ANDV Induced Responses of Hypoxic Endothelial Cells

ANDV 诱导缺氧内皮细胞的反应

• 批准号: 8264741
• 负责人: Erich R Mackow
• 金额: $ 23.55万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264741
• 关键词: Acute; Adherence; Adherens Junction; Altitude; Alveolar; Beds; Binding; Blood Platelets; Blood capillaries; Capillary Permeability; Cell Line; Cell physiology; Cells; Cytolysis; Disease; Edema; Endothelial Cells; Epithelial Cells; Extravasation; Fluorescence Microscopy; Gases; Genetic Transcription; Goals; Hantavirus; Hantavirus Pulmonary Syndrome; Human; Hyperbaric Oxygenation; Hypersensitivity; Hypoxia; Immunoperoxidase Technics; Infection; Integrins; Label; Liquid substance; Lung; Oxygen; Pathogenesis; Patients; Permeability; Persons; Platelet Activation; Play; Property; Prostacyclin synthase; Prostaglandins I; Proteins; Pulmonary Edema; Puromycin; Recruitment Activity; Reporting; Respiratory Insufficiency; Respiratory distress; Role; Signal Transduction; Staining method; Stains; Subfamily lentivirinae; Surface; System; Therapeutic; Thrombocytopenia; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Permeabilities; autocrine; capillary; fluorexon; hypoxia inducible factor 1; inhibitor/antagonist; medical attention; monolayer; mortality; paracrine; prevent; protein expression; public health relevance; receptor; response

DESCRIPTION (provided by applicant): ANDV causes hantavirus pulmonary syndrome (HPS), has a 40% mortality rate and is reportedly spread from person to person. ANDV primarily infects endothelial cells (ECs) and HPS is characterized by thrombocytopenia, hypoxia and acute pulmonary edema that leads to respiratory insufficiency. ANDV infection of ECs provides a primary means for hantaviruses to alter vascular permeability. Although ECs are not lysed by ANDV infection, ECs line capillaries of vast pulmonary alveolar beds and regulate capillary integrity. Hypoxia induces high altitude pulmonary edema through the induction of vascular endothelial growth factor (VEGF), and this response is transcriptionally directed by the hypoxia inducible factor 1? (HIF1?). ECs secrete VEGF and VEGF in turn activates VEGFR2 receptors on ECs in an autocrine and paracrine manner. VEGFR2 activation directs the disassembly of EC adherens junctions and induces capillary permeability. Interestingly, VEGF further induces transcription of HIF1?, forming a HIF1? -VEGF amplification loop that enhances EC responses to hypoxia and further increases capillary permeability. We have shown that ANDV infection of ECs dramatically enhances the permeability of ECs in response to VEGF. Consistent with this, we have recently determined that the pulmonary edema fluid of HPS patients contains high levels of VEGF. HPS patient hypoxia combined with the hypersensitivity of infected ECs to VEGF, provides a means for amplifying HIF1? -VEGF responses that contribute to pulmonary edema. We propose to study the role of hypoxia on ANDV infected ECs and define ANDV proteins and cellular responses that contribute to dramatic increases in EC permeability. Hantaviruses also cause thrombocytopenia and we have recently reported that quiescent platelets are selectively recruited to the surface of ANDV infected ECs, but not cells infected by the nonpathogenic hantavirus TULV. Platelet adherence to ANDV infected ECs is dependent on cell associated ANDV attachment to ?3 integrin receptors 3 days p.i. However, platelets are normally activated by adherence to ECs, and the mechanism by which platelets remain quiescent following binding to ANDV infected ECs remains to be determined. Interestingly, either inducing or preventing platelet activation can result in thrombocytopenia. Platelet activation is inhibited by platelet endothelial cell adherence molecule-1 (PECAM-1) and PECAM-1: 1) is activated by VEGF, 2) negatively regulates the activation of platelet integrins and 3) stimulates prostacyclin release from ECs. Curiously, hypoxia induces prostacyclin synthase in ECs and prostacyclin is a potent inhibitor of platelet activation. Further, adherence of platelets to the EC lining of alveolar capillaries could both sequester platelets and alter normal gas exchange, causing or exacerbating hypoxia. Although it is unclear whether ANDV activates PECAM-1 and induces prostacyclin, these findings are consistent with amplified HIF1? -VEGF responses of hypoxic ANDV infected ECs. Here we will investigate the mechanism by which ANDV and hypoxia induce EC responses that direct platelet adherence and quiescence. The goal of this project is to evaluate EC responses to hypoxia following ANDV infection or ANDV protein expression. A Lentivirus-puromycin selection system will be used to express ANDV proteins within primary human ECs and used to define the primary mechanisms by which ANDV and hypoxia increase permeability, alter EC adherence and induce platelet quiescence. These studies provide a principal understanding of the role of hypoxia on EC and platelet functions that contribute to ANDV induced HPS disease. PUBLIC HEALTH RELEVANCE: ANDV causes hantavirus pulmonary syndrome (HPS), resulting in fluid in the lungs that causes respiratory distress and has a 40% mortality rate. ANDV infects the endothelial cell lining of capillaries which normally limits fluid leakage termed edema. We investigate the mechanism by which ANDV causes fluid leakage and the role of low oxygen levels in inducing endothelial cell responses that increase leakage. These studies are likely to identify the mechanism of leakage and provide therapeutic approaches for reducing ANDV disease.

