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]     

MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

Pulmonary arterial hypertension (PAH) is featured by the increase in pulmonary vascular resistance and pulmonary arterial pressure. Despite that abnormal proliferation and phenotypic changes in human pulmonary artery smooth muscle cells (HPASMCs) contributing to the pathophysiology of PAH, the underlying molecular mechanisms remain unclear. In the present study, we detected the expression of miR‐629 in hypoxia‐treated HPASMCs and explored the mechanistic role of miR‐629 in regulating HPASMC proliferation, migration and apoptosis. Hypoxia time‐dependently induced up‐regulation of miR‐629 and promoted cell viability and proliferation in HPASMCs. Treatment with miR‐629 mimics promoted HPASMCs proliferation and migration, but inhibited cell apoptosis; while knockdown of miR‐629 suppressed the cell proliferation and migration but promoted cell apoptosis in HPASMCs. The bioinformatics prediction revealed FOXO3 and PERP as downstream targets of miR‐629, and miR‐629 negatively regulated the expression of FOXO3 and PERP via targeting the 3’ untranslated regions. Enforced expression of FOXO3 or PERP attenuated the miR‐629 overexpression or hypoxia‐induced enhanced effects on HPASMC proliferation and proliferation, and the suppressive effects on HPASMC apoptosis. Furthermore, the expression of miR‐629 was up‐regulated, and the expression of FOXO3 and PERP mRNA was down‐regulated in the plasma from PAH patients when compared to healthy controls. In conclusion, the present study provided evidence regarding the novel role of miR‐629 in regulating cell proliferation, migration and apoptosis of HPASMCs during hypoxia.     

IGF-1 induces iNOS expression via the p38 MAPK signal pathway in the anti-apoptotic process in pulmonary artery smooth muscle cells during PAH

Abstract

Apoptosis and cell proliferation are two important cellular processes that determine the accumulation of pulmonary artery smooth muscle cells (PASMC) during pulmonary arterial hypertension (PAH). Insulin-like growth factor 1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation, also tissue repair. Inducible nitric oxide synthase (iNOS) has been shown to serve many vasoprotective roles in vascular smooth muscle cells (VSMCs) including inhibition of VSMC proliferation and migration and stimulation of endothelial cell growth. In this study, we investigated the involvement of iNOS in the process of IGF-1-induced inhibition of PASMC apoptosis. We also examined the role of p38 mitogen-activated protein kinase (MAPK) in the IGF-1-induced iNOS activation. Our results show that exogenous IGF-1 induced the up-regulation of iNOS in PASMC. Immunofluorescence of IGF-1 and iNOS showed a decreased immunostaining of both IGF-1 and iNOS in the cytoplasm and the perinucleus under serum deprivation condition. iNOS inhibition in PASMC in vitro markedly induced IGF-1-mediated anti-apoptosis as assessed by the cell viability measurement, Western blot, mitochondrial potential analysis and nuclear morphology determination. A p38 MAPK inhibitor blocked all the effects of IGF-1 on iNOS. Our findings suggest that IGF-1 inhibits cells apoptosis in PASMC by activating the p38 MAPK–iNOS transduction pathway. This mechanism may contribute to the accumulation of PASMC in early human PAH.     

Hyperplastic Growth of Pulmonary Artery Smooth Muscle Cells from Subjects with Pulmonary Arterial Hypertension Is Activated through JNK and p38 MAPK

Smooth muscle in the pulmonary artery of PAH subjects, both idiopathic and hereditary, is characterized by hyperplasia. Smooth muscle cells (HPASMC) isolated from subjects with or without PAH retain their in vivo phenotype as illustrated by their expression of alpha-smooth muscle actin and expression of H-caldesmon. Both non PAH and PAH HPASMC display a lengthy, approximately 94h, cell cycle. The HPASMC from both idiopathic and hereditary PAH display an abnormal proliferation characterized by continued growth under non-proliferative, non-growth stimulated conditions. This effector independent proliferation is JNK and p38 MAP kinase dependent. Blocking the activation of either abrogates the HPASMC growth. HPASMC from non PAH donors under quiescent conditions display negligible proliferation but divide upon exposure to growth factors such as PDGF-BB or FGF2 but not EGF. This growth does not involve the MAP kinases. Instead it routes via the tyrosine kinase receptor through mTOR and then 6SK. In the PAH cells PDGF-BB and FGF2 augment the dysregulated cell proliferation, also through mTOR/6SK. Additionally, blocking the activation of mTOR also modulates the MAP kinase promoted dysregulated growth. These results highlight key alterations in the growth of HPASMC from subjects with PAH which contribute to the etiology of the disease and can clearly be targeted at various regulatory points for future therapies.     

Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia‐induced cell growth

Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia‐induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI‐treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia‐induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.     

Activation dynamics and signaling properties of Notch3 receptor in the developing pulmonary artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.     

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.     

Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.     

SCD1 activation impedes foam cell formation by inducing lipophagy in oxLDL‐treated human vascular smooth muscle cells

The formation of fat‐laden foam cells, which contributes to the fatty streaks in the plaques of atheromas, is an important process in atherosclerosis. Vascular smooth muscle cells (VSMCs) are a critical origin of foam cells. However, the mechanisms that underlie VSMC foam cell formation are not yet completely understood. Here, we demonstrated that oxidized low‐density lipoprotein (oxLDL) inhibited lipophagy by suppressing lipid droplet (LD)‐lysosome fusion and increased VSMC foam cell formation. Moreover, although oxLDL treatment inhibited lysosomal biogenesis, it had no significant effect on lysosomal proteolysis and lysosomal pH. Notably, through TMT‐based quantitative proteomic analysis and database searching, 94 differentially expressed proteins were identified, of which 54 were increased and 40 were decreased in the oxLDL group compared with those in the control group. Subsequently, SCD1, a protein of interest, was further investigated. SCD1 levels in the VSMCs were down‐regulated by exposure to oxLDL in a time‐dependent manner and the interaction between SCD1 and LDs was also disrupted by oxLDL. Importantly, SCD1 overexpression enhanced LD‐lysosome fusion, increased lysosomal biogenesis and inhibited VSMC foam cell formation by activating TFEB nuclear translocation and its reporter activity. Modulation of the SCD1/TFEB‐mediated lipophagy machinery may offer novel therapeutic approaches for the treatment of atherosclerosis.     

IGFBP6 regulates vascular smooth muscle cell proliferation and morphology via cyclin E–CDK2

Despite the high prevalence of varicose veins, the underlying pathogenesis of this disease remains unclear. The present study aims to explore the role of insulin-like growth factor binding protein 6 (IGFBP6) in vascular smooth muscle cells (VSMCs). Using a protein array approach, we identified several differentially expressed proteins between varicose great saphenous veins and normal great saphenous veins. Bioinformatic analysis showed that IGFBP6 was closely related to cell proliferation. Further validation confirmed that IGFBP6 was one of the most highly expressed proteins in varicose vein tissue. Knocking down IGFBP6 in VSMCs significantly attenuated cell proliferation and induced the S phase arrest during the cell cycle. Further experiments demonstrated that IGFBP6 knockdown increased cyclin E ubiquitination, which reduced expression of cyclin E and phosphorylation of CDK2. Furthermore, IGFBP6 knockdown arrested centrosome replication, which subsequently influenced VSMC morphology. Ultimately, IGFBP6 was validated to be involved in VSMC proliferation in varicose vein tissues. The present study reveals that IGFBP6 is closely correlated with VSMC biological function and provides unprecedented insights into the underlying pathogenesis of varicose veins.     

The hypoxia-induced microRNA-130a controls pulmonary smooth muscle cell proliferation by directly targeting CDKN1A

Excessive proliferation of human pulmonary artery smooth muscle cells (HPASMC) is one of the major factors that trigger vascular remodeling in hypoxia-induced pulmonary hypertension. Several studies have implicated that hypoxia inhibits the tumor suppressor p21 (CDKN1A). However, the precise mechanism is unknown.The mouse model of hypoxia-induced PH and in vitro experiments were used to assess the impact of microRNAs (miRNAs) on the expression of CDKN1A. In these experiments, the miRNA family miR-130 was identified to regulate the expression of CDKN1A. Transfection of HPASMC with miR-130 decreased the expression of CDKN1A and, in turn, significantly increased smooth muscle proliferation. Conversely, inhibition of miR-130 by anti-miRs and seed blockers increased the expression of CDKN1A. Reporter gene analysis proved a direct miR-130–CDKN1A target interaction. Exposure of HPASMC to hypoxia was found to induce the expression of miR-130 with concomitant decrease of CDKN1A. These findings were confirmed in the mouse model of hypoxia-induced pulmonary hypertension showing that the use of seed blockers against miR-130 restored the expression of CDKN1A.These data suggest that miRNA family miR-130 plays an important role in the repression of CDKN1A by hypoxia. miR-130 enhances hypoxia-induced smooth muscle proliferation and might be involved in the development of right ventricular hypertrophy and vascular remodeling in pulmonary hypertension.     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.