Endothelial Cell Activation by SARS-CoV-2 Spike Protein and Its RBD: Central Player of the Immunothrobotic Response in COVID-19

Abstract

COVID-19 has been associated with an active immunothrombotic process. The involvement of endothelial cells (ECs) in the feedback loop of the inflammatory and thrombotic process characteristic of COVID-19, as well as its differences with other infectious inflammatory conditions, remains an area requiring further elucidation. This study aimed to assess the immunothrombotic phenotype induced by the SARS-CoV-2 Spike (S) protein and its receptor-binding domain (RBD) in endothelial-derived cell lines. HUVEC and EA.hy926 cell lines were exposed to S protein and to its RBD. Inflammatory, thrombotic, and fibrinolytic mediators were quantified. Molecular docking assays were conducted to identify potential EC receptors for S protein. EC activation was dependent on both protein concentration and stimulation time. An increased release of immunothrombotic biomarkers were observed in endothelial-derived cells exposed to the S protein and to its RBD. The RBD induced a stronger endothelial response. Molecular docking demonstrated high affinity and a possible interaction between the S protein and endothelial receptors: CD-141, CD-147, IL-6R, TLR 2, 4, and 7. These findings confirm that the S protein and its RBD can induce an immunothrombotic phenotype in EC-derived cell lines, potentially exacerbating the disease pathology. We propose possible endothelial receptors mediating this response.

1. Introduction

COVID-19 has been circulating worldwide since 2020, affecting millions of people [1,2]. Vaccines have decreased morbidity and mortality [3,4,5,6] but not the ongoing spread of infection, which remains recurrent and intermittent across the population, with cases of severe illness and associated deaths reported [7,8].

SARS-CoV-2 uses the Spike (S) protein as the main route of entry to host cells [9]. The S protein is a homotrimer located in the viral membrane [10], and several mutations have been reported in this protein, leading to the different variants of the virus that have circulated around the world [11]. The S protein is structured in subunit 1 (S1) used by the virus to infect host cells and subunit 2 (S2) which is attached to the viral membrane [12,13]. The Spike protein possesses a domain located in the S1 subunit known as the receptor-binding domain (RBD) responsible for interacting with specific receptors on host cells. Mutations in the RBD have been linked to increased viral virulence and transmissibility [14]. The main receptor used by this virus is angiotensin-converting enzyme II (ACE-II) [9,10]. Since the vascular endothelium is an ACE2-rich tissue, endothelial cells are particularly vulnerable to infection, activation and subsequent dysfunction, which can compromise vascular homeostasis [15,16]. Additionally, alternative ACE-II receptors have been proposed in various cell types that can be used by the virus to achieve its internalization and infection [17,18].

Among the classical clinical manifestations, COVID-19 is also considered a vascular disease [19]. The link between hemostatic and endothelial abnormalities with the severity of the disease in COVID-19 patients has been extensively described [20]. Furthermore, previous studies have reported that “long COVID” has been associated with persistent endothelial dysfunction, coagulation disorders, and the continued circulation of the S protein, evidenced by elevated activation markers and ongoing vascular abnormalities after acute infection [20,21,22,23]. Studying the endothelial response to SARS-CoV-2 structural proteins may enhance our understanding of the pathophysiological role of ECs during the endothelial dysfunction observed in COVID-19. The aim of this work was to study the EC response to the SARS-CoV-2 full-length S protein and to the RBD.

2. Materials and Methods

2.1. SARS-CoV-2 Spike Protein and Receptor-Binding Domain

The recombinant SARS-CoV-2 full-length S protein (S1 + S2) was provided by BioVision Human CellExp^® (Waltham, MA, USA). Cat # P1525, size: P1525-50, and the SARS-CoV-2 S protein RBD was provided by GenScript^® (Piscataway, NY, USA). Cat # Z03491 SARS-CoV-2 Spike protein (RBD, mFc Tag). Both proteins belonged to the ancestral lineage (Wuhan-Hu-1).

2.2. Cell Culture

Two well-characterized endothelial cell-derived cell lines were used to analyze the effects of both viral proteins and to compare the response to other well-known stimuli. The human umbilical vein endothelial cell (HUVEC) (Lonza, Basel, Switzerland, Cat # CC-2519) was maintained in commercial complete endothelial growth medium-2 (EGM-2) (Lonza, Cat # CC-3121). The human endothelial cell-derived cell line EA.hy926 (ATCC, CRL-2922) was generously donated by Dr. Bruno Rivas Santiago from the Centro de Investigación Médica Zacatecas, Instituto Mexicano del Seguro Social (IMSS), Zacatecas, México. EA.hy926 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Green Island, NY, USA, Cat # 12800-017), supplemented with fetal bovine serum (FBS) (10% v/v), (Gibco, Cat # A3160402), penicillin (100 U/mL), streptomycin (100 µg/mL), amikacin sulphate (0.1 µg/mL) (Thermo Fisher Scientific, Waltham, WA, USA, Cat # 15140122), and vascular endothelial growth factor (VEGF) supplement from bovine pituitary (3 mg/mL) (Sigma-Aldrich, Inc., St. Louis, MO, USA, Cat #E2759).

