Mechanic Forces Promote Brain Endothelial Activation by SARS-CoV-2 Spike Protein.

Abstract

HomeStrokeVol. 52, No. 1Mechanic Forces Promote Brain Endothelial Activation by SARS-CoV-2 Spike Protein Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBMechanic Forces Promote Brain Endothelial Activation by SARS-CoV-2 Spike Protein Aaron Babendreyer, PhD and Andreas Ludwig, PhD Aaron BabendreyerAaron Babendreyer Institute of Molecular Pharmacology, RWTH Aachen University, Germany. Search for more papers by this author and Andreas LudwigAndreas Ludwig Correspondence to: Andreas Ludwig, PhD, Institute of Molecular Pharmacology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany. Email E-mail Address: [email protected] https://orcid.org/0000-0001-8536-4986 Institute of Molecular Pharmacology, RWTH Aachen University, Germany. Search for more papers by this author Originally published9 Nov 2020https://doi.org/10.1161/STROKEAHA.120.033119Stroke. 2021;52:271–273This article is a commentary on the followingFlow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 InfectionOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 9, 2020: Ahead of Print The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) by air-borne infection of nasal and lung-epithelial cells. COVID-19 is still increasing worldwide. Besides the development of antiviral therapy and immunization, it is essential to gain detailed understanding of the novel clinical presentations of COVID-19 that are often reported.See related article, p 260The spike protein within the lipid envelope of SARS-CoV-2 binds to ACE2 (angiotensin-converting enzyme 2).1 This interaction is crucial for viral infection.2 ACE2 is not only expressed in the nasal and lung epithelium but also endothelial cells of different organs including brain.3 Postmortem analysis revealed viral infection of endothelial cells accompanied by endothelitis in several organs of COVID-19 patients.4 Infection with SARS-CoV-2 is associated with an increased rate of cerebrovascular events including ischemic stroke and intracerebral hemorrhage. This is supported by increased ischemic rates even in young patients with COVID-19.5 The molecular and cellular mechanisms underlying this cerebrovascular risk still need to be better understood.In the present issue, Kaneko et al6 report that ACE2 mRNA and protein are expressed in cultured primary endothelial cells derived from the umbilical cord and the brain. Of note, in a 3-dimensional (3D) printed vascular culture model, the ACE2 expression is much higher than in monolayer culture. Moreover, perfusion of this 3D culture further upregulated ACE2-mRNA expression. This upregulation was associated with increased binding of liposomes carrying recombinant SARS-CoV-2 spike protein. Such binding was especially enhanced at sites of stenosis. Finally, binding of SARS-CoV-2 spike protein triggered gene transcription in human brain endothelial cells including upregulation of complement component C3 (Figure).Download figureDownload PowerPointFigure. Model for the influence of mechanical forces on the activation of brain endothelial cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Both shear stress and substrate properties promote expression of ACE2 (angiotensin-converting enzyme 2) in brain endothelial cells. This correlates with increased binding of SARS-CoV-2 spike protein, which then induces transcriptional changes, such as the gene induction of the complement factor C3. This process may not only be mediated by ACE2 alone but may also involve coreceptors, or the activation of proteases. For example, ADAM17 can be activated by the spike protein of SARS-CoV-2, which in turn leads to the shedding of surface molecules, such as TNFα (tumor necrosis factor alpha), which can lead to autocrine cell activation. ADAM17 can also shed ACE2 and switch off protective functions of ACE2.The study clearly highlights that it is of great importance to choose most physiological culture conditions when studying primary endothelial cells as a cardiovascular model. It is well known that moderate flow conditions causing physiological shear stress induce a number of physiologically relevant genes, whereas proinflammatory genes are supressed.7 For instance, physiological flow conditions downregulate the expression of endothelin-1 and prevent the expression of inflammatory chemokines and adhesion molecules.8 Vice versa, many of protective genes are directly or indirectly induced by the KLF2 (Kruppel-like factor 2) pathway. These include NO synthase, thrombomodulin, and the ADAM15 (A disintegrin and a metalloproteinase 15).9 The enzymatic activity of ACE2 counterregulates induced increase in blood pressure and reduces thrombogenicity. Thus, upregulation of ACE2 by physiological flow conditions can be considered a protective mechanism. This prompts further in depth investigation on the mode of ACE2 regulation. For example, which mechanosensitive signaling pathway is involved and which transcription factor binds the ACE2 promoter. Moreover, effects of pathophysiological turbulent flow in contrast to physiological laminar flow could be investigated in detail.Besides flow conditions, the substrate of the vascular bed has significant impact on the biology of endothelial cells.10 It is well described that substrate stiffness influences various signal transduction pathways, such as Rho signaling, in