利用FM4-64FX标记大鼠血管平滑肌细胞的囊泡运输

囊泡运输是大分子物质进入细胞的途径,血管平滑肌细胞(vascular smooth muscle cells,VSMCs)与外界存在频繁的信息和物质交换,该研究通过标识内吞囊泡来研究VSMCs的囊泡运输。体外培养大鼠胸主动脉VSMCs,用血管紧张素II(angiotensin II,Ang II)刺激,加入FM4-64FX短暂孵育后固定。通过免疫组化方法标记VSMCs血管紧张素II 1型受体(angiotensin II receptor type 1,AT1R),检测内吞囊泡和AR1R转运之间的关系。受到Ang II的激活后,VSMC快速形成内吞囊泡,将AT1R转运至胞质;存在血管紧张素受体阻断剂(angiotensin receptor blocker,ARB)时,内吞囊泡数量少,AT1R较少进入胞质。通过FM4-64 FX对胞内囊泡进行标识可以显示VSMCs的大分子物质运输,可观察特定的分子在内吞囊泡上的分布和运输情况。     

RhoA和血清反应因子(SRF)介导E1A激活基因阻遏子诱导人血管平滑肌细胞分化

为探讨E1A激活基因阻遏子(CREG)对人血管平滑肌细胞(VSMCs)分化的调控机制,应用重组逆转录病毒表达载体pLNCX2( )/CREG及pLXSN(-)/CREG制备稳定感染HITASY细胞模型,观察CREG蛋白过表达及表达抑制对人VSMCs分化的影响并探讨其调控机制.结果显示:pLNCX2( )/CREG稳定感染细胞呈分化表型,细胞细长变成组织样聚集生长趋势,细胞中CREG蛋白和平滑肌分化标志蛋白平滑肌α-肌动蛋白(SMα-actin)表达显著增加,同时SMα-actin相关调控因子——血清反应因子(SRF)入核转位,RhoA总蛋白表达上调,以Rho激酶特异性抑制剂Y-27632作用后,CREG诱导的SMα-actin表达下调的同时SRF出核转位;pLXSN(-)/CREG稳定感染的细胞体积变大,细胞极性消失,呈无序生长,细胞中CREG和SMα-actin蛋白表达显著降低,同时伴有SRF出核转位及RhoA总蛋白表达下调.免疫共沉淀分析发现,CREG蛋白能被分泌到VSMCs培养基中表达,并可与细胞膜受体6-磷酸甘露糖/胰岛素样生长因子Ⅱ型受体(M6P/IGF2R)发生直接相互作用.用蛋白磷酸酶PP2A特异性抑制剂okadaicacid减少M6P/IGF2R在细胞膜表面分布,可明显抑制CREG过表达引起的RhoA、SRF和SMα-actin表达.上述结果提示,在体外培养的人VSMCs中,CREG可能作为一种分泌型蛋白质通过与细胞膜受体M6P/IGF2R相互作用,依次激活SMα-actin蛋白相关调控因子RhoA和SRF引起SMα-actin表达增加,促进VSMCs向分化表型转换.     

小鼠皮肤黑色素瘤细胞在三种不同基质上的趋电性比较

微小直流电场具有指导细胞进行定向迁移的作用。各种细胞外基质的物理、化学性质会影响细胞的迁移。该研究以小鼠皮肤黑色素瘤细胞（B16-F10）为模型,比较微直流电场（250mV／mm）指导下细胞在平滑基底与两种不同市售基质Matrigel及FNC上的趋电性。结果显示,黑色素瘤细胞在三种基底上均有明显的向电场阴极迁移的趋电运动,但在不同基质上细胞趋电的方向性无显著差异,但细胞迁移速度及在细胞沿电场进行定向迁移的持续性有显著差异。     

