The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19. Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19. Dysfunction of endothelial cells (ECs) has emerged as a major driver of coronavirus disease 2019 (COVID-19) (1Teuwen L.-A. Geldhof V. Pasut A. Carmeliet P. COVID-19: The vasculature unleashed.Nat. Rev. Immunol. 2020; 20: 389-391Google Scholar, 2Jin Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar, 3Libby P. Lüscher T. COVID-19 is, in the end, an endothelial disease.Eur. Heart J. 2020; 41: 3038-3044Google Scholar). During resting state, ECs maintain their barrier function by limiting vasopermeability and preventing coagulation and inflammation. However, when activated in response to damage or infection, ECs produce chemoattractants, cytokines, and adhesion molecules, leading to vascular leakage, clot formation, inflammation, and leukocyte infiltration (4Godo S. Shimokawa H. Endothelial functions.Arterioscler. Thromb. Vasc. Biol. 2017; 37: e108-e114Google Scholar). Most people with severe COVID-19 die from acute respiratory distress syndrome, pulmonary edema, cytokine storm, multiple organ failure, and disseminated intravascular coagulation (5Elezkurtaj S. Greuel S. Ihlow J. Michaelis E.G. Bischoff P. Kunze C.A. Sinn B.V. Gerhold M. Hauptmann K. Ingold-Heppner B. Miller F. Herbst H. Corman V.M. Martin H. Radbruch H. et al.Causes of death and comorbidities in hospitalized patients with COVID-19.Sci. Rep. 2021; 11: 4263Google Scholar), all of which reflect EC dysfunction (1Teuwen L.-A. Geldhof V. Pasut A. Carmeliet P. COVID-19: The vasculature unleashed.Nat. Rev. Immunol. 2020; 20: 389-391Google Scholar, 2Jin Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar, 3Libby P. Lüscher T. COVID-19 is, in the end, an endothelial disease.Eur. Heart J. 2020; 41: 3038-3044Google Scholar). Moreover, severe cases or deaths because of COVID-19 are associated with chronic endothelial damage from comorbidities, such as aging, obesity, hypertension, diabetes, and cardiovascular disorders (6Vrints C.J.M. Krychtiuk K.A. Van Craenenbroeck E.M. Segers V.F. Price S. Heidbuchel H. Endothelialitis plays a central role in the pathophysiology of severe COVID-19 and its cardiovascular complications.Acta Cardiol. 2021; 76: 109-124Google Scholar, 7Bermejo-Martin J.F. Martín-Fernandez M. López-Mestanza C. Duque P. Almansa R. Shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease).J. Clin. Med. 2018; 7: 400Google Scholar). Potential mechanisms of vascular dysfunction in COVID-19 include EC death in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and replication (8Varga Z. Flammer A.J. Steiger P. Haberecker M. Andermatt R. Zinkernagel A.S. Mehra M.R. Schuepbach R.A. Ruschitzka F. Moch H. Endothelial cell infection and endotheliitis in COVID-19.Lancet. 2020; 395: 1417-1418Google Scholar), the binding of the spike protein of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor causing its downregulation and subsequent mitochondrial dysfunction (9Lei Y. Zhang J. Schiavon C.R. He M. Chen L. Shen H. Zhang Y. Yin Q. Cho Y. Andrade L. Shadel G.S. Hepokoski M. Lei T. Wang H. Zhang J. et al.SARS-CoV-2 spike protein impairs endothelial function via downregulation of ACE 2.Circ. 2021; Scholar), the activation of the proinflammatory and the of proinflammatory and Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar). In addition, the spike protein the expression of proinflammatory cytokines and S. M. C. R. H. SARS-CoV-2 spike protein inflammation via activation of the 2021; Scholar), the of K. T. R. SARS-CoV-2 spike protein and leukocyte adhesion in endothelial 2021; Scholar), and cell death F. J. Wang Yang J. J. T. T. Wang Q. M. Yang L. Y. Y. et al.SARS-CoV-2 spike inflammation and through by 2021; Scholar). However, the mechanisms of of these effects unclear. to ACE2, function as SARS-CoV-2 are cell adhesion molecules that on inflammation, and The spike protein an integrin-binding motif on the of the receptor-binding A. P. potential role for in cell entry by 2020; Scholar, V. A. and in associated to integrin motif Med. 2020; that binds to on pulmonary cells and S. S. A. A. M. The spike of SARS-CoV-2 binds to on the of 2021; Scholar). In of the binding of the spike protein to the integrin ⍺5β1 SARS-CoV-2 infection in W. The integrin binding as a for SARS-CoV-2 2020; and in G. In from SARS-CoV-2 infection by in 2021; Scholar), the therapeutic of integrin ⍺5β1 in COVID-19. Furthermore, of integrin ⍺5β1 in ECs activates the NF-κB responsible for the expression of in inflammation and S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar). These to the binding of spike to the integrin in ECs to the proinflammatory by the of ECs to a of the inflammatory blood leukocytes for with human umbilical vein endothelial cells for with spike, the receptor-binding of spike, the RGD or as a proinflammatory the adhesion of leukocytes to in a manner with and this the induced by a of EC proinflammatory of endothelial cells in Rev. Immunol. 7: and In addition, the receptor-binding of spike and the RGD tripeptide in to the by spike These results show that spike alone activates the proinflammatory in ECs and suggest that the RGD in the spike responsible for this RGD the integrin-binding motif of integrin the of integrin inflammation Z. A.J. M. Endothelial by Thromb. Vasc. Biol. 2018; and the EC inflammatory S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar). We to the binding of spike to integrin W. The integrin binding as a for SARS-CoV-2 2020; Scholar). with integrin and with spike or the receptor-binding of spike in the or the of the RGD tripeptide or integrin or the integrin and its receptor-binding integrin with the as the RGD tripeptide and the and prevented this binding of integrin the expression of adhesion molecules in ECs S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar), we the binding to integrin the of leukocyte The of or a of spike, spike domain, RGD or on leukocyte adhesion to to that with an integrin leukocyte adhesion in response to spike, the spike domain, and the RGD The of that integrin of the Rev. Immunol. Scholar). integrin activates NF-κB in ECs to inflammation S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar), we the by which spike leukocyte adhesion the activation of NF-κB a for in inflammation that in the by the with as The of for the nuclear translocation of NF-κB and its binding to and of target M. NF-κB The role of the Scholar). The NF-κB in and a the of In the of the of induced the of in the cell and this the induced by the nuclear of in response to the NF-κB the nuclear translocation of induced by spike and with these spike and induced the of the of in and prevented in response to spike in response to TNFα, prevented by spike and We that spike activates NF-κB through its with integrin the leukocyte adhesion to ECs dependent on NF-κB we the of spike RGD or of leukocyte adhesion to of NF-κB prevented the of leukocyte adhesion in response to all Furthermore, spike in a the of cell adhesion and vascular cell adhesion of which the adhesion of leukocytes to the of ECs of of The spike the expression of adhesion molecules for for the leukocyte Furthermore, the of and expression prevented by the of NF-κB and integrin These findings show that the binding of spike to integrin activates the NF-κB in ECs responsible for leukocyte of proinflammatory cytokines the and disseminated intravascular coagulation are of severe COVID-19 (1Teuwen L.-A. Geldhof V. Pasut A. Carmeliet P. COVID-19: The vasculature unleashed.Nat. Rev. Immunol. 2020; 20: 389-391Google Scholar, 2Jin Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar, 3Libby P. Lüscher T. COVID-19 is, in the end, an endothelial disease.Eur. Heart J. 2020; 41: 3038-3044Google Scholar), and integrin activation to of proinflammatory cytokines, and coagulation factors in ECs S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar). Here, we show that spike the of coagulation factors and proinflammatory cytokines and and and in of coagulation factors and proinflammatory cytokines prevented by integrin and the of the expression of prevented by the of NF-κB and integrin These findings that spike activates NF-κB that are dependent and of integrin to the vascular proinflammatory and of severe COVID-19. binding to an EC protein of SARS-CoV-2 protein in ECs via the which in to mitochondrial endothelial dysregulated and of COVID-19 (9Lei Y. Zhang J. Schiavon C.R. He M. Chen L. Shen H. Zhang