Journal: Frontiers in Immunology
Article Title: SARS−CoV−2 spike S1-mediated HIF−2α activation in retinal endothelial cells suggests a mechanism contributing to post−COVID endothelial dysfunction
doi: 10.3389/fimmu.2026.1770758
Figure Lengend Snippet: S1 and plasma from PCS patients influence ROS production, impair NO availability, and disrupt barrier integrity in HRECs, effects improved by belzutifan. (A) HRECs were mock-treated (control) or stimulated with S1 (100 ng/ml) for 0–6 h, and cellular ROS levels were measured using DCFDA/H 2 DCFDA (n = 3 independent experiments). (B) HRECs were mock-treated (control) or stimulated with S1 (100 ng/ml) for 4 h, and mitochondrial ROS production was measured by flow cytometric analysis using MitoSox Red (n = 4 independent experiments). (C, D) HRECs were mock-treated (control), stimulated with S1 (100 ng/mL) or CoCl 2 (100 µM), and treated with belzutifan (50 nM) for 72 h. Immunofluorescence staining was performed for F-actin (C, red ) and VE-cadherin (D, green ) , with nuclei counterstained with DAPI (blue). Images (left) were acquired at 20× magnification, and scale bars represent 100 µm. Graphs (right) illustrate the percentage of positive cells (C) and the corrected total cell fluorescence (CTCF) (D) (n = 3 independent experiments). (E) HRECs were cultured at confluence on ECIS electrodes and then stimulated with 100 ng/mL S1 or left untreated in the presence or absence of 50 nM belzutifan for 0–72 h. The loss of barrier integrity was determined by transendothelial electrical resistance (TEER). Values were normalized to time = 0 for easier comparisons (n = 3 independent experiments). (F) HRECs were treated with 2% plasma from healthy individuals (HC, n=8) or PCS patients (n=13) for 0–6 h, and cellular ROS levels were measured using DCFDA/H 2 DCFDA. (G) Mitochondrial ROS production in HRECs exposed to 2% plasma from HC (n=8) or PCS patients (n=13) for 4 h, measured by flow cytometric analysis using MitoSox Red. (H) Total NO levels in HRECs exposed to 2% plasma from HC (n=8) or PCS patients (n=13) for 4 h and 24 h, measured using a fluorometric assay for total nitrite/nitrate levels. (I) HRECs were cultured at confluence on ECIS electrodes and exposed to 2% plasma from HC or PCS patients in the presence or absence of 50 nM belzutifan for 0–48 h. The loss of barrier integrity was determined by transendothelial electrical resistance (TEER). Values were normalized to time = 0 for easier comparisons. Data are represented as means ± SD. Each dot represents one independent experiment for S1 studies or one individual donor for plasma studies. A p-value of <0.05 was considered statistically significant. P-values were determined by two-way ANOVA followed by Tukey’s post hoc test (A, E, F, I) , Mann–Whitney U test (B) , one-way ANOVA followed by Tukey’s post hoc test (C, D) , Student’s t-test (G) , and Kruskal–Wallis test followed by Dunn’s post hoc test (H) . .
Article Snippet: Primary human retinal endothelial cells (HRECs) were purchased from Innoprot and cultured in endothelial basal medium supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution (all from Innoprot).
Techniques: Clinical Proteomics, Control, Immunofluorescence, Staining, Fluorescence, Cell Culture, MANN-WHITNEY
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