Angiopoietin Mechanisms in Sepsis

描述（由申请人提供）：脓毒症每年影响美国超过30万人，治疗费用超过160亿美元/年，与30%的急性死亡率相关，预计发病率将增加，使该疾病成为一个主要的公共卫生问题。尽管进行了密集的基础和临床研究，但对这些患者的护理仍然主要是支持性的。迫切需要对其发病机制的新见解，可应用于患者护理。我们的实验室正在研究内皮细胞作为一种细胞类型，在脓毒症中受损，反过来又介导对宿主的进一步损害。具体来说，我们正在研究血管生成素，这是一类通过Tie-2受体作用于内皮细胞的分泌蛋白，作为这种疾病中血管通透性的调节剂。我们已经发现，循环血管生成素-2（Ang-2）在患有严重脓毒症的人类受试者中升高，并且过量的Ang-2足以促进穿过内皮细胞和在其他方面健康的成年小鼠中的渗漏。最近发表的Ang-2缺失小鼠的研究进一步验证了Ang-2作为炎症期间增加的渗透性的介质。我们还发现，血管生成素-1（Ang-1），其结构与Ang-2相似，但具有相反的信号传导作用，可防止小鼠内毒素血症中的血管渗漏。为了达到这种显著的保护作用，Ang-1必须激活Rho-GTdR家族中的一种调节蛋白，称为p190 RhoGAP。体内p190 RhoGAP的消耗足以消除Ang-1对内毒素的保护作用。这些发现使我们推测，通过过量的Ang-2和/或不足的Ang-1的血管生成素的紊乱有助于脓毒症的显著高通透性。为了探索这个中心问题，我们将啮齿动物脓毒症、盲肠结扎和穿孔的金标准模型应用于Ang- 2缺失小鼠和用有效的Ang-2中和抗体或用表达Ang-1的腺病毒处理的野生型小鼠，以评估存活率、渗透性和细胞应答（目的1）。鉴于Ang-1通过p190 RhoGAP激活来抵消内毒素介导的渗透性的意外能力，我们将进一步探索这种屏障防御机制（目的2）。最后，我们将返回到脓毒症患者中，在我们的急诊科进行一项前瞻性研究，以评估循环Ang-2和Ang-1的早期变化对死亡、休克和其他不良结局的预测价值（目的3）。目的1和3是高度翻译的，目的2将基本上阐明脓毒症中的内皮屏障防御机制。试点数据强烈支持每个目标，我们组建的团队在脓毒症领域的合作研究（基础科学，临床研究，病理学和流行病学）方面拥有丰富的经验，并且测试每个目标都具有成本效益。这些有针对性的研究将提高我们对脓毒症发病机制的理解，并可能为严重脓毒症患者提供新的诊断和治疗选择。脓毒症是一种常见、昂贵和致命的疾病，发病率不断上升;它可能已经占美国所有死亡人数的10%，使其成为一个主要的公共卫生威胁，其影响只可能增加。基于广泛的初步数据，我们认为血管生成素可能是关键参与这种疾病的发病机制。拟议的实验将使我们能够评估这些蛋白质在脓毒症中的诊断和治疗潜力。

DESCRIPTION (provided by applicant): Sepsis affects more than 300,000 individuals in the US annually, costs more than $16 billion/yr to treat, is associated with an acute mortality of 30%, and is expected to increase in incidence, making this disease a major public health concern. Care of these patients has remained primarily supportive despite intensive basic and clinical research efforts. Novel insights into its pathogenesis that can be applied to patient care are sorely needed. Our laboratory is studying the endothelium as a cell type that is injured in sepsis and that, in turn, mediates further damage to the host. Specifically, we are studying the angiopoietins a class of secreted proteins that act on endothelial cells through the Tie-2 receptor as regulators of vascular permeability in this disease. We have found that circulating angiopoietin-2 (Ang-2) is elevated in human subjects with severe sepsis, and that excess Ang-2 is sufficient to promote leak across endothelial cells and in otherwise healthy adult mice. More recently published studies of Ang-2 null mice have further validated Ang-2 as a mediator of increased permeability during inflammation. We have also found that angiopoietin-1 (Ang-1), which is similar in structure to Ang-2 but has opposite signaling effects, prevents vascular leak in murine endotoxemia. To achieve this remarkable protective effect, Ang-1 must activate a regulatory protein in the Rho-GTPase family called p190RhoGAP. In vivo depletion of p190RhoGAP is sufficient to abrogate the protection conferred by Ang-1 against endotoxin. These findings have led us to hypothesize that derangements in angiopoietins via excess Ang-2 and/or insufficient Ang-1 contribute to the marked hyperpermeability of sepsis. To explore this central question, we will apply the gold-standard model of rodent sepsis, cecal ligation and perforation, to Ang- 2 null mice, and wild-type mice treated either with a potent Ang-2 neutralizing antibody or with an Ang-1 expressing adenovirus to evaluate survival, permeability, and cellular responses (Aim 1). Given the unexpected ability of Ang-1 to counteract endotoxin-mediated permeability via p190RhoGAP activation, we will explore this mechanism of barrier defense further (Aim 2). Finally, we will return to human subjects with sepsis to perform a prospective study based in our emergency department to evaluate the predictive value of early changes in circulating Ang-2 and Ang-1 for death, shock, and other adverse outcomes (Aim 3). Aims 1 and 3 are highly translational, and Aim 2 will substantially elucidate endothelial barrier defense mechanisms in sepsis. Pilot data are strongly supportive of each aim, the team we have assembled has extensive experience in collaborative research (basic science, clinical research, pathology and epidemiology) in the area of sepsis, and testing each aim will be cost-efficient. These targeted studies will improve our understanding of the pathogenesis of sepsis, and potentially lead to novel diagnostic and therapeutic options for patients with severe sepsis. PUBLIC HEALTH RELEVANCE Sepsis is a common, costly, and lethal disease of increasing incidence; it may already account for 10% of all deaths in the US, making it a major public health threat whose impact is only likely to grow. Based on extensive preliminary data, we believe that angiopoietins may be critically involved in the pathogenesis of this disease. The proposed experiments will enable us to evaluate the diagnostic and therapeutic potential of these proteins in sepsis.