Direct endothelial ENaC activation mitigates vasculopathy induced by SARS-CoV2 spike protein

Abstract

IntroductionAlthough 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.MethodsIn this study, we use a combination of in vitro 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 in vivo studies in transgenic mice globally expressing human ACE2 and injected with RBD.ResultsWe 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.

Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC

“…However, PKC activation cannot be excluded as a relevant mechanism for ENaC inhibition in vivo since oocytes lack hACE2, which has been proposed to be an upstream mediator of this effect [ 14 ]. For instance, Romero et al have shown that treatment with the recombinant Receptor Binding Domain of the S protein can induce ENaC inhibition in human lung microvascular endothelial cells and this has been proposed to contribute to lung vasculature disfunction [ 14 ]. This inhibition correlates with a reduction in ACE2 surface expression and generation of reactive oxygen species.…”

“…Since then some authors have speculated that the competition between the S protein and ENaC for furin-mediated cleavage (see Fig 4), would lead to reduced alveolar fluid clearance and alveolar-capillary barrier dysfunction. This would provide possible pathophysiological mechanisms contributing to the development of lung edema in cases of severe COVID-19 [9,14,[21][22][23][24].…”

“…Grant and Lester have recently shown that inhibition of PKC with the Go ¨-6976 inhibitor did not prevent the inhibitory effect of the S protein of SARS-CoV2 on ENaC activity in X. laevis oocytes [29]. However, PKC activation cannot be excluded as a relevant mechanism for ENaC inhibition in vivo since oocytes lack hACE2, which has been proposed to be an upstream mediator of this effect [14]. For instance, Romero et al have shown that treatment with the recombinant Receptor Binding Domain of the S protein can induce ENaC inhibition in human lung microvascular endothelial cells and this has been proposed to contribute to lung vasculature disfunction [14].…”

“…In addition, the function of ENaC in endothelial cells is also relevant for the maintenance of the alveolar-capillary barrier. Thus, in these cells, its inhibition has been proposed to contribute to alveolar edema [ 13 , 14 ].…”

Zinc-Dependent Histone Deacetylases in Lung Endothelial Pathobiology

“…Given the well-established understanding that SARS-CoV-2 or its spike protein causes endothelial dysfunction [11], COVID-19 is widely recognized as an endothelial disease primarily affecting the microvasculature [12]. Overall, preventing EC barrier integrity is a critical clinical requirement for treating various acute and chronic lung diseases [13][14][15].…”