ANDV Induced Responses of Hypoxic Endothelial Cells

ANDV 诱导缺氧内皮细胞的反应

• 批准号: 8190126
• 负责人: Erich R Mackow
• 金额: $ 19.63万
• 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/8190126
• 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; 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%，据报道可在人与人之间传播。ANDV主要感染内皮细胞（ECs）， 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。内皮细胞的ANDV感染为汉坦病毒改变血管通透性提供了主要手段。尽管内皮细胞不会被ANDV感染溶解，但内皮细胞可以连接广大肺泡床的毛细血管并调节毛细血管的完整性。缺氧通过诱导血管内皮生长因子（VEGF）诱导高原肺水肿，而这种反应是由缺氧诱导因子1？(HIF1吗?)内皮细胞分泌VEGF， VEGF又以自分泌和旁分泌的方式激活内皮细胞上的VEGFR2受体。VEGFR2的激活指导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HIF1？，形成HIF1？-VEGF扩增环增强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感染的内皮细胞显著增强内皮细胞的通透性，以响应VEGF。与此一致，我们最近确定HPS患者的肺水肿液中含有高水平的VEGF。HPS患者缺氧合并感染ECs对VEGF的超敏反应，为HIF1？-VEGF反应导致肺水肿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，这些蛋白和细胞反应有助于显著增加EC的渗透性。汉坦病毒也引起血小板减少症，我们最近报道了安静的血小板被选择性地招募到ANDV感染的内皮细胞表面，而不是被非致病性汉坦病毒感染的细胞。血小板对ANDV感染的ECs的粘附依赖于细胞相关的ANDV附着？3整合素受体3天p.i。然而，血小板通常通过粘附到ECs而被激活，而血小板在与ANDV感染的ECs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。血小板内皮细胞粘附分子-1 （PECAM-1）抑制血小板活化，PECAM-1: 1)被VEGF激活，2)负调控血小板整合素的活化，3)刺激内皮细胞释放前列环素。奇怪的是，缺氧诱导内皮细胞中的前列环素合成酶，而前列环素是血小板活化的有效抑制剂。此外，血小板粘附在肺泡毛细血管的EC衬里既可以隔离血小板，也可以改变正常的气体交换，导致或加剧缺氧。虽然尚不清楚ANDV是否激活PECAM-1并诱导前列环素，但这些发现与放大的HIF1？-缺氧ANDV感染内皮细胞的vegf反应。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。该项目的目的是评估EC对ANDV感染或ANDV蛋白表达后缺氧的反应。慢病毒-嘌呤霉素选择系统将用于在原代人内皮细胞中表达ANDV蛋白，并用于确定ANDV和缺氧增加通透性、改变内皮粘附性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，这些功能有助于ANDV诱导的HPS疾病。