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

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: World Journal of Diabetes

Article Title: Effect of ranibizumab on diabetic retinopathy via the vascular endothelial growth factor/STAT3/glial fibrillary acidic protein pathway

doi: 10.4239/wjd.v16.i5.99473

Figure Lengend Snippet: Effect of ranibizumab on the viability of adult retinal pigment epithelial 19 cells and human retinal microvascular endothelial cells. Cell viability was assessed using the Cell Counting Kit-8 assay ( n = 3, independent experiments). A: Effect of ranibizumab (0 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, or 025 mg/mL) treatment on adult retinal pigment epithelial 19 (ARPE-19) cell viability; B: Effect of ranibizumab (0 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, or 025 mg/mL) treatment on human retinal microvascular endothelial cell (HRMEC) viability. All results are expressed as the mean ± SD. d P < 0.0001. 1 P vs 24 hour group. 2 P vs 48 hour group. NC: Untreated group.

Article Snippet: Human retinal microvascular endothelial cells (HRMECs) and primary human retinal endothelial cells were purchased from ScienCell (Carlsbad, CA, United States) and cultured in a low-glucose (5.5 mmol/L) primary endothelial cell culture medium (ScienCell, Wuhan, Hubei Province, China) in a humidified atmosphere containing 50 mL/L carbon dioxide at 37 °C.

Techniques: Cell Counting

Journal: World Journal of Diabetes

Article Title: Effect of ranibizumab on diabetic retinopathy via the vascular endothelial growth factor/STAT3/glial fibrillary acidic protein pathway

doi: 10.4239/wjd.v16.i5.99473

Figure Lengend Snippet: mRNA expression of vascular endothelial growth factor, interleukin 6, cluster of differentiation 18, intercellular adhesion molecule, tumor necrosis factor alpha, and signal transducer and activator of transcription 3 in adult retinal pigment epithelial 19 cells and human retinal microvascular endothelial cells. Untreated (NC) (5.5 mmol/L glucose), NC + ranibizumab (5.5 mmol/L glucose + 0.125 mg/mL ranibizumab), high glucose (Hg) (25 mmol/L glucose), and Hg + ranibizumab (25 mmol/L glucose + 0.125 mg/mL ranibizumab). A: Ratios of the mRNA expression of vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), cluster of 18 differentiation (CD18), intercellular adhesion molecule (ICAM), tumor necrosis factor alpha (TNF-α), and signal transducer and activator of transcription 3 (STAT3) in different adult retinal pigment epithelial 19 (ARPE-19) groups; B: Ratios of mRNA expression of VEGF, IL-6, CD18, ICAM, TNF-α, and STAT3 in different human retinal microvascular endothelial cell (HRMEC) groups. All results are expressed as the mean ± SD. a P < 0.05. b P < 0.01. 1 P vs NC group. 2 P vs Hg group.

Article Snippet: Human retinal microvascular endothelial cells (HRMECs) and primary human retinal endothelial cells were purchased from ScienCell (Carlsbad, CA, United States) and cultured in a low-glucose (5.5 mmol/L) primary endothelial cell culture medium (ScienCell, Wuhan, Hubei Province, China) in a humidified atmosphere containing 50 mL/L carbon dioxide at 37 °C.

Techniques: Expressing

Journal: World Journal of Diabetes

Article Title: Effect of ranibizumab on diabetic retinopathy via the vascular endothelial growth factor/STAT3/glial fibrillary acidic protein pathway

doi: 10.4239/wjd.v16.i5.99473

Figure Lengend Snippet: Expression of glial fibrillary acidic protein, signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 proteins in adult retinal pigment epithelial 19 cells and human retinal microvascular endothelial cells. Untreated (NC) (5.5 mmol/L glucose), NC + ranibizumab (5.5 mmol/L glucose + 0.125 mg/mL ranibizumab), high glucose (Hg) (25 mmol/L glucose), and Hg + ranibizumab (25 mmol/L glucose + 0.125 mg/mL ranibizumab). A: Expression of glial fibrillary acidic protein (GFAP), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (pSTAT3) proteins in adult retinal pigment epithelial 19 (ARPE-19) cells were detected by Western blot analysis; B: Expression of GFAP, STAT3, and pSTAT3 proteins in human retinal microvascular endothelial cells (HRMECs) were detected by Western blot analysis; C: Ratios of GFAP, STAT3, and pSTAT3 protein expression levels in different groups of ARPE-19 cells; D: Ratios of GFAP, STAT3, and pSTAT3 protein expression levels in different groups of HRMECs. All results are expressed as the mean ± SD. a P < 0.05. b P < 0.01. 1 P vs NC group. 2 P vs Hg group.

Article Snippet: Human retinal microvascular endothelial cells (HRMECs) and primary human retinal endothelial cells were purchased from ScienCell (Carlsbad, CA, United States) and cultured in a low-glucose (5.5 mmol/L) primary endothelial cell culture medium (ScienCell, Wuhan, Hubei Province, China) in a humidified atmosphere containing 50 mL/L carbon dioxide at 37 °C.

