Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats

Sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. Therapies that inhibit Rho-kinase (ROCK) and/or augment nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling can reverse or prevent progression of chronic pulmonary hypertension. Our objective in the present study was to determine whether sustained rescue treatment with inhaled CO(2) (therapeutic hypercapnia) would improve structural and functional changes of chronic hypoxic pulmonary hypertension. Spontaneously breathing pups were exposed to normoxia (21% O(2)) or hypoxia (13% O(2)) from postnatal days 1-21 with or without 7% CO(2) (Pa(CO(2)) elevated by ～25 mmHg) or 10% CO(2) (Pa(CO(2)) elevated by ～40 mmHg) from days 14 to 21. Compared with hypoxia alone, animals exposed to hypoxia and 10% CO(2) had significantly (P < 0.05) decreased pulmonary vascular resistance, right-ventricular systolic pressure, right-ventricular hypertrophy, and medial wall thickness of pulmonary resistance arteries as well as decreased lung phosphodiesterase (PDE) V, RhoA, and ROCK activity. Rescue treatment with 10% CO(2), or treatment with a ROCK inhibitor (15 mg/kg ip Y-27632 twice daily from days 14 to 21), also increased pulmonary arterial endothelial nitric oxide synthase and lung NO content. In contrast, cGMP content and cGMP-dependent protein kinase (PKG) activity were increased by exposure to 10% CO(2), but not by ROCK inhibition with Y-27632. In vitro exposure of pulmonary artery smooth muscle cells to hypercapnia suppressed serum-induced ROCK activity, which was prevented by inhibition of PKG with Rp-8-Br-PET-cGMPS. We conclude that sustained hypercapnia dose-dependently inhibited ROCK activity, augmented NO-cGMP-PKG signaling, and led to partial improvements in the hemodynamic and structural abnormalities of chronic hypoxic PHT in juvenile rats. Increased PKG content and activity appears to play a major upstream role in CO(2)-induced suppression of ROCK activity in pulmonary arterial smooth muscle.     

Brain natriuretic peptide inhibits hypoxic pulmonary hypertension in rats

Brainnatriuretic peptide (BNP) is a pulmonary vasodilator that is elevatedin the right heart and plasma of hypoxia-adapted rats. To test thehypothesis that BNP protects against hypoxic pulmonary hypertension, wemeasured right ventricular systolic pressure (RVSP), right ventricle(RV) weight-to-body weight (BW) ratio (RV/BW), and percentmuscularization of peripheral pulmonary vessels (%MPPV) in rats givenan intravenous infusion of BNP, atrial natriuretic peptide (ANP), orsaline alone after 2 wk of normoxia or hypobaric hypoxia (0.5 atm).Hypoxia-adapted rats had higher hematocrits, RVSP, RV/BW, and %MPPVthan did normoxic controls. Under normoxic conditions, BNP infusion(0.2 and 1.4 µg/h) increased plasma BNP but had no effect on RVSP,RV/BW, or %MPPV. Under hypoxic conditions, low-rate BNP infusion (0.2 µg/h) had no effect on plasma BNP or on severity of pulmonaryhypertension. However, high-rate BNP infusion (1.4 µg/h) increasedplasma BNP (69 ± 8 vs. 35 ± 4 pg/ml, P < 0.05),lowered RV/BW (0.87 ± 0.05 vs. 1.02 ± 0.04, P < 0.05), and decreased %MPPV (60 vs. 74%,P < 0.05). There was also a trend towardlower RVSP (55 ± 3 vs. 64 ± 2, P = not significant).Infusion of ANP at 1.4 µg/h increased plasma ANP in hypoxic rats (759 ± 153 vs. 393 ± 54 pg/ml, P < 0.05) but had noeffect on RVSP, RV/BW, or %MPPV. We conclude that BNP may regulatepulmonary vascular responses to hypoxia and, at the doses used in thisstudy, is more effective than ANP at blunting pulmonary hypertensionduring the first 2 wk of hypoxia.

     

Exaggerated hypoxic pulmonary hypertension in endothelin B receptor-deficient rats

Mechanisms by which endothelin (ET)-1 mediates chronic pulmonary hypertension remain incompletely understood. Although activation of the ET type A (ET(A)) receptor causes vasoconstriction, stimulation of ET type B (ET(B)) receptors can elicit vasodilation or vasoconstriction. We hypothesized that the ET(B) receptor attenuates the development of hypoxic pulmonary hypertension and studied a genetic rat model of ET(B) receptor deficiency (transgenic sl/sl). After 3 wk of severe hypoxia, the transgenic sl/sl pulmonary vasculature lacked expression of mRNA for the ET(B) receptor and developed exaggerated pulmonary hypertension that was characterized by elevated pulmonary arterial pressure, diminished cardiac output, and increased total pulmonary resistance. Plasma ET-1 was fivefold higher in transgenic sl/sl rats than in transgenic controls. Although mRNA for prepro-ET-1 was not different, mRNA for ET-converting enzyme-1 was higher in transgenic sl/sl than in transgenic control lungs. Hypertensive lungs of sl/sl rats also produced less nitric oxide metabolites and 6-ketoprostaglandin F(1alpha), a metabolite of prostacyclin, than transgenic controls. These findings suggest that the ET(B) receptor plays a protective role in the pulmonary hypertensive response to chronic hypoxia.     

Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary hypertension

Idiopathic pulmonary arterial hypertension (PAH) is characterized by proliferation of pulmonary vascular endothelial and smooth muscle cells causing increased vascular resistance and right heart failure. Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) are believed to cause the familial form of the disease. Reduced expression of BMPR2 is also noted in secondary PAH. Recent advances in the therapy of PAH have improved quality of life and survival, but many patients continue to do poorly. The possibility of treating PAH via improving BMPR2 signaling is thus a rational consideration. Such an approach could be synergistic with or additive to current treatments. We developed adenoviral vectors containing the BMPR2 gene. Transfection of cells in vitro resulted in upregulation of SMAD signaling and reduced cell proliferation. Targeted delivery of vector to the pulmonary vascular endothelium of rats substantially reduced the pulmonary hypertensive response to chronic hypoxia, as reflected by reductions in pulmonary artery and right ventricular pressures, right ventricular hypertrophy, and muscularization of distal pulmonary arterioles. These data provide further evidence for a role for BMPR2 in PAH and provide a rationale for the development of therapies aimed at improving BMPR2 signaling.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Altered pulmonary vasoreactivity in the chronically hypoxic lung

Prolonged exposure to alveolar hypoxia induces physiological changes in the pulmonary vasculature that result in the development of pulmonary hypertension. A hallmark of hypoxic pulmonary hypertension is an increase in vasomotor tone. In vivo, pulmonary arterial smooth muscle cell contraction is influenced by vasoconstrictor and vasodilator factors secreted from the endothelium, lung parenchyma and in the circulation. During chronic hypoxia, production of vasoconstrictors such as endothelin-1 and angiotensin II is enhanced locally in the lung, while synthesis of vasodilators may be reduced. Altered reactivity to these vasoactive agonists is another physiological consequence of chronic exposure to hypoxia. Enhanced contraction in response to endothelin-1 and angiotensin II, as well as depressed vasodilation in response to endothelium-derived vasodilators, has been documented in models of hypoxic pulmonary hypertension. Chronic hypoxia may also have direct effects on pulmonary vascular smooth muscle cells, modulating receptor population, ion channel activity or signal transduction pathways. Following prolonged hypoxic exposure, pulmonary vascular smooth muscle exhibits alterations in K+ current, membrane depolarization, elevation in resting cytosolic calcium and changes in signal transduction pathways. These changes in the electrophysiological parameters of pulmonary vascular smooth muscle cells are likely associated with an increase in basal tone. Thus, hypoxia-induced modifications in pulmonary arterial myocyte function, changes in synthesis of vasoactive factors and altered vasoresponsiveness to these agents may shift the environment in the lung to one of contraction instead of relaxation, resulting in increased pulmonary vascular resistance and elevated pulmonary arterial pressure.     

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

Grape seed procyanidin extract attenuates hypoxic pulmonary hypertension by inhibiting oxidative stress and pulmonary arterial smooth muscle cells proliferation

Hypoxia-induced oxidative stress and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Grape seed procyanidin extract (GSPE) possesses antioxidant properties and has beneficial effects on the cardiovascular system. However, the effect of GSPE on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 weeks to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio and median width of pulmonary arteries. GSPE attenuated the elevation of RVSP, RV/LV+S, and reduced the pulmonary vascular structure remodeling. GSPE also increased the levels of SOD and reduced the levels of MDA in hypoxia-induced HPH model. In addition, GSPE suppressed Nox4 mRNA levels, ROS production and PASMCs proliferation. Meanwhile, increased expression of phospho-STAT3, cyclin D1, cyclin D3 and Ki67 in PASMCs caused by hypoxia was down-regulated by GSPE. These results suggested that GSPE might potentially prevent HPH via antioxidant and antiproliferative mechanisms.     

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.     

Specific N-terminal CGRP fragments mitigate chronic hypoxic pulmonary hypertension in rats

