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.     

Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.     

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.     

Transforming growth factor-beta signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.     

Quantitative models of the rat pulmonary arterial tree morphometry applied to hypoxia-induced arterial remodeling.

Little is known about the constituent hemodynamic consequences of structural changes that occur in the pulmonary arteries during the onset and progression of pulmonary arterial remodeling. Many disease processes are known to be responsible for vascular remodeling that leads to pulmonary arterial hypertension, cor pulmonale, and death. Histology has been the primary tool for evaluating pulmonary remodeling, but it does not provide information on intact vascular structure or the vessel mechanical properties. This study is an extension of our previous work in which we developed an alternative imaging technique to evaluate pulmonary arterial structure. The lungs from Sprague-Dawley rats were removed, perfusion analysis was performed on the isolated lungs, and then an X-ray contrast agent was used to fill the arterial network for imaging. The lungs were scanned over a range of intravascular pressures by volumetric micro-computed tomography, and the arterial morphometry was mapped and measured in the reconstructed isotropic volumes. A quantitative assessment of hemodynamic, structural, and biomechanical differences between rats exposed for 21 days to hypoxia (10% O(2)) or normoxia (21.0% O(2)) was performed. One metric, the normalized distensibility of the arteries, is significantly (P < 0.001) larger [0.025 +/- 0.0011 (SE) mmHg(-1)] (n = 9) in normoxic rats compared with hypoxic [0.015 +/- 0.00077 (SE) mmHg(-1)] (n = 9). The results of the study show that these models can be applied to the Sprague-Dawley rat data and, specifically, can be used to differentiate between the hypoxic and the control groups.     

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.     

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.     

姜黄素改善慢性肺动脉高压肺血管重塑作用途径的研究

目的：探究姜黄素改善慢性低O2高CO2大鼠肺动脉高压肺血管重塑作用途径的研究。方法：建立慢低O2高CO2肺血管重塑模型,以24只雄性大鼠为受试对象,随机分为四组（n=6）：I组（常氧空白对照组）,Ⅱ（低O2高CO2模型组）,Ⅲ组（色甘酸钠对照组）,Ⅳ组（姜黄素实验组）。将后3组动物放入常压低O2高CO2舱中,吸入O2浓度8％～11％,CO2浓度3％～5％,每天8h,每周6d,连续4周,Ⅲ组给予色甘酸钠以20mg／kg体重腹注射处理,Ⅳ组给予姜黄素混悬液按150mg/kg体重灌胃处理。光镜下、透射电镜下观察肺动脉血管壁及其周围大细胞超微结构形态学改变,甲苯胺蓝染色法和免疫组织化学法对肺动脉周围的肥大细胞及其脱颗粒状态进行性定量测定。结果：①电镜下,Ⅱ组肺细小动脉中膜平滑肌增生,外膜胶原纤维密集,内弹力板扭曲,内皮细胞起,血管外肥大细胞内颗粒减少,胞膜不完整;光镜下,Ⅱ组相比1组肺细小动脉管腔／管总面积（WA／TA）明显升（P〈0．05）、管腔／管总面积（EA／TA）明显降低（P〈0．05）,甲苯胺蓝染色肥大细胞细胞数（NMC）、肥大细胞脱颗率（DR）及免疫组化检测类胰蛋白酶阳性细胞数（TBS）高于I组（P〈0．05）;②干预后,电镜下,Ⅲ组、Ⅳ组血管结基本正常,平滑肌增生及胶原增生较Ⅱ组轻,肥大细胞膜完整;光镜下,两干预组相比Ⅱ组WA／TA明显降低（P〈0．05）、ET／TA明显升高（P〈0．05）,甲苯胺蓝染色NMC、DR、TBS阳性细胞数分别低于Ⅱ组（P〈0．05）。结论：姜素可通过Mc途径抑制慢性低O2高CO2导致的大鼠肺血管重塑改变。     

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.     