Y. Yin Q. Cho Y. Andrade L. Shadel G.S. Hepokoski M. Lei T. Wang H. Zhang J. et al.SARS-CoV-2 spike protein impairs endothelial function via downregulation of ACE 2.Circ. 2021; Scholar). Here, we show that spike the expression of in and that this by and the of NF-κB These findings that spike on in ECs on binds to or activates Z. Y. Y. R. Y. Z. Q. Y. F. T. Z. J. H. et enzyme 2 from 5: Scholar), its by spike of a EC expression EC infection by SARS-CoV-2 the spike protein alone EC dysfunction in vivo, of spike to an in and 2 and tissues liver, kidney, and to expression of ICAM1, VCAM1, leukocyte of and proinflammatory cytokines (TNFα, IL-1β, and The that and in reflect a direct of spike on EC expression of proinflammatory and leukocyte to ECs in the spike administration to the expression of and in all tissues and this associated with of the leukocyte in the tissues and and to retinal for injection of spike in Furthermore, spike increased the expression of IL-1β, and IL-6 in the lung, liver, and The tissues are with the of blood and suggest a direct action of spike on EC dysfunction in proinflammatory effects of spike S. M. C. R. H. SARS-CoV-2 spike protein inflammation via activation of the 2021; Scholar, R. P. The SARS-CoV-2 acute in and barrier dysfunction in human endothelial J. 2021; or and administration and These reflect the action of spike on cell leukocyte vascular critical of inflammation Z. B. and in vascular and leukocyte acute J. Heart 2021; Scholar). with spike, the receptor-binding of spike, and the RGD tripeptide induced a in the of the of hyperpermeability The and Furthermore, the of EC and of with spike spike with the peripheral of of an adhesion protein that maintains the of ECs of endothelial These direct of in EC and are that the and EC barrier function T. The in endothelial cell Scholar). of causing EC activation S. M. in endothelial cells J. Scholar), and cell J. of and by cell Biol. Scholar). of with spike for in and and a in that by the of Furthermore, vasopermeability The and Scholar), and spike the of in in an manner In to the the protein of these of integrin prevented spike and RGD in these show that spike binding to integrin and to EC the spike protein alone EC hyperpermeability in vivo, 2 of alone or of spike the of and the retinal vasculature in intravitreal multiple retinal that in with In the of as an of retinal induced a in in the intravitreal injection These findings show that spike the barrier function of retinal in vascular of integrin as promising a for the of B. Y. S. and of an integrin in with and in and the of the integrin to Z. J. Zhang J. B. He W. Y. K. H. F. in by the integrin and SARS-CoV-2 W. The integrin binding as a for SARS-CoV-2 2020; Scholar, G. In from SARS-CoV-2 infection by in 2021; Scholar). Here, we show that and the binding of spike and the spike to in and the leukocyte adhesion to and hyperpermeability in response to spike, spike domain, and RGD has COVID-19 as a vascular disease (1Teuwen L.-A. Geldhof V. Pasut A. Carmeliet P. COVID-19: The vasculature unleashed.Nat. Rev. Immunol. 2020; 20: 389-391Google Scholar, 2Jin Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar, 3Libby P. Lüscher T. COVID-19 is, in the end, an endothelial disease.Eur. Heart J. 2020; 41: 3038-3044Google Scholar, P. COVID-19 a vascular Med. 2021; Scholar). damage and in severe COVID-19, to edema, intravascular coagulation, vascular inflammation, and inflammatory cell mechanisms for vascular dysfunction in COVID-19 (1Teuwen L.-A. Geldhof V. Pasut A. Carmeliet P. COVID-19: The vasculature unleashed.Nat. Rev. Immunol. 2020; 20: 389-391Google Scholar, 2Jin Y. Ji W. Yang H. Chen S. Zhang W. Duan G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches.Signal Transduct. Target. Ther. 2020; 5: 293Google Scholar, P. COVID-19 a vascular Med. 2021; the direct action of SARS-CoV-2 on ECs (9Lei Y. Zhang J. Schiavon C.R. He M. Chen L. Shen H. Zhang Y. Yin Q. Cho Y. Andrade L. Shadel G.S. Hepokoski M. Lei T. Wang H. Zhang J. et al.SARS-CoV-2 spike protein impairs endothelial function via downregulation of ACE 2.Circ. 2021; Scholar, K. T. R. SARS-CoV-2 spike protein and leukocyte adhesion in endothelial 2021; Scholar). the receptor for spike M. H. S. T. S. G. A. C. S. SARS-CoV-2 