Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hyp0Xic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological featureof hypoxic pulmonary vascular remodeling.However,the origin of the cells involved in this process is stillnot well understood.The present study was undertaken to test the hypothesis that transforming growthfactor-β1 (TGF-β1) can induce transdifferentiation of fibroblasts into myofibroblasts,which might play akey role in the muscularization of non-muscular pulmonary arterioles.It was found that mean pulmonaryarterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling index and rightventricular hypertrophy became evident after 14 d of hypoxia.The distribution of nonmuscular,partiallymuscular,and muscular vessels was significantly different after 7 d of hypoxia.Immunocytochemistryresults demonstrated that the expression of co-smooth muscle actin was increased in intra-acinar pulmonaryarteries with increasing hypoxic time.TGF-β1 mRNA expression in pulmonary arterial walls was increasedsignificantly after 14 d of hypoxia,but showed no obvious changes after 3 or 7 d of hypoxia.In pulmonarytunica adventitia and tunica media,TGF-β1 protein staining was poorly positive in control rats,but wasmarkedly enhanced after 3 d of hypoxia,reaching its peak after 7 d Of hypoxia.The myofibroblast phenotypewas confirmed by electron microscopy,which revealed microfilaments and a well-developed rough endo-plasmic reticulum.Taken together,our results suggested that TGF-β1 induces transdifferentiation of fibro-blasts into myofibroblasts,which is important in hypoxic pulmonary vascular remodeling.     

Hypoxia inducible factor-1 alpha correlates the expression of heme oxygenase 1 gene in pulmonary arteries of rat with hypoxia-induced pulmonary hypertension

AbstractTo test the hypothesis that hypoxia inducible factor-1 alpha (HIF-1α）up-regulated theexpression of heme oxygenase-1 (HO-1) gene in pulmonary arteries of rats with hypoxia-induced pulmonaryhypertension, 8 male Wistar rats in each of 5 groups were exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively.Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index weremeasured. Lungs were inflation fixed for immunohistochemistry, in situ hybridization; frozen for latermeasurement of HO-1 enzyme activity, mPAP increased significantly after 7 d of hypoxia [(18.4 ± 0.4)mmHg, P<0.05], reaching its peak after 14 d of hypoxia, then remained stable. Pulmonary artery remodeling became to develop significantly after 14 d of hypoxia. HIF-1αprotein in control was poorly positive (0.05 ±0.01), but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterialtunica media, the levels of HIF-la protein were markedly up-regulated after 3 d and 7 d of hypoxia(0.20±0.02; 0.22 ± 0.02, P<0.05), then declined after 14 d and 21 d of hypoxia. HIF-mRNA stainingwas poorly positive in control, hypoxia for 3 and 7 d, but enhanced significantly after 14 d of hypoxia(0.20±0.02, P<0.05), then remained stable. HO-1 protein increased after 7 d of hypoxia (0.10±0.01,P<0.05), reaching its peak after 14 d of hypoxia (0.21 0.02, P<0.05), then remained stable. HO-1 mRNA increased after 3 d of hypoxia, reaching its peak after 7 d of hypoxia (0.17 ± 0.01, P<0.05), then declined.Linear correlation analysis showed that HIF-lα mRNA, HO-1 protein and mPAP were associatedwith pulmonary remodeling. HIF-1 α protein (tunica intima) was conversely correlated with HIF-1α mRNA(r=0.921, P<0.01), HO-1 protein was conversely correlated with HIF-1α protein (tunica intima)(r=0.821, P<0.01 ). HIF-1αand HO-1 were both involved in the pathogenesis of hypoxia-induced pulmonaryhypertension in rat. Hypoxia inducible factor-1 alpha correlated the expression of heme oxygenase 1 genein pulmonary arteries of rat with hypoxia-induced pulmonary hypertension.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β1 （TGF-β1） can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and fight ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of α-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.     

