严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突蛋白在人血脑屏障的二维静态和三维微流控体外模型中改变屏障功能。

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood-brain barrier.

作者信息

Buzhdygan Tetyana P, DeOre Brandon J, Baldwin-Leclair Abigail, McGary Hannah, Razmpour Roshanak, Galie Peter A, Potula Raghava, Andrews Allison M, Ramirez Servio H

出版信息

bioRxiv. 2020 Jun 15:2020.06.15.150912. doi: 10.1101/2020.06.15.150912.

Abstract

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

摘要

随着全球研究人员将注意力集中在了解严重急性呼吸综合征冠状病毒2(SARS-CoV-2)上,逐渐浮现的情况是,这种病毒会对包括脑血管系统在内的多个器官系统的脉管系统产生严重影响。观察到的对中枢神经系统的影响包括神经症状(头痛、恶心、头晕)、致命微血栓形成,以及罕见的脑炎病例。然而,我们对该病毒如何导致这些从轻度到重度的神经症状以及脑血管系统如何受到影响仍不清楚。因此,本报告展示的结果探讨了是否能观察到SARS-CoV-2病毒刺突蛋白对原代人脑血管内皮细胞(hBMVECs)产生有害影响。首先,利用死后脑组织,我们发现血管紧张素转换酶2即ACE2(SARS-CoV-2刺突蛋白已知的结合靶点)在额叶皮质的各种管径血管中均有表达。此外,在细胞培养条件下维持的原代人脑血管内皮细胞(hBMVEC)中也可检测到ACE2。细胞活力分析显示,在48小时的暴露窗口内,S1、S2或仅含受体结合域(RBD)的S1截短形式对hBMVEC活力的影响都微乎其微。然而,当将病毒刺突蛋白引入重现血脑屏障(BBB)基本特征的模型系统时,根据所测试的刺突蛋白亚基不同,屏障出现了不同程度的明显破坏。我们研究结果的关键在于证明了S1在人类血脑屏障的先进三维微流控模型中会促使屏障完整性丧失,该平台与这个中枢神经系统界面处的人类生理状况最为相似。后续分析还表明,SARS-CoV-2刺突蛋白有能力在脑内皮细胞上引发促炎反应,这可能导致血脑屏障功能状态改变。总之,这些结果首次表明了SARS-CoV-2刺突蛋白可能对脑内皮细胞产生的直接影响;从而为新冠肺炎患者出现的神经后果提供了一个合理的解释。

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