Function and Regulation of TSPAN2 in Vascular Disease

TSPAN2在血管疾病中的功能和调控

• 批准号: 10543854
• 负责人: Xiaochun Long
• 金额: $ 51.48万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-08 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543854
• 关键词: Adult; Allografting; Apolipoprotein E; Arteriovenous fistula; Atherosclerosis; Attenuated; Blood Vessels; CD44 gene; CRISPR/Cas technology; Cell Differentiation process; Cell membrane; Cell surface; Data; Dependence; Development; Disease; Docking; Electrophoretic Mobility Shift Assay; Endocytosis; Epigenetic Process; Exhibits; Failure; Gene Expression; Genes; Genetic Transcription; Goals; High Fat Diet; Homeostasis; Human; Impairment; In Vitro; Injury; Integrin Binding; Integrin alpha3beta1; Integrins; Knockout Mice; Knowledge; Laminin; Lesion; Link; Luciferases; Maintenance; Medial; Mediator; Membrane; Modeling; Molecular; Mus; Mutant Strains Mice; Operative Surgical Procedures; Pathologic; Pathology; Pathway interactions; Phenotype; Proliferating; Proteins; RNA; Receptor Protein-Tyrosine Kinases; Regulation; Role; Sampling; Serum Response Factor; Signal Transduction; Smooth Muscle Myocytes; Stimulus; Testing; Therapeutic; Translating; Vascular Diseases; Vascular Smooth Muscle; blood pressure control; cell motility; effective therapy; genome editing; genome wide association study; improved; in vitro Assay; in vivo; loss of function; member; migration; mouse model; myocardin; neointima formation; novel; programs; protein activation; protein degradation; response; restenosis; screening; vascular abnormality; vascular smooth muscle cell proliferation

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.

项目概要 分化的血管平滑肌细胞 (VSMC) 经过基因编程，可在以下时间增殖和迁移： 在进行成体血管稳态收缩时的低速率。 VSMC没有终末分化 并且可以响应各种不同的反应而进行表型转换至去分化的合成模式 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。 由于细胞表面可及性，VSMC 分化所需的细胞膜调节因子可能会存在 有吸引力的治疗潜力。然而，关于关键跨膜调节因子的信息很少 控制 VSMC 分化。 TSPAN 拥有独特的信号平台，称为富含四跨膜蛋白的信号平台 微结构域 (TEM) 与 TSPAN 和其他跨膜伙伴（包括受体）顺式组织 酪氨酸激酶和整合素。 TSPAN 作为膜“对接”分子，在 不同的信号级联。最近的全基因组关联研究已明确将 TSPAN2 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 TSPAN2 是唯一的 TSPAN 成员，在 VSMC 表型调节和病变血管。 TSPAN2 在 VSMC 中富集并表现出血清 反应因子（SRF）/心肌素（MYOCD）依赖性体外表达。新的功能数据表明 VSMC 中 TSPAN2 的缺失会减弱收缩基因表达，同时促进 VSMC 增殖 和移民； TSPAN2 的强制表达可阻止球囊损伤模型中新内膜的形成。 TSPAN2 与 CD44 蛋白相互作用并促进其降解，CD44 蛋白是血管中重要的病理介质 疾病。此外，TSPAN2 与层粘连蛋白结合整合素 α3β1 相互作用，而 β1 整合素对于 维持 VSMC 收缩表型。这些初步发现支持了一个新的假设 TSPAN2 通过两种不同的方式稳定 VSMC 收缩表型并抑制血管病理 途径：通过促进 CD44 蛋白的降解，使合成的 VSMC 表型失活， 与整合素α3β1相互作用，促进收缩表型。提出了三个具体目标 检验这个假设。目标 1 将确定 TSPAN2 在动静脉瘘和动脉粥样硬化中的功能 鼠标模型。目标 2 将阐明 TSPAN2 稳定 VSMC 收缩的机制 表型涉及通过促进 CD44 蛋白降解和合成 VSMC 表型失活 通过与整合素 α3β1 相互作用激活收缩表型。目标 3 将阐明分子 病理血管条件下 TSPAN2 基因表达受损的基础 SRF/MYOCD 途径。该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， 这将有可能带来更有效的血管疾病治疗方法。