Rho-GDIα抑制尼古丁诱导的血管平滑肌细胞迁移

研究尼古丁对大鼠胸主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)迁移的影响和机制,为探讨吸烟加速动脉粥样硬化等心血管疾病的机制研究提供实验依据。用终浓度为100μmol/L的尼古丁作用于体外培养的大鼠VSMCs 24 h,以不加尼古丁的细胞为对照组,采用迁移小室法(Transwell)和划痕法检测有或无尼古丁条件下VSMCs的迁移情况,实时定量RT-PCR检测Rho家族活性调节因子Rho-GDIα的表达;RNA干扰抑制Rho-GDIα的表达,探讨其在尼古丁诱导的VSMCs迁移中的作用。迁移小室法和划痕法分别显示了尼古丁组的VSMCs迁移数量和距离明显比对照组的多和远;尼古丁显著抑制Rho-GDIα的mRNA表达;RNA干扰Rho-GDIα后,VSMCs的迁移能力明显提高。结果提示,尼古丁可能部分通过抑制Rho-GDIα的表达促进VSMCs的迁移。     

血小板衍生生长因子受体结构与功能的研究

血小板衍生生长因子受体(platelet-derived growth factor receptor, PDGFR),包括PDGFRα和PDGFRβ,属于第三类受体酪氨酸激酶家族.它们参与血管再生和创伤修复等重要生理过程,并能够促进肿瘤细胞的增殖、迁移与存活.目前,在多种肿瘤、纤维化以及心血管疾病中均检测到高表达或突变的PDGFR受体以及高表达的血小板衍生生长因子(platelet-derived growth factor, PDGF), PDGF/PDGFR已成为基础生物学与转化医学研究所关注的重要靶点.本文总结了当前关于PDGFR的结构与功能研究,对PDGF/PDGFR信号传导机制以及PDGF/PDGFR信号异常与疾病的关系进行了梳理,论述了针对该信号通路的多种靶向药物的治疗机制,并对今后研究方向进行了展望.     

血小板源性生长因子对血管平滑肌细胞效应的机制研究进展

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖及迁移是动脉粥样硬化(arteriosclerosis,AS)、支架后再狭窄(in-stent restenosis,ISR)发生、发展中共同的病理过程。血小板源性生长因子(platelet-derived growth factor,PDGF)是一种强效的促有丝分裂剂,在VSMCs增殖及迁移过程中具有重要作用。本文综述PDGF的生物学作用及其促进VSMCs增殖及迁移的机制研究新进展,特别是通过抑制该靶点对VSMCs增殖及迁移的意义。     

血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。     

PIEZO1影响HaCaT细胞的趋电性迁移

目的 伤口中心产生的内源性电场是指导细胞定向迁移促进伤口愈合的重要因子。PIEZO1是机械门控阳离子通道家族成员之一,它参与细胞迁移,影响细胞的趋化迁移,并且受到电压的调节,在伤口愈合过程中发挥重要作用。但PIEZO1是否参与影响电场指导的细胞定向迁移过程尚不清晰,本文以HaCaT细胞为模型探讨PIEZO1及其下游相关蛋白质对细胞趋电性迁移的影响。方法 应用活细胞工作站追踪HaCaT细胞在微直流电场中的迁移,使用抑制剂及RNAi技术调控PIEZO1功能和表达,研究PIEZO1对于细胞趋电性迁移的影响;用蛋白质印迹（Western blot）检验细胞的整合素（integrin） β1与FAK磷酸化水平在电场作用下的变化情况,探讨PIEZO1与integrin β1及FAK等对电场信号的响应及细胞趋电性迁移的影响。结果 PIEZO1广谱抑制剂钌红、GsMTx4和RNAi处理显著抑制了HaCaT细胞向正极趋电性迁移的能力;电场和GsMTx4单独作用升高FAK磷酸化和integrin β1表达,GsMTx4阻止电场进一步升高FAK的磷酸化水平和integrin β1表达;siRNA干扰PIEZ...     

