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: Theranostics

Article Title: System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy.

doi: 10.7150/thno.72947

Figure Lengend Snippet: Figure 6. ADAM10’s activator EGCG inhibits the activation of human retinal endothelial cells (HRECs) in vitro and vessel outgrowth in ex vivo models. (A and B) ADAM10 activity impairment in ocular disease models are shown. (A) ADAM10 activity in retinae from control mice and OIR mice. n = 3. (B) ADAM10 activity in choroid/RPE from control mice and CNV mice. n = 4. Data are presented as mean ± s.e.m. Statistical analysis was determined by unpaired, two-tailed Student’s t-test; * p < 0.05. (C) AlamarBlue assay demonstrated inhibition of HRECs viability by 50 µM EGCG following 24 h treatment (n = 3). (D) DAPI staining demonstrated inhibition of HRECs migration by 50 µM EGCG following 4 h treatment (n = 3). (E) EGCG inhibits choroidal vessel outgrowth in a dose-dependent manner. Representative images (left) and quantitative analysis (right) of microvessel formation from mouse choroidal explants demonstrating a significant inhibitory effect of EGCG (n = 3 independent experimental groups, n ≥ 6 explants per treatment group). (F) EGCG inhibits aortic vessel sprouts at the dosage of 50 µM. Representative images (left) and quantitative analysis (right) of macrovessel formation from mouse aortic explants demonstrating a significant inhibitory effect of EGCG (n = 3 independent experimental groups, n ≥ 6 explants per treatment group). Scale bar: 100 µm. All images shown are representative, and data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test or unpaired, two-tailed Student’s t-test; * p < 0.05 and *** p < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) were purchased from Angioproteomie (Boston, MA, USA) and maintained in endothelial growth media 2 (EGM2), which contained EBM2 basal media and Endothelial Cell Growth Medium BulletKitsTM (Lonza, Basel, Switzerland) or endothelial basal media 2 (EBM2), which contained 0.2 % FBS according to the supplier’s instruction.

Techniques: Activation Assay, In Vitro, Ex Vivo, Activity Assay, Control, Two Tailed Test, Alamar Blue Assay, Inhibition, Staining, Migration, Comparison

Journal: Theranostics

Article Title: System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy.

doi: 10.7150/thno.72947

Figure Lengend Snippet: Figure 7. AXL is ADAM10’s downstream substrate and ADAM10’s function is dependent on the activity of AXL. (A) Full-length AXL protein levels in HREC with 50 µM ADAM10 activator EGGC or 10 µM ADAM10 inhibitor GI 254023X is shown at both 4- and 24-h post treatment. GAPDH is used as loading control (B) Combination treatment of AXL inhibitor R428 and ADAM10 activator EGCG have no additive effects on HRECs viability. AlamarBlue assay demonstrated inhibition of HRECs viability by 50 µM EGCG, 1 µM R428 or combination treatment of 50 µM EGCG, 1 µM R428 following 24 h treatment (n = 3). (C) Combination treatment of AXL inhibitor R428 and ADAM10 activator EGCG have no additive effects on HRECs migration. DAPI staining demonstrated inhibition of HRECs migration by 50 µM EGCG, 5 µM R428 or combination treatment of 50 µM EGCG, 5 µM R428 following 4 h treatment (n = 3). (D) R428 reverses the promoting effect of EGCG inhibitor GI 254023X on HRECs viability. AlamarBlue assay demonstrated effects of HRECs viability by 10 µM GI 254023X, 1 µM R428 or combination treatment of 10 µM GI 254023X, 1 µM R428 following 24 h treatment (n = 3). (E) R428 reverses the promoting effect of GI 254023X on HRECs migration. DAPI staining demonstrated effects of HRECs migration by 10 µM GI 254023X, 5 µM R428 or combination treatment of 10 µM GI 254023X, 5 µM R428 following 4 h treatment (n = 3). Scale bar: 100 µm. All images shown are representative, and data are presented as means ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test; ** p < 0.01 and *** p < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) were purchased from Angioproteomie (Boston, MA, USA) and maintained in endothelial growth media 2 (EGM2), which contained EBM2 basal media and Endothelial Cell Growth Medium BulletKitsTM (Lonza, Basel, Switzerland) or endothelial basal media 2 (EBM2), which contained 0.2 % FBS according to the supplier’s instruction.

Techniques: Activity Assay, Control, Alamar Blue Assay, Inhibition, Migration, Staining, Comparison

