Angiopoietin Mechanisms in Sepsis

描述（由申请人提供）：在美国，败血症每年影响超过30万人，每年治疗费用超过160亿美元，急性死亡率为30%，预计发病率将增加，使这种疾病成为主要的公共卫生问题。尽管进行了大量的基础和临床研究，但对这些患者的护理仍然主要是支持性的。对其发病机制的新见解，可以应用于病人护理是迫切需要的。我们的实验室正在研究内皮细胞作为一种在败血症中受损的细胞类型，进而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，这是一类分泌蛋白，通过Tie-2受体作用于内皮细胞，作为血管通透性的调节剂。我们发现，在患有严重脓毒症的人类受试者中，循环血管生成素-2 （Ang-2）升高，并且过量的Ang-2足以促进内皮细胞和其他健康成年小鼠的渗漏。最近发表的对Ang-2缺失小鼠的研究进一步证实了Ang-2是炎症期间通透性增加的介质。我们还发现血管生成素-1 （ang1）在结构上与ang2相似，但具有相反的信号作用，可防止小鼠内毒素血症中的血管泄漏。为了实现这种显著的保护作用，Ang-1必须激活Rho-GTPase家族中的一种调节蛋白p190RhoGAP。体内p190RhoGAP的耗竭足以消除Ang-1对内毒素的保护作用。这些发现使我们假设血管生成素通过过量的Ang-2和/或不足的Ang-1紊乱导致脓毒症的明显高通透性。为了探索这个核心问题，我们将应用金标准模型的啮齿动物败血症，盲肠结扎和穿孔，Ang-2缺失小鼠和野生型小鼠，用有效的Ang-2中和抗体或表达Ang-1的腺病毒治疗，以评估存活率，渗透性和细胞反应（目的1）。考虑到Ang-1通过激活p190RhoGAP来抑制内毒素介导的通透性的能力，我们将进一步探索这种屏障防御机制（Aim 2）。最后，我们将回到脓毒症患者中，在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）。Aims 1和3具有高度的翻译性，Aims 2将实质性地阐明脓毒症的内皮屏障防御机制。试点数据强烈支持每个目标，我们组建的团队在败血症领域的合作研究（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，并且测试每个目标将具有成本效益。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，并有可能为严重脓毒症患者带来新的诊断和治疗选择。脓毒症是一种常见的、昂贵的、致命的疾病，发病率越来越高；它可能已经占到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，其影响只会越来越大。基于广泛的初步数据，我们认为血管生成素可能在该疾病的发病机制中起关键作用。提出的实验将使我们能够评估这些蛋白在败血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.