Senescent endothelial cells are predisposed to SARS-CoV-2 infection and subsequent endothelial dysfunction

Figures

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  Figure 1. SARS-CoV-2 infects ECs. (A) Quantitative PCR analysis for ACE2 in early passage (P3) and replicative senescent (P20) HUVECs (n = 4 each). Target genes expression was normalized to 18S expression levels. (B) Quantitative PCR analysis for ACE2 in Calu-3 cells and early passage or replicative senescent ECs (n = 3 each). ACE2 expression was normalized to 18S expression levels. (C) Quantitative PCR analysis for SARS-CoV-2 ORF1ab in early passage and replicative senescent ECs infected with SARS-CoV-2 at 1 MOI (n = 4 for senescent EC-no infection; n = 6 each for others). ORF1ab expression was normalized to 18S expression levels. (D) Representative images of immunocytochemistry for SARS-CoV-2 spike protein (red fluorescence) in early passage and replicative senescent HUVECs infected with SARS-CoV-2 (50 MOI) at 6 hpi. Bars: 100 μm. Spike-staining was quantitatively analyzed (n = 12 each). (E) SARS-CoV-2 attachment on early passage and replicative senescent HUVECs was analyzed using quantitative PCR for ORF1ab (n = 6 each). Statistical analyses were performed using two-tailed unpaired Student’s t-test (A,B), while Mann Whitney U-test were used for D and E. One-way ANOVA with Fisher’s LSD…

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  Figure 2. SARS-CoV-2 enters into ECs through endocytosis. (A) Representative electron microscopy images (× 80,000) for senescent HUVECs infected with SARS-CoV-2 at 50 MOI (2 hpi). Arrows indicate SARS-CoV-2 virions. Bars: 200 nm. (B) Representative electron microscopy images (× 80,000) for early passage and senescent ECs infected with SARS-CoV-2 at 1 MOI (6 hpi). Arrows indicate SARS-CoV-2 entry via caveolae-like structures. Bars: 200 nm. (C) Representative electron microscopy images (× 80,000) for early passage and senescent ECs infected with SARS-CoV-2 at 1 MOI (24 and 48 hpi). Arrows indicate SARS-CoV-2 virions. Bars: 200 nm. (D) Representative images of immunocytochemistry for SARS-CoV-2 spike protein (red fluorescence) and Rab5A (green fluorescence) in replicative senescent HUVECs infected with SARS-CoV-2 (50 MOI) at 6 hpi. SARS-CoV-2 spike proteins were largely co-localized with Rab5A. Bars: 100 μm. Spike and Rab5A double-positive areas were quantitatively analyzed (n = 6 each). Statistical analysis was performed using Mann Whitney U-test. Data are presented as mean ± SE **P < 0.01.

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  Figure 3. SARS-CoV-2 infection affects various genes expression in ECs. (A) Quantitative PCR analysis for tissue factor (TF), intercellular adhesion molecule-1 (ICAM-1), interleukin-1a (IL-1a), and IL-6 in early passage and senescent HUVECs with or without SARS-CoV-2 infection (1 or 50 MOI) at 72 hpi (n = 4 each for control cells; n = 6 each for infected cells). Statistical analyses were performed using one-way ANOVA with Fisher’s LSD post hoc analysis for TF, ICAM-1, and IL-6, while Kruskal-Wallis test by ranks was used for IL-1a. Data are presented as mean ± SE *P < 0.05, **P < 0.01, and ****P < 0.0001. (B) Comprehensive RNA-Seq data analysis in ECs infected with SARS-CoV-2 or INFA was performed. Volcano plots for genes whose expression was significantly altered (P < 0.05) by SARS-CoV-2 or INFA infection in early passage and senescent ECs were shown (n = 3 each). Red plots indicate the genes significantly increased more than 2-fold after the virus infection. Blue plots indicate the genes significantly (P < 0.05) decreased less than half after the virus infection. (C,D) Venn Diagram analysis of the genes whose expression was significantly altered by SARS-CoV-2 or INFA infection (> 2 or <…

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  Figure 4. Pathway analysis for the genes whose expression was significantly altered by the virus infection. Pathways affected by the virus infection were analyzed using iPathwayGuide. Dots representing the top 10 impacted pathways are positioned by their p-values from two different analyses; an Impact analysis measuring total perturbation accumulation (pAcc) and an Over-representation analysis (pORA). The size of each dot denotes the total number of genes in the corresponding pathway.