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 induces VEGF mRNA expression in HRECs but not immune activation markers. HRECs were mock-treated (control, n=4), stimulated with S1 (100 ng/mL, n=4), or LPS (100 ng/mL, n=4) for 4 h. mRNA expression of (A) VEGF , (B) IL-6 , (C) TNF , (D) IL-8 , (E) MCP-1 , (F) CXCL1 , (G) ICAM-1 , and (H) CXCL10 was quantified by RT-qPCR and normalized to GAPDH . Each dot represents one independent experiment. A p-value of <0.05 was considered statistically significant. P-values were determined by one-way ANOVA followed by Tukey’s post hoc test (A, B, D, F-H) or Kruskal– Wallis test followed by Dunn’s post hoc test (C, E) .

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: Expressing, Activation Assay, Control, Quantitative RT-PCR

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 induces high HIF-1/2α nuclear translocation and VEGFR2 upregulation in HRECs. HRECs were mock-treated (control), stimulated with S1 (100 ng/mL), or treated with CoCl 2 (100 µM) for immunofluorescence analysis. (A) HIF-1α nuclear translocation after 8 h. (B) PDK-1, BNIP-3, and GLUT-1 expression after 24 h. (C) HIF-2α nuclear translocation after 24 h and 72 h. (D) VEGFR2 expression after 24 h or 72 h. All the primary antibodies were labelled with FITC (green), and the nuclei were counterstained with DAPI (blue). Images (left) were acquired at 20× magnification, and scale bars represent 20 µm (A, D) or 100 µm (B, C) . Graphs (right) illustrate the percentage of nuclear translocation (A, C) , the corrected total cell fluorescence (B) , and the percentage of positive cells (D) . Data are represented as means ± SD. Each dot represents one independent experiment. A p-value of <0.05 was considered statistically significant. P-values were determined by one-way ANOVA followed by Tukey’s post hoc test.

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: Translocation Assay, Control, Immunofluorescence, Expressing, Fluorescence

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

Journal: Nature Communications

Article Title: Endothelial RAB5IF is required for pathological and developmental retinal angiogenesis

doi: 10.1038/s41467-025-66212-x

Figure Lengend Snippet: A – E The human retinal microvascular endothelial cells (hRMECs) expressing the applied RAB5IF shRNAs (shRAB5IF#1, shRAB5IF#2 and shRAB5IF#3, with different shRNA sequences against RAB5IF ) or the scramble control shRNA (shC) were established, expression of RAB5IF mRNA and protein was shown ( n = 5 biological repeats, 15 μg protein/lane for WB) ( A ); Cells were further cultured for the indicated time periods, sprouting ( B ), cell proliferation (by testing EdU-positive nuclei ratio, C ), migration (Transwell assays, D ) and the capillary tube formation ( E ) were tested by the listed assays. F – J Stable hRMECs expressing the listed lentiviral CRISPR/Cas9-RAB5IF-KO construct (koRAB5IF#1/koRAB5IF#2, with different sgRNA sequences against RAB5IF ) or the control construct with non-sense sgRNA (koC) were established, and RAB5IF protein expression was tested ( n = 5 biological repeats, 15 μg protein/lane for WB) ( F ); Cells were further cultured for the indicated time periods, sprouting ( G ), cell proliferation (by testing EdU-positive nuclei ratio, H ), migration (Transwell assays, I ) and the capillary tube formation ( J ) and were tested. K , L Stable hRMECs with the designated genetic modifications on RAB5IF were cultured and subjected to Seahorse analyses to evaluate oxidative phosphorylation ( K ) and glycolysis ( L ). M – R Stable hRMECs with the lentiviral RAB5IF-expressing construct (oeRAB5IF-Sl1/oeRAB5IF-Sl2, two stable cell selections) or the empty vector (Vec) were established, expression of RAB5IF mRNA and protein was shown ( n = 5 biological repeats, 15 μg protein/lane for WB) ( M ); Cells were further cultured for the indicated time periods, sprouting ( N ), cell proliferation (by testing EdU-positive nuclei ratio, O ) and migration (Transwell assays, P ) were tested, with results quantified. Seahorse assays were also carried out to evaluate ATP contents ( Q ) and basal glycolysis levels ( R ). Data are presented as means ± S.D., n = 5 (biological repeats). “N.S.” indicates no statistical difference ( P > 0.05); One-way ANOVA with Bonferroni’s post hoc test. Scale bars = 100/200 μm. Source data are provided as a Source Data file.

Article Snippet: Human retinal microvascular endothelial cells (hRMECs, cat no. CP-H130) were procured from the Procell (Shanghai, China).

Techniques: Expressing, shRNA, Control, Cell Culture, Migration, CRISPR, Construct, Phospho-proteomics, Stable Transfection, Plasmid Preparation

Journal: PLOS One

Article Title: Circular RNA FTO functions as a hsa-miR-141-3p sponge to regulate the growth and migration abilities of human retinal endothelial cells via up-regulating ZEB1

doi: 10.1371/journal.pone.0338208

Figure Lengend Snippet: The circFTO levels in cataract patients, non-PDR and PDR patients (A) and HG treated HRECs (B) were determined with RT-qPCR. ***P<0.001 VS cataract group. &&&P<0.001 VS non-PDR group. (C) The genomic locus and sequence of circFTO. RT-qPCR was performed to detect the expressions of liner FTO and liner FTO after RNase R (D) and Actinomycin D (E) treatment. (A) The differences were detected using One-way ANOVA (n=30). (B, D, E) The differences were detected using Student-T test (n=3).

Article Snippet: Human retinal microvascular endothelial cells (HRECs) were obtained from Innoprot® (P10880; Derio–Bizkaia, Spain).

Techniques: Quantitative RT-PCR, Sequencing

Journal: PLOS One

Article Title: Circular RNA FTO functions as a hsa-miR-141-3p sponge to regulate the growth and migration abilities of human retinal endothelial cells via up-regulating ZEB1

doi: 10.1371/journal.pone.0338208

Figure Lengend Snippet: A-B: The target hsa-miR-141-3p and binding sites of circFTO was predicted by starbase software and validated by the dual-luciferase reporter. The hsa-miR-141-3p expression in HRECs was determined by RT-qPCR after si- circFTO (C) and HG (D) treatment. The differences were detected using Student-T test (n=3).

Article Snippet: Human retinal microvascular endothelial cells (HRECs) were obtained from Innoprot® (P10880; Derio–Bizkaia, Spain).

Techniques: Binding Assay, Software, Luciferase, Expressing, Quantitative RT-PCR

Journal: PLOS One

Article Title: Circular RNA FTO functions as a hsa-miR-141-3p sponge to regulate the growth and migration abilities of human retinal endothelial cells via up-regulating ZEB1

doi: 10.1371/journal.pone.0338208

Figure Lengend Snippet: A-B: The target hsa-miR-141-3p and binding sites of ZEB1 was confirmed by the targetscan database and validated by the dual-luciferase reporter gene system. The ZEB1 expressions in HRECs were measured with RT-qPCR after hsa-miR-141-3p mimic (C). The mRNA (D) and protein (E) levels of ZEB1 were detected by RT-qPCR and western blot in HG-treated and circFTO knockdown HRECs. (A-C) The differences were detected using Student-T test (n=3). (D) The differences were detected using One-way ANOVA (n=3).

Article Snippet: Human retinal microvascular endothelial cells (HRECs) were obtained from Innoprot® (P10880; Derio–Bizkaia, Spain).

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Western Blot, Knockdown