injury in our neonatal rat models is similar to that described for humans with PPHN, injury in the human is often antenatal in onset rather than postnatal. Therefore, our models may more closely resemble human neonates with chronic lung disease or congenital heart disease exposed to hypoxia rather than PPHN. Second, we could not assess systemic vascular resistance with our methodology and therefore, despite a lack of change in heart rate and LV performance following treatment with ROCK inhibitors, the effects on systemic blood flow and pressure remain unknown. Finally, Y-27632 and fasudil are only selective inhibitors of ROCK and therefore could have effects on other pathways that may play a role in sustained pulmonary vasoconstriction. Nevertheless, the above limitations decrease neither the importance of the current insights gained in our neonatal rat models nor the potential utility of selective ROCK inhibitors as a promising new class of therapeutic agents for human neonates and infants with PHT.

In conclusion, we provide evidence for the utility of ROCK inhibitors in NO-unresponsive experimental chronic neonatal PHT, which may have important clinical implications for a subgroup of human neonates and infants with PHT that is refractory to conventional therapy. Further studies are indicated in models of neonatal PHT to establish whether ROCK inhibitors are safe and effective over a longer duration.


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This work was funded by Operating (R. P. Jankov and B. P. Kavanagh) and Equipment (P. J. McNamara) Grants from the Canadian Institutes of Health Research (CIHR). R. P. Jankov is supported by a Career Development Award through the Canadian Child Health Clinician Scientist Program, a CIHR Strategic Training Initiative. B. P. Kavanagh holds the Dr. Geoffrey Barker Research Chair in Critical Care Medicine at the Hospital for Sick Children.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. P. Jankov, Dept. of Newborn and Developmental Paediatrics, Sunnybrook Health Sciences Centre, 76 Grenville St., Toronto, Ontario, Canada M5S 1B2 (e-mail: robert.jankov{at}sunnybrook.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* P. J. McNamara and P. Murthy contributed equally to this study. Back


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