Both cell lines were maintained in 25 cm² tissue-culture-treated flasks in an atmosphere of 5% CO₂ at 37 °C. Medium renewal was performed every 2 days and cells were subcultured when they became 80% confluent. All the experiments were performed using cells between passages 5 and 8, and performed by triplicate.

2.3. Inflammatory and Thrombotic Stimulus

The following proteins were used as known inflammatory stimuli: tumor necrosis factor (TNF-α) from (Invitrogen Thermo Fisher Scientific, Waltham, WA, USA, KHC3011), interleukin 6 (IL-6) from (Novex Lifetech, Thermo Fisher Scientific, Waltham, WA, USA), and lipopolysaccharide (LPS) was obtained from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Vascular endothelial growth factor (VEGF) (Sigma -Aldrich, Inc., St. Louis, MO, USA) was used as an endothelial cell activation protein, and von Willebrand factor (vWF) from (IMUBIND BioMedica Diagnostics, Windsor, NS, Canada), adenosine diphosphate (ADP), and epinephrine (EPI) (Chrono-log), were used as prothrombotic stimuli.

Dengue virus serotype 2 non-structural protein 1 (DENV2 NS1) was generously donated by Oxford University and used as viral protein stimuli. The production and purification of DENV-2 NS1 protein was carried out by standard polyethyleneimine (PEI) transfection at 37 °C as previously described [24].

2.4. EA.hy926 and HUVEC Stimulation Assays

Briefly, cells (1 × 10⁴ cell/well) were seeded and grown in a 96-well cell-treated plate for 24 h in the conditions previously mentioned. Further, 100 µL of fresh medium was added. In individual assays, cells were incubated directly with SARS-CoV-2 full-length S protein and RBD protein at different concentrations (0.25, 0.5, 1.0, and 2.0 µg/mL) for different times (30, 60 and 120 min). After selecting the best endothelial stimulation condition, in independent assays, ECs were stimulated for 60 min with the following stimuli: S protein (0.25 µg/mL), RBD (1.0 µg/mL). TNF-α (20 ng/µL), IL-6 (20 ng/µL), LPS (100 ng/µL), and DENV2 NS1 (2.5 µg/mL) were used as inflammatory controls. VEGF (10 ng/µL) was used as EC’s known activation control, and vWF (10 µg/µL), ADP (20 µM), and EPI (100 µM) were used as thrombotic stimuli. Different stimuli were assessed in order to determine the differential endothelial cells’ responses and compare them to the S and the RBD response. Concentrations of viral proteins and inflammatory and procoagulant controls were based on previous reports [24,25,26,27,28]. All experiments were performed in a 5% CO₂ humidified incubator at 37 °C. After the incubation, the supernatant of the endothelial cells was obtained and stored at −80 °C until use for immunothrombotic biomarker determination.

2.5. Assessment of Immunothrombotic Biomarkers

The following immunothrombotic biomarkers were assessed: D-dimer, type 1 plasminogen activator inhibitor (PAI-1), tissue factor (TF), tissue plasminogen (tPA), coagulation factor IX (F IX), interleukin 6 (IL-6), interleukin 8 (IL-8), P-selectin (P-sel), P-selectin glycoprotein ligand-1 (PSGL-1), soluble CD40 ligand (sCD40L), and monocyte chemoattractant protein-1 (MCP-1). Soluble biomarkers determinations were performed by flow cytometry using the LEGENDplex Kit™ Human Thrombosis Panel Standard (Cat #740894) and the LEGENDplex Kit™ Human Inflammation Panel 1 (Cat #740811), both from BioLegend^®, San Diego, CA, USA, following the instructions suggested by the supplier. The samples were read using CytoFLEX equipment, Beckman Coulter Inc^®, Brea, CA, USA.

Briefly, samples were incubated with beads that are differentiated by size. Each bead set is conjugated with specific antibodies on its surface and acts as the capture bead for that particular analyte. After washing, a biotinylated detection antibody cocktail is added forming a capture bead–analyte–detection antibody sandwich. Streptavidinphycoerythrin (SA-PE) was subsequently added, and samples were taken to the CytoFLEX equipment for analysis.

2.6. Von Willebrand Factor Determination

The von Willebrand factor was assessed by using the ab223864 Human von Willebrand Factor SimpleStep ELISA^® Kit (Cat #ab223864) following specifications and recommendations issued by the supplier.