Differential and Reciprocal Regulation between Hypoxia-inducible Factor-α Subunits and Their Prolyl Hydroxylases in Pulmonary Arteries of Rat with Hypoxia-induced Hypertension

Hypoxia-inducible factor (HIF)-α subunits (HIF-1α,HIF-2α and HIF-3α),which play a pivotalrole during the development of hypoxia-induced pulmonary hypertension (HPH),are regulated through post-U'anslational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD 1,PHD2 and PHD3).PHDs could also be regulated by HIF.But differential and reciprocal regulation between HIF-α and PHDs duringthe development of HPH remains unclear.To investigate this problem,a rat HPH model was established.Meanpulmonary arterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling indexand right ventricular hypertrophy became evident after 14 d of hypoxia.HIF-1α and HIF-2α mRNA increasedslightly after 7 d of hypoxia,but HIF-3α increased significantly after 3 d of hypoxia.The protein expressionlevels of all three HIF-α were markedly upregulated after exposure to hypoxia.PHD2 mRNA and proteinexpression levels were upregulated after 3 d of hypoxia;PHD 1 protein declined after 14 d of hypoxia withoutsignificant mRNA changes.PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia,then themRNA remained at a high level,but the protein declined after 14 d of hypoxia.In hypoxic animals,HIF-lotproteins negatively correlated with PHD2 proteins,whereas HIF-2α and HIF-3α proteins showed negativecorrelations with PHD3 and PHD 1 proteins,respectively.All three HIF-α proteins were positively correlatedwith PHD2 and PHD3 mRNA.In the present study,HIF-α subunits and PHDs showed differential andreciprocal regulation,and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.     

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.     

Up-regulation of hypoxia-inducible factor-1α enhanced the cardioprotective effects of ischemic postconditioning in hyperlipidemic rats

Hyperlipidemia is an independent risk factor in the devel- opment of ischemic heart disease, which can increase myo- cardial susceptibility to ischemia/reperfusion （I/R） injury. Ischemic postconditioning （PostC） has now been demon- strated as a novel strategy to harness nature＇s protection against myocardial I/R injury in normal conditions. However, the effect of PostC on hyperlipidemic animals remains elusive. It has been shown in our previous study that PostC reduces the myocardial I/R injury, and hypoxia- inducible factor-1α （HIF-1α） may play an important role in the cardioprotective mechanisms of PostC on normal rats. Here, we tested the hypothesis that the cardioprotec- tion of PostC on hyperlipidemic rats is associated with the up-regulated HIF-1α expression. Male Wistar rats were fed with a high-fat diet for 8 weeks, and then randomly div- ided into five groups： sham, I/R, dimethyloxalylglycine （DMOG） ＋ I/R, PostC, and DMOG ＋ PostC group. The detrimental indices induced by I/R injury included infarct size, plasma creatine kinase （CK） activity and caspase-3 ac- tivity. The results showed that PostC could reduce the infarct size, when compared with the I/R group, which was consistent with the significant lower levels of plasma CK activity and caspase-3 activity, and that it increased the ex- pression of HIF-1α in hyperlipidemic rats. When DMOG was given before PostC to up-regulate HIF-1α protein level, the degree of I/R injury was attenuated. In conclu- sion, these data suggested that the up-regulation of HIF-1α may be one of the cardioprotective mechanisms of PostC against I/R injury in hyperlipidemic rats.     

Chronic intermittent hypoxia accelerates liver fibrosis in rats with combined hypoxia and nonalcoholic steatohepatitis via angiogenesis rather than endoplasmic reticulum stress

