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

严重急性呼吸综合征冠状病毒-2(SARS-CoV2)是2019年12月发现的一种新型冠状病毒，它现在是一场不断扩大的大流行的罪魁祸首，迄今已导致全球30多万人死亡。虽然SARS-CoV2肺炎引起的缺氧性呼吸衰竭是严重疾病最常见的表现，但休克、凝血障碍、静脉血栓栓塞症、中风和肺动脉高压(PH)等心血管表现似乎是死亡的主要原因。最近出现的以川崎病和休克为特征的危重儿童病例也突显了SARS-CoV2感染可能导致以广泛的血管炎症为特征的急性或延迟综合征。重要的是，对冠状病毒病2019(新冠肺炎)患者的尸检研究表明，有证据表明内皮细胞中存在病毒包涵体，内皮细胞凋亡，血管周围炎症浸润和远端肺小动脉的微血管血栓。此外，首例美国新冠肺炎危重患者报告称，几乎所有需要机械通气的患者都发生了血管升压剂依赖性休克。由于在这些患者中没有细菌合并感染，作者暗示了对血管系统的直接、病毒介导的影响。然而，对这些心血管和肺血管并发症的机制了解还很欠缺。 我们的实验室专门研究与肺动脉高压(PAH)相关的内皮功能障碍的细胞模型(Awad KS和Elinoff JM等人)。Am J Physiol肺细胞Mol Physiol 2016)。PAH的病理性血管重构，虽然是慢性的，但不是急性的，其特点是增殖、促血栓形成和炎症细胞表型。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。我们的实验室还拥有研究肾素-血管紧张素-醛固酮系统和炎症之间的接口的相关专业知识。我们最近证明螺内酯通过促进TFIIH亚单位XPB的蛋白酶体降解来广泛抑制人肺内皮细胞中的炎症信号(Elinoff JM等人)。心脏血管紧张症2018年)。螺内酯抑制血管炎症的这一先前未知的盐皮质激素受体非依赖性机制可能是改善新冠肺炎多系统循环表现的有用辅助手段。在这里，我们计划研究SARS-CoV-2感染对人原代内皮细胞的细胞、分子和转录影响。在平行研究中，将调查临床相关干预和治疗的影响，如氧疗、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。 具体目标： 1.用活SARS-CoV2感染临床相关血管腔的原代人内皮细胞和人Ea.hy926内皮细胞，并对其细胞表型结果进行表征。重症新冠肺炎患者的循环炎症介质和内皮功能障碍的标志物将与感染SARS-CoV2的内皮细胞单层的条件培养液进行比较。 2.检测SARS-CoV2假型慢病毒颗粒和重组Spike蛋白以及编码SARS-CoV2基因的哺乳动物表达载体对人内皮细胞的影响。 3.测试临床相关治疗药物对SARS-CoV2内皮模型系统的影响。

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.