血管紧张素Ⅱ通过其1型受体诱导胰岛β细胞TXNIP的表达

本研究旨在探讨血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对INS-1胰岛细胞凋亡及硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)表达的影响,并分析其可能的作用机制。体外常规培养大鼠胰岛细胞株INS-1,使用CCK-8试剂盒检测不同浓度和不同作用时间的Ang Ⅱ对细胞存活率的影响,确定最佳作用浓度及作用时间为1×10~(-6) mol/L和24 h;在上述浓度及作用时间条件下,使用流式细胞术及Western blot检测细胞凋亡;Western blot检测Ang Ⅱ对TXNIP、碳水化合物反应元件结合蛋白(carbohydrate response element-binding protein,ChREBP)及血管紧张素Ⅱ 1型受体(angiotensin Ⅱ type 1 receptor,AT1R)蛋白表达的影响;Real-time PCR检测TXNIP及ChREBP mRNA表达;IF/ICC法观察TXNIP、ChREBP及AT1R的表达变化。结果显示Ang Ⅱ可浓度依赖性及时间依赖性地降低细胞活力(P0.05,n=6)并上调TXNIP的表达(P0.05,n=6);与对照组相比,Ang Ⅱ组细胞凋亡率、ChREBP及AT1R的表达均明显增高(P0.05,n=6)。使用AT1R受体抑制剂替米沙坦(telmisartan,TM)后,Ang Ⅱ对INS-1细胞TXNIP及ChREBP的诱导作用被抑制(P0.05,n=6),Ang Ⅱ诱导的细胞活力降低及细胞凋亡升高被逆转。上述结果表明,Ang Ⅱ可通过AT1R增加ChREBP活化而上调TXNIP的表达,促进细胞凋亡,提示TXNIP可能在糖尿病时Ang Ⅱ诱发胰岛细胞凋亡中发挥作用,并有望成为糖尿病新的治疗靶点。     

血管平滑肌细胞在自发性高血压大鼠颈动脉重构中的作用及替米沙坦的干预研究

观察血管平滑肌细胞(VSMCs)在自发性高血压大鼠(SHR)颈动脉重构中的作用及替米沙坦的干预效果.将30只12周龄的SHR随机分为高血压组(SHR)、替米沙坦高剂量组(TelH)、替米沙坦低剂量组(TelL),另设同性别、周龄的WKY大鼠为对照组(n=10),干预18周.观察各组大鼠收缩压(SBP)、颈动脉中膜厚度(MT)、中膜横截面积(MCSA)、中膜细胞平均核面积、颈动脉 VSMCs增殖指数(PI)及凋亡指数(AI)等的变化.结果显示: ①两周后TelH组SBP明显低于SHR组(P＜0.01),其降压作用持续至实验结束,而TelL组SBP与SHR组无显著性差异(P＞0.05);②SHR组的MT、MCSA分别明显高于WKY组(P＜0.01),TelH组的MT、MCSA分别明显低于SHR组(P＜0.01),TelL组的MT明显低于SHR组(P＜0.05);③SHR组中膜VSMCs平均核面积明显大于WKY组(P＜0.01),而TelH、TelL组分别小于SHR组(P＜0.05);④各组颈动脉中膜VSMCs的PI均无明显差异(P＞0.05);SHR组颈动脉中膜VSMCs的AI明显低于WKY组(P＜0.01),而TelH、TelL组明显高于SHR组(P＜0.01);SHR组颈动脉中膜VSMCs的PI/AI明显高于WKY组(P＜0.01),而TelH、TelL组明显低于SHR组(P＜0.01);⑤颈动脉中膜VSMCs的AI与中膜MCSA呈显著负相关(r = -0.871,P＜0.01 ).说明VSMCs的肥大和增殖/凋亡失衡可能在SHR颈动脉重构中起重要作用,替米沙坦除降压作用外,能通过减轻VSMCs肥大,增加VSMCs凋亡,使增殖/凋亡趋于平衡而减轻其重构.     

