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.     

Involvement of RhoA/Rho kinase signaling in pulmonary hypertension of the fawn-hooded rat.

The fawn-hooded rat (FHR) develops severe pulmonary hypertension (PH) when raised for the first 3-4 wk of life in the mild hypoxia of Denver's altitude (5,280 ft.). The PH is associated with sustained pulmonary vasoconstriction and pulmonary artery remodeling. Furthermore, lung alveolarization and vascularization are reduced in the Denver FHR. We have recently shown that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in animal models of hypoxic PH. In this study, we investigated the role of RhoA/Rho kinase signaling in the PH of Denver FHR. In alpha-toxin permeabilized pulmonary arteries from Denver FHR, the contractile sensitivity to Ca2+ was increased compared with those from sea-level FHR. RhoA activity and Rho kinase I protein expression in pulmonary arteries of Denver FHR (10-wk-old) were higher than in those of sea-level FHR. Acute inhalation of the Rho kinase inhibitor fasudil selectively reduced the elevated pulmonary arterial pressure in Denver FHR in vivo. Chronic fasudil treatment (30 mg.kg-1.day-1, from birth to 10 wk old) markedly reduced the development of PH and improved lung alveolarization and vascularization in Denver FHR. These results suggest that Rho kinase-mediated sustained vasoconstriction, through increased Ca2+ sensitivity, plays an important role in the established PH and that RhoA/Rho kinase signaling contributes significantly to the development of PH and lung dysplasia in mild hypoxia-exposed FHR.     

Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats

Recent evidence suggests that Rho/Rho kinase signaling plays an important role in the sustained vasoconstriction induced by many agonists and is involved in the pathogenesis of systemic vascular diseases. However, little is known about its role in increased vascular tone in hypoxic pulmonary hypertension (PH). The purpose of this study was to examine whether Rho/Rho kinase-mediated Ca2+ sensitization contributed to sustained vasoconstriction and increased vasoreactivity in hypoxic PH in rats. Acute intravenous administration of Y-27632, a Rho kinase inhibitor, nearly normalized the high pulmonary arterial blood pressure and total pulmonary resistance in chronically hypoxic rats. In contrast to nifedipine, Y-27632 also markedly decreased elevated basal vascular tone in hypertensive blood-perfused lungs and isolated pulmonary arteries. Y-27632 and another Rho kinase inhibitor, HA-1077, completely reversed nitro-L-arginine-induced vasoconstriction in physiological salt solution-perfused hypertensive lungs, whereas inhibitors of myosin light chain kinase (ML-9), protein kinase C (GF-109203X), phosphatidylinositol 3-kinase (LY-294002), and tyrosine kinase (tyrphostin A23) caused only partial or no reversal of the vasoconstriction. Vasoconstrictor responses to KCl were augmented in hypertensive physiological salt solution-perfused lungs and pulmonary arteries, and the augmentation was eliminated by Y-27632. These results suggest that Rho/Rho kinase-mediated Ca2+ sensitization plays a central role in mediating sustained vasoconstriction and increased vasoreactivity in hypoxic PH.     

Hypoxic pulmonary vasoconstriction: role of ion channels.

Acute hypoxia induces pulmonary vasoconstriction and chronic hypoxia causes structural changes of the pulmonary vasculature including arterial medial hypertrophy. Electro- and pharmacomechanical mechanisms are involved in regulating pulmonary vasomotor tone, whereas intracellular Ca(2+) serves as an important signal in regulating contraction and proliferation of pulmonary artery smooth muscle cells. Herein, we provide a basic overview of the cellular mechanisms involved in the development of hypoxic pulmonary vasoconstriction. Our discussion focuses on the roles of ion channels permeable to K(+) and Ca(2+), membrane potential, and cytoplasmic Ca(2+) in the development of acute hypoxic pulmonary vasoconstriction and chronic hypoxia-mediated pulmonary vascular remodeling.     

