Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, United States.
Department of Pharmacology and Toxicology, Medical College of Georgia at Augusta University, Augusta, GA, United States.
Front Immunol. 2023 Aug 10;14:1241448. doi: 10.3389/fimmu.2023.1241448. eCollection 2023.
Although both COVID-19 and non-COVID-19 ARDS can be accompanied by significantly increased levels of circulating cytokines, the former significantly differs from the latter by its higher vasculopathy, characterized by increased oxidative stress and coagulopathy in lung capillaries. This points towards the existence of SARS-CoV2-specific factors and mechanisms that can sensitize the endothelium towards becoming dysfunctional. Although the virus is rarely detected within endothelial cells or in the circulation, the S1 subunit of its spike protein, which contains the receptor binding domain (RBD) for human ACE2 (hACE2), can be detected in plasma from COVID-19 patients and its levels correlate with disease severity. It remains obscure how the SARS-CoV2 RBD exerts its deleterious actions in lung endothelium and whether there are mechanisms to mitigate this.
In this study, we use a combination of studies in RBD-treated human lung microvascular endothelial cells (HL-MVEC), including electrophysiology, barrier function, oxidative stress and human ACE2 (hACE2) surface protein expression measurements with studies in transgenic mice globally expressing human ACE2 and injected with RBD.
We show that SARS-CoV2 RBD impairs endothelial ENaC activity, reduces surface hACE2 expression and increases reactive oxygen species (ROS) and tissue factor (TF) generation in monolayers of HL-MVEC, as such promoting barrier dysfunction and coagulopathy. The TNF-derived TIP peptide (a.k.a. solnatide, AP301) -which directly activates ENaC upon binding to its a subunit- can override RBD-induced impairment of ENaC function and hACE2 expression, mitigates ROS and TF generation and restores barrier function in HL-MVEC monolayers. In correlation with the increased mortality observed in COVID-19 patients co-infected with S. pneumoniae, compared to subjects solely infected with SARS-CoV2, we observe that prior intraperitoneal RBD treatment in transgenic mice globally expressing hACE2 significantly increases fibrin deposition and capillary leak upon intratracheal instillation of S. pneumoniae and that this is mitigated by TIP peptide treatment.
虽然 COVID-19 和非 COVID-19 性 ARDS 都可能伴有循环细胞因子水平的显著升高，但前者与后者的显著不同之处在于前者肺毛细血管中的血管病变更为严重，其特征是氧化应激和凝血功能障碍增加。这表明存在 SARS-CoV2 特异性的因素和机制，使内皮细胞容易出现功能障碍。虽然病毒很少在内皮细胞或循环中检测到，但它的刺突蛋白的 S1 亚单位，包含与人 ACE2（hACE2）结合的受体结合域（RBD），可以在 COVID-19 患者的血浆中检测到，其水平与疾病严重程度相关。目前尚不清楚 SARS-CoV2 RBD 如何在肺内皮细胞中发挥其有害作用，以及是否有减轻这种作用的机制。
在这项研究中，我们使用了一系列方法，包括用 SARS-CoV2 RBD 处理人肺微血管内皮细胞（HL-MVEC）的研究，包括电生理学、屏障功能、氧化应激和人 ACE2（hACE2）表面蛋白表达的测量，以及在全身表达 hACE2 的转基因小鼠中的研究，并注射 RBD。
我们表明，SARS-CoV2 RBD 可损害内皮细胞 ENaC 活性，降低表面 hACE2 的表达，并增加 HL-MVEC 单层中的活性氧物种（ROS）和组织因子（TF）的产生，从而促进屏障功能障碍和凝血功能障碍。TNF 衍生的 TIP 肽（也称为 solnatide，AP301）-当与 a 亚单位结合时可直接激活 ENaC-可逆转 RBD 诱导的 ENaC 功能和 hACE2 表达的损害，减轻 ROS 和 TF 的产生，并恢复 HL-MVEC 单层的屏障功能。与 COVID-19 患者与 S. pneumoniae 混合感染时观察到的死亡率增加相关，与仅感染 SARS-CoV2 的患者相比，我们发现全身表达 hACE2 的转基因小鼠腹腔内预先给予 RBD 治疗后，经气管内滴注 S. pneumoniae 后，肺毛细血管中的纤维蛋白沉积和毛细血管渗漏显著增加，而 TIP 肽治疗可减轻这种情况。