For both immunothrombotic biomarkers and vWF determinations, standard curves were generated in each assay from serial dilutions of the standards provided by the manufacturer. Background correction was performed by subtracting the absorbance or fluorescence values of the blank from each reading, and R² coefficients (>0.98) were accepted for the validation of each curve.

2.7. Molecular Docking Assays

The structures of the following molecules: S full-length ancestral lineage, ACE-II, thrombomodulin (CD-141), basigin (CD-147), neuropilin 1, interleukin 6 receptor (IL-6R), integrin α5β1, and Toll-like receptor (TLR) 2, 4, and 7 were obtained from RCSB Protein Data Bank (https://www.pdb.org) and imported into AutoDockTools 1.5.6 for docking. We used AutoDockTools to process the proteins. The modifications we made were removing ligands, correcting the S protein structure, and eliminating the water molecules associated with the S protein structure. The PDBQT format was used to perform the docking by AutoDock-Vina and the blank screen CMD of the system. Structures were visualized using PyMol (version 1.7.2.1). Metformin and omeprazole structures were considered as negative controls.

2.8. Statistical Analysis

All experiments were independently repeated at least three times. The normality of data distribution was assessed using the Shapiro–Wilk test. The results of multiple experiments are expressed as mean ± standard deviation (S.D). Multiple group comparisons were made using a one-way analysis of variance (ANOVA). Tukey’s test was used as a post hoc test for pairwise comparisons. p < 0.05 values were considered statistically significant in all cases. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA).

2.9. Ethics

Not applicable, since the study does not involve humans, human data, or animals.

3. Results

EC-derived cell line exposure to the SARS-CoV-2 proteins increases the release of proinflammatory, prothrombotic, and antifibrinolytic proteins in a concentration- and time-dependent manner.

3.1. EA.hy926 and HUVEC Activation by the S Protein and Its RBD Are Concentration- and Time-Dependent

In order to assess whether the S protein and the RBD domain could induce endothelial cell-derived cell line activation, EA.hy926 cells were incubated with different concentrations of each protein (0.25, 0.5, 1.0, 2.0 μg/mL) for different times (30, 60, 120 min) to perform an endothelial cell stimulation kinetics. Subsequently, the concentration of IL-6, PAI-1, and tPA, known immunothrombotic biomarkers, was quantified in the culture supernatants. According to our results, it is possible to observe that the S protein is able to induce the release of IL-6 at all concentrations assessed and during the different time periods, maintaining consistency in the results (p < 0.01). On the other hand, the RBD was able to induce the highest expression of IL-6 at 1.0 μg/mL at 60 and 120 min (p < 0.001). Regarding PAI-1, S protein at a concentration of 0.25 µg/mL for 60 min showed an increase in the concentration secreted by endothelial cells, without showing a statistical difference. On the other hand, tPA showed the highest concentration when stimulated with S protein at a concentration of 0.5 µg/mL (p < 0.001) for 60 min. The concentration of protein S of 0.25 µg/mL for a stimulation time of 60 min and 1.0 µg/mL for 120 min also showed an increase in the secreted concentration of this interleukin.

In contrast, the RBD required 1.0 µg/mL for 60 min to achieve the highest release of PAI-1 (p < 0.01). No differences were observed in tPA (Figure 1A–C).

Figure 1.

Stimulation of EA.hy926 endothelial-derived cells with SARS-CoV-2 S protein and its RBD. Endothelial cells were incubated with different protein concentrations 0.25, 0.5, 1.0, 2.0 µg/mL for 30, 60, and 120 min at 37 °C, 5% CO₂. Concentration of IL-6 (A) PAI-1 (B), and tPA (C) released from EA.hy926 after incubation with both proteins under different incubation conditions. * p < 0.05, ** p < 0.01, *** p < 0.001, ANOVA post hoc Tukey.

Similar results were observed using the HUVEC endothelial-derived cell line. Based on our results, it can be observed that protein S is able to induce high expression of IL-6, the level of this expression is constant in all four protein concentrations and during the different stimulation times (p < 0.001). Similarly, the RBD showed an increase in IL-6 concentrations starting at 0.25 µg/mL, reaching the highest concentration at 1.0 µg/mL for 60 and 120 min (p < 0.0001) (Figure 2A). However, when assessing PAI-1, high levels were induced when ECs were treated with S protein at 0.25 µg/mL for 60 min (p < 0.05), and RBD at 1.0 µg/mL for 60 min (p < 0.0001) (Figure 2B). No significant differences were observed in tPA levels (Figure 2C). Based on the results obtained, we decided to continue with a concentration of 0.25 for protein S and 1.0 for the RBD for 60 min stimulation, since these conditions showed consistency in the induction of immunothrombotic biomarker expression in ECs.