Animal models of pulmonary hypertension: Rho kinase inhibition

Pulmonary Hypertension is a terminology encompassing a range of etiologically different pulmonary vascular diseases. The most common is that termed pulmonary arterial hypertension or PAH; a rare but often fatal disease characterized by a mean pulmonary arterial pressure of >25?mmHg. PAH is associated with a complex etiology highlighted by core characteristics of increased pulmonary vascular resistance and elevation of mean pulmonary artery pressure. When sustained, pulmonary vascular remodeling occurs and eventually patients pass away due to right heart failure. Hypoxic pulmonary vasoconstriction is an early event occurring in pulmonary hypertension due to chronic exposure to hypoxia. While the underlying mechanisms of hypoxic pulmonary vasoconstriction may be controversial, a role for RhoA/Rho kinase mediated regulation of intracellular Ca(2+) has been recently identified. Further study suggests that RhoA may have an integral role in other pathophysiological processes such as cell proliferation and migration occurring in all forms of PH. Indeed Rho proteins are known to play essential roles in actin cytoskeleton organization in all eukaryotic cells and thus Rho and Rho-GTPases are implicated in fundamental cellular processes such as cellular proliferation, migration, adhesion, apoptosis and gene expression. This review focuses on providing an overview of the role of RhoA/Rho kinase in currently available animal models of pulmonary hypertension.     

Platelet-derived growth factor (PDGF) induces pulmonary vascular remodeling through 15-LO/15-HETE pathway under hypoxic condition

15-lipoxygenase (15-LO) is known to play an important role in chronic pulmonary hypertension. Accumulating evidence for its down-stream participants in the vasoconstriction and remodeling processes of pulmonary arteries, while how hypoxia regulates 15-LO/15-hydroxyeicosatetraenoic acid (15-HETE) to mediate hypoxic pulmonary hypertension is still unknown. Platelet-derived growth factor (PDGF) is an important vascular regulator whose concentration increases under hypoxic condition in the lungs of both humans and mice with pulmonary hypertension. The present study was carried out to determine whether hypoxia advances the pulmonary vascular remodeling through the PDGF/15-LO/15-HETE pathway. We found that pulmonary arterial medial thickening caused by hypoxia was alleviated after a treatment of the hypoxic rats with imatinib, which was associated with down-regulations of 15-LO-2 expression and 15-HETE production. Moreover, the increases in cell proliferation and endogenous 15-HETE content by hypoxia were attenuated by the inhibitors of PDGF-β receptor in pulmonary artery smooth muscle cells (PASMCs). The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of 15-LO inhibitors or 15-LO RNA interference. These results suggest that hypoxia promotes PASMCs proliferation and survival, contributing to pulmonary vascular medial hypertrophy, which is likely to be mediated via the PDGF-BB/15-LO-2/15-HETE pathway.     

Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity.

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).     

Acute and chronic hypoxic pulmonary hypertension in guinea pigs

To determine whether the strength of acute hypoxic vasoconstriction predicts the magnitude of chronic hypoxic pulmonary hypertension, we performed serial studies on guinea pigs. Unanesthetized, chronically catheterized guinea pigs increased mean pulmonary arterial pressure (PAP) from 11 +/- 0.5 to 13 +/- 0.7 Torr in acute hypoxia (10% O2 for 65 min). The response was maximal at 5 min, remained stable for 1 h, and was reversible on return to room air. Cardiac index did not change with acute hypoxia or recovery. Guinea pigs exposed to chronic hypoxia increased PAP, measured in room air 1 h after removal from the hypoxic chamber, to 18 +/- 1 Torr by 5 days with little further increase in PAP to 20 +/- 1 Torr after 21 days. Cardiac index fell from 273 +/- 12 to 206 +/- 7 ml.kg-1.min-1 (P less than 0.05) after 21 days of hypoxia. Medial thickness of pulmonary arteries adjacent to terminal bronchioles and alveolar ducts increased significantly by 10 days. The magnitude of the pulmonary vasoconstriction to acute hypoxia persisted and was unabated during the development and apparent stabilization of chronic hypoxic pulmonary hypertension, suggesting that if vasoconstriction is the stimulus for remodeling, then the importance of the stimulus lessens with duration of hypoxia. In individual animals followed serially, we found no correlation between the magnitude of the acute vasoconstrictor response before chronic hypoxia and the severity of chronic pulmonary hypertension that subsequently developed either because the initial response was small and variable or because vasoconstriction may not be the sole stimulus for vascular remodeling in the guinea pig.     