Techniques: Expressing, Western Blot

Journal: Nature Communications

Article Title: Development of graphitic carbon nitride quantum dots-based oxygen self-sufficient platforms for enhanced corneal crosslinking

doi: 10.1038/s41467-024-49645-8

Figure Lengend Snippet: a Cell viability of HCEC cells incubated with g-C 3 N 4 QDs at different concentrations (biologically independent samples, two-way ANOVA multiple comparison test, mean ± SD, n = 6, the absence of a P -value indicates no statistical difference between the groups ( p > 0.05)). b Cell viability of hRMEC cells incubated with g-C 3 N 4 QDs at different concentrations (biologically independent samples, two-way ANOVA multiple comparison test, mean ± SD, n = 6, the absence of a P -value indicates no statistical difference between the groups ( p > 0.05)). c Cell viability of ARPE−19 cells incubated with g-C 3 N 4 QDs at different concentrations (biologically independent samples, two-way ANOVA multiple comparison test, mean ± SD, n = 6, the absence of a P -value indicates no statistical difference between the groups ( p > 0.05)). d Calcein-AM/PI double staining of HCEC cells incubated with g-C 3 N 4 QDs at different concentrations (The experiment was repeated three times independently with similar results). e , f Flow-cytometric analysis of HCEC cells incubated with g-C 3 N 4 QDs at different concentrations. (biologically independent samples, two-way ANOVA multiple comparison test, mean ± SD, n = 3, living cell (0 vs 100, p < 0.0001; 0 vs 200, p < 0.0001; 0 vs 400, p < 0.0001; 100 vs 400, p < 0.0001; 200 vs 400, p < 0.0001), Early apoptotic cell (0 vs 100, p < 0.0001; 0 vs 200, p < 0.0001; 0 vs 400, p < 0.0001); Late Apoptotic Cell (0 vs 100, p = 0.0027; 0 vs 200, p < 0.0001; 0 vs 400, p < 0.0001; 100 vs 200, p = 0.0044; 100 vs 400, p < 0.0001; 200 vs 400, p < 0.0001), Necrosis Cell (0 vs 400, p = 0.0019; 200 vs 400, p = 0.0009), the absence of a P -value indicates no statistical difference between the groups ( p > 0.05)).

Article Snippet: The human retinal microvascular endothelial cells (HRMEC), human retinal epithelial cells (ARPE-19), human corneal epithelial cells (HCEC) were provided by the Global Bioresource Center.

Techniques: Incubation, Comparison, Double Staining

Journal: Scientific Reports

Article Title: PPARα activation directly upregulates thrombomodulin in the diabetic retina

doi: 10.1038/s41598-020-67579-1

Figure Lengend Snippet: PPARα directly transactivates THBD expression. ( a , b ) Immunoblot analysis showing the expression of TM in HUVECs ( a ) and HRMECs ( b ) treated with pemafibrate (10 μM) or vehicle for 24 h. Expression of TM was increased by pemafibrate treatment in both HUVECs and HRMECs. ( c , d ) HUVECs ( c ) and HRMECs ( d ) were transfected with siRNA targeted to human PPARα (5 nM) or control siRNA and treated with pemafibrate or vehicle for 24 h. THBD mRNA was measured using RT-qPCR. Cyclophilin mRNA was used as the invariant control. Knockdown of PPARα canceled pemafibrate-mediated upregulation of THBD in HUVECs and HRMECs. ( e ) PPARα binding on the THBD promoter was evaluated by ChIP-qPCR. ChIP signals are presented as fold enrichment. Cyclophilin was used as a negative binding region. PPARα was bound approximately 100 bp upstream from the TSS of THBD . ( f ) Alignment of promoter sequences of human THBD , mouse Thbd , and rat Thbd containing two putative DR1 motifs. The DR1 motifs at positions – 1,135 to – 1,123 bp and – 189 to – 177 bp of human THBD are conserved in mouse and rat Thbd . ( g ) Alignment of the two putative DR1 motifs in the human THBD promoter. At the consensus site, the consensus nucleotide found in the PPARα binding sequences is represented by black letters. ( h ) Luciferase reporter analysis using the human THBD promoter. HUVECs were transfected with the luciferase reporter containing wild-type THBD promoter or indicated deletion and mutations in DR1 motifs together with PPARα and RXRα expression plasmids. After transfection, HUVECs were treated with fenofibric acid for 24 h and subjected to the luciferase reporter assay. The luciferase activity with fenofibric acid was divided by the activity without fenofibric acid and is presented as fold activation. (Results are expressed as mean ± SD of three independent experiments. *p < 0.05. Mann–Whitney U test).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and human retinal microvascular endothelial cells (HRMECs) were purchased from Lonza (Walkersville, MD, USA) and cultured in EGM-2 MV medium (Lonza) containing 5% FBS at 37 °C in 5% CO 2 .

Techniques: Expressing, Western Blot, Transfection, Control, Quantitative RT-PCR, Knockdown, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Activity Assay, Activation Assay, MANN-WHITNEY