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: Advanced healthcare materials

Article Title: Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

doi: 10.1002/adhm.202400388

Figure Lengend Snippet: Figure 1. a) Schematic summary of precision culture scaling (PCS-X) to customize high-throughput 3D tissue and disease models. PCS-X comprises design of experiments (DOE) methods to systematically adjust the composition and automated and parallelized preparation of cultures, the collection and analysis of quantitative readouts, and multiple linear regression (MLR) modeling of the data to identify individual and interactive effects of the investigated parameters to instruct further adjustment. Exemplifying the approach, high-throughput 3D vasculogenesis models were developed from hydrogel culture-based protocols using human umbilical vein endothelial cells or human retinal microvascular endothelial cells in mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes, respectively. The hydrogels were made of multi-armed poly(ethylene glycol) and the sulfated glycosaminoglycan heparin (starPEG-sGAG hydrogels), functionalized with covalently bound cell-adhesive RGDSP peptides (to sGAG) and sGAG-complexed growth factors. b) Parallelized hydrogel culture fabrication: The stock solutions, containing the starPEG-peptide conjugate and the sGAG (heparin-maleimide)/RGDSP peptide/growth factor/cell mixture, respectively, were automatically transferred and mixed in a 96-well plate with V-shaped wells, then transferred into a low-volume 384-well plate. c) Image analysis of vasculogenesis was performed by a dedicated 3D image analysis routine (exemplary shown image displays a HUVEC monoculture stained for F-Actin): Confocal input images were filtered (Gaussian filter) to minimize over-quantification of subcellular features, a 3D rendition of these structures was computed, masked, and subjected to a filament algorithm generating skeletonized trajectories of cellular structures. Scale bar 200 μm. d) DOE methods were used to assess effects of three crucial culturing parameters on vasculogenesis of hydrogel-embedded endothelial cells in balanced experimental designs (central composite designs). The experimental data were analyzed by MLR, generating robust models to predict endothelial cell vasculogenesis across the parameter space studied.

Article Snippet: Human telomerase reverse transcriptase (hTert) immortalized human retinal microvascular cells (HRMVECs) and hTert immortalized GFPexpressing human retinal microvascular pericytes (HRMVPs), as well as non-fluorescent hTert immortalized HRMVPs (Angio-Proteomie, US), were cultured on tissue culture flasks pre-coated with quick coating solution (Angio-Proteomie, US) in endothelial growth medium and pericyte growth medium (Angio-Proteomie, US) at 37 °C and 5% CO2 in a humidified incubator, respectively.

Techniques: High Throughput Screening Assay, Adhesive, Staining

Journal: Advanced healthcare materials

Article Title: Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

doi: 10.1002/adhm.202400388

Figure Lengend Snippet: Figure 5. Effects of vasculogenesis inhibitors on interactions between HRMVECs and HRMVPs. a) 3D image analysis routine to quantify cell-cell contacts (Imaris, Oxford Instruments). 1) The relevant channels are filtered (Gaussian), 2) a channel-specific surface algorithm is performed, 3) the Imaris Xtension ‘surface surface contact area’ is applied for creating a surface at the junction of both cell types. 4) Close-up of contacts (yellow) between HRMVECs (CellTracker Orange, green) and HRMVPs (GFP, orange) (scale bar 1–3. 200 μm and 4. 50 μm). Total filament lengths for b) HRMVECs and c) cocultured HRMVPs. d) Percentage of all HRMVECs surfaces in contact with HRMVPs. e) Total number of contacts. f) Total surface area of contacts. Data presented as violin plots with horizontal lines indicating quartiles 1–3 (n = 4–6). Asterisks indicate multiplicity adjusted P values of one-way ANOVA with post hoc Tukey test, comparing inhibitor treatments to respective vehicle controls; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human telomerase reverse transcriptase (hTert) immortalized human retinal microvascular cells (HRMVECs) and hTert immortalized GFPexpressing human retinal microvascular pericytes (HRMVPs), as well as non-fluorescent hTert immortalized HRMVPs (Angio-Proteomie, US), were cultured on tissue culture flasks pre-coated with quick coating solution (Angio-Proteomie, US) in endothelial growth medium and pericyte growth medium (Angio-Proteomie, US) at 37 °C and 5% CO2 in a humidified incubator, respectively.

Techniques:

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