cell entry on and and by a 2020; Scholar, M. A. V. of cell entry and receptor for SARS-CoV-2 and 2020; 5: Scholar, J. Y. C. G. Q. A. F. entry mechanisms of S. A. 2020; Scholar), spike cell that is, B. K. Chen C. L. M. R. P. A. et a for SARS-CoV-2 2020; Scholar), S. M. C. R. H. SARS-CoV-2 spike protein inflammation via activation of the 2021; Scholar, K. T. SARS-CoV-2 spike protein via receptor in and human 2021; Scholar), and S. S. A. A. M. The spike of SARS-CoV-2 binds to on the of 2021; Scholar, W. The integrin binding as a for SARS-CoV-2 2020; Scholar). In integrin an integrin spike SARS-CoV-2 entry and infection of cells and in W. The integrin binding as a for SARS-CoV-2 2020; and and inflammation in G. In from SARS-CoV-2 infection by in 2021; Scholar). of integrin by RGD motif activates the expression of proinflammatory in ECs S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar), the binding of spike to in ECs and its on the EC inflammatory response has In this we show that spike binding to integrin activates the inflammatory of the expression of adhesion molecules and and the of leukocytes to EC TNFα, a of EC inflammatory of endothelial cells in Rev. Immunol. 7: Scholar). the of spike on the protein of and a between and protein has for adhesion molecules in human vascular ECs with inflammatory K. T. T. Y. K. R. effects of and on vascular endothelial new cell Scholar, R. C. C.A. M. Vascular cell adhesion gene and expression are through an in human vascular endothelial Clin. Scholar). In addition, a between the of spike on and and that on leukocyte adhesion. that spike the of and in with this the intravenous administration of spike the expression of and and that of the leukocyte in tissues to that spike leukocyte vascular the vascular as in retinal capillaries. The that the RGD tripeptide and the receptor-binding of spike to of spike, the RGD tripeptide and spike to and prevented the proinflammatory in ECs that spike, through its RGD motif in its receptor-binding domain, binds to integrin ⍺5β1 in ECs to inflammation Furthermore, we show that the NF-κB a to in ECs in response to on ECs to nuclear translocation of expression of adhesion molecules and coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and leukocyte and with the of NF-κB and prevented all These findings are with a that NF-κB the by which to proinflammatory in ECs S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar). NF-κB a activated in ECs by proinflammatory cytokines, such as via an of endothelial cells in Rev. Immunol. 7: Scholar). and cells to spike S. M. C. R. H. SARS-CoV-2 spike protein inflammation via activation of the 2021; Scholar, K. T. SARS-CoV-2 spike protein via receptor in and human 2021; and to the protein M. R. Yang P. the SARS-CoV-2 protein to produce inflammatory Immunol. 2021; via the activation of cytokine a the cytokine in severe COVID-19 R. P. The SARS-CoV-2 acute in and barrier dysfunction in human endothelial J. 2021; Scholar). ⍺5β1 in cells S. P. L. A. V. integrin activates an of gene expression for and Biol. Scholar), and with to their T. G. The the in response to and Scholar). However, human ECs or on their S. L. B. A. T. expression of receptor 2 in human Biol. Scholar), that between and integrin ⍺5β1 to the proinflammatory of spike in the by which the activation of in ECs by spike inflammation the of that by binding to the receptor in spike the downregulation of protein and the subsequent of mitochondrial function and leading to EC damage (9Lei Y. Zhang J. Schiavon C.R. He M. Chen L. Shen H. Zhang Y. Yin Q. Cho Y. Andrade L. Shadel G.S. Hepokoski M. Lei T. Wang H. Zhang J. et al.SARS-CoV-2 spike protein impairs endothelial function via downregulation of ACE 2.Circ. 2021; Scholar). Z. Y. Y. R. Y. Z. Q. Y. F. T. Z. J. H. et enzyme 2 from 5: Scholar), and its severe COVID-19 M. C. J. et between and expression and COVID-19 respiratory Rep. 2021; 11: Scholar), downregulation of inflammation to we that the spike activation of in ECs that this protein, these findings suggest of expression a EC and of expression infection of ECs by SARS-CoV-2 receptor for vasopermeability leading to of severe COVID-19. 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