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.     

Hypoxia-responsive growth factors upregulate periostin and osteopontin expression via distinct signaling pathways in rat pulmonary arterial smooth muscle cells.

This study tested the hypothesis that expression of the novel adhesion molecule periostin (PN) and osteopontin (OPN) is increased in lung and in isolated pulmonary arterial smooth muscle cells (PASMCs) in response to the stress of hypoxia and explored the signaling pathways involved. Adult male rats were exposed to 10% O2 for 2 wk, and growth-arrested rat PASMCs were incubated under 1% O2 for 24 h. Hypoxia increased PN and OPN mRNA expression in rat lung. In PASMCs, hypoxia increased PN but not OPN expression. The hypoxia-responsive growth factors fibroblast growth factor-1 (FGF-1) and angiotensin II (ANG II) caused dose- and time-dependent increases in PN and OPN expression in PASMCs. FGF-1-induced PN expression was blocked by the FGF-1 receptor antagonist PD-166866 and by inhibitors of phosphatidylinositol 3-kinase (PI3K) (LY-294002, wortmannin), p70S6K (rapamycin), MEK1/2 (U-0126, PD-98059), and p38MAPK (SB-203580) but not of JNK (SP-600125). ANG II-induced PN expression was blocked by the AT(1)-receptor antagonist losartan and by inhibitors of PI3K and MEK1/2. In contrast, FGF-1-induced OPN expression was blocked by inhibitors of JNK or MEK1/2 but not of PI3K, p70S6K, or p38MAPK. Activation of p70S6K and p38MAPK by anisomycin robustly stimulated PN but not OPN expression. This study is the first to demonstrate that growth factor-induced expression of PN in PASMCs is mediated through PI3K/p70S6K, Ras/MEK1/2, and Ras/p38MAPK signaling pathways, whereas the expression of OPN is mediated through Ras/MEK1/2 and Ras/JNK signaling pathways. These differences in signaling suggest that PN and OPN may play different roles in pulmonary vascular remodeling under pathophysiological conditions.     

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.     

Genistein rescues hypoxia-induced pulmonary arterial hypertension through estrogen receptor and β-adrenoceptor signaling

Pulmonary vascular remodeling is an important pathological feature of pulmonary arterial hypertension (PAH), which is characterized by thickening of the medial smooth muscle layer. Hypertrophy of pulmonary artery smooth muscle cells (PASMCs) participates in the development of medial thickening. Genistein can attenuate PAH and inhibit medial thickening of pulmonary arteries. Since hypoxia is one of the main causes of pulmonary hypertension, this study aims to investigate the mechanism of genistein in inhibiting hypertrophic responses in PASMCs induced by hypoxia. Cells isolated from the chick embryo were cultured with or without genistein and subjected to hypoxia or not. The increase of cell surface area and α-smooth muscle actin (α-SMA) of PASMCs was significantly suppressed by genistein during hypoxia. This result was confirmed by the incorporation of puromycin into peptide chains and flow cytometry analysis. Constrained mRNA and protein hypoxia-inducible factor (HIF)-1α expression was improved by genistein under hypoxia condition. Genistein restored redox homeostasis by fluorescent probe determination. The effect of genistein on hypertrophic response was blocked by estrogen receptor inhibitor, β1-adrenoceptor agonist and β2-adrenoceptor antagonist. In conclusion, genistein potently attenuates hypoxia-induced hypertrophy of PASMCs, which may enable a novel therapy for PAH.     

Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension

Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.     

Atrial natriuretic peptide in hypoxia

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. Among them, atrial natriuretic peptide (ANP) synthesis and secretion is increased during hypoxic exposure and plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases, including chronic hypoxia-induced pulmonary hypertension and vascular remodeling, and right ventricular hypertrophy and right heart failure. This review discusses the roles of ANP and its receptors in hypoxia-induced pulmonary hypertension. We and other investigators have demonstrated that ANP gene expression is enhanced by exposure to hypoxia and that the ANP so generated protects against the development of hypoxic pulmonary hypertension. Results also show that hypoxia directly stimulates ANP gene expression and ANP release in cardiac myocytes in vitro. Several cis-responsive elements of the ANP promoter are involved in the response to changes in oxygen tension. Further, the ANP clearance receptor NPR-C, but not the biological active NPR-A and NPR-B receptors, is downregulated in hypoxia adapted lung. Hypoxia-sensitive tyrosine kinase receptor-associated growth factors, including fibroblast growth factor (FGF) and platelet derived growth factor (PDGF)-BB, but not hypoxia per se, inhibit NPR-C gene expression in pulmonary arterial smooth muscle cells in vitro. The reductions in NPR-C in the hypoxic lung retard the clearance of ANP and allow more ANP to bind to biological active NPR-A and NPR-B in the pulmonary circulation, relaxing preconstricted pulmonary vessels, reducing pulmonary arterial pressure, and attenuating the development of hypoxia-induced pulmonary hypertension and vascular remodeling.     

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.     

miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling

Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases.     

