Damian Sendler: In order to keep the body’s homeostasis intact, vascular endothelial cells (ECs) play an important role. COVID-19 suffers from the classic symptoms of this deadly illness due to damage to the EC barrier: swelling, inflammation of the blood vessels, and abnormalities in blood clotting. However, it is not known how COVID-19 affects ECs. SARS-CoV-2 spike protein alone stimulates the EC inflammatory phenotype in a way reliant on integrin 51 signaling, as shown in this study. To induce nuclear translocation of NF-B and the subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNF, IL-1, and IL-6), as well as adhesion of peripheral blood leukocytes and EC monolayer hyperpermeability, researchers incubated human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the In addition, integrin 51 activation inhibitors were shown to have no impact. It was also found that these vascular effects occur in vivo when spike was administered intravenously and found to increase lung, liver, kidney, and eye expression of ICAM1, VCAM1, CD45 and TNF, IL-1 and IL-6 as well as to disrupt retinal capillary barrier function when spike was administered intravitreally. Our findings suggest that the spike protein, through its RGD motif in the receptor-binding domain of the receptor, is capable of binding and activating the NF-B target gene expression programs responsible for vascular leakage and leukocyte adhesion in endothelial cells (ECs), which in turn promotes leukocyte migration. A novel direct effect of SARS-CoV-2 has been discovered on EC dysfunction, and this discovery introduces the potential for using integrin 51 to treat COVID-19 inflammation.
Damian Jacob Sendler: COVID-19 has been linked to endothelial cell dysfunction (EC dysfunction) (1, 2, 3). Vasopermeability and coagulation and inflammation are prevented, and ECs retain their barrier function when at rest. Because of the damage or infection, endothelial cells (EC) are stimulated and release chemoattractants (cytokines), adhesion molecules (integrins), and leukocyte infiltration (4). Acute respiratory distress syndrome, pulmonary edema, cytokine storm, multiple organ failure, and disseminated intravascular coagulation (5) are the most common causes of death in persons with severe COVID-19, all of which are indicative of EC failure (1, 2, 3). A number of chronic endothelial diseases, such as aging, obesity, hypertension, diabetes, and cardiovascular disease, are linked to severe COVID-19 instances or fatalities (6, 7).
Damian Sendler
Dr. Sendler: When SARS-CoV-2 enters and replicates, it may cause the EC death mechanism described above (8), which may then result in mitochondrial dysfunction (9), as well as the downregulation of the SARS-CoV-2 spike protein and activation of the proinflammatory ACE2 receptor, as well as a buildup of proinflammatory and vasoconstrictor angiotensin II (2). Proinflammatory cytokines and chemokines may be activated by the spike protein, as well as harmful reactive oxygen species and cell death (12). However, the underlying mechanisms of many of these impacts remain a mystery to this day.
Integrins may play a role in modulating SARS-CoV-2 infection in place of ACE2. Blood clotting, inflammation, and angiogenesis are all affected by the hemostatic, heterodimeric, transmembrane adhesion molecules known as integrins. One of the receptor-binding domains of the spike protein has an integrin-binding RGD motif that is exposed and binds to 1 integrins on pulmonary epithelial cells and monocytes (13, 14). (15). COVID-19’s therapeutic effectiveness is shown by blocking the binding of the spike protein to the integrin 51, which prevents the infection of SARS-CoV-2 in vitro (16) and in vivo (17). NF-B, the transcription factor that regulates the production of proteins involved in inflammation and angiogenesis, is activated by the ligation of integrin 51 in ECs by fibronectin (18). As a result of these findings, we investigated the role of spike in inducing the endothelial proinflammatory phenotype via binding to the integrin 51.
The capacity of ECs to attach leukocytes, a characteristic of the inflammatory process, may be used to measure inflammatory alterations. Human umbilical vein endothelial cells (HUVEC) were pretreated for 16 hours with spike, the receptor-binding domain of spike, the RGD tripeptide, or TNF as a pro-inflammatory control, and peripheral blood leukocytes were incubated for 1 hour (Figure 1 ). Leukocyte adhesion to HUVEC was enhanced by Spike with great efficacy (EC50 = 1.6 nM) and resembled that generated by TNF, which is a major EC pro-inflammatory change initiator (19) (19) (Figure 1a, b). It was also shown that spike and the RGD tripeptide had comparable dose-response curves (EC50=1.8 nM) because of their receptor-binding domains (Figure 1c). According to these findings, the RGD sequence found in the spike receptor-binding domain is responsible for triggering the ECs’ proinflammatory response when used alone.