Enhanced alpha1-adrenergic trophic activity in pulmonary artery of hypoxic pulmonary hypertensive rats

Mechanisms that induce the excessive proliferation of vascular wall cells in hypoxic pulmonary hypertension (PH) are not fully understood. Alveolar hypoxia causes sympathoexcitation, and norepinephrine can stimulate alpha(1)-adrenoceptor (alpha(1)-AR)-dependent hypertrophy/hyperplasia of smooth muscle cells and adventitial fibroblasts. Adrenergic trophic activity is augmented in systemic arteries by injury and altered shear stress, which are key pathogenic stimuli in hypoxic PH, and contributes to neointimal formation and flow-mediated hypertrophic remodeling. Here we examined whether norepinephrine stimulates growth of the pulmonary artery (PA) and whether this is augmented in PH. PA from normoxic and hypoxic rats [9 days of 0.1 fraction of inspired O(2) (Fi(O(2)))] was studied in organ culture, where wall tension, Po(2), and Pco(2) were maintained at values present in normal and hypoxic PH rats. Norepinephrine treatment for 72 h increased DNA and protein content modestly in normoxic PA (+10%, P < 0.05). In hypoxic PA, these effects were augmented threefold (P < 0.05), and protein synthesis was increased 34-fold (P < 0.05). Inferior thoracic vena cava from normoxic or hypoxic rats was unaffected. Norepinephrine-induced growth in hypoxic PA was dose dependent, had efficacy greater than or equal to endothelin-1, required the presence of wall tension, and was inhibited by alpha(1A)-AR antagonist. In hypoxic pulmonary vasculature, alpha(1A)-AR was downregulated the least among alpha(1)-AR subtypes. These data demonstrate that norepinephrine has trophic activity in the PA that is augmented by PH. If evident in vivo in the pulmonary vasculature, adrenergic-induced growth may contribute to the vascular hyperplasia that participates in hypoxic PH.     

p53 Gene deficiency promotes hypoxia-induced pulmonary hypertension and vascular remodeling in mice

Chronic hypoxia induces pulmonary arterial remodeling, resulting in pulmonary hypertension and right ventricular hypertrophy. Hypoxia has been implicated as a physiological stimulus for p53 induction and hypoxia-inducible factor-1α (HIF-1α). However, the subcellular interactions between hypoxic exposure and expression of p53 and HIF-1α remain unclear. To examine the role of p53 and HIF-1α expression on hypoxia-induced pulmonary arterial remodeling, wild-type (WT) and p53 knockout (p53KO) mice were exposed to either normoxia or hypoxia for 8 wk. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as measured by the ratio of the right ventricle to the left ventricle plus septum weights, and vascular remodeling. However, the right ventricular systolic pressures, the ratio of the right ventricle to the left ventricle plus septum weights, and the medial wall thickness of small vessels were significantly greater in the p53KO mice than in the WT mice. The p53KO mice had lower levels of p21 and miR34a expression, and higher levels of HIF-1α, VEGF, and PDGF expression than WT mice following chronic hypoxic exposure. This was associated with a higher proliferating cell nuclear antigen expression of pulmonary artery in p53KO mice. We conclude that p53 plays a critical role in the mitigation of hypoxia-induced small pulmonary arterial remodeling. By interacting with p21 and HIF-1α, p53 may suppress hypoxic pulmonary arterial remodeling and pulmonary arterial smooth muscle cell proliferation under hypoxia.     