In the present study,we aimed to investigate the role of endoplasmic reticulum stress(ERS)and its related inflammation and angiogenesis in liver fibrosis in a rat model of combined hypoxia and nonalcoholic steatohepatitis(NASH)and to confirm whether the intervention of hypoxia-inducible factor 1α(HIF1α)can improve fibrosis.Liver histological changes and biochemical indices,HIF1α,inflammatory factors,ERS-related parameters(GRP78,CHOP,caspase-3,and caspase-12),and angiogenesis indices(VEGFA,VEGFR2,and CD34)were evaluated.Compared with the control rats,the liver tissue of rats with hypoxia and NASH had obvious NASH characteristics and hepatic fibrosis was significantly aggravated,including bridging fibrosis in some rats.The mRNA expression levels of HIF1α,VEGFA,and VEGFR2 and total immunohistochemical staining scores of VEGFR2 and CD34 were significantly increased.In addition,HIF1α silencing significantly decreased HIF1α,biochemical indices(ALT,AST,and TG),inflammatory factors(TNFα,IL6,and IL1β),and angiogenesis indices(CD34 and VEGFR2),consequently,improved the hepatic fibrosis score in the rat model of combined hypoxia and NASH.Taken together,chronic intermittent hypoxia accelerates liver fibrosis in rats with combined hypoxia and NASH via angiogenesis rather than ERS and HIF1α intervention can improve liver fibrosis,angiogenesis,inflammatory factors,and biochemical indices.Therefore,HIF1α is a key regulatory factor of liver fibrosis in rats with combined hypoxia and NASH.     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

SENP1在大鼠低氧性肺动脉高压形成中肺血管壁中表达及可能作用

目的:探讨大鼠低氧性肺动脉高压(HPH)形成过程中SENP1在肺小动脉的动态表达变化及作用。方法:40只成年雄性Wistar大鼠随机分为5组(n=8):对照组和缺氧3 d、7 d、14 d2、1 d组,常压间断低氧复制HPH大鼠模型。测各组大鼠平均肺动脉压(mPAP)、右心室肥大指数(RVHI)、血管形态学指标;原位杂交、逆转录-聚合酶链反应(RT-PCR)检测肺内SUMO特异性蛋白酶-1(SUMO-specific proteases-1,SENP1)mRNA表达,免疫组化、Westernblot检测其蛋白质水平。结果:①缺氧7 d后,肺小动脉出现血管重塑,且mPAP明显上升;低氧14 d后,肺小动脉重塑更明显,mPAP达高峰。RVHI在低氧14 d后明显增加。②原位杂交显示,SENP1 mRNA在对照组肺小动脉壁呈阳性表达,低氧后其相对量无明显变化。RT-PCR显示肺组织SENP1 mRNA表达与原位杂交所观察到的肺小动脉壁SENP1 mRNA变化趋势一致;SENP1蛋白在对照组呈阳性表达,低氧7 d后其表达量开始呈进行性下降。Western blot显示肺组织内SENP1蛋白表达与免疫组化观察到的肺小动脉壁SENP1蛋白变化趋势一致。③SENP1蛋白与mPAP、重塑指数、RVHI均呈负相关。结论:慢性低氧诱导肺小动脉壁SENP1蛋白降解,进而可能在HPH发病过程中发挥一定的作用。     

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.     

低氧诱导因子脯氨酰羟化酶与OS－9在大鼠低氧肺动脉高压中的协同作用

目的：研究HIF-1α、PHDs及OS-9的表达变化在低氧性肺动脉高压（HPH）中的作用和意义。方法：SD大鼠随机分5组（n=8）;对照组（C组）和低氧3、7、14和21d组,常压低氧复制HPH大鼠模型。原位杂交、RT-PCR检测mRNA表达,免疫组化、Westernblot检测蛋白质表达。结果：①HIF-1αmRNA对照纽和低氧3d无明显差异,低氧14d后表达明显增高;HIF-1α蛋白质低氧3d组表达明显增高,7d达高峰;②对照组PHD1mRNA呈阳性表达,各低氧组与对照组比较差异不显著,PHD1蛋白质在对照组强阳性表达,低氧14d下降,低氧21d保持较低水平;对照组PHD2mRNA呈阳性表达,低氧3d增高,14d达到高峰,21d维持高水平,其蛋白质表达趋势与mRNA相同;对照组PHD3mRNA和蛋白质表达不明显,低氧3dmRNA明显增高,蛋白质低氧3d明显增高,低氧7d保持高水平,低氧14d和21d下降。③OS-9mRNA在对照组呈强阳性表达,低氧3d后迅速降低,14d达到最低水平;其蛋白质表达趋势与mRNA相同。相关分析表明,肺小动脉壁OS-9蛋白质表达水平与OS-9mRNA呈正相关,与RVHI、mPAP、WA％及LA％呈负相关。结论：HIF-1α、PHDs及OS-9均在大鼠HPH的发病机制中发挥作用。OS-9可能通过增强PHDs的活性来调节HIF-1α的表达,从而在HPH的发生和发展中发挥作用。     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子-1α和细胞增殖的关系

