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.     

Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.     

Thymoquinone suppresses platelet‐derived growth factor‐BB–induced vascular smooth muscle cell proliferation,migration and neointimal formation

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.     

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.     

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.     

Prolonged endothelin B receptor blockade causes pulmonary hypertension in the ovine fetus

Endothelin (ET)-1 contributes to regulation of pulmonary vascular tone and structure in the normal ovine fetus and in models of perinatal pulmonary hypertension. The hemodynamic effects of ET-1 are due to activation of its receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, whereas the ET(B) receptor mediates vasodilation. In a lamb model of chronic intrauterine pulmonary hypertension, ET(B) receptor activity and gene expression are decreased. To determine whether prolonged ET(B) receptor blockade causes pulmonary hypertension, we studied the hemodynamic effects of selective ET(B) receptor blockade with BQ-788. Animals were treated with an infusion of either BQ-788 or vehicle for 7 days. Prolonged BQ-788 treatment increased pulmonary arterial pressure and pulmonary vascular resistance (P < 0.05). The pulmonary vasodilator response to sarafotoxin 6c, a selective ET(B) receptor agonist, was attenuated after 7 days of BQ-788 treatment, demonstrating pharmacological blockade of the ET(B) receptor. Animals treated with BQ-788 had greater right ventricular hypertrophy and muscularization of small pulmonary arteries (P < 0. 05). Lung ET-1 levels were threefold higher in the animals treated with BQ-788 (P < 0.05). We conclude that prolonged selective ET(B) receptor blockade causes severe pulmonary hypertension and pulmonary vascular remodeling in the late-gestation ovine fetus.     

Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions

Pulmonary veins show greater sensitivity to endothelin (ET)-1-induced vasoconstriction than pulmonary arteries, and remodeling was observed in pulmonary veins under hypoxic conditions. We examined, using an immunohistochemical method, the expression of Big ET-1, ET-converting enzyme (ECE), and ET(A) and ET(B) receptors in rat pulmonary veins under normoxic and hypoxic conditions. In control rats, Big ET-1 and ECE were coexpressed in the intima and media of the pulmonary veins, with an even distribution along the axial pathway. ET(A) and ET(B) receptors were expressed in the pulmonary veins, with a predominant distribution in the proximal segments. The expression of Big ET-1 was more abundant in the pulmonary veins than in the pulmonary arteries. After exposure to hypoxia for 7 or 14 days, the expression of Big ET-1, ECE, and ET receptors increased in small pulmonary veins. Increases in the medial thickness, wall thickness, and immunoreactivity for alpha-smooth muscle actin were also observed in the small pulmonary veins under hypoxic conditions. The upregulation of ET-1 and ET receptors in the small pulmonary veins is associated with vascular remodeling, which may lead to the development of hypoxic pulmonary hypertension.     

Cultured primate aortic smooth muscle cells express both the PDGF-A and PDGF-B genes but do not secrete mitogenic activity or dimeric platelet-derived growth factor protein

Proliferation of smooth muscle cells (SMC) in the arterial intima of man and experimental animals is important in the pathogenesis of atherosclerosis. Vascular SMC proliferation in vitro is stimulated by a number of agents, including the potent protein mitogen, platelet-derived growth factor (PDGF). Recent studies on rat arterial SMC indicate that these cells may, under certain circumstances, synthesize PDGF protein mitogens, suggesting that the regulation of SMC proliferation in vivo may have an autocrine or paracrine component. In this study we demonstrate that cultured nonhuman primate (baboon) aortic SMC transcribe both the PDGF-A and PDGF-B genes but do not secrete detectable mitogenic activity characteristic of native PDGF. The absence of this activity was not due to the presence in the cell conditioned medium of factors inhibitory for PDGF-mediated mitogenic activity. Metabolic labeling of the cells and immunoprecipitation with specific antibodies to human PDGF did not detect a dimeric (30 kDa) PDGF protein in either the intracellular or extracellular compartments, but instead identified PDGF-related proteins of molecular weight 12 kDa and 100 kDa. These data suggest the presence in vascular SMC of a mechanism regulating the translation of PDGF mRNA that may play an important role in the control of SMC proliferation in vivo.     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH‐SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or ?1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH‐SMC (fold change 1.5≤ or ?1.5≥, p < 0.05). HUASMC expressed increased amount of α‐smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH‐SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH‐SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH‐SMC. There was a trend toward reduced proliferation of PAH‐SMC with paxillin‐si‐RNA and increased proliferation with ELAVL1‐siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH‐SMC proliferation.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Prenyltransferase inhibitors block superoxide production by pulmonary vascular smooth muscle

We recently showed that the farnesyltransferase inhibitor FTI-277 blocks interleukin 1beta (IL-1beta)-induced nitric oxide production in pulmonary vascular smooth muscle cells (SMC), whereas the geranylgeranyltransferase inhibitor GGTI-298 enhances this effect. Here we show that IL-1beta and platelet-derived growth factor (PDGF) stimulate superoxide production by pulmonary vascular SMC and that this effect is blocked by both FTI-277 and GGTI-298, suggesting that farnesylated and geranylgeranylated proteins are required for superoxide production. We also show that FTI-277 and GGTI-298 block superoxide production stimulated by constitutively active mutant H-Ras. Furthermore, superoxide production by IL-1beta, PDGF factor, and constitutively activated Ras is blocked by diphenyleneiodonium, implicating NAD(P)H oxidase as the generating enzyme. Given the role of oxidant radicals in vascular reactivity and injury, the action of both FTI-277 and GGTI-298 in suppressing superoxide generation by an inflammatory cytokine as well as by a potent smooth muscle mitogen may be therapeutically useful.     

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

The Flavonoid Quercetin Reverses Pulmonary Hypertension in Rats

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT_2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT_2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.