Cellular and Molecular Consequences of SARS-VoV2 Infection in Pulmonary Vscular Endothelium

肺血管内皮中 SARS-VoV2 感染的细胞和分子后果

• 批准号: 10262653
• 负责人: Jason Matthew Elinoff
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262653
• 关键词: 2019-nCoV; Acute; Affect; Apoptosis; Ascorbic Acid; Autopsy; Biological Models; Blood Coagulation Disorders; Blood Vessels; Blood specimen; COVID-19; Cardiovascular Manifestation; Cardiovascular system; Case Series; Cause of Death; Cell model; Cells; Cessation of life; Chronic; Clinical; Critical Illness; Critically ill children; Disease; Distal; ERCC3 gene; Endothelial Cells; Endothelium; Functional disorder; Genes; Human; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Infiltrate; Injury; Interferons; Intervention; Laboratories; Lung; Mechanical ventilation; Mediating; Mineralocorticoid Receptor; Molecular; Mutation; Nitric Oxide; Oxygen Therapy Care; Pathologic; Patients; Phenotype; Pneumonia; Proteins; Pulmonary Embolism; Pulmonary Hypertension; Recombinants; Renin-Angiotensin-Aldosterone System; Reporting; Respiratory Failure; Severe Acute Respiratory Syndrome; Shock; Signal Transduction; Spironolactone; Stroke; Syndrome; Testing; Therapeutic Effect; Thrombus; Translations; United States National Institutes of Health; Vascular Diseases; Vascular remodeling; Vasoconstrictor Agents; Viral; arteriole; clinical center; clinical phenotype; clinically relevant; co-infection; cohort; endothelial dysfunction; expression vector; in vitro Model; in vivo; inflammatory marker; insight; monolayer; mortality; novel coronavirus; pandemic disease; particle; pulmonary arterial hypertension; respiratory hypoxia; transcription factor TFIIH; transcriptomics; vascular inflammation; venous thromboembolism

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2), a novel coronavirus identified in December 2019, is now responsible for an expanding pandemic resulting so far in more than 300,000 deaths worldwide. While hypoxic respiratory failure due to SARS-CoV2 pneumonia is the most common presentation of severe illness, cardiovascular manifestations including shock, coagulopathy, venous thromboembolism, stroke and pulmonary hypertension (PH) appear to be major causes of mortality. Recent cases of critically ill children presenting with features of Kawasakis Disease and shock also highlight the potential of SARS-CoV2 infection to result in an acute or delayed syndrome characterized by widespread vascular inflammation. Importantly, autopsy studies in patients with coronavirus disease 2019 (COVID-19) demonstrated evidence of viral inclusions in endothelial cells, endothelial cell apoptosis, perivascular inflammatory infiltrates and microvascular thrombi in distal pulmonary arterioles. Furthermore, the first US case series of critically ill patients with COVID-19 reported that vasopressor-dependent shock developed in nearly all patients that required mechanical ventilation. Due to the absence of a bacterial co-infection in these patients, the authors implicated a direct, virally-mediated affect on the vasculature. Yet, a mechanistic understanding of these cardiovascular and pulmonary vascular complications is lacking. Our laboratory specializes in cellular models of endothelial dysfunction in the context of pulmonary arterial hypertension (PAH) (Awad KS and Elinoff JM et al. Am J Physiol Lung Cell Mol Physiol 2016). Pathologic vascular remodeling in PAH, while chronic rather than acute, is characterized by a proliferative, pro-thrombotic and inflammatory cellular phenotype. Notably, interferon driven-inflammation has been a prominent feature both in vivo and in some of our in vitro models of PAH-associated genetic defects. Our lab also has relevant expertise investigating the interface between the renin-angiotensin-aldosterone system and inflammation. We recently demonstrated that spironolactone broadly suppresses inflammatory signaling in human pulmonary endothelial cells by promoting the proteasomal degradation of the TFIIH subunit, XPB (Elinoff JM et al. Cardiovasc Res 2018). This previously unrecognized mineralocorticoid receptor-independent mechanism by which spironolactone suppresses vascular inflammation may be a useful adjunct for ameliorating the multisystem circulatory manifestations of COVID-19. Here, we plan to investigate the cellular, molecular and transcriptomic consequences of SARS-CoV-2 infection in human primary endothelial cells. In parallel studies, the impact of clinically relevant interventions and treatments, such as oxygen therapy, nitric oxide, ascorbic acid and renin-angiotensin-aldosterone system modulators including spironolactone will be investigated for their effects on SARS-CoV-2-induced endothelial cell dysfunction and injury. Specific Aims: 1. Infect primary human endothelial cells from clinically relevant vascular compartments and the human Ea.hy926 endothelial line with viable SARS-CoV2 and characterize the cellular phenotypic consequences. Circulating inflammatory mediators and markers of endothelial dysfunction in subjects with severe COVID-19 will be compared to conditioned media from endothelial cell monolayers infected with SARS-CoV2. 2. Examine the effects of SARS-CoV2 pseudotyped lentiviral particles and recombinant spike protein, as well as mammalian expression vectors encoding SARS-CoV2 genes, on human endothelial cells. 3. Test clinically relevant therapeutics for effects on SARS-CoV2 endothelial model systems.

