Function and Regulation of TSPAN2 in Vascular Disease

项目摘要 分化的血管平滑肌细胞（VSMCs）在遗传上被编程为增殖和迁移， 对成人血管进行收缩时的心率较低。VSMC不是终末分化的 并且可以响应于各种细胞因子而经历向去分化合成模式的表型转换。 病理生理刺激，导致动脉粥样硬化和再狭窄等多种血管疾病。 由于细胞表面的可接近性，VSMC分化所需的细胞膜调节因子可能保持不变。 有吸引力的治疗潜力。然而，关于关键的跨膜调节子的信息很少 控制VSMC分化。TSPAN具有独特的信号传导平台，称为富含四跨膜蛋白的TSPAN。 微结构域（TEM）与TSPAN和其他跨膜配偶体（包括受体）顺式组织 酪氨酸激酶和整联蛋白。TSPAN充当膜“对接”分子，在细胞膜的形成和细胞膜的形成中发挥关键作用。 不同的信号级联最近的全基因组关联研究已经将TSPAN 2与 动脉粥样硬化和血压控制。受这些发现的启发，我们询问了来自多个RNA的数据， 筛选，并一致发现，TSPAN 2是唯一的TSPAN成员显着下降， VSMC表型调节和病变血管。TSPAN 2富集于VSMC中并表现出血清 反应因子（SRF）/心肌素（MYOCD）依赖性表达。新的功能数据显示， VSMC中TSPAN 2的缺失减弱收缩基因表达，同时促进VSMC增殖 和迁移; TSPAN 2的强制表达阻断了球囊损伤模型中的新生内膜形成。TSPAN 2 与CD 44蛋白相互作用并促进CD 44蛋白的降解，CD 44蛋白是血管病变中的一种重要病理介质， 疾病此外，TSPAN 2与层粘连蛋白结合整联蛋白α3β1相互作用，而β1整联蛋白对于TSPAN 2的表达是必需的。 维持VSMC收缩表型。这些初步发现支持了一个新的假设 TSPAN 2稳定VSMC收缩表型并通过两种不同的途径抑制血管病理学， 途径：通过促进CD 44蛋白的降解，使合成的VSMC表型失活， 与促进收缩表型的整合素α3β1相互作用。提出了三个具体目标， 测试这个假设。目的1探讨TSPAN 2在动静脉瘘和动脉粥样硬化中的作用 小鼠模型。目的2将阐明TSPAN 2稳定VSMC收缩的机制， 表型涉及通过促进CD 44蛋白降解使合成的VSMC表型失活， 通过与整合素α3β1相互作用激活收缩表型。目的3将阐明分子 在病理性血管条件下，通过干扰TSPAN 2基因表达， SRF/MYOCD通路。这项提案的成功完成将揭示以前未知的分子 涉及关键跨膜调节因子作用的VSMC表型可塑性调控机制， 这将潜在地导致对血管疾病的更有效的治疗。

PROJECT SUMMARY Differentiated vascular smooth muscle cells (VSMCs) are genetically programmed to proliferate and migrate at low rate while carrying out contractility for adult vessel homeostasis. VSMCs are not terminally differentiated and can undergo phenotypic switching to a dedifferentiated synthetic mode in response to various pathophysiological stimuli, contributing to diverse vascular diseases such as atherosclerosis and restenosis. Due to cell surface accessibility, cell membrane regulators required for VSMC differentiation may hold attractive therapeutic potential. However, there is a paucity of information on key transmembrane regulators governing VSMC differentiation. TSPANs possess a unique signaling platform, termed as tetraspanin-enriched microdomains (TEMs) organized in cis with TSPANs and other transmembrane partners including receptor tyrosine kinases and integrin(s). TSPANs serve as membrane “docking” molecules, exerting critical roles in diverse signal cascades. Recent genome wide association studies have specifically linked TSPAN2 to atherosclerosis and blood pressure control. Inspired by these findings, we interrogated data from multiple RNA screenings and consistently found that TSPAN2 is the sole TSPANs member significantly decreased during VSMC phenotypic modulation and in diseased vessels. TSPAN2 is enriched in VSMCs and exhibits serum response factor (SRF) /Myocardin (MYOCD)-dependent expression in vitro. New functional data shows that depletion of TSPAN2 in VSMCs attenuates contractile gene expression, while promoting VSMC proliferation and migration; forced expression of TSPAN2 blocks neointima formation in a balloon injury model. TSPAN2 interacts with and promotes degradation of CD44 protein, a prominent pathological mediator in vascular disease. In addition, TSPAN2 interacts with the laminin-binding integrin α3β1, and β1 integrins are essential to the maintenance of the VSMC contractile phenotype. These preliminary findings support a novel hypothesis that TSPAN2 stabilizes the VSMC contractile phenotype and suppresses vascular pathology via two distinct pathways: by promoting degradation of CD44 protein which inactivates the synthetic VSMC phenotype and interaction with integrin α3β1 which facilitates the contractile phenotype. Three specific aims are proposed to test this hypothesis. Aim 1 will determine functions of TSPAN2 in arteriovenous fistula and atherosclerosis mouse models. Aim 2 will elucidate the mechanisms through which TSPAN2 stabilizes the VSMC contractile phenotype involving inactivation of the synthetic VSMC phenotype by promoting CD44 protein degradation and activation of the contractile phenotype by interacting with integrin α3β1. Aim 3 will elucidate the molecular basis for the impaired TSPAN2 gene expression under pathological vascular conditions via perturbation of SRF/MYOCD pathway. Successful completion of this proposal will reveal previously unknown molecular mechanisms governing VSMC phenotypic plasticity involving the actions of key transmembrane regulators, which will potentially lead to more effective therapies for vascular diseases.