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.     

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.     

AQP1促进肺动脉平滑肌细胞的增殖与迁移（应作者要求撤稿）

该文应作者要求已撤稿。肺动脉平滑肌细胞（PASMCs）的迁移和增殖是肺动脉重塑进而造成肺动脉高压的主要病理基础。水通道蛋白1（AQP1）具有促进上皮细胞、内皮细胞迁移的作用,但机制不清。由于AQP1也表达于血管平滑肌细胞,推测AQP1可能参与缺氧诱导的PASMCs增殖及迁移。通过PCR和免疫印迹分析,检测AQP的表达以及缺氧对AQP表达水平的影响,并通过细胞迁移以及增殖实验观察AQP1在缺氧诱导的PASMCs迁移与增殖中的作用。AQP1在PASMCs和主动脉平滑肌细胞（AoSMCs）均表达,但缺氧只增加PASMCs中AQP1的表达,以及促进PASMCs的迁移与增殖。敲除AQP1可抑制PASMCs的增殖以及缺氧诱导的细胞增殖和迁移。过表达AQP1促进PASMCs的增殖和迁移。缺氧促进β联蛋白在PASMCs内的表达。敲除β联蛋白后,抑制AdAQP1所介导的PASMCs迁移与增殖。这些结果表明,缺氧可促进AQP1在肺动脉内的表达,AQP1可通过β联蛋白对PASMCs的增殖和迁移进行调节。     

Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies

Activation of AMP-activated protein kinase (AMPK) plays an important role in cardiovascular protection. It can inhibit arterial smooth muscle cell proliferation and cardiac fibroblast collagen synthesis induced by anoxia. However, the role of AMPK-dependent signalling cascades in the pulmonary vascular system is currently unknown. This study aims to determine the effects of AMPK on pulmonary hypertension and pulmonary vessel remodelling induced by hypoxia in rats using in vivo and in vitro studies. In vivo study: pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodelling were found in hypoxic rats. Meanwhile, AMPKα₁ and phosphorylated AMPKα₁ were increased markedly in pulmonary arterioles and lung tissues. Mean pulmonary arterial pressure, index of right ventricular hypertrophy and parameters of pulmonary vascular remodelling, including vessel wall area/total area, density of nuclei in medial smooth muscle cells, and thickness of the medial smooth muscle cell layer were markedly suppressed by AICAR, an AMPK agonist. In vitro study: the expression of AMPKα₁ and phosphorylated AMPKα₁ was increased in pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. The effects of PASMC proliferation stimulated by hypoxia were reinforced by treatment with Compound C, an AMPK inhibitor. AICAR inhibited the proliferation of PASMCs stimulated by hypoxia. These findings suggest that AMPK is involved in the formation of hypoxia-induced pulmonary hypertension and pulmonary vessel remodelling. Up-regulating AMPK can contribute to decreasing pulmonary vessel remodelling and pulmonary hypertension induced by hypoxia.     

Oxymatrine prevents hypoxia- and monocrotaline-induced pulmonary hypertension in rats

Pulmonary hypertension is a progressive disease characterized by marked pulmonary arterial remodeling and increased vascular resistance. Inflammation and oxidative stress promote the development of pulmonary hypertension. Oxymatrine, one of the main active components of the Chinese herb Sophora flavescens Ait. (Kushen), plays anti-inflammatory and antioxidant protective roles, which effects on pulmonary arteries remain unclear. This study aimed to investigate the effects of oxymatrine on pulmonary hypertension development. Sprague–Dawley rats were exposed to hypoxia for 28 days or injected with monocrotaline, to develop pulmonary hypertension, along with administration of oxymatrine (50 mg/kg/day). Hemodynamics and pulmonary arterial remodeling data from the rats were then obtained. The antiproliferative effect of oxymatrine was verified by in vitro assays. The inflammatory cytokine mRNA levels and leukocyte and T cell accumulation in lung tissue were detected. The antioxidative effects of oxymatrine were explored in vitro. Our study shows that oxymatrine treatment attenuated right-ventricular systolic pressure and pulmonary arterial remodeling induced by hypoxia or monocrotaline and inhibited proliferation of pulmonary arterial smooth muscle cells (PASMCs). Increased expression of inflammatory cytokine mRNA and accumulation of leukocytes and T cells around the pulmonary arteries were suppressed with oxymatrine administration. Under hypoxic conditions, oxymatrine significantly upregulated Nrf2 and antioxidant protein SOD1 and HO-1 expression, but downregulated hydroperoxide levels in PASMCs. In summary, this study indicates that oxymatrine may prevent pulmonary hypertension through its antiproliferative, anti-inflammatory, and antioxidant effects, thus providing a promising pharmacological avenue for treating pulmonary hypertension.     

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.     

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.     

Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.     

Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells

We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1-20 ng/ml for 1-24 h); or with ANG II (1-100 nM), endothelin-1 (ET-1, 0.1 microM), ANP (0.1 microM), sodium nitroprusside (SNP, 0.1 microM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.