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 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.     

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.     

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.     

Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts

In heart disease, transforming growth factor-β1(TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide(H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1(10 ng m L-1) for 24 h with or without sodium hydrosulfide(Na HS, 100 μmol L-1, 30 min pretreatment) treatment. Na HS, an exogenous H2 S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, Na HS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2 S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2 S may play significant roles in cardiac remodeling associated with heart failure.     

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.     

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。     

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.     

Pulmonary vascular remodelling in a high-altitude Aymara Indian

A histological study of the pulmonary vasculature in a young male high-altitude Aymara Indian revealed four aspects of interest. There was muscularization of the terminal portion of the pulmonary arterial tree to involve pulmonary arterioles as small as 15 m in diameter, thus forming a basis for the slightly increased pulmonary vascular resistance of native highlanders. Intimal longitudinal muscle was found in pulmonary arteries and arterioles and thought to be due to chronic alveolar hypoxia. Inner muscular tubes similar to those found in chronic obstructive lung disease were present. Pulmonary veins and venules also showed intimal muscularization suggesting that alveolar hypoxia affects vascular smooth muscle cells per se irrespective of their situation. The nature of the remodelling in a pulmonary blood vessel depends on a combination of hypoxia and haemodynamics.     

Metalloproteinase inhibition by Batimastat attenuates pulmonary hypertension in chronically hypoxic rats

Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 +/- 3 mmHg in hypoxic controls, 24 +/- 1 mmHg in Batimastat-treated hypoxic rats, and 16 +/- 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.     

Pulmonary vascular remodeling in isolated mouse lungs: effects on pulsatile pressure-flow relationships

Chronic hypoxia causes pulmonary vasoconstriction and pulmonary hypertension, which lead to pulmonary vascular remodeling and right ventricular hypertrophy. To determine the effects of hypoxia-induced pulmonary vascular remodeling on pulmonary vascular impedance, which is the right ventricular afterload, we exposed C57BL6 mice to 0 (control), 10 and 15 days of hypobaric hypoxia (n=6, each) and measured pulmonary vascular resistance (PVR) and impedance ex vivo. Chronic hypoxia led to increased pulmonary artery pressures for flow rates between 1 and 5ml/min (P<0.01), and increased PVR, 0-Hz pulmonary vascular impedance and the index of wave reflection (P<0.05) as well as a more negative impedance phase angle for low frequencies (P<0.05). The increases in resistance and 0-Hz impedance correlated with increased muscularization of small arterioles measured with quantitative immunohistochemistry (P<0.01). The increases in wave reflection and decreases in phase angle are likely due to increased proximal artery stiffness. These results confirm that chronic hypoxia causes significant changes in steady and pulsatile pressure-flow relationships in mouse lungs and does so via structural remodeling. They also provide important baseline data for experiments with genetically engineered mice, with which molecular mechanisms of pulmonary vascular remodeling can be investigated.     

eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia

Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.