Regulation of PDGF production and ERK activation by estrogen is associated with TSC2 gene expression

Mechanisms that regulate the growth response to estrogen (17beta-estradiol, E2) are poorly understood. Recently, loss of function of the tuberous sclerosis complex 2 (TSC2) gene has been associated with E2-related conditions that are characterized by benign cellular proliferation. We examined the growth response to E2 in vascular smooth muscle cells (VSMCs) that possess wild-type TSC2 and compared them with ELT-3 smooth muscle cells that do not express TSC2. In TSC2-expressing VSMCs, growth inhibition in response to E2 was associated with downregulation of platelet-derived growth factor (PDGF), PDGF receptor (PDGFR), and limited activation of extracellular signal-regulated kinase (ERK). In contrast, the growth-promoting effect of E2 in TSC2-null ELT-3 cells was associated with induction of PDGF, robust phosphorylation of PDGFR, and sustained activation of ERK. Furthermore, in ELT-3 cells, cellular growth and ERK activation by E2 were inhibited by the PDGFR inhibitor tyrphostin AG 17 and by PDGF-neutralizing antibody. These results demonstrate that autocrine production of PDGF and augmentation of the ERK pathway leads to estrogen-induced cellular proliferation in TSC2-null cells, a pathway that was downregulated in cells that express TSC2. Understanding the mechanisms that regulate the diverse responses to the steroid hormone estrogen could lead to novel approaches to the treatment of estrogen-related diseases that are characterized by aberrant cell proliferation.     

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.     

Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells.

IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross-linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A naturally occurring carotenoid,lutein, reduces PDGF and H<Subscript>2</Subscript>O<Subscript>2</Subscript> signaling and compromises migration in cultured vascular smooth muscle cells

Background  

Platelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs.     

Both AT₁ and AT₂ receptors mediate proliferation and migration of porcine vascular smooth muscle cells

Angiotensin receptor antagonists have shown clinical promise in modulating vascular disease, in part by limiting smooth muscle cell proliferation and migration. The majority of studies examining the contribution of these receptors have been undertaken in cells derived from rat aorta, which primarily express the ANG II type 1 (AT(1)) receptor. This investigation studied the relative contribution of AT(1) and ANG II type 2 (AT(2)) receptors to the mitogenic program of porcine smooth muscle cells. Smooth muscle cells were derived from porcine coronary artery explants. The presence of both AT(1) and AT(2) receptors was demonstrated through ligand binding and RT-PCR analysis. Biochemical and cellular markers of proliferation were monitored in the presence of selective receptor antagonists. Smooth muscle cell migration was measured using both wound healing and Boyden chamber migration assays. Visualization of the AT(1) and AT(2) receptors in growing and quiescent porcine smooth muscle cells with epifluorescence microscopy demonstrated that their subcellular distribution varied with growth state. An examination with several growth assays revealed that both AT(1)-specific losartan and AT(2)-specific PD-123319 receptor antagonists inhibited ANG II-stimulated RNA and DNA synthesis, PCNA expression, and hyperplasia. ANG II induced both directional and nondirectional cell migration. AT(1) receptor antagonist treatment significantly decreased ANG II-induced directional migration only, whereas AT(2) receptor antagonist treatment significantly reduced both modes of migration. Interestingly, the focal adhesion kinase inhibitor PF-573228 also blocked migration but not proliferation. Furthermore, focal adhesion kinase activation in response to ANG II was prevented only by PD-123319, indicating that this activation is downstream of the AT(2) receptor. The observed role of the AT(2) receptor in ANG II-induced migration was confirmed with smooth muscle cells depleted of the AT(2) receptor with short hairpin RNA treatment.     

The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is a highly hydrophobic protein which appears to transform cells through the activation of growth factor receptors. To investigate the specificity of E5-growth factor receptor interactions required for mitogenic signaling, we utilized a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin-3 (IL-3) for sustained proliferation in culture. This IL-3 dependence can be functionally substituted by the expression of a variety of surrogate growth factor receptors and the addition of the corresponding ligand. Several receptor cDNAs for the alpha- and beta-type platelet-derived growth factor receptors [alpha PDGFR and beta PDGFR], the epidermal growth factor receptor, and the colony-stimulating factor 1 receptor) were transfected into 32D cells constitutively expressing the E5 protein to test for IL-3-independent growth. Only beta PDGFR was capable of abrogating the IL-3 dependence of 32D cells. The proliferative signal induced by the coexpression of beta PDGFR and E5 was accompanied by stable complex formation between these proteins, constitutive tyrosine phosphorylation of the receptor, and tumorigenicity in nude mice. The lack of cooperative interaction between E5 and the epidermal growth factor receptor, the colony-stimulating factor 1 receptor, and the highly related alpha PDGFR was paralleled by the inability of E5 to bind to these receptors and failure to increase receptor tyrosine phosphorylation. Thus, these data indicate that the ability of E5 to induce sustained proliferation and transformation of 32D cells is a direct consequence of specific interaction between the E5 protein and the beta PDGFR signaling complex and the subsequent stimulation of receptor tyrosine phosphorylation.     