Figure 2.

Time- and concentration-dependent activation of HUVEC endothelial-derived cells with S protein and its RBD. HUVECs were incubated with different protein concentrations 0.25, 0.5, 1.0, 2.0 µg/mL for 30, 60 and 120 min at 37 °C, 5% CO₂ with S protein and RBD. Release of IL-6 (A), PAI-1 (B), and tPA (C) from the HUVEC cell line after incubation with both proteins under different protein concentrations and times are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ANOVA post hoc Tukey.

3.2. SARS-CoV-2 Proteins Stimulate the Release of Proinflammatory Molecules in EA.hy926

After establishing the optimal stimulation conditions, we decided to look further and try to characterize the immunothrombotic phenotype induced in EA.hy926 and HUVEC endothelial-derived cells, and compare it with other known inflammatory, procoagulant, and infectious drivers.

Our results show an increase in IL-6 secretion in ECs treated with the RBD, but not with the S protein, when compared to the basal state (p < 0.05). Interestingly, ECs stimulated with LPS, IL-6, and VEGF also showed an increased secretion of IL-6 (p < 0.0001), similar to that observed when incubated with the RBD (Figure 3A). RBD was also able to induce IL-8 release (p < 0.001), but this was not observed when ECs were exposed to S protein. VEGF and IL-6 induced higher release of IL-8 when compared to the basal state or with another inflammatory or thrombotic stimulus (p < 0.0001) (Figure 3B). When assessing P-sel, PSGL-1 and sCD40L, SARS-CoV-2 viral proteins did not induce a significant differential release of these proteins. Only cells treated with IL-6 showed higher concentrations of these proteins (p < 0.05) (Figure 3C–E). Finally, only VEGF induces the release of MCP-1 by EA.hy296 cells (p < 0.05) (Figure 3F).

Figure 3.

EA.hy926 cells were incubated with 0.25 µg/mL of S protein, 1.0 µg/mL of RBD, or known inflammatory, activation, and thrombotic agonists. The release of each inflammatory biomarker was assessed: (A) IL-6, (B) IL-8, (C) P-selectin, (D) PSGL-1, (E) sCD40L and (F) MCP-1. Data presented as means ± S.D. Differences were considered significant when compared against control. * p < 0.05, *** p < 0.001, **** p < 0.0001, ANOVA post hoc Tukey.

3.3. S and RBD Promote the Expression of Antifibrinolytic Factors in EA.hy926 ECs

Next, to assess the prothrombotic response of EA.hy926, we measured D-dimer, coagulation factor IX, PAI-1, tPA, TF, and vWF levels (Figure 4A–F). The amount of D-dimer was significantly increased upon exposure to IL-6 (p < 0.001). Viral proteins induced an increase in D-dimer concentrations, but non-significant differences were found. No differences in FIX concentrations were observed. Under the same conditions, the concentration of PAI-1 and tPA show a very characteristic behavioral pattern. Viral proteins and inflammatory stimuli such as TNF-α, as well as VEGF, induced an increase in PAI-1 release (p < 0.001). On the other hand, tPA concentrations induced by viral proteins, TNF-α, and VEGF are observed to be decreased compared to the control group. These results suggest an antifibrinolytic state. No differences were observed when comparing TF concentrations. Finally, vWF was also higher when ECs were stimulated with TNF-α (p < 0.05).

Figure 4.

S protein and its RBD induce the expression and release of procoagulant and antifibrinolytic proteins in EA.hy926. ECs were treated with 0.25 µg/mL of SARS-CoV-2 S protein, 1.0 µg/mL of RBD or known activation agonists. The release of each thrombotic biomarker was assessed: (A) D-dimer, (B) FIX, (C) PAI-1, (D) tPA, (E) TF and (F) vWF. Data presented as mean ± S.D. * p < 0.05; *** p < 0.001; **** p < 0.0001. ANOVA post hoc Tukey.