Linked opening angle and histological and mechanical aspects of the proximal pulmonary arteries of healthy and pulmonary hypertensive rats and calves

Understanding how arterial remodeling changes the mechanical behavior of pulmonary arteries (PAs) is important to the evaluation of pulmonary vascular function. Early and current efforts have focused on the arteries' histological changes, their mechanical properties under in vitro mechanical testing, and their zero-stress and no-load states. However, the linkage between the histology and mechanical behavior is still not well understood. To explore this linkage, we investigated the geometry, residual stretch, and histology of proximal PAs in both adult rat and neonatal calf hypoxic models of pulmonary hypertension (PH), compared their changes due to chronic hypoxia across species, and proposed a two-layer mechanical model of artery to relate the opening angle to the stiffness ratio of the PA outer to inner layer. We found that the proximal PA remodeling in calves was quite different from that in rats. In rats, the arterial wall thickness, inner diameter, and outer layer thickness fraction all increased dramatically in PH and the opening angle decreased significantly, whereas in calves, only the arterial wall thickness increased in PH. The proposed model predicted that the stiffness ratio of the calf proximal PAs changed very little from control to hypertensive group, while the decrease of opening angle in rat proximal PAs in response to chronic hypoxia was approximately linear to the increase of the stiffness ratio. We conclude that the arterial remodeling in rat and calf proximal PAs is different and the change of opening angle can be linked to the change of the arterial histological structure and mechanics.     

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.     

Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism

Cell-free hemoglobin (Hb) exposure may be a pathogenic mediator in the development of pulmonary arterial hypertension (PAH), and when combined with chronic hypoxia the potential for exacerbation of PAH and vascular remodeling is likely more pronounced. We hypothesized that Hb may contribute to hypoxia-driven PAH collectively as a prooxidant, inflammatory, and nitric oxide (NO) scavenger. Using programmable micropump technology, we exposed male Sprague-Dawley rats housed under room air or hypoxia to 12 or 30 mg per day Hb for 3, 5, and 7 wk. Blood pressure, cardiac output, right ventricular hypertrophy, and indexes of pulmonary vascular remodeling were evaluated. Additionally, markers of oxidative stress, NO bioavailability and inflammation were determined. Hb increased pulmonary arterial (PA) pressure, pulmonary vessel wall stiffening, and right heart hypertrophy with temporal and dose dependence in both room air and hypoxic cohorts. Hb induced a modest increase in plasma oxidative stress markers (malondialdehyde and 4-hydroxynonenal), no change in NO bioavailability, and increased lung ICAM protein expression. Treatment with the antioxidant Tempol attenuated Hb-induced pulmonary arterial wall thickening, but not PA pressures or ICAM expression. Chronic exposure to low plasma Hb concentrations (range = 3-10 μM) lasting up to 7 wk in rodents induces pulmonary vascular disease via inflammation and to a lesser extent by Hb-mediated oxidation. Tempol demonstrated a modest effect on the attenuation of Hb-induced pulmonary vascular disease. NO bioavailability was found to be of minimal importance in this model.     

Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.

The pulmonary vasoconstrictor, thromboxane, may contribute to the development of pulmonary hypertension. Our objective was to determine whether a combined thromboxane synthase inhibitor-receptor antagonist, terbogrel, prevents pulmonary hypertension and the development of aberrant pulmonary arterial responses in newborn piglets exposed to 3 days of hypoxia. Piglets were maintained in room air (control) or 11% O(2) (hypoxic) for 3 days. Some hypoxic piglets received terbogrel (10 mg/kg po bid). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. A cannulated artery technique was used to measure responses to acetylcholine. Pulmonary vascular resistance for terbogrel-treated hypoxic piglets was almost one-half the value of untreated hypoxic piglets but remained greater than values for control piglets. Dilation to acetylcholine in preconstricted pulmonary arteries was greater for terbogrel-treated hypoxic than for untreated hypoxic piglets, but it was less for pulmonary arteries from both groups of hypoxic piglets than for control piglets. Terbogrel may ameliorate pulmonary artery dysfunction and attenuate the development of chronic hypoxia-induced pulmonary hypertension in newborns.     