2019年12月发现的一种新型冠状病毒--严重急性呼吸综合征冠状病毒2型（SARS-CoV 2），目前正在导致一场不断扩大的大流行，迄今为止已导致全球30多万人死亡。虽然SARS-CoV 2肺炎引起的缺氧性呼吸衰竭是严重疾病的最常见表现，但心血管表现包括休克、凝血病、静脉血栓栓塞、中风和肺动脉高压（PH）似乎是死亡的主要原因。最近出现的具有川崎病和休克特征的危重儿童病例也突出表明了SARS-CoV 2感染导致以广泛血管炎症为特征的急性或延迟综合征的可能性。重要的是，对2019年冠状病毒病（COVID-19）患者的尸检研究证明了内皮细胞中存在病毒包涵体、内皮细胞凋亡、血管周围炎性浸润和远端肺小动脉微血管血栓的证据。此外，美国首个COVID-19重症患者病例系列报告称，几乎所有需要机械通气的患者都出现了血管加压素依赖性休克。由于这些患者中没有细菌合并感染，作者认为对血管系统有直接的病毒介导的影响。然而，缺乏对这些心血管和肺血管并发症的机制理解。 我们的实验室专门研究肺动脉高压（PAH）背景下内皮功能障碍的细胞模型（Awad KS and Elinoff JM et al. Am J Physiol Lung Cell Mol Physiol 2016）。PAH的病理性血管重塑（慢性而非急性）的特征是增殖性、血栓形成前和炎性细胞表型。值得注意的是，干扰素驱动的炎症在体内和我们的一些PAH相关遗传缺陷的体外模型中都是一个突出的特征。我们的实验室也有相关的专业知识，研究肾素-血管紧张素-醛固酮系统和炎症之间的接口。我们最近证明，螺内酯通过促进TFIIH亚基XPB的蛋白酶体降解，广泛抑制人肺内皮细胞中的炎症信号传导（Elinoff JM et al. Aprivasc Res 2018）。这种以前未被认识到的盐皮质激素受体非依赖性机制，通过螺内酯抑制血管炎症，可能是改善COVID-19多系统循环表现的有用辅助手段。在这里，我们计划调查的细胞，分子和转录组的后果SARS冠状病毒-2感染人原代内皮细胞。在平行研究中，将研究临床相关干预和治疗（如氧疗、一氧化氮、抗坏血酸和包括螺内酯在内的肾素-血管紧张素-醛固酮系统调节剂）对SARS-CoV-2诱导的内皮细胞功能障碍和损伤的影响。 具体目标： 1. 用活的SARS-CoV 2感染来自临床相关血管区室的原代人内皮细胞和人Ea.hy926内皮细胞系，并表征细胞表型结果。将严重COVID-19受试者的循环炎症介质和内皮功能障碍标志物与来自SARS-CoV 2感染的内皮细胞单层的条件培养基进行比较。 2. 检测SARS-CoV 2假型慢病毒颗粒和重组刺突蛋白以及编码SARS-CoV 2基因的哺乳动物表达载体对人内皮细胞的影响。 3. 测试临床相关疗法对SARS-CoV 2内皮模型系统的影响。