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.     

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.     

Effect of L-arginine on pulmonary artery smooth muscle cell apoptosis in rats with hypoxic pulmonary vascular structural remodeling

This study investigated the effect of L-arginine (L-Arg) on the apoptosis of pulmonary arterysmooth muscle cells (PASMC) in rats with hypoxic pulmonary vascular structural remodeling,and itsmechanisms.Seventeen Wistar rats were randomly divided into a control group (n=5),a hypoxia group(n=7),and a hypoxia L-Arg group (n=5).The morphologic changes of lung tissues were observed underoptical microscope.Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay,the apoptosis of PASMC was examined.Fas expression in PASMC wasexamined using immunohistochemistry.The results showed that the percentage of muscularized artery insmall pulmonary vessels,and the relative medial thickness and relative medial area of the small and medianpulmonary muscularized arteries in the hypoxic group were all significantly increased.Pulmonary vascularstructural remodeling developed after hypoxia.Apoptotic smooth muscle cells of the small and median pul-monary arteries in the hypoxia group were significantly less than those in the control group.After 14 d ofhypoxia,Fas expression by smooth muscle cells of median and small pulmonary arteries was significantlyinhibited.L-Arg significantly inhibited hypoxic pulmonary vascular structural remodeling in association withan augmentation of apoptosis of smooth muscle cells as well as Fas expression in PASMC.These resultsshowed that L-Arg could play an important role in attenuating hypoxic pulmonary vascular structural remod-eling by upregulating Fas expression in PASMC,thus promoting the apoptosis of PASMC.     

Sandostatin inhibits development of medial proliferation of pulmonary arteries in a rat model of pulmonary hypertension

We investigated the effects of subcutaneous administration of 50 and 100 μg/kg/day of sandostatin on monocrotaline-induced medial proliferation of pulmonary arteries and right ventricular overload in rats. In a dosage of 100 μg/kg/day, sandostatin significantly reduced right ventricular systolic pressure, the mass ratio of the right ventricular free wall to the left ventricle, the right ventricular wall thickness, the right ventricular myofiber diameter, the percent medial pulmonary artery thickness, the percent area of smooth muscle cell, and proliferating cell nuclear antigen activity. Our results suggest that sandostatin inhibits development of medial proliferation of pulmonary arteries and right ventricular overload in a dosage of 100 μg/kg/day.     

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.     

Effects of endogenous carbon monoxide on collagen synthesis in pulmonary artery in rats under hypoxia

To study the role of endogenous carbon monoxide (CO) in collagen metabolism during hypoxic pulmonary vascular remodeling, a total of 18 Wistar rats were used in the study and they were randomly divided into three groups: hypoxia group (n = 6), hypoxia with zinc protoporphyrin-IX (ZnPP-IX) group (n = 6) and control group (n = 6). The measurement of mean pulmonary artery pressure (mPAP) and carboxyhemoglobin (HbCO) formation in lung tissue homogenates was measured. A morphometric analysis of pulmonary vessels was performed, in which the percentage of muscularized arteries (MA); partially muscularized arteries (PMA) and nonmuscularized arteries (NMV) in small and median pulmonary vessels, relative medial thickness (RMT) and relative medial area (RMA) of pulmonary arteries were analyzed. Collagen type I and III and transforming growth factor-beta3 (TGF-beta3) expressions were detected by immunohistochemical assay. The expressions of procollagen type I and III and TGF-beta3 mRNA were detected by in situ hybridization. The results showed that ZnPP-IX significantly increased mPAP and markedly decreased HbCO formation in lung tissue homogenates in rats under hypoxia (P < 0.01). In the hypoxia rats treated with ZnPP-IX, the percentage of muscularized arteries of small and median pulmonary vessels was obviously increased, and RMT and RMA of intra-acinar muscularized pulmonary arteries were markedly increased compared with hypoxic rats. Ultrastructural changes, such as hyperplasia and hypertrophy of endothelial cells (ECs) and smooth muscle cells (SMCs) and the increased number of SMCs in synthetic phenotype were found in intra-acinar pulmonary muscularized arteries of hypoxic rats treated with ZnPP-IX. Meanwhile, ZnPP-IX promoted the expression of collagen type I and III and TGF-beta3 protein in pulmonary arteries of rats under hypoxia (P < 0.01). Furthermore, ZnPP-IX elevated obviously the expressions of procollagen type I and III mRNA, and TGF-beta3 mRNA in pulmonary arteries of rats under hypoxia (P < 0.01). The results of this study suggested that ZnPP-IX played an important role in promoting collagen synthesis in pulmonary arteries of rats with hypoxic pulmonary structural remodeling by increasing the expression of TGF-beta3. The above findings also suggested a possible role of endogenous CO in the pathogenesis of chronic hypoxic pulmonary hypertension.     

