The role of HMGB-1 and innate immunity in promoting angiogenesis after ischemia

描述(由申请人提供)：本提案描述了Ulka Sachdev博士的职业目标和研究计划，他是匹兹堡大学血管外科的外科助理教授。Sachdev博士在纽约西奈山医学院完成了她的普通外科和血管外科培训，在整个培训和早期的教职生涯中，她一直活跃在基础科学研究领域。她的目标是成为一名独立资助的外科医生兼科学家，并最终进行翻译研究，以帮助患者。这一建议源于四年来对缺血诱导的血管生成以及HMGB-1在这一现象中的作用的研究。外周动脉疾病是导致严重的功能残疾和疾病的原因之一。随着腿部动脉闭塞疾病的日益严重，严重的肢体缺血可能会导致高达40%的不可重建疾病的患者截肢。在肢体缺血的情况下，促进血管生成的机制还需要进一步的研究，以优化不能进行手术血管重建术的患者的药物治疗。炎症是血管生成的一个组成部分，最近的证据表明，核蛋白HMGB-1在介导炎症的全身效应中发挥作用，也可能是促血管生成的。HMGB-1激活先天免疫受体，包括Toll样受体(TLR)2和4，以及晚期糖基化终产物受体(RAGE)。该建议的目的是评估HMGB-1和TLRs在促进缺血反应中血管生成的作用。Sachdev博士初步证明，HMGB-1促进内皮细胞(EC)管的形成，这是一种血管生成表型，当与低氧刺激相结合时，增加EC的增殖，并可能通过自噬介导。她还表明，HMGB-1的抗体可以阻止内皮管的形成，当给予接受股动脉结扎的小鼠时，会导致更大的肌肉坏死，这是公认的血管生成模型。抑制自噬也会扰乱内皮管的形成。与对照组相比，缺乏TLR4的小鼠也表现出更大的肌肉坏死面积，TLR2KO小鼠也是如此。最后，她证明，与常氧心肌细胞相比，缺血心肌细胞表现出细胞核中HMGB-1的丢失，这表明组织缺血可能导致HMGB-1释放到周围环境中，使其可用于介导血管生成事件。该建议有三个主要目的，旨在了解HMGB-1在体外对血管细胞的影响，以及在体内缺血诱导的血管生成。第一个目的是确定在EC血管生成行为中自噬和HMGB1释放之间的时间关系。第二个目的是确定HMGB-1和自噬在体内骨骼肌血管生成中的作用。第三个目的是确定TLRs在HMGB1的血管生成作用中的作用。 与公共健康相关：以下建议旨在阐明在组织缺血的背景下，由坏死或应激的细胞及其受体释放的危险信号HMGB-1是否有助于通过血管生成过程促进新血管的形成。该项目将有助于了解控制新生血管的机制，并可能帮助无法通过搭桥或血管内治疗进行血管重建术的周围血管疾病患者。

DESCRIPTION (provided by applicant): This proposal describes the career goals and research plan for Dr. Ulka Sachdev, who is an Assistant Professor of Surgery in the Division of Vascular Surgery at the University of Pittsburgh. Dr. Sachdev completed her General Surgery and Vascular Surgery training at Mount Sinai School of Medicine in New York, and has been active in basic science research throughout her training and early faculty career. Her goal is to become an independently funded surgeon-scientist, and eventually perform translational research to assist patients. This proposal culminates from four years of research on ischemia-induced angiogenesis, and the role of HMGB-1 within that phenomenon. Peripheral artery disease is a cause of significant functional disability and morbidity. With increasing severity of arterial occlusive disease in the leg, critical limb ischemia can occur resulting in amputation in up to 40% of patients with non-reconstructable disease. Further study is required to evaluate the mechanisms that promote angiogenesis in the setting of limb ischemia, in order to optimize medical treatments for patients who can not undergo surgical revascularization. Inflammation is a component of angiogenesis, and recent evidence suggests that the nuclear protein HMGB-1 which is known to play a role in mediating systemic effects of inflammation, may also be pro-angiogenic. HMGB-1 activates receptors of innate immunity, including Toll-like Receptors (TLR) 2 and 4, as well as the Receptor for Advanced Glycation End-products (RAGE). The purpose of this proposal is to evaluate the role of HMGB-1 and TLRs in promoting angiogenesis in response to ischemia. Preliminarily, Dr. Sachdev has shown that HMGB-1 enhances endothelial cell (EC) tube formation, which is an angiogenic phenotype, increases EC proliferation when coupled with hypoxic stimulus, and may be mediated by autophagy. She has also shown that antibody to HMGB-1 blocks endothelial tube formation, and results in greater muscle necrosis when given to mice that have undergone femoral artery ligation, which is a recognized model of angiogenesis. Inhibition of autophagy also disrupts endothelial tube formation. Mice deficient in TLR4 also demonstrate greater areas of muscle necrosis than controls, as do TLR2KO mice. Finally, she has shown that ischemic myocytes demonstrate loss of HMGB-1 from nuclei compared to normoxic myocytes, suggesting that tissue ischemia may lead to release of HMGB-1 into the surrounding milieu, rendering it available to mediate angiogenic events. There are three main aims of this proposal, directed at understanding the effect of HMGB-1 on both vascular cells in vitro, and ischemia-induced angiogenesis in vivo. The first aim is to determine the temporal relationship between autophagy and HMGB1 release during EC angiogenic behavior. The second aim is to determine the function of HMGB-1 and autophagy in skeletal muscle angiogenesis in vivo. The third aim is to identify a role for the TLRs in mediating the angiogenic actions of HMGB1. PUBLIC HEALTH RELEVANCE: The following proposal aims to elucidate if the danger signal HMGB-1 which is released by necrotic or stressed cells, as well as its receptors, helps promote the formation of new blood vessels through the process of angiogenesis in the setting of tissue ischemia. This project will help understand the mechanisms that govern neovascularization, and may help patients with peripheral vascular disease who can not undergo revascularization by bypass or endovascular therapies.