Mechanisms for Maintaining Endothelial Integrity During Inflammation

炎症期间维持内皮完整性的机制

• 批准号: 8322572
• 负责人: CHRISTOPHER V CARMAN
• 金额: $ 43.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-20 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322572
• 关键词: Actins; Address; Anti-Inflammatory Agents; Anti-inflammatory; Blood; Blood Clot; Blood Vessels; Blood coagulation; Cardiovascular system; Cells; Confocal Microscopy; Data; Disease; Electron Microscopy; Endothelium; Equilibrium; Exhibits; Exposure to; Extravasation; Genetic; Goals; Healed; Homeostasis; Image; Immune; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Leukocyte Trafficking; Leukocytes; Liquid substance; Mediating; Mediator of activation protein; Modeling; Molecular; NADPH Oxidase; Oxidative Stress; Oxygen; Pathologic; Pathology; Physiological; Plasma; Process; Property; Proteins; Reactive Oxygen Species; Recruitment Activity; Research; Role; Self-control as a personality trait; Signal Pathway; Signal Transduction; Structure; Surface; Therapeutic; Thrombosis; Time; Tissues; Wound Healing; Xanthine Oxidase; base; design; healing; human EMS1 protein; improved; in vivo; inhibitor/antagonist; loss of function; novel; novel therapeutics; restoration; tissue culture; wound

DESCRIPTION (provided by applicant): A central component of inflammatory pathology is breakdown of endothelial integrity and the resulting uncontrolled plasma leakage, leukocyte trafficking and thrombosis. Normal protective inflammatory responses exhibit micron-scale endothelial remodeling dynamics that balance disruptive activities with proactive self-restorative ones, thereby preserving overall integrity. Thus, recruited inflammatory cells breach the endothelial barrier by forming micron-scale para- and trans-cellular discontinuities, which serve as passageways for leukocyte transmigration (i.e., diapedesis). Normally, the endothelium closes these 'micro-wounds' immediately following passage of the leukocyte, thereby largely uncoupling them from plasma leak. In addition, soluble inflammatory mediators cause the endothelium itself to both create and (after minute-scale durations) close similar micro-wounds, thereby inducing controlled, self-limited vascular leak. Thus, the endothelium exhibits dynamic and proactive functions that efficiently 'heal' micro-wounds in order to maintain overall integrity and homeostasis during inflammation. Importantly, in pathologic conditions, such as presence of oxidative stress, homeostasis is lost and leukocyte diapedesis and soluble mediators can cause breakdown of vascular integrity and uncontrolled plasma leak and thrombosis. Formally, pathologic breakdown of integrity could arise from either excessive disruption or inadequate restoration. However, only the former has been studied in detail and neither the normal self-restorative remodeling mechanisms nor basis for their putative perturbation in endothelium are understood. This proposal is aimed at addressing, directly, both of these issues for the first time. Based on our extensive preliminary data we hypothesize that endothelial micro-wounds formed by leukocyte transmigration (as well as those formed by soluble mediators) are closed by novel actin lamellipodial activities that are regulated by intracellular Rac and reactive oxygen species (ROS) signaling pathways and are susceptible to perturbation by oxidative stress. The specific goal of this proposal is to elucidate the detailed mechanisms responsible for closing para- and trans-cellular leukocyte diapedesis passageways. First we will determine the functional role of Rac1 (and associated proteins such as cortactin and IQGAP) in healing diapedesis micro-wounds in cultured endothelium. Then in similar settings we will determine the role for Rac1-associated NADPH oxidase signaling (e.g., p47phox) in micro-wound closure and its sensitivity to perturbation by oxidative stress. Finally, we will establish the physiologic role of these Rac/ROS lamellipodial activities in micro-wound healing via both ex vivo vascular tissue culture models and a combination of in vivo intravital, tissue whole-mount confocal and electron microscopy imaging along with genetic and pharmacologic function perturbation. These studies are will improve our understanding of, and likely reveal new therapeutic strategies for, inflammatory pathology.

描述（由申请人提供）：炎症病理学的一个核心成分是内皮完整性的崩溃以及由此产生的不受控制的血浆泄漏，白细胞运输和血栓形成。正常的保护性炎症反应表现出微米尺度的内皮重塑动力学，可以平衡破坏性活动与主动的自我调查，从而保持整体完整性。因此，招募的炎症细胞通过形成微小尺度的偏见和细胞细胞不连续性来泄露内皮屏障，这些不连续性是白细胞透射术的通道（即尿布）。通常，内皮在白细胞通过后立即关闭这些“微钩”，从而在很大程度上使它们与等离子体泄漏相结合。此外，可溶性炎症介质会导致内皮本身产生和（钟表后持续时间之后）闭合相似的微型扭曲，从而引起受控的，自限的血管泄漏。因此，内皮表现出动态和主动的功能，可有效地“治愈”微链，以维持炎症期间的整体完整性和稳态。重要的是，在病理状况下，例如存在氧化应激，稳态失去，白细胞尿和可溶性介体可能导致血管完整性的分解以及不受控制的血浆泄漏和血栓形成。正式地，完整性的病理分解可能是由于过度破坏或恢复不足而引起的。但是，只有对前者进行了详细的研究，并且既不了解正常的自我调整的重塑机制，也不了解其在内皮中推定的扰动的基础。该提案旨在首次直接解决这两个问题。基于我们广泛的初步数据，我们假设白细胞迁移（以及可溶性介体形成的迁移）形成的内皮微扭曲是由新型的肌动蛋白层状型活性封闭的，这些活性由细胞内RAC和反应性氧气（ROS）信号通路和反应性途径和反应性强度均呈现氧化的阳性型lamelipodial活性。该提案的具体目标是阐明负责关闭para和细胞细胞白细胞二尿症的详细机制。首先，我们将确定RAC1（以及相关蛋白（例如Cortactin和IQGAP））在培养的内皮细胞中的尿腹膜肌中的功能作用。然后，在类似的环境中，我们将确定与Rac1相关的NADPH氧化酶信号传导（例如P47phox）在微扭结闭合中的作用及其对氧化应激对扰动的敏感性。最后，我们将通过体内血管组织培养模型以及体内插入术，组织全安装整体共聚焦和电子显微镜成像以及遗传和药理功能的结合，建立这些RAC/ROS层状型活性在微链愈合中的生理作用。这些研究将改善我们对炎症病理学的新治疗策略，并可能揭示出新的治疗策略。