3.4. HUVEC Releases Inflammatory Cytokines When Stimulated with SARS-CoV-2 Proteins

The SARS-CoV-2 S protein and RBD significantly induced IL-6 release in HUVECs. These results are similar to those observed when treated with other inflammatory and procoagulant stimuli (p < 0.001) (Figure 5A). On the other hand, S protein enhanced IL-8 release (p < 0.001) RBD also induced the release of this interleukin (p < 0.01). The IL-8 concentrations induced by S protein were similar to those observed when HUVECs were treated with LPS and DENV2 NS1; however, they were lower than those generated by IL-6 and VEGF, which are both agonists of endothelial activation (Figure 5B). Figure 5C,D shows the concentrations of P-sel and the soluble fraction of its ligand (PSGL1) released by HUVECs treated with the different proteins in contrast to the basal state. It is possible to observe that SARS-CoV-2 proteins promoted the generation of these proteins. In the case of P-sel, they induce the generation of a higher concentration when compared to other inflammatory, viral, and procoagulant stimuli (p < 0.0001). On the other hand, the PSGL1 concentrations released when cells are treated with RBD are similar to those observed when HUVECs are stimulated with another viral protein such as DENV2 NS1 (p < 0.01). Only the RBD and VEGF stimulated a significant production of MCP-1 (p < 0.0001) (Figure 5E). Finally, when measuring the concentrations of sCD40L, Figure 5F shows that only the RBD and the proteins LPS, TNF-α, and IL-6 promoted its generation (p < 0.0001).

Figure 5.

SARS-CoV-2 Spike protein and its RBD induces an inflammatory response in HUVECs. HUVEC cell line was incubated with 0.25 µg/mL of Spike protein, or 1.0 µg/mL of RBD for 60 min and compared against untreated (media) and treated with EC known activation factors. The release of each inflammatory biomarker was assessed: (A) IL-6, (B) IL-8, (C) P-selectin, (D) PSGL-1, (E) sCD40L and (F) MCP-1. Data are expressed as media ± S.D; p differences were considered significant when compared against control * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ANOVA post hoc Tukey.

3.5. SARS-CoV-2 Proteins Induce a Prothrombotic and Antifibrinolytic Profile in HUVEC ECs

Subsequently, we decided to explore whether the thrombotic and fibrinolytic response was affected.

Figure 6A shows that only the S protein promoted the generation of D-dimer (p < 0.5). On the other hand, the RBD did not promote the generation of this molecule; however, inflammatory stimuli such as LPS, IL-6, and VEGF did enhance their genesis (p < 0.0001). No changes were observed in the concentrations of FIX (Figure 6B). Interestingly, Figure 6C,D demonstrate a dysfunction in the fibrinolytic mechanisms. It is possible to determine that SARS-CoV-2 proteins favor the release of PAI-1, similar to that observed with other proinflammatory proteins (p < 0.0001). When assessing tPA, which is inhibited by PAI-1 acts, untreated cells showed similar results when compared with the release induced by the viral proteins. Neither the S protein nor the RBD induced the expression of TF or vWF. Only the dengue NS1 protein, bacterial LPS, and IL-6 favored the release of TF (Figure 6E). No significant differences were observed in the concentrations of vWF (Figure 6F). The results are summarized in Table 1.

Figure 6.

SARS-CoV-2 S protein and RBD exacerbate the expression and release of procoagulant and antifibrinolytic proteins in the HUVEC cell line. ECs were treated with 0.25 µg/mL of SARS-CoV-2 Spike protein or 1.0 µg/mL of RBD and compared against untreated cells and known activation agonists. The release of each thrombotic biomarker was assessed: (A) D-dimer, (B) FIX, (C) PAI-1, (D) tPA, (E) TF and (F) vWF. Data presented as mean ± S.D. * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001. ANOVA post hoc Tukey.

Table 1.

Summary of biomarker expression in endothelial cell lines EA.hy926 and HUVEC.

Biomarker	EA.hy296	HUVEC
IL-6	+	+++
IL-8	++	+++
P-selectin	+	++++
PSGL-1	+	++
sCD40-L	+	+++
MCP-1	+	++++
D-dimer	+	+
FIX	-	-
PAI-1	+++	++++
tPA	+	+
TF	+	+
vWF	+	+

The qualitative symbols represent differential expression levels of each biomarker when compared to the basal expression of each protein in untreated cells following experimental treatment: “+” low expression, “++” moderate expression, “+++” high expression, and “++++” very high expression, “-” no expression.

3.6. S Protein and Its RBD Could Interact with Different Endothelial Cell Receptors in Addition to ACE-II

Molecular docking analysis revealed potential interactions between the S protein and several endothelial receptors besides ACE-II. The docking score determines the possible interaction between molecules. A more negative value means a significant probability of interaction between molecules. Another relevant value in molecular docking assays is the confidence score, in which values close to 1 suggest that the process occurs under the in silico conditions used. Thrombomodulin (CD-141), basigin (CD-147), interleukin 6 receptor, and TLRs 2, 4, and 7 showed values of docking score with the S protein even better than those reported with ACE-II, a receptor used as internal control. All proteins also showed good confidence scores, all above 0.9. On the other hand, interaction between the viral protein with neuropilin 1 and integrin α5β1 showed values similar to those found when matching with ACE-II. Likewise, the confidence scores showed results higher than 0.86 (Table 2). Possible interaction sites between S protein and endothelial cell receptors are shown (Figure 7). Metformin and omeprazole were considered as negative controls. These molecules exhibited considerably lower binding affinities against S protein when compared to the proteins evaluated in our study. This supports the specificity and relevance of our docking result. This information can be found in the Supplementary File (Figures S1 and S2 and Table S1).

