RETINAL MICROVASCULAR CELLS, MATRIX AND OCULAR DISORDERS

视网膜微血管细胞、基质和眼部疾病

• 批准号: 2628985
• 负责人: IRA M HERMAN
• 金额: $ 25.96万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2628985
• 关键词: affinity chromatography; angiogenesis; cell cell interaction; cell differentiation; cell growth regulation; extracellular matrix; fibroblast growth factor; gel mobility shift assay; gene complementation; gene expression; genetic regulatory element; histogenesis; immunoprecipitation; laboratory mouse; laboratory rabbit; polymerase chain reaction; protein tyrosine kinase; retina disorder; tissue /cell culture; transcription factor; transforming growth factors; vascular endothelium; vascular smooth muscle; western blottings

DESCRIPTION (Adapted from applicant's abstract): Experiments carried out in the investigator's laboratory have tested the general hypothesis that cell-cell and cell-matrix interactions modulate retinal microvascular growth and differentiation. Specifically, it addressed whether the endothelial-derived and matrix-associated growth regulators fibroblast growth factor (FGF-2) and transforming growth factor beta-1 (TGF-beta-1) influence retinal pericyte growth and contractile phenotype by signaling through molecular regulators of cell cycle progression and myogenic determination. Through the creation of in vitro and in vivo models, advantage will be taken of dominant-negative mutations in pericyte receptor tyrosine and serine/threonine kinases (RTKs, STKs), and knock-out mice harboring targeted disruptions in TGF-beta-1 and/or the cell cycle inhibitor p27 to directly establish the molecular mechanisms regulating the pericyte recruitment and differentiation during retinal microvascular remodeling. Using molecular, biochemical and cell biologic approaches, retroviral and plasmid-mediated gene delivery systems will be used to stably express high levels of truncation and point mutant RTK and STK in neonatal and adult pericyte cultures. Promoter-luciferase, in vitro kinase and receptor complementation analyses, and immunoprecipitation/Western blot will conclusively establish those downstream signaling components and cell cycle regulators required for promoting FGF and TGF-beta-1 mediated signal transduction. In addition, based on recent evidence accumulating in the applicant's laboratory which indicate that novel relationships may exist between the regulation of pericyte myogenic determination and cell cycle arrest, it is proposes to identify the molecular events controlling pericyte myogenic determination and contractile phenotype by characterizing the cis-regulatory and trans-activating factors responsible for regulating pericyte smooth muscle contractile protein expression, beginning with the vascular smooth muscle actin (VSMA) gene as a model. Promoter-luciferase, electrophoretic mobility shift and super-shift assays as well as DNA sequence-specific affinity chromatography will not only reveal the transcriptional regulators of pericyte contractile phenotype, but may also help to provide important new insights regarding apoptosis protection during myogenic commitment/differentiation. This knowledge will prove invaluable in order to unravel the molecular events controlling retinal microvascular morphogenesis during normal development or in association with retinal vasoproliferative disorders.

描述（改编自申请人的摘要）： 研究人员的实验室已经验证了一般假设， 细胞-细胞和细胞-基质相互作用调节视网膜微血管生长 和差异化。 具体而言，它涉及是否 内皮源性和基质相关生长调节因子成纤维细胞 生长因子（FGF-2）和转化生长因子β-1（TGF-β-1） 信号转导对视网膜周细胞生长和收缩表型影响 通过细胞周期进程和肌源性的分子调节剂 保持战略定力 通过建立体外和体内模型， 将利用周细胞受体中的显性负突变 酪氨酸和丝氨酸/苏氨酸激酶（RTK，STKs），以及敲除小鼠 携带TGF-β-1和/或细胞周期抑制剂的靶向破坏 p27直接建立调节周细胞的分子机制 在视网膜微血管重塑过程中的募集和分化。 利用分子、生物化学和细胞生物学方法， 质粒介导的基因递送系统将用于稳定表达高表达的 新生儿和成人中截断和点突变RTK和STK的水平 周细胞培养。 启动子-荧光素酶、体外激酶和受体 互补分析和免疫沉淀/Western印迹将 最终确定那些下游信号成分和细胞周期 促进FGF和TGF-β 1介导的信号所需的调节剂 转导 此外，根据最近积累的证据， 申请人的实验室，表明可能存在新的关系 周细胞生肌决定的调控与细胞周期之间的关系 逮捕，它建议确定控制周细胞的分子事件 生肌决定和收缩表型， 顺式调节因子和反式激活因子 周细胞平滑肌收缩蛋白的表达，从 血管平滑肌肌动蛋白（VSMA）基因作为模型。 启动子-荧光素酶， 电泳迁移率变动和超变动分析以及DNA 序列特异性亲和层析不仅可以揭示 周细胞收缩表型的转录调节因子，但也可能 有助于提供重要的新的见解，关于细胞凋亡的保护， 生肌定型/分化。 这些知识将证明是无价的 为了解开控制视网膜微血管的分子事件 在正常发育过程中或与视网膜相关的形态发生 血管增生性疾病。