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.

严重的急性呼吸综合症冠状病毒-2（SARS-COV2）是2019年12月确定的新型冠状病毒，目前负责全球超过300,000人死亡的大流行。尽管SARS-COV2肺炎引起的低氧呼吸衰竭是最常见的严重疾病表现，而心血管表现形式，包括休克，凝血病，静脉血栓栓塞，中风和肺动脉高压（PH），似乎是死亡率的主要原因。最近出现野生川崎疾病特征和冲击特征的重症儿童的病例也突出了SARS-COV2感染的潜力，导致急性或延迟的综合征，其特征是广泛的血管炎症。重要的是，2019年冠状病毒病患者的尸检研究（COVID-19）证明了内皮细胞，内皮细胞凋亡，血管周围炎性浸润性浸润和微血管血栓的病毒夹杂物的证据。此外，美国第一个病例疾病患者COVID-19的第一个病例系列报告说，几乎所有需要机械通气的患者都会在几乎所有需要机械通气的患者中产生了加压剂依赖性休克。由于这些患者没有细菌共感染，作者暗示了对脉管系统的直接，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械理解。 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和Elinoff JM等人Am J Physiol j Physiol Lung Lung Lung Lung Lung Lung Lung Lung Physiol 2016）。 PAH中的病理血管重塑，虽然慢性而不是急性，但其特征是增生，促血栓形成和炎症性细胞表型。值得注意的是，干扰素驱动的炎症一直是体内和某些与PAH相关遗传缺陷的体外模型的重要特征。我们的实验室还具有相关的专业知识，研究了肾素 - 血管紧张素 - 醛固酮系统与炎症之间的界面。我们最近证明，螺内酯通过促进TFIIH亚基的蛋白酶体降解（Elinoff JM JM等人Cardiovasc Res 2018），通过促进人类肺部内皮细胞的炎症信号传导。这种先前无法识别的矿物皮质受体独立的机制，螺内酯抑制血管炎症的机制可能是改善COVID-19的多系统循环表现的有用辅助。在这里，我们计划研究人类原发质内皮细胞中SARS-COV-2感染的细胞，分子和转录组合后果。在平行研究中，将研究临床相关的干预措施和治疗，例如氧疗法，一氧化氮，抗坏血酸和肾素 - 血管紧张素 - 醛固酮系统调节剂，包括螺旋酮（包括螺内酯）对SARS-COV-2诱导的内皮细胞功能障碍和损伤的影响。 具体目的： 1。从临床相关的血管室和人类EA.Hy926内皮线中感染原发性人内皮细胞，具有可行的SARS-COV2，并表征了细胞表型后果。将COVID-19的受试者中循环炎症介质和内皮功能障碍的标记与感染SARS-COV2感染的内皮细胞单层的条件培养基相比，将进行比较。 2。检查SARS-COV2伪型慢病毒颗粒和重组峰值蛋白的影响，以及编码SARS-COV2基因的哺乳动物表达矢量对人内皮细胞的影响。 3。测试对SARS-COV2内皮模型系统的影响的临床相关治疗剂。