Table 2.

Spike–endothelial cell receptors affinity obtained from molecular docking.

Endothelial Cell Receptor	Docking Score	Confidence Score
ACE-II	−291.62	0.9444
Thrombomodulin	−335.25	0.9760
Basigin	−302.45	0.9547
Neuropilin 1	−250.09	0.8810
Integrin α5β1	−241.22	0.8611
IL-6R	−328.49	0.9726
TLR-2	−340.98	0.9785
TLR-4	−332.87	0.9748
TLR-7	−398.33	0.9931

Figure 7.

Molecular docking representation of SARS-CoV-2 S protein docked with (A) ACE-II, (B) thrombomodulin, (C) basignin, (D) neuropilin 1, (E) integrin α5β1, (F) IL-6R, (G) TLR-2, (H) TLR-4, and (I) TLR-7. S protein is represented by the structure in yellow in figures: (B,C,F). In figures: (A,D,E) the S protein is represented by the structure represented in brown. In figures (G–I) the S protein is represented by the blue structures. The remained structures are the ligands.

4. Discussion

Endothelial cells are known to play an important role in the pathophysiology of severe COVID-19. Vascular endothelial damage involves severe inflammatory processes, active thrombosis, and fibrinolytic dysfunction [29,30,31,32]. In this study, we hypothesized that the SARS-CoV-2 S protein and its RBD directly activate endothelial cells, promoting an immunothrombotic phenotype.

Endothelial parenchyma is a rich source of angiotensin-converting enzyme 2 (ACE-II) [17]. This places ECs as targets for SARS-CoV-2 viral entry into the organism. It has been suggested that S protein may promote endothelial cell activation [33]. In this work, we demonstrate that both the S protein and the RBD can induce endothelial cell activation in a protein-concentration- and time-of-stimulation-dependent manner. Our results align with previous studies reporting the ability of Spike protein to induce endothelial activity and dysfunction [34,35].

Based on our results, it is possible to determine that these viral proteins enhance IL-6 generation in ECs, similar to those observed when cells are treated with proinflammatory agonists. Our results are in agreement with reports suggesting that S protein favors the release of this cytokine and places IL-6 as a driver of endothelial inflammation [28,36]. There are reports proposing the activation of the JAK/STAT3, PI3K/AKT and NF-κβ factor pathway, associating inflammation and endothelial dysfunction [36,37]. Similarly, IL-6 expression by ECs has been linked to an increased release of MCP-1 in these cells [34,37,38]. Consistent with our data, it is possible to observe that the viral proteins increase the concentrations of this chemotactic protein [26].

IL-8 cytokine is associated with an acute inflammatory process and can be stored in the Weibel–Palade bodies (WPBs) of endothelial cells [39]. Based on our results, it is possible to suggest that SARS-CoV-2 viral proteins are potent inducers of the inflammatory phenotype in the endothelium. These results are in agreement with those reported by Haffke et al., who observed elevated concentrations of IL-8 released by injured endothelium [40]. Similarly, it has been reported that endothelial cells stimulated with SARS-CoV-2 virus induce the expression of this cytokine [41].

P-selectin (P-sel) is another marker of EC activity. P-sel is a protein associated with a pro-adhesive endothelial phenotype for leukocytes, promoting the local inflammatory state [42]. We also determined values of the soluble fraction of its ligand, the PSGL-1 protein, which is expressed mainly by leukocytes and endothelial cells and is directly related to leukocyte–endothelial cell interaction [43]. We found that in the HUVEC, but not in the EA.hy926 cell line, viral proteins led to the expression of this protein. These results are consistent with previous reports linking their endothelial expression to inflammatory challenges [39]. An alternative pathway for P-sel expression independent of the classical WPB degranulation pathway proposes an increase in cAMP and intracellular calcium concentrations [44]. The differential expression observed between EA.hy926 and HUVEC may reflect inherent differences in cellular metabolism. Similar results were observed when assessing PSGL-1 concentrations. Our results agree with those reported by Bhargavan et al., who reported the association of these proteins with coagulopathy and NETosis [45]. The upregulation of P-sel and PSGL-1 reinforces the role of endothelial–leukocyte interactions in COVID-19-associated vasculopathy. Similar results were observed when assessing sCD40-L, a highly expressed protein in ECs which is associated with activation of the adaptive immune response [46]. Also, this protein has been associated with immunosuppression and a poor prognosis in subjects affected by COVID-19 [47,48].

