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.     

Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. The transforming growth factor (TGF)-beta superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-beta1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38(MAPK) or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-beta1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-beta1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-beta superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.     

Transforming growth factor-β regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure

We have previously shown that inhibition of transforming growth factor-β (TGF-β) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-β signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-β neutralizing antibody), or vehicle. Lung function and development and TGF-β and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-β synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-β signaling in hypoxia-exposed animals. Inhibition of TGF-β signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-β signaling indicates that TGF-β is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.     

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.     

Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.     

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.     

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.     

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.     

Macrophage migration inhibitory factor mediates hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mif ^+/+) and MIF-knockout (mif ^−/−) mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mif ^−/− cells when they were supplemented with media from mif ^+/+ cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.     

Hypoxia Induces Transforming Growth Factor-β1 Gene Expression in the Pulmonary Artery of Rats via Hypoxia-inducible Factor-1α

The present study was undertaken to investigate the dynamic expression of hypoxia induciblefactor-1 α (HIF-1α) and transforming growth factor-β1 (TGF-β1) in hypoxia-induced pulmonary hypertensionof rats.It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d ofhypoxia.Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d ofhypoxia.HIF-1α mRNA staining was less positive in the control,hypoxia for 3 d and hypoxia for 7 d,butbegan to enhance significantly after 14 d of hypoxia,then remained stable.Expression of HIF-1 α protein inthe control was less positive,but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats.TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, butshowed no obvious changes after 3 or 7 d of hypoxia.In pulmonary tunica adventitia and tunica media,TGF-β1 protein staining was less positive in control rats,but was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d of hypoxia,and then weakening after 14 and 21 d of hypoxia.Western blottingshowed that HIF- 1α protein levels increased significantly after 7 d of hypoxia and then remained at a highlevel. TGF-β1 protein level was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d ofhypoxia,and then decreasing after 14 and 21 d of hypoxia.Linear correlation analysis showed that HIF-1αmRNA, TGF-β1 mRNA, TGF-β1 protein were positively correlated with mPAP,vessel morphometry andright ventricular hypertrophy index.TGF-β1 protein (tunica adventitia) was negatively correlated withHIF-lα mRNA.Taken together,our results suggest that changes in HIF-lα and TGF-β1 expression afterhypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.     

Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.     

Protective effects of S-nitrosoalbumin on lung injury induced by hypoxia-reoxygenation in mouse model of sickle cell disease

Nitric oxide (NO) is a potential new therapeutic agent for sickle cell disease (SCD). We investigated the effects of NO donor on hypoxia-induced acute lung injury that occurs when transgenic sickle cell SAD mice are exposed to chronic hypoxia, a model for lung vasoocclusive sickle cell events. In wild-type and SAD mice, intraperitoneal injection of S-nitrosoalbumin (NO-Alb) produced no significant hematologic changes under room air conditions, whereas it induced mild temporary hypotension and inhibition of platelet aggregation. NO-Alb administration (300 mg/kg ip twice a day, equivalent to 7.5 microM NO) in wild-type and SAD mice exposed to 46 h of hypoxia (8% oxygen) followed by 2 h of normoxia resulted in 1) reduction of the hypoxia-induced increase in blood neutrophil count, 2) prevention of hypoxia-induced increased IL-6 and IL-1beta levels in bronchoalveolar lavage, 3) reduction of the lung injury induced by hypoxia-reoxygenation, 4) prevention of thrombus formation, and 5) prevention of hypoxia-induced increase of lung matrix metalloproteinase-9 gene expression. These effects provide new insights into the possible use of NO-Alb in the treatment of acute lung injury in SCD.     

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.