endothelial cells. For example, stiffer substrates found under fibrotic conditions are more likely to lead to an inflammatory phenotype in endothelial cells.11 Another factor is the increased deposition of fibronectin, which induces inflammatory processes through activation of integrins. In the design of the present study, these aspects could explain the inductive effect on ACE2 expression by cultivation in a 3D vascular model alone. On one hand, substrate stiffness was significantly reduced by using polydimethylsiloxane compared with classical cell culture plastic, and on the other hand, integrin signaling was changed due to fibronectin coating. Therefore, it is of great importance not only to mimic flow conditions but also biomechanical properties of the environment. The 3D culture model described in this study fulfills these issues and is clearly more appropriate for the study of endothelial SARS-CoV-2 interaction than conventional endothelial cell culture.At sites of stenosis, the upregulation of ACE2 could not only confer protection but could also allow more binding of SARS-CoV-2 via its spike protein. This is indicated in the presented study by binding analysis of liposomes carrying the spike protein. It is not yet clear whether this increased binding would in fact lead to an increased infection of endothelial cells. Moreover, other mechanisms may also contribute to SARS-CoV-2 binding, and further interactions can be required for infection. Of note, CD147 (basignin) is discussed to act as another receptor for SARS-CoV-2 in epithelial cell lines.12 However, recent investigations using sensitive binding assays could not confirm the role of basigin/CD147 as an alternative receptor for recombinant spike protein.13 Nevertheless, it might be that ACE2 and CD147 are parts of a larger complex rather than independent alternative receptors.14 Moreover, spike protein is known to undergo proteolytic processing by TMPRSS2 (transmembrane protease, serine 2), and this is a critical event for cell entry of the virus.2 It is not clear whether these additional or accessory proteins are coregulated in endothelial cells by flow conditions or substrate stiffness. This could be further studied in the 3D flow culture setting.Importantly, spike protein itself can activate endothelial responses, and for this, a cellular infection by the virus is not necessary. Taken that ACE2 is the only relevant receptor for the spike protein, further research is warranted to elucidate the underlying signaling pathway including the activity of protein kinases and transcription factors. Spike protein with a D614G mutation found in new virus variants can bind ACE2 better than that of the original variant,15 but it needs to be clarified whether this also leads to increased cell activation via ACE2. It has been noted that a soluble form of ACE2 exists, which is generated by limited proteolysis (shedding) at the cell surface. Members of the surface-expressed disintegrin and metalloproteinases (ADAMs) are known to mediate the shedding of membrane-expressed cytokines, adhesion molecules, growth factors, and their receptors.16 In vitro experiments indicate that ACE2 can be cleaved by ADAM17.17 Even more interesting, spike protein of SARS-CoV-1 has been found to activate ADAM17-mediated shedding of ACE2.18 This mechanism can be relevant for the infection. Additionally, activation of ADAM17 would lead to the cleavage of junctional adhesion molecules and thereby promote increased vascular permeability. Moreover, cleavage and release of TNF (tumor necrosis factor) or growth factors would further activate endothelial cells in a positive feedback loop (Figure). These indirect mechanisms could represent additional or alternative pathways leading to changes of gene transcription that were observed by the authors. Furthermore, shedding of ACE2 would reduce the surface expression of the protease, and this would likely affect its protective function. Besides acting as protective protease, ACE2 is known to interact with the sodium-dependent amino acid transporter SIT1/B(0)AT1.19 Structural modeling suggested that the ACE2-B0AT1 complex could bind to the spike protein of SARS-CoV-2 simultaneously.20 By modifying the activity of the complex, the spike protein may be able to affect physiological functions in endothelial cells leading to a stress response including the transcription of proinflammatory genes.In conclusion, novel data with brain endothelial cells in 3D flow culture indicate that upregulation of ACE2 associates with increased SARS-CoV-2 spike protein binding and transcriptional cell activation. These effects can be mediated by the spike protein independently of cellular infection by the virus. This may involve direct cell activation via ACE2 or potentially via other accessory receptors or even activation of proinflammatory shedding enzymes. These different pathways may all contribute to the increased cardiovascular risk in COVID19 patients.Sources of FundingThis study was supported, in part, by Bundesministerium für Bildung und Forschung (project 01KI20207) and Deutsche Forschungsgemeinschaft 363055819/GRK2415 [ME3T] project C1).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.For Disclosures, see page 273.Correspondence to: Andreas Ludwig, PhD, Institute of Molecular Pharmacology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany. Email [email protected]de