RAPID ENDOTHELIAL RESPONSE TO REOXYGENATION INJURY

内皮细胞对复氧损伤的快速反应

• 批准号: 2750566
• 负责人: DAVID SHEPRO
• 金额: $ 25.27万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750566
• 关键词: actin binding protein; actins; cardiovascular disorder prevention; cellular pathology; cyclic AMP; digital imaging; free radical oxygen; gap junctions; gel electrophoresis; immunofluorescence technique; ischemia; microcirculation; microfilaments; neutrophil; phosphatidylinositols; physiologic stressor; prostaglandins; protein kinase C; reperfusion; second messengers; tissue /cell culture; vascular endothelium; vascular endothelium permeability; western blottings

DESCRIPTION: (Adapted from the Applicant's Abstract) The long term objective of the laboratory is to examine the physiology of microvessel endothelial cells (MEC) as it relates to the post-translational regulation of microvascular paracellular permeability. The underlying premise is that interendothelial junctional pores are not fixed, and the degree of patency is modulated by actin and actin-associated proteins. The applicant has developed an in vitro physiological model using cultured pulmonary MEC to investigate signal transduction cascades and to identify potential cytoskeletal targets associated with regulation of the microvascular barrier. Hypoxia/reoxygenation (H/R) injury mimicking, in part, ischemia-reperfusion injury is used to create an inflammatory setting. The overall hypothesis to be tested is that reactive oxygen species generated in reoxygenation injury act principally through activation of inflammatory second messenger pathways (e.g. protein kinase C, phosphatidylinositol 4,5-biphosphate) or inhibition of barrier promotion pathways (cAMP-dependent protein kinase) and not merely as nonspecific toxins. Through these pathways, the reactive oxygen species disrupt membrane-cytoskeleton interactions. This in turn destabilizes the plasma membrane and by so doing not only increases paracellular permeability but also increases MEC intracellular susceptibility to the cascade of reactive oxygen species. Conversely, the restorative response of junctional integrity, primarily acting through increases in cAMP, reestablishes membrane-cytoskeleton linkages. In parallel with a bubble chamber model for H/R injury in MEC, a simple chemical model is used to produce injury employing ATP-depleting agents and exogenously added reactive oxygen species, as well as inhibitors and activators of second messenger cascades. The question of whether repeated, minor exposures to H/R protect MEC against a major H/R event by the production of specific cytoskeletal proteins will also be examined. Lastly, assays will be performed to: (1.) Compare pulmonary MEC to other MEC phenotypes to ascertain unique injury responses; and (2.) Determine if the induced H/R injuries and repair coincide with changes in MEC shape and contractility associated with monolayer junctional permeability to macromolecules and neutrophil diapedesis. Hence, the regulation of paracellular permeability by membrane- cytoskeletal proteins may provide for the development of novel strategies for therapeutic or prophylactic intervention.

描述：（改编自申请人的摘要）长期 实验室的目的是检查微血管的生理学 内皮细胞（MEC），因为它与翻译后相关 微血管旁细胞通透性的调节。 底层的 前提是内皮间连接孔不是固定的， 通畅程度由肌动蛋白和肌动蛋白相关蛋白调节。 申请人开发了一种体外生理模型 培养肺 MEC 以研究信号转导级联 识别与调控相关的潜在细胞骨架靶点 微血管屏障。缺氧/复氧（H/R）损伤 部分模仿缺血再灌注损伤来创建 炎症环境。 要检验的总体假设是 复氧损伤作用中产生的活性氧 主要通过激活炎症第二信使途径 （例如蛋白激酶 C、磷脂酰肌醇 4,5-二磷酸）或 抑制屏障促进途径（cAMP 依赖性蛋白激酶） 而不仅仅是非特异性毒素。 通过这些途径， 活性氧破坏膜-细胞骨架相互作用。 这反过来又破坏了质膜的稳定性，这样做不仅 增加细胞旁通透性，同时也增加细胞内 MEC 对活性氧级联的敏感性。 反过来， 连接完整性的恢复反应，主要作用 通过增加 cAMP，重建膜-细胞骨架连接。 与 MEC 中 H/R 损伤的气泡室模型并行，一个简单的 化学模型用于利用 ATP 消耗剂产生损伤 和外源添加的活性氧，以及抑制剂和 第二信使级联的激活剂。 是否的问题 重复、轻微地接触 H/R 可保护 MEC 免受重大 H/R 事件的影响 通过产生特定的细胞骨架蛋白也将 检查了。 最后，将进行分析以： (1.) 比较肺部 MEC 与其他 MEC 表型的比较，以确定独特的损伤反应；和 (2.) 确定诱发的 H/R 损伤和修复是否与 与单层相关的 MEC 形状和收缩性的变化 对大分子的连接通透性和中性粒细胞渗出。 因此，细胞旁通透性的调节是通过膜- 细胞骨架蛋白可能有助于开发新的 治疗或预防性干预策略。