描述（由申请人提供）：ANDV导致汉坦病毒肺综合征（HPS）的死亡率为40％，据报道是人到人的传播。 ANDV主要感染内皮细胞（EC）和HP的特征是血小板减少症，缺氧和急性肺水肿，导致呼吸不足。 ECS的ANDV感染为汉坦病毒改变了血管渗透性提供了主要手段。尽管EC并未因ANDV感染而裂解，但ECS线毛细血管具有巨大的肺肺泡床并调节毛细血管完整性。缺氧通过诱导血管内皮生长因子（VEGF）诱导高海拔肺水肿，而这种反应是由低氧诱导因子1指导的转录？ （HIF1？）。 ECS分泌VEGF和VEGF反过来以自分泌和旁分泌方式激活EC上的VEGFR2受体。 VEGFR2激活指导EC粘附连接的拆卸，并诱导毛细管渗透性。有趣的是，VEGF进一步诱导HIF1的转录，形成HIF1？ - VEGF扩增环，可增强EC对缺氧的反应并进一步增加毛细管渗透性。 我们已经表明，EC的ANDV感染显着提高了EC对VEGF的渗透性。与此一致，我们最近确定HPS患者的肺水肿液含有高水平的VEGF。 HPS患者缺氧与感染ECS对VEGF的超敏反应相结合，提供了放大HIF1的手段？ - 有助于肺水肿的VEGF反应。我们建议研究缺氧对ANDV感染EC的作用，并定义ANDV蛋白以及细胞反应，这有助于急剧增加EC渗透性。 汉坦病毒还引起血小板减少症，我们最近报告说，静态血小板被选择性地募集到ANDV感染的EC表面，而不是被非病毒性汉塔病毒TULV感染的细胞。血小板遵守ANDV感染的ECS取决于3天P.I的细胞和V附着的细胞附着和V的附着。但是，通常通过遵守EC的粘附而激活血小板，并且在与ANDV感染的EC结合后，血小板保持静止的机制仍有待确定。有趣的是，要么诱导或预防血小板激活会导致血小板减少症。血小板激活受到血小板内皮细胞粘附分子-1（PECAM-1）和PECAM-1：1）的抑制，VEGF被激活，2）负调节血小板整合素的激活和3）刺激从EC中释放的前列腺素蛋白。奇怪的是，缺氧在ECS中诱导前列环蛋白合酶，前列环蛋白是血小板激活的有效抑制剂。此外，血小板遵守肺泡毛细血管的EC衬里可能会隔离血小板并改变正常的气体交换，从而导致或加剧缺氧。尽管尚不清楚ANDV是否激活PECAM-1并诱导前列环蛋白，但这些发现与放大的HIF1一致？ -VEGF缺氧和V感染的EC的反应。在这里，我们将研究ANDV和缺氧引起的EC反应的机制，该反应直接粘附和静止。 该项目的目的是评估EC在ANDV感染或ANDV蛋白表达后对缺氧的反应。慢病毒 - 假病毒选择系统将用于在原代人EC中表达和V蛋白，并用于定义ANDV和缺氧增加渗透性，改变EC的依从性并引起血小板静脉的主要机制。这些研究提供了对缺氧对EC和血小板功能的作用的主要理解，这有助于ANDV诱导的HPS疾病。 公共卫生相关性：ANDV导致汉塔病毒肺综合征（HPS），导致肺部液体导致呼吸窘迫，并具有40％的死亡率。 ANDV感染了毛细血管的内皮细胞衬里，这通常限制流体泄漏称为水肿。我们研究了和V导致液体泄漏的机制以及低氧水平在诱导内皮细胞反应中的作用，从而增加泄漏。这些研究可能会确定泄漏的机理，并提供减少和V疾病的治疗方法。