These results confirm that the exacerbated inflammatory response observed in COVID-19 is being fueled in part by ECs that are activated directly by the virus proteins. This confirms that endothelial inflammation provides positive feedback, perpetuating endothelial activation, favoring the adhesion and activation of leukocytes, and inducing the release of more inflammatory factors by the endothelium.

Once the inflammatory response of the endothelium was determined, we decided to evaluate the thrombotic profile and the fibrinolytic response in these cells. D-dimer levels, a marker of fibrin degradation and thrombotic activity, were modestly increased by viral proteins, and have been found to be elevated in patients with severe COVID-19 [49,50]. We suggest fibrin formation is a consequence of the conversion of fibrinogen found in the culture medium. When we assessed the fibrinolytic state, we observed that both SARS-CoV-2 proteins increase PAI-1 expression in both cell lines. On the other hand, we studied the concentrations of tPA, a substrate of PAI-1. Interestingly, an inverse correlation is evident between PAI-1 and tPA in the ECs that were stimulated with viral proteins, suggesting a dysfunctional fibrinolytic process [51]. Our results are in agreement with those reported by Han and Pandey, who observed a high expression of PAI-1 and associated the metallopeptide ZMPSTE24 as a regulator of this pathway [52]. Abnormalities in the fibrinolytic status of patients affected by COVID-19 have been previously reported [47,53]

Finally, when assessing the concentrations of coagulation modulating proteins, we observed that only TNF-α induced the expression of vWF in the EA.hy926 cell line. vWF is a protein stored in WPBs, highly procoagulant, and released upon endothelial activation and injury [39]. Our results are in agreement with Guo and Kanamarlapudi, who reported that S protein induces an increase in vascular permeability, accompanied by vWF release, in an ACE-II receptor-dependent pathway [54]. High vWF concentrations have been directly associated with disease severity and active immunothrombotic states in patients affected by COVID-19 [47,53].

Other proteins that we assessed were coagulation factor IX, in which we found no differences in its expression in any cell line in response to any stimulus. This is explained by the fact that this factor is not stored nor generated by endothelial cells [55]. TF expression indicates endothelial cell activation and injury. This protein is procoagulant and activates the proteolytic cascade of coagulation [56]. SARS-CoV-2 proteins did not favor the expression of this protein, only inflammatory insults in HUVECs. TF expression by endothelial cells have been reported after exposure to the RBD to endothelial cells, generating coagulopathy [57].

In this study it is possible to observe that the RBD induces a stronger immunothrombotic response when compared to the complete S protein. This may be due to differences in the three-dimensional conformation of the S protein, protein stability, aggregation state, and accessibility of functional domains in the solution, which could affect receptor engagement and downstream signaling. In the case of the RBD, since it is more exposed in its domain conformation, it exhibits greater affinity for receptors in the absence of the stabilizing domains of the complete protein (S2) [58]. On the other hand, the presence of glycosylation sites on the S protein can modulate the interaction with receptors when it is in its three-dimensional conformation [59]. Similarly, the RBD shows a smaller size than the complete protein, this leads to a higher concentration and, therefore, a greater possibility of ligand–receptor interaction.

Furthermore, different endothelial surface receptors alternative to the ACE-II protein have been proposed for SARS-CoV-2 [16,60]. For this reason, we decided to assess by bioinformatic molecular docking the possible interactions and affinities that exist between the S protein and the proposed receptors, using ACE-II as an internal molecular docking control. Molecular docking analysis revealed that there are alternative endothelial receptors to ACE-II which may serve as pathways of virus infection and may be associated with the infectious capacity of SARS-CoV-2. We report that thrombomodulin has a high affinity for S protein, as does basigin. These results support the hypothesis that inhibition of the thrombomodulin–thrombin-activated protein C pathway axis is associated with the endothelial dysfunction observed in COVID-19 [61]. TM is a membrane glycoprotein whose ligand is thrombin, acting as a natural anticoagulant by neutralizing the proteolytic capacity of this molecule. This protein also naturally regulates the inflammatory response in ECs, so its blockade influences the immunothrombotic response of the endothelium to proinflammatory stimuli [62]. On the other hand, our results support Wang et al., who proposed basigin as an alternative route of infection of endothelial cells by SARS-CoV-2 virus [63]. This receptor has even been proposed as a therapeutic target in COVID-19 [64].

Other receptors shown to be targets for S protein were neuropilin 1, a protein associated with angiogenic processes in response to VEGF. This protein has been linked to the maintenance of endothelial adhesion and has been reported to participate in endothelial activation in SARS-CoV-2 virus infection [65,66,67]. Glycoprotein α5β1 is another endothelial receptor with good affinity for S protein. Robles et al. confirmed that the virus can infect endothelium through this receptor [28].

