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Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.

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We provide evidence that growth differentiation factor 15 (GDF-15)- and stromal cell-derived factor 1 (SDF-1)-induced angiogenesis strongly depends on the presence of Nrf2 protein but does not rely on its transcriptional activity. Instead, Nrf2 serves as a protein restraining Keap1 (Kelch-like ECH-associated protein 1), its known transcriptional repressor. Angiogenic response is abrogated in Nrf2-deficient endothelial cells but not in cells expressing dominant negative form or Keap1-binding fragment of Nrf2. Deficiency of Nrf2 protein available for Keap1 leads to the overabundance of RhoGAP1 (Rho GTPase-activating protein 1), the protein regulating cell division cycle 42 (Cdc42) activity. This impairs podosome assembly and disrupts actin rearrangements, thereby preventing angiogenesis. Effects of Nrf2 deficiency can be rescued by concomitant knockdown of RhoGAP1 or Keap1. Importantly, in the established murine model of Nrf2 deficiency, the N-terminal fragment of Nrf2 containing Keap1 binding domain is preserved. Thus, this model can be used to characterize Nrf2 as a transcription factor, but not as a Keap1-sequestering protein. Innovation and Conclusion: To date, the significance of Nrf2 in cell function has been ascribed solely to the regulation of transcription. We demonstrate that Nrf2 serves as a protein tethering Keap1 to allow podosome assembly and angiogenesis. Moreover, we emphasize that the new Nrf2 function of a Keap1 scavenger implies revisiting the interpretation of some of the previous data on the Nrf2-Keap1 system. Antioxid. Redox Signal. 00, 000-000.

Heme oxygenase-1 (HO-1), encoded by HMOX1 gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin (N = 28) or without statins (N = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes HMOX1, NQO1, and GCLM expression remained unchanged in AAA. In vitro studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although HMOX1 expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs in vitro is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including HMOX1, in AAA tissue denotes Nrf2 independency.