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.     

Cofilin,a hypoxia‐regulated protein in murine lungs identified by 2DE: Role of the cytoskeletal protein cofilin in pulmonary hypertension

Chronic alveolar hypoxia induces vascular remodeling processes in the lung resulting in pulmonary hypertension (PH). However, the mechanisms underlying pulmonary remodeling processes are not fully resolved yet. To investigate functional changes occurring during hypoxia exposure we applied 2DE to compare protein expression in lungs from mice subjected to 3 h of alveolar hypoxia and those kept under normoxic conditions. Already after this short‐time period several proteins were significantly regulated. Subsequent analysis by MALDI‐MS identified cofilin as one of the most prominently upregulated proteins. The regulation was confirmed by western blotting and its cellular localization was determined by immunohisto‐ and immunocytochemistry. Interestingly, enhanced cofilin serine 3 phosphorylation was observed after short‐term and after chronic hypoxia‐induced PH in mice, in pulmonary arterial smooth muscle cells (PASMC) from monocrotaline‐induced PH in rats, in lungs of idiopathic pulmonary arterial hypertension patients and in hypoxic or platelet‐derived growth factor BB‐treated human PASMC. Furthermore, elevated cofilin phosphorylation was attenuated by curative treatment of monocrotaline‐induced PH in rats and hypoxia‐induced PH in mice with the PDGF‐BB receptor antagonist imatinib. In conclusion, short‐term hypoxic exposure induced prominent changes in lung protein regulation. These very early changes allowed us to identify potential triggers of PH. Thus, respective 2DE analysis can lead to the identification of new target proteins for the possible treatment of PH.     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH.     

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.     

Hypoxic pulmonary hypertension is prevented in rats with common bile duct ligation.

Biliary cirrhosis in the rat triggers intrapulmonary vasodilatation and gas-exchange abnormalities that characterize the hepatopulmonary syndrome. This vasodilatation correlates with increased levels of pulmonary microcirculatory endothelial NO synthase (eNOS) and hepatic and plasma endothelin-1 (ET-1). Importantly, during cirrhosis, the pulmonary vascular responses to acute hypoxia are blunted. The purpose of this work was to examine the pulmonary vascular responses and adaptations to the combination of liver cirrhosis and chronic hypoxia (CH). In addition to hemodynamic measurements, we investigated whether pulmonary expression changes of eNOS, ET-1 and its receptors (endothelin A and B), or heme oxygenase 1 in experimental cirrhosis affect the development of hypoxic pulmonary hypertension. We induced cirrhosis in male Sprague-Dawley rats using common bile duct ligation (CBDL) and exposed them to CH (inspired PO2 approximately 76 Torr) or maintained them in Denver (Den, inspired PO2 approximately 122 Torr) for 3 wk. Our data show 1) CBDL-CH rats had a persistent blunted hypoxic pulmonary vasoconstriction similar to CBDL-Den; 2) the development of hypoxic pulmonary hypertension was completely prevented in the CBDL-CH rats, as indicated by normal pulmonary arterial pressure and lack of right ventricular hypertrophy and pulmonary arteriole remodeling; and 3) selective increases in expression of ET-1, pulmonary endothelin B receptor, eNOS, and heme oxygenase 1 are potential mechanisms of protection against hypoxic pulmonary hypertension in the CBDL-CH rats. These data demonstrate that unique and undefined hepatic-pulmonary interactions occur during liver cirrhosis and chronic hypoxia. Understanding these interactions may provide important information for the prevention and treatment of pulmonary hypertension.