Furthermore, since ECs express a wide variety of innate immune receptors, such as the Toll-like receptor (TLR) family [68,69,70], we decided to assess the affinity of TLR-2, TLR-4, and TLR-7. All three receptors showed an affinity even higher than that observed with the natural virus receptors, suggesting that these membrane and intracellular receptors, respectively, are not a gateway for the virus. TLR activation initiates NF-κβ and MAPK signaling through MyD88 and/or RIG-1, favoring the establishment of an inflammatory phenotype in the endothelium [71]. Normally, TLR7 are absent in resting ECs, but are inducible under inflammatory activation. It is well known that these TLRs are associated as receptors for PAMPs in other infectious processes that cause vascular injury such as COVID-19 [71]. Different authors have shown that these receptors can be used by the virus to infect human cells, including platelets, kidney, neurons, and endothelial cells [72,73,74]. Our results confirm this and support the imperativeness of testing them as therapeutic targets.

Finally, observing the exacerbated inflammatory state of COVID-19 and based on our results where we reported that IL-6 favors endothelial activation, we decided to evaluate whether the IL-6 receptor (IL-6R) could act as a receptor for S protein. Our results show that there is a high affinity between this IL-6R and the S protein. This suggests that the IL-6 receptor plays an important role in perpetuating the inflammatory state and endothelial dysfunction observed in SARS-CoV-2 infection [75]. These results remain predictive and require future experimental validation.

HUVEC and EA.hy926 cell lines were used to examine the stability and consistency of inflammatory and procoagulant responses in endothelial cells independently of cell line transformation. The use of the HUVEC cell line provides physiological rigor, and more closely recapitulate the physiological phenotype of vascular endothelium, including the regulated expression of adhesion molecules such as P-selectin and integrins. While EA.hy926 provides reproducibility in repeatable and scalable experiments. The EA.hy926 cell line has been recently used in experiments to characterize the response of endothelial cells to various stimuli such as shear forces, inflammatory insults, and plant-derived peptides [26,76]. Since the EA.hy926 cell line is a hybridoma derived from the fusion of HUVECs with A549 lung carcinoma cells, this hybrid origin may result in significant alterations in gene and protein expression profiles, including integrins, selectins and other adhesion molecules, reflecting the partial retention of epithelial or tumor-associated transcriptional programs, potentially affecting endothelium interactions, and immunothrombotic signaling as previously reported [77]. The intrinsic phenotypic characteristics of the EA.hy926 line may also play a key role in the expression of immunothrombotic biomarkers. Further research is essential to elucidate the complexities of this protein–endothelial cell interaction, as well as the molecules and signaling pathways that drive the functional differences between HUVECs and EA.hy926 cells. Understanding these differences is imperative to determine the impact of vascular ECs in the setting of immunothrombotic infectious diseases, such as COVID-19, that specifically affect different vascular sites. However, the results suggest that both cell lines may constitute a representative model for the study of venous immunothrombotic phenomena and ultimately allow the development of more precise and specific strategies to study pathologies that may affect the venous vascular niche.

5. Conclusions

Our findings demonstrate that the SARS-CoV-2 Spike protein and its RBD induce a concentration- and time-dependent activation of endothelial cells, promoting a proinflammatory and antifibrinolytic phenotype. Both proteins stimulate the release of cytokines and the expression of immunothrombotic mediators in EA.hy926 and HUVECs, suggesting that their interaction with endothelial cells extends beyond ACE2 receptor binding. These results support the role of the Spike and RBD proteins in endothelial dysfunction and highlight their potential contribution to the immunothrombotic complications observed in COVID-19.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cimb48020161/s1.

Author Contributions

M.E.V.-S. designed the research; M.E.V.-S. and A.O.-Z. conceived and designed the experiments; A.C.-M., J.V.-C. and A.E.H.-M. performed the cell culture experiments and data analysis; N.G.-L. participated in research design; A.C.-M., Y.A.-M. and R.V.-A. performed immunothrombotic and vWF determination experiments; A.C.-M. and G.V.-M. designed and performed the molecular docking assays. M.E.V.-S. and N.G.-L. performed data analysis; A.C.-M. wrote the original manuscript and M.E.V.-S., N.G.-L., and A.O.-Z. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by CIC-UMSNH and SECIHTI México in its call for proposals “Ciencia de Frontera: Paradigmas y controversias 2021”, grant number 320085. A. Cano-Méndez, Jennifer Velez Chávez and Rogelio Vega Agavo are recipients of SECIHTI-Mexico fellowships for post-graduate students. The sponsors of this study are public or non-profit organizations that support science in general and have no role in gathering, analyzing, or interpreting the data.