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: Identification of ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) in diabetic retinopathy (DR) and high‐glucose‐treated high‐glucose‐treated human retinal endothelial cells (HRECs). (a) The heatmap of the differentially expressed genes in low glucose (LG) and high glucose (HG). Upregulated genes and downregulated genes are shown in red and blue. (b) Volcano plots showing long non‐coding ribonucleic acids expression in the LG and HG groups. The red dots show the significant expressed genes. (c) Reverse transcription quantitative polymerase chain reaction was carried out to detect ATP2B1‐AS1 levels in 5 mmol/L or 25 mmol/L glucose treated HRECs. (d) Reverse transcription quantitative polymerase was carried out to distinguish the level of ATP2B1‐AS1 in blood samples obtained from DR patients ( n = 30) and healthy individuals. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) prevents cell proliferation, migration, angiogenesis and permeability. (a) Reverse transcription quantitative polymerase chain reaction was made to measure the expression of ATP2B1‐AS1 after transfecting plasmid cloning deoxyribonucleic acid (pcDNA)‐long non‐coding ribonucleic acids (lncRNA) ATP2B1‐AS1 (pcDNA‐lnc) and (short hairpin RNA‐lncRNA ATP2B1‐AS1; shR‐lnc) into high‐glucose‐treated human retinal endothelial cells (HRECs). (b) The level of ATP2B1‐AS1 was detected by reverse transcription polymerase chain reaction after transfecting pcDNA‐lnc and shR‐lnc into HRECs by Cell Counting Kit‐8 assay. (c) Proliferation of HRECs was detected by Cell Counting Kit‐8 assay. (d, e) Migration ability was measured by wound healing migration assay and transwell assay. (f) Tube formation assay was used to distinguish angiogenesis ability in HRECs. (g) Cell junctional assembly formation of CDH5 staining. (h) Vascular permeability was detected by using evans blue injection. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Migration, Permeability, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Cloning, shRNA, Polymerase Chain Reaction, Cell Counting, Transwell Assay, Tube Formation Assay, Staining, Injection, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) sponges microRNA (miR)‐4729. The microRNAs lists and scores on predicted by using the MicroRNA Target Prediction Database. (b) Predicted miR‐4729 binding sites in 3′UTR of ATP2B1‐AS1 and dual luciferase report assay in ATP2B1‐AS1‐wild type (WT) or ATP2B1‐AS1‐mutation (MUT) co‐transfected with miR negative control (NC) or miR‐4729 mimics. (c) Level of miR‐4729 in high‐glucose‐treated human retinal endothelial cells (HRECs) transfected with shR‐lnc or pcDNA‐lnc. (d) miR‐4729 expression in blood from diabetes retinopathy (DR) patients ( n = 30) and non‐DR individuals. (e) Pearson's correlation analysis was used to check the relationship between ATP2B1‐AS1 and miR‐4729. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Luciferase, Mutagenesis, Transfection, Negative Control, Expressing, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) reduced high glucose‐treated high‐glucose‐treated human retinal endothelial cells (HRECs) cell proliferation, migration, angiogenesis and permeability through regulating microRNA (miR)‐4729–IQ motif‐containing GTPase‐activating protein 2 (IQGAP2) axis. (a) Schematic indicating the miR‐4729 sites in IQGAP2 and dual luciferase assay in IQGAP2‐wild type (WT) or IQGAP2‐mutation (MUT) treated HRECs co‐transfected with miR‐NC or miR‐4729 mimics. (b) The protein IQGAP2 level was detected by WB after transfection. (c) HRECs proliferation was detected by Cell Counting Kit‐8 assay after transfection. (d, e) Migration ability was measured by wound healing migration assay and transwell assay after transfection. (f) Tube formation assay was used to detect the ability of angiogenesis in HRECs after transfection. (g) Cell junctional assembly formation of VE‐cadherin staining after transfection. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Migration, Permeability, Luciferase, Mutagenesis, Transfection, Cell Counting, Transwell Assay, Tube Formation Assay, Staining, Standard Deviation

Journal: Regenerative engineering and translational medicine

Article Title: CD140b (PDGFRβ) signaling in adipose-derived stem cells mediates angiogenic behavior of retinal endothelial cells

doi: 10.1007/s40883-018-0068-9

Figure Lengend Snippet: Representative images of HREs co-cultured with control siRNA treated ASCs or CD140b siRNA treated ASCs for 6 days. (A) Upper panel shows colored images of HREs, stained with Isolectin B4 (red); ASCs, stained for α-SMA (green), and co-cultures counter-stained with DAPI (blue). Lower panel shows Red-only channels representing angiogenic tubes stained with Isolectin B4 (4× magnification). (B) Representative high magnification images of ASCs and HREs co-culture. (C) Image analysis of vascular tube length calculated by image J software as pixels/field. Data represent Mean ± SEM performed in triplicates. *, p<0.05 using unpaired Student T-test; n=3 donors.

Article Snippet: Co-culture of ASCs and human retinal endothelial cells (HREs) Human Retinal Endothelial Cells (HREs; ACBRI 181, Cell Systems Corporation, Kirkland, WA) were co-cultured with Cd140b+ or CD140b− ASCs as described previously ( 8 , 15 ).

Techniques: Cell Culture, Control, Staining, Co-Culture Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Macrophage-Myofibroblast Transition Contributes to Myofibroblast Formation in Proliferative Vitreoretinal Disorders

doi: 10.3390/ijms241713510

Figure Lengend Snippet: Identification of CD206 + cells in epiretinal membranes. Immunohistochemical staining for the M2 macrophage marker CD206 showing immunoreactivity in ( A ) vascular endothelial cells (arrows), ( B ) stromal monocytes/macrophages (arrows) and ( C ) stromal spindle-shaped cells (arrows) in membranes from patients with proliferative diabetic retinopathy (PDR). ( D ) CD206 surface expression was detected in human retinal microvascular endothelial cells (HRMECs) using flow cytometry. Results are presented as the mean percent of CD206 + cells ± SEM and are derived from four independent experiments. Immunoreactivity for CD206 was detected ( E ) in monocytes/macrophages (arrows) and ( F ) spindle-shaped cells (arrows) in membranes from patients with proliferative vitreoretinopathy (PVR). Note that some of the CD206 + cells in the PVR membrane contained pigment. Representative figures are provided for 1 patient out of a total of 12 PDR or 12 PVR patients. Each panel is from a different patient (scale bar, 10 µm).

Article Snippet: Human retinal Müller glial cells (MIO-M1) (a generous gift from Prof. A. Limb, Institute of Ophthalmology, University College, London, UK) and human retinal microvascular endothelial cells (HRMECs) (Cell Systems Corporation, Kirkland, WA, USA) were cultured as described previously [ ].

Techniques: Immunohistochemical staining, Staining, Marker, Expressing, Flow Cytometry, Derivative Assay, Membrane