The role of endogenous lung neuropeptides in regulation of the pulmonary circulation

Vascular resistance in the mammalian pulmonary circulation is affected by many endogenous agents that influence vascular smooth muscle, right ventricular myocardium, endothelial function, collagen and elastin deposition, and fluid balance. When the balance of these agents is disturbed, e.g. by airway hypoxia from high altitude or pulmonary obstructive disorders, pulmonary hypertension ensues, as characterized by elevated pulmonary artery pressure (P(PA)). Among neuropeptides with local pulmonary artery pressor effects are endothelin-1 (ET-1), angiotensin II (AII), and substance P, and among mitigating peptides are calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), atrial natriuretic peptide (ANP), vasoactive intestinal peptide (VIP) and ET-3. Moreover, somatostatin28 (SOM28) exacerbates, whereas SOM14 decreases P(PA) in hypoxic rats, with lowering and increasing of lung CGRP levels, respectively. Pressure can also be modulated by increasing or decreasing plasma volume (VIP and ANP, respectively), or by induction or suppression of vascular tissue remodeling (ET-1 and CGRP, respectively). Peptide bioavailability and potency can be regulated through hypoxic up- and down- regulation of synthesis or release, activation by converting enzymes (ACE for AII and ECE for ET-1), inactivation by neutral endopeptidase and proteases, or by interaction with nitric oxide (NO). Moreover, altered receptor density and affinity can account for changed peptide efficacy. For example, upregulation of ET(A) receptors and ET-1 synthesis occurs in the hypoxic lung concomitantly with reduced CGRP release. Also, receptor activity modifying protein 2 (RAMP2) has been shown to confer ADM affinity to the pulmonary calcitonin-receptor-like receptor (CRLR). We recently detected the mRNA encoding for RAMP2, CRLR, and the CGRP receptor RDC-1 in rat lung. The search for an effective, lung selective treatment of pulmonary hypertension will likely benefit from exploring the imbalance and restoring the balance between these native modulators of intrapulmonary pressure. For example, blocking of the ET-1 receptor ET(A) and vasodilation by supplemental CGRP delivered i. v. or via airway gene transfer, have proven to be useful experimentally.     

Effects of hydrogen sulfide on hypoxic pulmonary vascular structural remodeling

To study the role of hydrogen sulfide (H2S) in hypoxic pulmonary vascular structural remodeling (HPVSR), a total of 24 Wistar rats were randomly divided into three groups: control group (n = 8), hypoxia group (n = 8) and hypoxia with sodium hydrosulfide (hy + NaHS) group (n = 8). The mean pulmonary artery pressure (mPAP), plasma H2S and the percentage of muscularized arteries (MA), partially muscularized arteries (PMA) and nonmuscularized arteries (NMA) in small pulmonary vessels were measured. Collagen I and III, elastin, transforming growth factor-beta3 (TGF-beta3), proliferative cell nuclear antigen (PCNA) and human urotensin II(U-II) expressions were detected by immunohistochemical assay. The mRNA expressions of procollagen I and III, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinease-1 (TIMP-1) were detected by in situ hybridization. The results showed that NaHS significantly increased plasma H2S, decreased mPAP and the percentage of MA and PMA of small pulmonary vessels in rats under hypoxia. Meanwhile, NaHS inhibited the proliferation of pulmonary artery smooth muscle cells (PASMCs) represented by a decrease in the expressions of PCNA and human U-II in pulmonary artery wall. NaHS reduced the expression of collagen I and III, elastin and TGF-beta3 protein and decreased the expressions of procollagen I and III mRNA in pulmonary arteries of rats under hypoxia, but it did not impact the ratio of TIMP-1 mRNA to MMP-1mRNA in pulmonary arteries of rats under hypoxia. These data suggested that H2S played an important role in the development of HPVSR.     