c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.     

Endothelial and Smooth Muscle-derived Neuropilin-like Protein Regulates Platelet-derived Growth Factor Signaling in Human Vascular Smooth Muscle Cells by Modulating Receptor Ubiquitination

Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is up-regulated in the neointima of remodeling arteries and modulates vascular smooth muscle cell (VSMC) growth. Platelet-derived growth factor (PDGF) is the prototypic growth factor for VSMCs and plays a key role in vascular remodeling. Here, we sought to further define ESDN function in primary human VSMCs. ESDN down-regulation by RNA interference significantly enhanced PDGF-induced VSMC DNA synthesis and migration. This was associated with increased ERK1/2, Src, and PDGF receptor (PDGFR)β phosphorylation, without altering total PDGFRβ expression levels. In binding assays, ESDN down-regulation significantly increased ¹²⁵I-PDGF maximum binding (B_max) to PDGF receptors on VSMCs without altering the binding constant (K_d), raising the possibility that ESDN regulates PDGFR processing. ESDN down-regulation significantly reduced ligand-induced PDGFRβ ubiquitination. This was associated with a significant reduction in the expression level of c-Cbl, an E3 ubiquitin ligase that ubiquitinylates PDGFRβ. Thus, ESDN modulates PDGF signaling in VSMCs via regulation of PDGFR surface levels. The ESDN effect is mediated, at least in part, through effects on PDGFRβ ubiquitination. ESDN may serve as a target for regulating PDGFRβ signaling in VSMCs.Vascular injury initiates a cascade of events that ultimately leads to vascular remodeling and often intimal hyperplasia. Vascular smooth muscle cell (VSMC)² proliferation and migration are key cellular events in this process. Platelet-derived growth factor (PDGF)-BB is released by platelets, endothelial cells, VSMCs, and inflammatory cells at the sites of vascular injury and is a particularly potent regulator of VSMC proliferation and migration (1). PDGF binding to PDGF receptor (PDGFR)β in VSMCs leads to receptor dimerization, autophosphorylation, and activation of downstream signaling pathways, including MAPK. The ligand-bound receptor is internalized through the endocytotic pathway and may either recycle to the membrane or undergo ubiquitination and lysosomal degradation (2). A number of endogenous stimulatory and inhibitory regulators, including the E3 ubiquitin ligase, c-Cbl (3), tightly regulate the mitogenic stimulus by modulating the duration and intensity of the signal.We have identified endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN, also called CLCP1 or DCBLD2) as a marker and regulator of cell proliferation in vascular remodeling (4). ESDN is a transmembrane protein with a domain structure similar to neuropilins (5, 6). ESDN can be induced by PDGF-BB and serum and is highly expressed in the neointima of injured rat (5), mouse (4), and human (4) arteries. ESDN expression parallels cell proliferation in the vessel wall in vivo (4). Furthermore, ESDN is up-regulated in proliferating VSMCs, and ESDN overexpression inhibits VSMC growth (4). Here, we expand the scope of our previous studies to demonstrate that ESDN regulates PDGF-induced VSMC migration and inhibits PDGF signaling in VSMCs. We further establish that this effect is mediated, at least in part, through changes in the surface expression of PDGF receptors. Finally, our study indicates that ESDN mediates PDGFRβ ubiquitination by regulating c-Cbl gene expression.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction