VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL PROTEIN KINASE C

血管平滑肌和内皮蛋白激酶 C

• 批准号: 2415610
• 负责人: J Anthony Ware
• 金额: $ 29.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415610
• 关键词: angiogenesis; biological signal transduction; cell migration; enzyme inhibitors; enzyme structure; gene expression; integrins; intracellular; isozymes; laboratory rat; mitogens; phosphorylation; polymerase chain reaction; protein kinase C; receptor binding; receptor expression; tissue /cell culture; transfection; vascular endothelium; vascular smooth muscle; western blottings

The purpose of the proposed study is to define completely the role of the intracellular enzyme protein kinase C (PKC) in proliferation and migration of endothelial cells, which are key processes in both the formation of new blood vessels (angiogenesis) as well as the repair of damaged endothelium. PKC, rather than being a single enzyme, is a family of related but structurally distinct isoenzymes encoded by different genes and with different cofactor requirements and substrates. Thus, it is possible that each PKC isoenzyme may selectively mediate separate effects on proliferation or migration of vascular cells, which is a principal hypothesis that will be tested in this proposal. The first specific aim is to create and characterize three classes of molecular inhibitors that selectively inhibit the function or expression of individual PKC isoenzymes normally expressed in endothelium. These three classes include pseudosubstrate, antisense, and kinase negative (dominant negative) inhibitors of each isoenzyme, and will be overexpressed to create lines of endothelial cells deficient in one or more of these isoenzymes. In the second specific aim, an isoenzyme or subset of isoenzymes of PKC that mediate migration, proliferation and vessel-forming capability of endothelium will be identified by testing the ability of endothelial cells that overexpress the PKC inhibitors created as part of Specific Aim #1 to respond to mitogens. Those lines of endothelial cells that exhibit altered proliferation or migration will be tested in a three dimensional angiogenesis assay. These functional phenotypes will be compared with phosphorylation of intracellular substrates for PKC in these cells. In the third specific aim, the mechanism by which individual PKC isoenzymes might modulate endothelial proliferation or migration will be explored by testing a model for endothelial response to mitogens. In this model, activation of individual PKC isoenzymes affects, and is affected by, the function of the endothelial integrin receptor for adhesive proteins and the activity of kinases and protooncogenes that mediate distal steps in the signal transduction pathway leading to endothelial proliferation and migration. Together, these three interrelated projects comprise a research program that should yield novel information regarding the physiologic and possible therapeutic role of the PKC gene family in endothelial and vascular cells and may provide details critical to our understanding of endothelial repair following injury and angiogenesis during disease states.

拟议研究的目的是完全定义 细胞内酶蛋白激酶C（PKC）在增殖和迁移中 内皮细胞，这是新形成的关键过程 血管（血管生成）以及受损内皮的修复。 PKC而不是单一酶，是一个相关的家族，但 结构上不同的同工酶由不同的基因编码， 不同的辅因子要求和基材。因此，有可能 每个PKC同工酶可以选择性地介导对 血管细胞的增殖或迁移，这是主要的 该提议将在该提议中进行检验的假设。第一个具体目的是 创建和表征三类的分子抑制剂 有选择地抑制单个PKC的功能或表达 通常在内皮中表达的同工酶。这三个课程包括 假底物，反义和激酶阴性（主要阴性） 每个同工酶的抑制剂，并将过表达以创建线条 内皮细胞缺乏其中一个或多个这些同工酶。在 第二个特定目标，同工酶或PKC的同工酶的子集 介导迁移，增殖和船舶形成能力 内皮将通过测试内皮细胞的能力来鉴定 过表达PKC抑制剂作为特定目标＃1的一部分创建的 回应有丝分裂剂。表现出改变的内皮细胞的线条 扩散或迁移将在三维中测试 血管生成测定。这些功能表型将与 这些细胞中PKC的细胞内底物的磷酸化。在 第三个特定目的，单个PKC同工酶可能的机制 调节内皮的增殖或迁移将由 测试一个模型，以实现对有丝分裂剂的内皮反应。在这个模型中， 单个PKC同工酶的激活会影响，并受到影响，并受到影响 内皮整合素受体的功能，可用于粘合蛋白和 激酶和原子基因介导远端步骤的活性 信号转导途径导致内皮增殖和 迁移。这三个相互关联的项目一起组成了一项研究 应该产生有关生理和生理学的新信息的程序 PKC基因家族在内皮和 血管细胞，可能会为我们理解至关重要 疾病后的内皮修复和疾病血管生成后 国家。