内质网应激在大鼠低氧高二氧化碳性肺动脉高压中的作用

目的：观察低氧高二氧化碳性肺动脉高压大鼠的肺血管重塑并探讨内质网应激（ERS）在肺动脉高压中的作用。方法：将40只SD大鼠随机分为四组：常氧对照组（N）、低氧高二氧化碳组（HH）、ERS通路抑制剂4-苯基丁酸（4-phenylbutyric acid）组（4-PBA）、ERS通路激动剂衣霉素（tunicamycin）组（TM）,n=10。测量各组大鼠的肺动脉平均压（mPAP）、颈动脉平均压以及右心室肥大指数,免疫荧光α-SMA标记法鉴定各组肺中小动脉平滑肌细胞,电镜观察肺组织及肺中小动脉形态学变化,原位末端标记法（TUNEL）检测各组肺动脉平滑肌细胞的凋亡指数,采用RT-PCR和Western blot分别检测各组大鼠葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、c-Jun氨基末端激酶（JNK）、天冬氨酸特异性半胱氨酸蛋白酶-12（caspase-12）mRNA及蛋白质表达。结果：①与N组相比,HH组、4-PBA组、TM组mPAP、右心室游离壁重量/左心室加心室间隔重量[RV/（LV+S）]、肺动脉管壁面积/管总面积（WA/TA）比值增加（P<0.0 1）,肺动脉管腔面积/管总面积（LA/TA）比值减小（P<0.01）,细胞凋亡指数降低（P <0.05或P<0.01）。ERS相关蛋白质及mRNA的表达量升高,各差异均有统计学意义。②与HH组相比,4-PB A组mPAP和[RV/（LV+S）]、WA/TA值减小（P<0.01）,LA/TA值和细胞凋亡指数上升（P<0.05或P<0.01）,ERS相关蛋白质和mRNA的表达量均下调（P<0.05或P<0.01）;③与HH组相比,TM组mPAP、[RV/（LV+S）]、WA/TA值升高（P<0.05或P<0.01）;肺动脉中膜层增厚,LA/TA值和细胞凋亡指数降低（P<0.01）。ERS相关蛋白质及mRNA的表达量均升高,除GRP78蛋白质表达量无明显变化外,其余各差异均有统计学意义。结论：低氧高二氧化碳诱导的肺动脉高压大鼠肺血管重塑可能与肺动脉平滑肌细胞增殖过度及凋亡过少有关;ERS相关因子（JNK、caspase-12和CHOP）参与低氧高二氧化碳性肺动脉高压的调控。     

A role for platelet-derived growth factor beta-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.     

Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs

We tested the hypothesis that pulmonary endothelial nitric oxide synthase (eNOS) gene expression is primarily regulated by hemodynamic factors and is thus increased in rats with chronic hypoxic pulmonary hypertension. Furthermore, we examined the role of endothelin (ET)-1 in this regulatory process, since ET-1 is able to induce eNOS via activation of the ET-B receptor. Therefore, chronic hypoxic rats (10% O(2)) were treated with the selective ET-A receptor antagonist LU-135252 (50 mg x kg(-1) x day(-1)). Right ventricular systolic pressure and cross-sectional medial vascular wall area of pulmonary arteries rose significantly, and eNOS mRNA levels increased 1.8- and 2.6-fold after 2 and 4 wk of hypoxia, respectively (each P < 0.05). Pulmonary ET-1 mRNA and ET-1 plasma levels increased significantly after 4 wk of hypoxia (each P < 0.05). LU-135252 reduced right ventricular systolic pressure, vascular remodeling, and eNOS gene expression in chronic hypoxic rats (each P < 0.05), whereas ET-1 production was not altered. We conclude that eNOS expression in chronic hypoxic rat lungs is modified predominantly by hemodynamic factors, whereas the ET-B receptor-mediated pathway and hypoxia seem to be less important.     

