Chronic hypercapnia inhibits hypoxic pulmonary vascular remodeling

Chronic hypercapnia is commonly found in patients with severe hypoxic lung disease and is associated with a greater elevation of pulmonary arterial pressure than that due to hypoxia alone. We hypothesized that hypercapnia worsens hypoxic pulmonary hypertension by augmenting pulmonary vascular remodeling and hypoxic pulmonary vasoconstriction (HPV). Rats were exposed to chronic hypoxia [inspiratory O(2) fraction (FI(O(2))) = 0.10], chronic hypercapnia (inspiratory CO(2) fraction = 0.10), hypoxia-hypercapnia (FI(O(2)) = 0.10, inspiratory CO(2) fraction = 0.10), or room air. After 1 and 3 wk of exposure, muscularization of resistance blood vessels and hypoxia-induced hematocrit elevation were significantly inhibited in hypoxia-hypercapnia compared with hypoxia alone (P < 0.001, ANOVA). Right ventricular hypertrophy was reduced in hypoxia-hypercapnia compared with hypoxia at 3 wk (P < 0.001, ANOVA). In isolated, ventilated, blood-perfused lungs, basal pulmonary arterial pressure after 1 wk of exposure to hypoxia (20.1 +/- 1.8 mmHg) was significantly (P < 0.01, ANOVA) elevated compared with control conditions (12.1 +/- 0.1 mmHg) but was not altered in hypoxia-hypercapnia (13.5 +/- 0.9 mmHg) or hypercapnia (11.8 +/- 1.3 mmHg). HPV (FI(O(2)) = 0.03) was attenuated in hypoxia, hypoxia-hypercapnia, and hypercapnia compared with control (P < 0.05, ANOVA). Addition of N(omega)-nitro-L-arginine methyl ester (10(-4) M), which augmented HPV in control, hypoxia, and hypercapnia, significantly reduced HPV in hypoxia-hypercapnia. Chronic hypoxia caused impaired endothelium-dependent relaxation in isolated pulmonary arteries, but coexistent hypercapnia partially protected against this effect. These findings suggest that coexistent hypercapnia inhibits hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy, reduces HPV, and protects against hypoxia-induced impairment of endothelial function.     

Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.     

Chronic hypoxia attenuates nitric oxide-dependent hemodynamic responses to acute hypoxia

Alterations in the nitric oxide (NO) pathway have been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension. Chronic hypoxia can either suppress the NO pathway, causing pulmonary hypertension, or increase NO release in order to counteract elevated pulmonary arterial pressure. We determined the effect of NO synthase inhibitor on hemodynamic responses to acute hypoxia (10% O(2)) in anesthetized rats following chronic exposure to hypobaric hypoxia (0.5 atm, air). In rats raised under normoxic conditions, acute hypoxia caused profound systemic hypotension and slight pulmonary hypertension without altering cardiac output. The total systemic vascular resistance (SVR) decreased by 41 +/- 5%, whereas the pulmonary vascular resistance (PVR) increased by 25 +/- 6% during acute hypoxia. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg) attenuated systemic vasodilatation and enhanced pulmonary vasoconstriction. In rats with prior exposure to chronic hypobaric hypoxia, the baseline values of mean pulmonary and systemic arterial pressure were significantly higher than those in the normoxic group. Chronic hypoxia caused right ventricular hypertrophy, as evidenced by a greater weight ratio of the right ventricle to the left ventricle and the interventricular septum compared to the normoxic group (46 +/- 4 vs. 28 +/- 3%). In rats which were previously exposed to chronic hypoxia (half room air for 15 days), acute hypoxia reduced SVR by 14 +/- 6% and increased PVR by 17 +/- 4%. Pretreatment with L-NAME further inhibited the systemic vasodilatation effect of acute hypoxia, but did not enhance pulmonary vasoconstriction. Our results suggest that the release of NO counteracts pulmonary vasoconstriction but lowers systemic vasodilatation on exposure to acute hypoxia, and these responses are attenuated following adaptation to chronic hypoxia.     

Role of calcitonin gene-related peptide (CGRP) in chronic hypoxia-induced pulmonary hypertension in the mouse. Influence of gene transfer in vivo

Calcitonin gene-related peptide (CGRP) is believed to play an important role in maintaining low pulmonary vascular resistance (PVR) and may be involved in modulating the pulmonary vascular response to chronic hypoxia. In the present study, an adenoviral vector encoding CGRP (AdRSVCGRP) was used to examine the effects of in vivo gene transfer of CGRP to the lung on increases in PVR, right ventricular mass, and pulmonary vascular remodeling that occurs in chronic hypoxia in the mouse. Following intratracheal administration of AdRSVCGRP or reporter gene mice were exposed to 16 days of chronic hypoxia (FIO(2) 0.10). The increase in pulmonary arterial pressure (PAP), PVR, right ventricular mass, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing CGRP, whereas systemic arterial pressure was not altered. Following exposure to hypoxia, a subgroup of mice were treated with capsaicin, which did not significantly alter CGRP expression in the mouse lung. These data show that in vivo transfer of the CGRP gene to the lung attenuates the increase in PVR, right ventricular mass, and pulmonary vascular remodeling in chronically hypoxic mice with little effect on the systemic circulation. Moreover, these data suggest that adenoviral gene transfer of CGRP to the lung results in expression of the gene product in non-neural tissue.     

