Signaling pathways in human vascular smooth muscle cells

人血管平滑肌细胞的信号通路

• 批准号: 6527527
• 负责人: Debbie Beasley
• 金额: $ 27.65万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-13 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527527
• 关键词: antisense nucleic acid; cell proliferation; cyclins; gel mobility shift assay; gene expression; gene induction /repression; genetic promoter element; human tissue; hypoxia; pulmonary hypertension; tissue /cell culture; transcription factor; transfection; vascular smooth muscle; western blottings

It has long been thought that hypoxia is a stimulus for human vascular smooth muscle cell (HVSMC) proliferation occurring in pulmonary hypertension, however, the mechanisms involved are unknown. Based on our novel finding that hypoxia directly stimulates proliferation in cultured HVSMC, the main objective of this proposal is to determine the role of hypoxia-inducible transcription factors (HIFs) in the O2-dependent regulation of HVSMC proliferation. Numerous studies have demonstrated a crucial role of HIF-1, a dimer consisting of alpha and beta subunits, in hypoxia-inducible gene transcription. Our preliminary data indicate that hypoxia induces increased nuclear levels of HIF-1alpha and HIF-2alpha protein (by Western blot analysis), increased nuclear HIF complexes which bind to a hypoxia response element (by EMSA), and enhanced HIF-dependent reporter gene expression, in HVSMC. Furthermore, transient overexpression of HIF-1alpha or HIF-2alpha stimulates HVSMC proliferation. Based on these findings, the proposed studies will test the hypothesis that hypoxia induces human VSMC proliferation via upregulation of HIFs, and will further explore the role of downstream signaling pathways in their mitogenic effect. Transient and stable transfection techniques, adenoviral vectors, antisense oligonucleotides, electrophoretic mobility shift assays, Western blot and promoter activity analysis will be used to address the following specific aims: to determine whether HIF transcription factors mediate the proliferative responses of human VSMC to hypoxia; to characterize HIF-dependent gene promoters that are involved in the subsequent mitogenic effect. Elucidating the roles of hypoxia-inducible transcription factors in human VSMC proliferation will enhance our understanding of the pathogenesis of vascular diseases such as pulmonary hypertension, and may lead to identification of novel therapeutic targets for pharmacologic intervention in this disease.

长期以来，人们一直认为缺氧是肺动脉高压中人类血管平滑肌细胞（HVSMC）增殖的刺激因素，但其机制尚不清楚。 基于我们的新发现，即缺氧直接刺激培养的 HVSMC 增殖，本提案的主要目的是确定缺氧诱导转录因子 (HIF) 在 HVSMC 增殖的 O2 依赖性调节中的作用。 大量研究表明，HIF-1（一种由 α 和 β 亚基组成的二聚体）在缺氧诱导的基因转录中发挥着至关重要的作用。 我们的初步数据表明，缺氧会导致 HIF-1α 和 HIF-2α 蛋白的核水平增加（通过蛋白质印迹分析）， 增加 HVSMC 中与缺氧反应元件结合的核 HIF 复合物（通过 EMSA），并增强 HIF 依赖性报告基因表达。 此外，HIF-1α 或 HIF-2α 的瞬时过度表达会刺激 HVSMC 增殖。 基于这些发现，拟议的研究将检验缺氧通过上调 HIF 诱导人 VSMC 增殖的假设，并将进一步探讨下游信号通路在其促有丝分裂作用中的作用。 瞬时和稳定转染技术、腺病毒载体、反义寡核苷酸、电泳迁移率变动分析、蛋白质印迹和启动子活性分析将用于解决以下具体目标：确定HIF转录因子是否介导人VSMC对缺氧的增殖反应；表征参与后续有丝分裂效应的 HIF 依赖性基因启动子。 阐明缺氧诱导转录因子在人 VSMC 增殖中的作用将增强我们对血管疾病发病机制的理解，例如 肺动脉高压，并可能导致确定对该疾病进行药物干预的新治疗靶点。