Many ligands for integrins include RGD as the integrin-binding motif, such as fibronectin, which is the primary ligand for the integrin 51. The EC inflammatory program is activated by the upregulation of fibronectin during inflammation (20). (18). In order to establish that spike binds to integrin 51, we performed ELISA (16). When spike or the receptor-binding domain of spike was treated with integrin 51 or neutralizing antibodies against integrin 51 or the integrin 5 subunit, the plates were coated with RGD tripeptide or neutralizing antibodies. RGD tripeptide and anti-5 antibodies blocked integrin binding to Spike and its receptor-binding domain (Kd = 200pM) as predicted (Figure 2 a) (Figure 2b). The promotion of leukocyte attachment by spikes may be explained if fibronectin ligation of integrin 51 results in the production of adhesion molecules in endothelial cells (ECs) (18). A dosage (100 nM) of spike, spike receptor-binding domain, RGD tripeptide, or TNF (1 nM) with maximum stimulatory impact on leukocyte adherence to HUVEC was examined to determine that evoked effects were successfully taking place before anti-51 or anti-5 antibodies were used (Figure 2c). Both antibodies prevented leukocyte adherence in response to spike, the spike receptor-binding domain, and the RGD tripeptide, consistent with an integrin 51-dependent action. TNF’s action was not altered, indicating that it is not reliant on integrins (21).
Spike’s ability to increase leukocyte adherence was examined since the activation of NF-B in endothelial cells (ECs) causes inflammation (18). A protein known as inhibitors of kappa B (IB) inhibits the activity of NF-kappa B, a transcriptional factor for a number of genes associated with inflammation, in the cytoplasm. For NF-B to be transported into the nucleus and bind to target genes’ promoters and enhancers, it must first be phosphorylated or degraded (22). Fluorescence cytochemistry and a monoclonal antibody against the p65 component of NF-B were used to investigate the cellular distribution of NF-B in HUVECs (Figure 3 a). In the absence of treatment, cells’ cytoplasms were evenly dispersed with p65. This redistribution of p65 was similar to that caused by TNF, which was triggered by Spike. Nuclear translocation of p65 in response to spike and TNF was inhibited by anti-5 antibodies, but was not inhibited by same antibodies when spike was combined with anti-NF-B inhibitor BAY11-7085 (Figure 3a). Anti-5 antibodies inhibited the degradation of IB in response to spike, but not in reaction to TNF, while BAY11-7085 protected IB degradation by both spike- and TNF-induced stimuli (Figure 3b). Spike seems to activate NF-B through integrin 51, according to our findings.
COVID-19 has been classified as a vascular disease as a result of growing evidence (1, 2, 3, 34). Because of the edema, intravascular coagulation, vascular inflammation, and unregulated inflammation cell infiltration that result from blood vessel injury in severe COVID-19, progressive lung damage and multi-organ failure are also possible outcomes. Vascular dysfunction in COVID-19 has been linked to a number of different pathways (1, 2, 34), but little is known about how SARS-CoV-2 interacts directly with ECs (9, 11).
It has been shown that ACE2 is the best-known host receptor for spike (35, 36, 37), while alternative spike cell surface receptors have been discovered, including neuropilin-1, toll-like receptors (10), and RGD-binding integrins (39). (15, 16). It is known that the RGD-binding integrin 51 facilitates the entrance of SARS-CoV-2 into epithelial cells and monocytes in vitro and enhances lung viral load and inflammation in vivo after spike binding to it (16). (17). Proinflammatory genes in ECs may be activated by the binding of fibronectin RGD to integrin 51 in ECs (18), but the influence of spike on the EC response has not been examined.
Damian Jacob Sendler
Damian Jacob Markiewicz Sendler: EC inflammation is activated when spikes are bound to the integrin 51 receptor, as shown in this study. TNF, a well-known EC inflammatory response-inducing agent, was shown to enhance the production of ICAM1 and VCAM1 and the attachment of leukocytes to the EC monolayers (Figure 8). (19). No impact of spike on ICAM1 or VCAM1 proteins was detected, however mRNA and protein levels for both adhesion molecules were shown to be closely correlated in human vascular endothelial cells treated with several inflammatory mediators.. (40, 41). In addition, the increase in VCAM1 and ICAM1 mRNA levels and leukocyte adhesion had a strong dose-response relationship. An increase in VCAM1 and ICAM1 activity in EC may be linked to spikes, according to this association. ICAM1 and VCAM1 and leukocyte marker CD45 expression were elevated in retinal capillaries after intravenous injection of spike. This suggests that spike increases leukocyte adhesion across the various capillary beds.
RGD tripeptide and receptor binding domain of spike elicited similar responses to those of a spike, and that the RGD tripeptide itself blocked spike- and spike receptor-binding domain-binding to integrin 5-1, and that spike-induced proinflammatory effect in ECs was prevented by integrin 5-1 neutralizing antibodies indicated that spike, through its RGD motif in its receptor-binding domain, binds the receptor 5-1 in ECs to promote inflammation (Figure 8). ECs’ 51 signaling in response to spike is heavily reliant on the transcription factor NF-B, as well. Anti-NF-B and anti-5 antibodies blocked all of Spike’s activities on ECs, which included IB breakdown, nuclear translocation of NF-B, production of adhesion molecules, coagulation factors (TF and FVIII), proinflammatory cytokines (TNF, IL-1, and IL-6), and leukocyte adherence (Figure 8). In ECs, ligation of fibronectin to 51 upregulates the expression of proinflammatory genes through the NF-B signaling pathway, according to this research (18). In fact, proinflammatory cytokines like TNF activate NF-B as a major signaling molecule in ECs through an integrin-independent pathway (19).