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.     

Atrial natriuretic peptide-dependent modulation of hypoxia-induced pulmonary vascular remodeling

Hypoxic stress upsets the balance in the normal relationships between mitogenic and growth inhibiting pathways in lung, resulting in pulmonary vascular remodeling characterized by hyperplasia of pulmonary arterial smooth muscle cells (PASMCs) and fibroblasts and enhanced deposition of extracellular matrix. Atrial natriuretic peptide (ANP) reduces pulmonary vascular resistance and attenuates hypoxia-induced pulmonary hypertension in vivo and PASMC proliferation and collagen synthesis in vitro. The current study utilized an ANP null mouse model (Nppa-/-) to test the hypothesis that ANP modulates the pulmonary vascular and alveolar remodeling response to normobaric hypoxic stress. Nine-10 wk old male ANP null (Nppa-/-) and wild type nontransgenic (NTG) mice were exposed to chronic hypoxia (10% O(2), 1 atm) or air for 6 wks. Measurement: pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial and alveolar remodeling were assessed. Hypoxia-induced pulmonary arterial hypertrophy and muscularization were significantly increased in Nppa-/- mice compared to NTG controls. Furthermore, the stimulatory effects of hypoxia on alveolar myofibroblast transformation (8.2 and 5.4 fold increases in Nppa-/- and NTG mice, respectively) and expression of extracellular matrix molecule (including osteopontin [OPN] and periostin [PN]) mRNA in whole lung were exaggerated in Nppa-/- mice compared to NTG controls. Combined with our previous finding that ANP signaling attenuates transforming growth factor (TGF)-beta-induced expression of OPN and PN in isolated PASMCs, the current study supports the hypothesis that endogenous ANP plays an important anti-fibrogenic role in the pulmonary vascular adaptation to chronic hypoxia.     

Adiponectin ameliorates hypoxia-induced pulmonary arterial remodeling

We have demonstrated that adiponectin has anti-atherosclerotic properties. We also reported hypoadiponectinemia and nocturnal reduction in circulating adiponectin concentrations in patients of severe obstructive sleep apnea-hypopnea syndrome (OSAS). OSAS is often complicated with pulmonary hypertension. In this study, we investigated the effect of adiponectin on chronic hypoxia-induced pulmonary arterial remodeling in mice. Exposure of mice to 3-weeks sustained hypoxia (10% O₂) resulted in significant accumulation of adiponectin in pulmonary arteries. The percentage media wall thickness (%MT), representing pulmonary arterial remodeling, under hypoxic condition, was greater in adiponectin-knockout mice than wild-type mice. Overexpression of adiponectin significantly decreased hypoxia-induced pulmonary arterial wall thickening and right ventricular hypertrophy. These findings demonstrate for the first time that overexpression of adiponectin suppresses the development of hypoxic-induced pulmonary remodeling, and that adiponectin may combat a new strategy for pulmonary vascular changes that underlie pulmonary hypertension in OSAS.     

Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats

Xanthine oxidase (XO)-derived reactive oxygen species (ROS) formation contributes to experimental chronic hypoxic pulmonary hypertension in adults, but its role in neonatal pulmonary hypertension has received little attention. In rats chronically exposed to hypoxia (13% O(2)) for 14 days from birth, we examined the effects of ROS scavengers (U74389G 10 mg.kg(-1).day(-1) or Tempol 100 mg.kg(-1).day(-1) ip) or a XO inhibitor, Allopurinol (50 mg.kg(-1).day(-1) ip). Both ROS scavengers limited oxidative stress in the lung and attenuated hypoxia-induced vascular remodeling, confirming a critical role for ROS in this model. However, both interventions also significantly inhibited somatic growth and normal cellular proliferation in distal air spaces. Hypoxia-exposed pups had evidence of increased serum and lung XO activity, increased vascular XO-derived superoxide production, and vascular nitrotyrosine formation. These changes were all prevented by treatment with Allopurinol, which also attenuated hypoxia-induced vascular remodeling and partially reversed inhibited endothelium-dependent arterial relaxation, without affecting normal growth and proliferation. Collectively, our findings suggest that XO-derived superoxide induces endothelial dysfunction, thus impairing pulmonary arterial relaxation, and contributes to vascular remodeling in hypoxia-exposed neonatal rats. Due to the potential for adverse effects on normal growth, targeting XO may represent a superior "antioxidant" strategy to ROS scavengers for neonates with pulmonary hypertension.     

Dominant negative mutation of the TGF-beta receptor blocks hypoxia-induced pulmonary vascular remodeling.

The present study utilized a novel transgenic mouse model that expresses an inducible dominant negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII mouse) to test the hypothesis that TGF-beta signaling plays an important role in the pathogenesis of chronic hypoxia-induced increases in pulmonary arterial pressure and vascular and alveolar remodeling. Nine- to 10-wk-old male DnTGFbetaRII and control nontransgenic (NTG) mice were exposed to normobaric hypoxia (10% O2) or air for 6 wk. Expression of DnTGFbetaRII was induced by drinking 25 mM ZnSO4 water beginning 1 wk before hypoxic exposure. Hypoxia-induced increases in right ventricular pressure, right ventricular mass, pulmonary arterial remodeling, and muscularization were greatly attenuated in DnTGFbetaRII mice compared with NTG controls. Furthermore, the stimulatory effects of hypoxic exposure on pulmonary arterial and alveolar collagen content, appearance of alpha-smooth muscle actin-positive cells in alveolar parenchyma, and expression of extracellular matrix molecule (including collagen I and III, periostin, and osteopontin) mRNA in whole lung were abrogated in DnTGFbetaRII mice compared with NTG controls. Hypoxic exposure had no effect on systemic arterial pressure or heart rate in either strain. These data support the hypothesis that endogenous TGF-beta plays an important role in pulmonary vascular adaptation to chronic hypoxia and that disruption of TGF-beta signaling attenuates hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, pulmonary arterial hypertrophy and muscularization, alveolar remodeling, and expression of extracellular matrix mRNA in whole lung.     

Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation, arrested lung growth, and pulmonary hypertension (PHT), as observed in human infants with severe bronchopulmonary dysplasia. Inhalation of CO(2) (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. Spontaneously breathing rat pups were treated with bleomycin (1 mg/kg/d ip) or saline vehicle from postnatal days 1-14 while being continuously exposed to 5% CO(2) (Pa(CO(2)) elevated by 15-20 mmHg), 7% CO(2) (Pa(CO(2)) elevated by 35 mmHg), or normocapnia. Bleomycin-treated animals exposed to 7%, but not 5%, CO(2), had significantly attenuated lung tissue macrophage influx and PHT, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. The level of CO(2) neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased numbers of peripheral arteries. Bleomycin led to increased expression and content of lung tumor necrosis factor (TNF)-α, which was found to colocalize with tissue macrophages and to be attenuated by exposure to 7% CO(2). Inhibition of TNF-α signaling with the soluble TNF-2 receptor etanercept (0.4 mg/kg ip from days 1-14 on alternate days) prevented bleomycin-induced PHT without decreasing tissue macrophages and, similar to CO(2), had no effect on arrested alveolar development. Our findings are consistent with a preventive effect of therapeutic hypercapnia with 7% CO(2) on bleomycin-induced PHT via attenuation of macrophage-derived TNF-α. Neither tissue macrophages nor TNF-α appeared to contribute to arrested lung development induced by bleomycin. That 7% CO(2) normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of PHT in this model.     

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.     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

Pulmonary vascular remodeling in isolated mouse lungs: effects on pulsatile pressure-flow relationships

Chronic hypoxia causes pulmonary vasoconstriction and pulmonary hypertension, which lead to pulmonary vascular remodeling and right ventricular hypertrophy. To determine the effects of hypoxia-induced pulmonary vascular remodeling on pulmonary vascular impedance, which is the right ventricular afterload, we exposed C57BL6 mice to 0 (control), 10 and 15 days of hypobaric hypoxia (n=6, each) and measured pulmonary vascular resistance (PVR) and impedance ex vivo. Chronic hypoxia led to increased pulmonary artery pressures for flow rates between 1 and 5ml/min (P<0.01), and increased PVR, 0-Hz pulmonary vascular impedance and the index of wave reflection (P<0.05) as well as a more negative impedance phase angle for low frequencies (P<0.05). The increases in resistance and 0-Hz impedance correlated with increased muscularization of small arterioles measured with quantitative immunohistochemistry (P<0.01). The increases in wave reflection and decreases in phase angle are likely due to increased proximal artery stiffness. These results confirm that chronic hypoxia causes significant changes in steady and pulsatile pressure-flow relationships in mouse lungs and does so via structural remodeling. They also provide important baseline data for experiments with genetically engineered mice, with which molecular mechanisms of pulmonary vascular remodeling can be investigated.     

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.     

Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice

Background

Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.

Methodology/Principal Findings

Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3−/− hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3−/− mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3−/− mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3−/− mice.

Conclusions

Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.     

Metalloproteinase inhibition by Batimastat attenuates pulmonary hypertension in chronically hypoxic rats

Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 +/- 3 mmHg in hypoxic controls, 24 +/- 1 mmHg in Batimastat-treated hypoxic rats, and 16 +/- 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.