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引起汉坦病毒肺综合征（HPS），死亡率为40%，据报道在人与人之间传播。ANDV主要感染内皮细胞（EC），HPS的特征是血小板减少、缺氧和急性肺水肿，导致呼吸功能不全。 ANDV感染内皮细胞为汉坦病毒改变血管通透性提供了主要手段。尽管ANDV感染不裂解EC，但EC排列在巨大肺泡床的毛细血管中并调节毛细血管的完整性。低氧通过诱导血管内皮生长因子（VEGF）诱导高原肺水肿，这种反应是由低氧诱导因子1？(HIF1?).内皮细胞分泌VEGF，而VEGF又以自分泌和旁分泌的方式激活内皮细胞上的VEGFR 2受体。VEGFR 2活化指导EC粘附连接的分解并诱导毛细血管通透性。有趣的是，VEGF进一步诱导HIF 1？，形成HIF 1？-VEGF扩增环，其增强EC对缺氧的反应并进一步增加毛细血管通透性。 我们已经表明，ANDV感染的内皮细胞显着增强内皮细胞的通透性，响应VEGF。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患者缺氧与受感染的内皮细胞对VEGF的超敏反应相结合，提供了一种放大HIF 1？- 导致肺水肿的VEGF反应。我们建议研究缺氧对ANDV感染的EC的作用，并确定ANDV蛋白和细胞反应，有助于EC通透性的急剧增加。 汉坦病毒也引起血小板减少症，我们最近报道，静止的血小板被选择性地募集到ANDV感染的EC的表面，而不是被非致病性汉坦病毒TULV感染的细胞。血小板粘附ANDV感染的内皮细胞是依赖于细胞相关的ANDV附着？3整联蛋白受体，p.i.然而，血小板通常通过粘附于EC而被活化，并且血小板在与ANDV感染的EC结合后保持静止的机制仍有待确定。有趣的是，诱导或阻止血小板活化都可能导致血小板减少症。血小板活化被血小板内皮细胞粘附分子-1（PECAM-1）抑制，PECAM-1：1）被VEGF激活，2）负调节血小板整联蛋白的活化，3）刺激前列环素从EC释放。奇怪的是，缺氧诱导内皮细胞中的前列环素合酶，而前列环素是血小板活化的有效抑制剂。此外，血小板粘附到肺泡毛细血管的EC衬里可以隔离血小板并改变正常的气体交换，引起或加剧缺氧。虽然目前还不清楚是否ANDV激活PECAM-1和诱导前列环素，这些发现是一致的扩增HIF 1？- 缺氧ANDV感染的EC的VEGF应答。在这里，我们将研究ANDV和缺氧诱导EC反应，直接血小板粘附和静止的机制。 本项目的目的是评估ANDV感染或ANDV蛋白表达后EC对缺氧的反应。慢病毒-嘌呤霉素选择系统将用于在原代人EC内表达ANDV蛋白，并用于确定ANDV和缺氧增加渗透性、改变EC粘附和诱导血小板静止的主要机制。这些研究提供了缺氧对EC和血小板功能的作用的主要理解，有助于ANDV诱导的HPS疾病。 公共卫生相关性：ANDV引起汉坦病毒肺综合征（HPS），导致肺部积液，引起呼吸窘迫，死亡率为40%。ANDV感染毛细血管的内皮细胞衬里，其通常限制称为水肿的液体渗漏。我们研究了ANDV引起液体渗漏的机制，以及低氧水平在诱导内皮细胞反应增加渗漏中的作用。这些研究有可能确定渗漏的机制，并提供减少ANDV疾病的治疗方法。