在低氧条件下,观察大鼠肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells,PASMCs）中活性氧（reactive oxygen species,ROS）的变化,探讨ROS的变化是否通过调控低氧诱导因子-4α（hypoxia-inducible factor 1α, HIF-1α）的表达影响PASMCs的增殖。采用组织块法原代培养大鼠PASMCs,分成3组：常氧组（21％O2,24h）,低氧组（5％O2,24h）,低氧＋Mn-TBAP组（5％O2,24h,Mn-TBAP是一种ROS清除剂）。用激光共聚焦显微镜荧光染色法检测细胞内ROS的变化;用RT-PCR和免疫组织化学方法分别测定HIF-1α mRNA和蛋白的表达;用MTT法检测细胞增殖程度。结果显示：（1）低氧组PASMCs内ROS水平明显高于常氧组（P〈0．05）,低氧＋Mn-TBAP组ROS水平明显低于低氧组（P〈0．05）,但仍高于常氧组（P〈0．05）;（2）低氧组及低氧＋Mn-TBAP组的HIF-1α mRNA和蛋白表达均高于常氧组（P〈0．05）,且低氧组表达高于低氧＋Mn-TBAP组（P〈0．05）;（3）低氧组细胞增殖明显高于常氧组和低氧＋Mn-TBAP组（P〈0．05）,低氧＋Mn-TBAP组细胞增殖高于常氧组（P〈0．05）。结果表明：在低氧条件下大鼠PASMCs中ROS水平明显升高,RROS的变化能够调节HIF-1α的表达,进而影响平滑肌细胞的增殖,提示ROS可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。     

游泳运动对低氧性肺动脉高压大鼠肺组织apelin及其受体表达的影响

目的:探讨游泳运动对大鼠肺组织新的小分子活性肽apelin及其受体(APJ)表达的影响。方法:45只雄性大鼠随机分成三组:正常对照组、低氧组(七周)和游泳组(低氧+游泳锻炼七周组,低氧3周后,于每天入低氧舱前行无负重游泳运动60 min,每天1次)。七周后测定各组大鼠平均肺动脉压(mPAP)、右心室与左心室加室间隔的重量比[RV/(LV+S)]、肺细小动脉管壁面积/管总面积(WA/TA)、管腔面积/管总面积(CA/TA)及中膜厚度(PAMT)。免疫蛋白印迹与免疫组化法测定肺组织apelin/APJ的蛋白表达。结果:①低氧组mPAP和RV/(LV+S)比正常对照组分别高73.6%和31.2%(P均<0.01),而游泳组比低氧组分别低21.1%和8.9%(P均<0.05)。②低氧组WA/TA和PAMT较正常对照组分别高70.8%和102%,而游泳组较低氧组分别低24.8%和40.1%(P均<0.01)。低氧组CA/TA较正常对照组低15.1%,而游泳组较低氧组高10.3%(P均<0.01)。③低氧组肺组织apelin蛋白表达较正常对照组上调374%(P<0.01),而APJ蛋白表达下调87.1%(P均<0.01);游泳组肺组织apelin蛋白表达较低氧组下调48%,而APJ蛋白表达上调287%(P均<0.01)。④apelin蛋白主要在血管外膜及炎症细胞胞浆内表达,APJ蛋白主要在血管内膜、外膜及炎症细胞上表达。结论:游泳运动减缓肺动脉高压和肺血管重塑作用可能与调节肺组织apelin/APJ系统的表达有关。