Down-regulation of lncRNA MEG3 promotes hypoxia-induced human pulmonary artery smooth muscle cell proliferation and migration via repressing PTEN by sponging miR-21

Hypoxia-induced pulmonary hypertension is a life-threatening disease arising from a progressive increase in pulmonary vascular resistance, irreversible pulmonary vascular remodeling and resulting in right ventricular failure. Recent studies suggested that pulmonary artery smooth muscle cell proliferation and migration played an important role in the pathogenesis of hypoxia-induced pulmonary hypertension. However, the mechanisms of hypoxia-induced pulmonary hypertension are complicated and largely unclear. In this study, we discovered that lncRNA MEG3 was down-regulated in human pulmonary artery smooth muscle cell in hypoxia, and inhibition of MEG3 promoted the cell proliferation and cell migration in both normal and hypoxia condition. Further study demonstrated that MEG3 exerted its function via regulation of miR-21 expression in both normal and hypoxia condition. In addition, we displayed the modulation of PTEN by miR-21 and their role in hypoxia. Ultimately, our study illustrated that MEG3 exerts its role via miR-21/PTEN axis in human pulmonary artery smooth muscle cell under both normal and hypoxia conditions.     

Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.     

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

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

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

Grape seed procyanidin extract attenuates hypoxic pulmonary hypertension by inhibiting oxidative stress and pulmonary arterial smooth muscle cells proliferation

Hypoxia-induced oxidative stress and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Grape seed procyanidin extract (GSPE) possesses antioxidant properties and has beneficial effects on the cardiovascular system. However, the effect of GSPE on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 weeks to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio and median width of pulmonary arteries. GSPE attenuated the elevation of RVSP, RV/LV+S, and reduced the pulmonary vascular structure remodeling. GSPE also increased the levels of SOD and reduced the levels of MDA in hypoxia-induced HPH model. In addition, GSPE suppressed Nox4 mRNA levels, ROS production and PASMCs proliferation. Meanwhile, increased expression of phospho-STAT3, cyclin D1, cyclin D3 and Ki67 in PASMCs caused by hypoxia was down-regulated by GSPE. These results suggested that GSPE might potentially prevent HPH via antioxidant and antiproliferative mechanisms.     

LRP1 promotes synthetic phenotype of pulmonary artery smooth muscle cells in pulmonary hypertension

Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways.Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a β1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures.In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.     

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.     

红花注射液对慢性低O2高CO2肺动脉高压的影响

目的：探讨红花注射液对大鼠在慢性低O2高CO2下肺动脉高压的抑制作用。方法：将SD大鼠分为对照组,慢性低O2高CO2组,慢性低O2高CO2＋红花注射液组。用电镜、放免等方法,观察各组大鼠肺动脉平均压、颈动脉平均压、肺细小动脉显微结构、血浆和肺匀浆TXB2及6-keto-PGF1a含量的变化。结果：①慢性低O2高CO2组mPAP比对照组显著增高,红花注射液组的mPAP比慢性低O2高CO2组显著降低,3组间mCAP比较差异无显著性。②慢性低O2高CO2组与对照组相比血浆和肺匀浆TXB2浓度、TXB2／6-keto-PGF1a比值显著增高,6-keto-PGF1a浓度显著下降;红花注射液组与慢性低P2高CO2相比血浆和肺匀浆TXB2浓度、TXB2／6-keto-PGF1a显著下降,6-keto-PGF1a显著升高。③光镜下慢性低O2高CO2组与对照组相比,肺细小动脉管壁面积／管总面积（WA／TA）和肺细小动脉中膜厚度（PAMT）均显著增高。红花注射液组WA／TA和PAMT显著降低。④电镜下慢性低O2高CO2组大鼠肺细小动脉内皮细胞吞饮小泡增多,血管壁增厚,中膜平滑肌细胞增生,纤维细胞增多,肺泡Ⅱ型上皮细胞微绒毛脱落;红花注射液组肺细小动脉中膜平滑肌细胞增生减轻,纤维细胞少,胶原纤维减少,肺泡Ⅱ型上皮细胞微绒毛丰富、结构清。结论：红花注射液有减轻慢性低O2高CO2性肺动脉高压和肺血管结构重建的作用,可能与抑制TXA2的合成,保护血管内皮细胞,使TXA2/PGI2比值降低有关.