Toll-like receptor-induced cytokine release by neutrophils, macrophages, and lung epithelial cells in response to spike (10, 39) and the envelope protein (42) provides a mechanism for the cytokine storm found in severe COVID-19 (25). When toll-like receptors are activated, they may work in tandem with integrins like 5 to enhance their signaling pathways (43). There are few or no toll-like receptors on the surface of human endothelial cells, indicating that interactions between toll receptors and integrin 51 do not contribute to the proinflammatory impact of spike on the endothelium. Additionally, ACE2 is upregulated in the ECs after the activation of 51-NF-B signaling by spike (Figure 8). By binding to EC’s ACE2 receptor, spike reduces ACE2 protein levels, inhibiting mitochondrial function and eNOS activity, and ultimately damaging the EC (9). A decrease in the expression of ACE2 would lead to an increase in inflammation, as ACE2 is anti-inflammatory and its overexpression protects against severe COVID-19 symptoms (45). Interestingly, we discovered that spiking NF-B signaling in ECs boosts ACE2 mRNA levels in an anti-parallel manner. If this impact is translated into an increase in the amount of ACE2 protein, these data imply two different scenarios: By supplying more SARS-CoV-2 receptors for infection, elevation of ACE2 expression increases viral infection of endothelial cells (ECs) in a protective manner.
Another characteristic of severe COVID-19 is excessive vasopermeability, which results in edema. In this study, we demonstrated that spike binding to integrin 51 increased EC monolayer hyperpermeability through the RGD motif (Figure 8). The receptor-binding domain and the RGD tripeptide imitated spike-induced hyperpermeability in monolayers, while antibodies against the 51 integrin and the 5-integrin subunit inhibited it. Furthermore, intravitreal injection of spike resulted in several hemorrhagic regions and accelerated extravasation of Evans-blue-linked albumin in retinal capillaries, demonstrating spike-induced promotion of EC hyperpermeability. Disruption in barrier function of retinal capillaries caused by a spike is similar to that caused by intravitreal injections of an anti-inflammatory agent known as vascular endothelial growth factor (VEGF) (46, 47).
Damien Sendler: EC-cell junctions are known to be stabilized by 1-integrins throughout development (48), but inflammation may potentially lead to their breakdown (26). One of the most important mechanisms for inducing inflammation is the activation of integrins, such as those found on the cell membranes of endothelial cells (ECs) (49). We found that the spike signals via 51 promoted the production of stress fibers, endothelial cell retraction, and inter-endothelial gap creation in accordance with this finding (Figure 8). Spike increased RhoA and Cdc42 GTPases and downregulated Rac1 in EC, which are all Rho family GTPases that govern these activities With respect to EC junctions and EC matrix adhesion, Rho GTPases act in two ways, depending on the situation in which they are activated. RhoA is linked to the breakdown of the barrier, Rac1 to its upkeep, and Cdc42 to its stability and recovery during inflammation (50).
According to the observations presented above, spike interfered with the peripheral distribution of CD31 in stable EC monolayers (Figure 8). CD31’s altered location in ECs is compatible with enhanced vasopermeability since it shows sticky capabilities and is mostly localized at junctions between neighboring cells (27). EC junctional proteins are downregulated in both normal and diabetic animals when spike is administered, which is consistent with a previous study revealing that spike compromises the endothelium barrier’s integrity (51). After that, spike phosphorylated/activated eNOS in the endothelial cells (EC), and NO is a major vasorelaxant and vasopermeability factor (31, 52) (Figure 8).
We have shown that by binding to 51, spike alters the EC phenotype to induce vascular inflammation in vitro and in vivo (Figure 8). To regulate permeability or quiesce leukocytes is lost when spike-mediated 51 activation occurs, which are both signs of EC failure (19). As a result of these discoveries, we can better understand why COVID-19 is a vascular illness and how to treat it.
There is a possibility that vaccination with the spike protein in COVID-19 vaccines might lead to EC dysfunction. According to this research, the EC50 value of spike was discovered to be [300ng-1], which is substantially lower than that observed in the COVID-19 severe COVID-19 (53), which had spike protein levels of [30 ng mL-1]. Because the majority of the spike stays attached to cells, it does not disperse much from the injection site. Moreover (54). Because antibodies created by a previous immunization rapidly and efficiently eliminate any trace quantities of spike that may have entered the blood, spike is no longer detectable following the second vaccination (54). So immunization should assist avoid EC damage in COVID-19 rather than aggravate it.
