In Vitro SARS-CoV-2 Infection of Microvascular Endothelial Cells: Effect on Pro-Inflammatory Cytokine and Chemokine Release

Abstract

In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.

Keywords: Human Microvascular Endothelial Cells (HMEC-1); SARS-CoV-2; inflammatory mediators.

Figure 1

Detection of ACE2 protein in VERO E6 and HMEC-1 cells. (A) Western blot analysis showing ACE2 (about 120 kDalton) in VERO E6 and HMEC-1 cell lines. (B) Quantification of ACE2 intensity. ACE2 expression was normalized versus β-actin expression. Statistical significance was determined using t-test, between HMEC-1 and VERO E6 (*** p value < 0.0001).

Figure 2

SARS-CoV-2 variants growth curves in HMEC-1 and VERO E6 cell lines. At 4, 6, 24, 48, 72 h and 7 days p.i., SARS-CoV-2 N1 gene expression in cell supernatants (panel (A): viral load expressed as copies/mL) and cellular RNA (panel (B): viral load expressed as copies/µg) of infected HMEC-1 and VERO E6 cell lines was evaluated by means of qRT-PCR.

Figure 3

SARS-CoV-2 variant B.1 N1 gene ΔCt (sgRNA-gRNA) values. The sgRNA and gRNA cycle threshold values at 24, 48, 72 h and 7 days p.i. were obtained by means of two qRT-PCRs targeting the N1 gene. Plots whiskers indicate down to the minimum and up to the maximum value for each time point.

Figure 4

Localization of SARS-CoV-2 in HMEC-1 cell line, analyzed by TEM. (A) HMEC-1 presenting SARS-CoV-2 infection (arrow) and a severe degeneration (scale bar: 500 nm). (B) Enlargement of previous figure, showing spherical viral electron-dense particles within vacuoles (scale bar: 100 nm). (C) Spherical viral particles located in the cytosol (scale bar: 500 nm). (D) Spherical viral particles located in the cytosol, near the RER. (E) Aggregate of spherical viral particles outside the cells (scale bar: 1 µm). (F) Enlargement of panel (E), showing spherical viral particles, containing black dots on the inside, within structures with smooth membrane (scale bar: 200 nm).

Figure 5

IL-6, IL-8, TNF-α, IFN-α, and hTF transcription in SARS-CoV-2 B.1 infected HMEC-1. log₁₀ fold-change values above 0 express upregulation of target gene. Cytokines and hTF mRNA levels were assessed by reverse transcription, SYBR Green Real Time PCR, and Fold Difference analysis between uninfected and infected cells at 24, 48, 72 h and 7 days p.i. The cytokines and hTF mRNA expression levels were normalized with GAPDH mRNA level and reported in the figure as log₁₀ fold difference. Significant difference of IL-6 expression at 24 h and IFN-α at 24 h and 48 h in infected cells compared to uninfected cells was observed; * p < 0.05.

Figure 6

IL-6 and IL-8 evaluation in supernatant of SARS-CoV-2 infected HMEC-1 by ELISA test. IL-6 concentration (pg/mL) in SARS-CoV-2 infected supernatants at 4 h, 6 h, 24 h, 48 h, 72 h, and 7 days p.i. for the B.1 strain (panel (A)). Significant IL-6 higher expression was observed in infected cells at 24 h, 48 h, and 72 h p.i. (*** p < 0.001) compared to non-infected cells. IL-6 expression at 4 h, 6 h, and 24 h p.i. for the B.1.617.2 and B.1.1.529 strains. Significant IL-6 higher expression was observed at 24 h for both strains (*** p < 0.001 and * p < 0.05 for B.1.617.2 and B.1.1.529, respectively) (panel (B)). IL-8 concentration (pg/mL) in uninfected and SARS-CoV-2 infected supernatants. Significant IL-8 lower expression was observed in infected cells at 24 h p.i.; *** p < 0.001 (panel (C)).