Signaling pathways in human vascular smooth muscle cells

人血管平滑肌细胞的信号通路

• 批准号: 6399101
• 负责人: Debbie Beasley
• 金额: $ 32.85万
• 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/6399101
• 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时血管平滑肌细胞（HVSMC）增殖的刺激因素，但其机制尚不清楚。 基于我们的新发现，缺氧直接刺激增殖培养的HVSMC，这一建议的主要目的是确定缺氧诱导的转录因子（HIF）的作用，在O2依赖性调节HVSMC增殖。 许多研究已经证明了HIF-1（由α和β亚基组成的二聚体）在缺氧诱导的基因转录中的关键作用。 我们的初步数据表明，缺氧诱导HIF-1 α和HIF-2 α蛋白的核水平增加（通过Western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-1 α或HIF-2 α的瞬时过表达刺激HVSMC增殖。 基于这些发现，拟议的研究将测试缺氧通过上调HIF诱导人VSMC增殖的假设，并将进一步探索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被用来解决以下具体目标：以确定是否HIF转录因子介导的人VSMC的增殖反应缺氧;表征HIF依赖的基因启动子，参与随后的促有丝分裂的效果。 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， 肺动脉高压，并可能导致确定新的治疗目标，药物干预这种疾病。