Linked mechanical and biological aspects of remodeling in mouse pulmonary arteries with hypoxia-induced hypertension

Supravalvular aortic stenosis (SVAS) is associated with decreased elastin and altered arterial mechanics. Mice with a single deletion in the elastin gene (ELN(+/-)) are models for SVAS. Previous studies have shown that elastin haploinsufficiency in these mice causes hypertension, decreased arterial compliance, and changes in arterial wall structure. Despite these differences, ELN(+/-) mice have a normal life span, suggesting that the arteries remodel and adapt to the decreased amount of elastin. To test this hypothesis, we performed in vitro mechanical tests on abdominal aorta, ascending aorta, and left common carotid artery from ELN(+/-) and wild-type (C57BL/6J) mice. We compared the circumferential and longitudinal stress-stretch relationships and residual strains. The circumferential stress-stretch relationship is similar between genotypes and changes <3% with longitudinal stretch at lengths within 10% of the in vivo value. At mean arterial pressure, the circumferential stress in the ascending aorta is higher in ELN(+/-) than in wild type. Although arterial pressures are higher, the increased number of elastic lamellae in ELN(+/-) arteries results in similar tension/lamellae compared with wild type. The longitudinal stress-stretch relationship is similar between genotypes for most arteries. Compared with wild type, the in vivo longitudinal stretch is lower in ELN(+/-) abdominal and carotid arteries and the circumferential residual strain is higher in ELN(+/-) ascending aorta. The increased circumferential residual strain brings the transmural strain distribution in ELN(+/-) ascending aorta close to wild-type values. The mechanical behavior of ELN(+/-) arteries is likely due to the reduced elastin content combined with adaptive remodeling during vascular development.     

PNPLA3 mediates hepatocyte triacylglycerol remodeling

The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3^I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3^WT) and PNPLA3^I148M on HuH7 cell lipidome after [¹³C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3^I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [¹³C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3^WT and PNPLA3^I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3^WT associated in PCA with newly synthesized [¹³C]TAGs, particularly 52:1 and 50:1, while PNPLA3^I148M associated with similar preexisting TAGs. PNPLA3^WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3^I148M. PNPLA3^I148M localized more extensively to lipid droplets (LDs) than PNPLA3^WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3^I148M.     

Rapamycin attenuates hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in mice

Background

Chronic hypoxia induces pulmonary arterial hypertension (PAH). Smooth muscle cell (SMC) proliferation and hypertrophy are important contributors to the remodeling that occurs in chronic hypoxic pulmonary vasculature. We hypothesized that rapamycin (RAPA), a potent cell cycle inhibitor, prevents pulmonary hypertension in chronic hypoxic mice.

Methods

Mice were held either at normoxia (N; 21% O₂) or at hypobaric hypoxia (H; 0.5 atm; ~10% O₂). RAPA-treated animals (3 mg/kg*d, i.p.) were compared to animals injected with vehicle alone. Proliferative activity within the pulmonary arteries was quantified by staining for Ki67 (positive nuclei/vessel) and media area was quantified by computer-aided planimetry after immune-labeling for α-smooth muscle actin (pixel/vessel). The ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was used to determine right ventricular hypertrophy.

Results

Proliferative activity increased by 34% at day 4 in mice held under H (median: 0.38) compared to N (median: 0.28, p = 0.028) which was completely blocked by RAPA (median HO+RAPA: 0.23, p = 0.003). H-induced proliferation had leveled off within 3 weeks. At this time point media area had, however, increased by 53% from 91 (N) to 139 (H, p < 0.001) which was prevented by RAPA (H+RAPA: 102; p < 0.001). RV/[LV+S] ratio which had risen from 0.17 (N) to 0.26 (H, p < 0.001) was attenuated in the H+RAPA group (0.22, p = 0.041). For a therapeutic approach animals were exposed to H for 21 days followed by 21 days in H ± RAPA. Forty two days of H resulted in a media area of 129 (N: 83) which was significantly attenuated in RAPA-treated mice (H+RAPA: 92). RV/[LV+S] ratios supported prevention of PH (N 0.13; H 0.27; H+RAPA 0.17). RAPA treatment of N mice did not influence any parameter examined.

Conclusion

Therapy with rapamycin may represent a new strategy for the treatment of pulmonary hypertension.