MIcrophysiological systems to model vascular malformations

项目摘要 COVID 19是由SARS-CoV-2引起的，SARS-CoV-2是人类冠状病毒家族的一个新成员， 相关的SARS-CoV（SARS）和MERS-CoV（MERS）病毒。SARS-CoV-2病毒刺突蛋白与人的结合 血管紧张素转换酶-2（ACE 2）在细胞表面上，然后由丝氨酸/苏氨酸引发 蛋白酶TMPRS 22，于是整个复合物被靶细胞内化。ACE 2表达为 身体的多种细胞类型，包括肺和肠上皮细胞（可能是初始感染的主要部位）， 多器官血管内皮细胞（EC）。除了描述得很好的肺炎样疾病 其特征是肺功能受损，随后血液中pO 2水平降低，患者通常还 显示出多器官受累的迹象，包括肠道、肾脏、肝脏、心脏和大脑。最近， 许多儿科患者已经显示出川崎病（一种全身性血管炎症）的迹象。 超过30%的COVID 19 ICU患者也显示出血栓形成的迹象，25%的患者患有静脉栓塞。脑 还报道了可能由于凝块形成引起的局部缺血。目前尚不清楚的是， 参与是由于这些组织的继发性感染，或者这些都是全身性感染的结果。 过度炎症可能导致全身炎症反应过度的一系列事件源于SARS- CoV-2感染机制血管紧张素II（AngII）是一种重要的血管收缩剂， 在生理条件下，其水平通过ACE 2的快速降解而受到严格控制，然而，SARS-CoV- 2进入细胞清除ACE 2从细胞表面，潜在地延长AngII的作用。初级 其后果是双重的：肺血管收缩延长（加重氧合不良 血液的已经受损的肺）和向促炎状态的转变，因为它是好的， 确定AngII可以驱动局部血管炎症，在很大程度上通过诱导EC中的IL-6， 平滑肌细胞（SMC）。IL-6是全身性炎症过度的主要驱动因素之一，并且在其最常见的情况下， 严重的形式，所谓的“细胞因子风暴”。IL-6也与血栓形成密切相关，可能通过 EC和巨噬细胞上组织因子的上调，以及EC上血栓调节蛋白的下调。 通过父母奖，我们产生了血管化微器官（VMO），由灌注的 人类脉管系统和周围的基质，并进一步发展成血管化的微型大脑 （VMB）和血管化微肝（VML）。其他细胞，包括 巨噬细胞和SMC也被并入VMO。利用这些平台，我们将解决三个 假设：1）VMO可用于评估恢复期血清、可溶性ACE 2或小分子血管紧张素Ⅱ（ACE Ⅱ）的能力， 分子抑制剂来阻断SARS-CoV-2假型病毒的进入; 2）持续的AngII表达可以 引发EC和巨噬细胞的过度炎症反应;以及3）过度炎症可以 危及外周和BBB血管系统以及肝功能。

PROJECT SUMMARY COVID19 is caused by SARS-CoV-2, a novel member of the human coronavirus family that includes the closely related SARS-CoV (SARS) and MERS-CoV (MERS) viruses. SARS-CoV-2 viral spike protein binds to human angiotensin converting enzyme-2 (ACE2) on the surface of cells and is then primed by the serine/threonine protease TMPRS22, whereupon the entire complex is internalized by the target cell. ACE2 is expressed by multiple cell types of the body, including lung and gut epithelium (likely the primary sites of initial infection) and vascular endothelial cells (EC) of multiple organs. In addition to the well-described pneumonia-like disease characterized by compromised lung function with subsequent depressed pO2 levels in blood, patients often also show signs of multi-organ involvement, which can include gut, kidney, liver, heart, and brain. Most recently, numerous pediatric patients have been showing signs of Kawasaki disease, a systemic vascular inflammation. Over 30% of COVID19 ICU patients also show signs of thrombosis and 25% suffer venous embolism. Cerebral ischemia, likely due to clot formation, has also been reported. What is not clear is whether this multi-organ involvement is due to secondary infection of these tissues or whether these are all a consequence of systemic hyperinflammation. The sequence of events that could drive systemic hyperinflammation stems from the SARS- CoV-2 mechanism of infection. Angiotensin II (AngII) is an important vasoconstrictor and under normal physiological conditions its level is closely controlled through rapid degradation by ACE2, however, SARS-CoV- 2 entry into cells clears ACE2 from the cell surface, potentially prolonging the action of AngII. Primary consequences of this would be two-fold: prolonged vasoconstriction in the lung (exacerbating poor oxygenation of the blood by the already compromised lungs) and a shift toward a pro-inflammatory state, as it is well established that AngII can drive local vascular inflammation, in large part through the induction of IL-6 in EC and smooth muscle cells (SMC). IL-6 is one of the major drivers of systemic hyperinflammation, and in its most severe form, a so-called “cytokine storm”. IL-6 is also strongly correlated with thrombosis, likely through upregulation of tissue factor on EC and macrophages, and by downregulation of thrombomodulin on EC. Through the parent award we have generated Vascularized Micro-Organs (VMOs), comprised of perfused human vasculature and a surrounding stroma, and have further developed these into Vascularized Micro-Brains (VMBs) incorporating a Blood-Brain Barrier, and Vascularized Micro-Livers (VMLs). Additional cells, including macrophages and SMC have also been incorporated into the VMO. Using these platforms we will address three hypotheses: 1) That the VMO can be used to assess the ability of convalescent serum, soluble ACE2 or small molecule inhibitors to block entry of a SARS-CoV-2 pseudotyped virus; 2) That sustained AngII expression can trigger a hyperinflammatory response from EC and macrophages; and 3) That hyperinflammation can compromise peripheral and BBB vasculature as well as liver function.