Chronic hypoxic pulmonary hypertension (HPH) is characterized by elevated pulmonary arterial pressure (P(PA)), right ventricular hypertrophy (RVH), pulmonary vascular remodeling, pulmonary edema and polycythemia. Currently, there is no safe and effective treatment for HPH. Calcitonin gene-related peptide (CGRP) is the most potent peptide vasodilator discovered thus far. We previously demonstrated that exogenous CGRP reversed HPH in rats. However, the CGRP1 receptor antagonist CGRP(8-37) and smaller inhibitory C-terminal CGRP fragments that can be formed by enzymatic cleavage in vivo may compromise the beneficial effects of endogenous or exogenous CGRP. We here examine the agonistic efficacy of N-terminal rat alpha-CGRP peptides containing the disulfide bridge (Cys(2)-Cys(7)) with amidated C-terminal in prevention of HPH. Chronic infusion of CGRP(1-8), CGRP(1-13), or CGRP(1-14) at 7 nmol/h/rat via the right jugular vein during 14 days of hypobaric hypoxia (10% inspired O(2)) significantly decreased the P(PA), RVH and pulmonary arterial medial thickness in comparison with controls, suggesting that these CGRP sequences can mitigate chronic HPH in rats. Systemic pressure was unchanged by infused peptides indicating no carry-over effect. In conclusion, N-terminal CGRP fragments (CGRP(1-8), CGRP(1-13) and CGRP(1-14)) may have a protective role in hypoxic pulmonary hypertension.     

Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca(2+) concentration ([Ca(2+)](i)). We hypothesized that CH decreases EC [Ca(2+)](i) associated with membrane depolarization and reduced Ca(2+) entry. To test this hypothesis, we assessed 1) basal endothelial Ca(2+) in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (E(m)), 3) store-operated Ca(2+) current (I(SOC)), and 4) store-operated Ca(2+) (SOC) entry in arteries from control and CH rats. We found that basal EC Ca(2+) was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. I(SOC) activation by 1 muM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn(2+) quenching revealed a parallel reduction in Ca(2+) entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca(2+)](i). In contrast, basal EC Ca(2+) influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that E(m) may not play a major role in determining basal EC [Ca(2+)](i) following CH.     

Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats

Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10-100 nM) decreased the resting [Ca2+]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]i response induced by caffeine. Sildenafil (0.1 nM-1 microM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+ signaling pathway involved in this model of pulmonary hypertension.     

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.     

Temporal increase in the reactivity of pulmonary vasculature to substance P in chronically hypoxic rats

We previously demonstrated that the pulmonary vascular response to substance P (SP) increased in chronically hypoxic rats. This study explored the temporal increase in reactivity of the pulmonary vascular response to SP and its underlying mechanisms. First, young female Wistar rats were exposed to sea level (SL) or simulated high altitude (HA) for 15 h/day for 3 days, 1 wk, 2 wk, and 4 wk. Lungs were isolated and perfused with 4% bovine serum albumin in Krebs-Henseleit buffer solution. SP (1.5 x 10(-4) M) induced significant increases in pulmonary arterial pressure (P(pa)), venous pressure (P(v)), capillary pressure (P(c)), arterial resistance (R(a)), and filtration coefficient (K(fc)) in SL lungs. Increases in P(pa) and R(a) were significantly augmented in HA lungs, with a temporal increase trend peaking at 2 wk of HA exposure. The selective neurokinin (NK) type 1 (NK1) receptor antagonist SR-14033 significantly attenuated SP-induced increases in P(pa), P(v), P(c), R(a), and K(fc) in SL lungs. In lungs exposed to HA for 2 wk, SR-14033 suppressed the effect of SP on P(pa). Also, chronic hypoxia induced significant increases in NK1 receptors and NK1 receptor mRNA, with a temporal trend. We conclude that chronic hypoxia temporally augments SP-induced vascular responses, which are closely associated with increases in NK1 receptors and gene expression.     

Attenuation of chronic hypoxic pulmonary hypertension by simvastatin

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to improve multiple normal endothelial cell functions and inhibit vascular wall cell proliferation. We hypothesized that one such agent, simvastatin, would attenuate chronic hypoxic pulmonary hypertension. Male adult Sprague-Dawley rats were exposed (14 days) to normoxia (N), normoxia plus once-a-day administered simvastatin (20 mg/kg ip) (NS), hypoxia (10% inspired O2 fraction) (H), or hypoxia plus simvastatin (HS). Mean pulmonary artery pressure, measured in anesthetized, ventilated rats with an open-chest method, was reduced from 25 +/- 2 mmHg in H to 18 +/- 1 in HS (P < 0.001) but did not reach normoxic values (12 +/- 1 mmHg). Similarly, right ventricular/left ventricular plus interventricular septal weight was reduced from 0.53 +/- 0.02 in the H group to 0.36 +/- 0.02 in the HS group (P < 0.001). The increased hematocrit in H (0.65 +/- 0.02) was prevented by simvastatin treatment (0.51 +/- 0.01, P < 0.001). Hematocrit was similar in N versus NS. Alveolar vessel muscularization and medial thickening of vessels 50-200 microM in diameter induced by hypoxia were also significantly attenuated in the HS animals. Lung endothelial nitric oxide synthase (eNOS) protein expression in the HS group was less than H (P < 0.01) but was similar in N versus NS. We conclude that simvastatin treatment potently attenuates chronic hypoxic pulmonary hypertension and polycythemia in rats and inhibits vascular remodeling. Enhancement of lung eNOS expression does not appear to be involved in mediating this effect.