Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: Bioinformatics analysis reveals downregulated UNC5B expression in the retina of DR model mice, predominantly in endothelial cells. The DR mice retinal single-cell sequencing dataset ( GSE178121 ) was obtained from the Gene Expression Omnibus database. (A-C) Single-cell RNA sequencing data were processed with Seurat software, showing (A) the tSNE visualization of cell clusters, (B) the initial subpopulation annotation based on known cell markers and (C) the final identification of 11 retinal cell types after marker-based annotation. (D) tSNE plot illustrating the global expression pattern of UNC5B across retinal cells. (E) Dot plots comparing UNC5B expression between control and DR mouse retinas. (F) GSVA enrichment analysis of endothelial cells classified as UNC5B-positive and UNC5B-negative, showing differentially enriched biological processes. (G) GSVA enrichment analysis of endothelial cells classified as UNC5B-positive and UNC5B-negative, showing differentially enriched KEGG pathways. DR, diabetic retinopathy; GSVA, gene set variation analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NORM, normal; p_adj, adjusted P-value; RGC, retinal ganglion cell; RPE, retinal pigment epithelium; STZ, streptozotocin; tSNE, t-distributed stochastic neighbor embedding; UNC5B, unc-5 netrin receptor B.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Expressing, Single Cell, Sequencing, Gene Expression, RNA Sequencing, Software, Marker, Control

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B expression is downregulated in DR and RVO models. Retinal tissues from DR model mice (DR) were collected at 2, 4 and 8 weeks post-induction and compared with those from age-matched untreated mice (WT). (A) RT-qPCR and (B) western blot analysis of UNC5B expression in the retinas of both groups. Endothelial cells were stimulated with 30 mM glucose for 24 and 48 h and compared with untreated cells from the same batch (0 h). (C) RT-qPCR and (D) western blot analysis of UNC5B expression in these three groups. Retinal tissues were collected from RVO model mice 1 day after successful induction (RVO) and compared with those from age-matched untreated mice (WT). (E) RT-qPCR and (F) western blot analysis of UNC5B expression in the retinas of both groups. (G) Immunofluorescence staining of whole-mounted retinas from untreated normal mice to show UNC5B localization (green) and IB4-labeled vasculature (red). Scale bar, 100 μ m. Aqueous humor samples were collected from patients diagnosed with senile cataract, DR and RVO, with 8 individuals per group. (H) UNC5B expression was assayed by ELISA in aqueous humor samples from patients with DR and RVO, and age-related cataract controls. Data are presented as the mean ± SD. n=5 per group, except for western blot experiments (n=4) and ELISA (n=8). Multi-group comparisons between the WT/0 h/Control group and each of the other groups were performed using one-way ANOVA followed by Dunnett's multiple comparisons test, while two-group comparisons between the WT and RVO groups were performed using two-tailed unpaired Student's t-tests. * P<0.05 vs. WT/0 h/Control. DME, diabetic macular edema; DR, diabetic retinopathy; IB4, isolectin B4; NPDR, non-proliferative DR; PDR, proliferative DR; RT-qPCR, reverse transcription-quantitative PCR; RVO, retinal vein occlusion; UNC5B, unc-5 netrin receptor B; w, weeks; WT, wild-type.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B maintains the normal barrier function of endothelial cells. (A) Fluorescence microscopy image of HRMECs transfected with green fluorescence-labeled lentivirus-coated UNC5B shRNA. Scale bar, 100 μ m. HRMECs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMECs. UNC5B (B) mRNA and (C) protein expression was examined by reverse transcription-quantitative PCR and western blotting. (D) Live and apoptotic cells were assessed using PI/Calcein-AM staining (green, live cells; red, dead or dying cells). Scale bar, 100 μ m. (E) mRNA and (F) protein expression levels of barrier regulators were examined. (G) HRMECs were incubated with RBITC-BSA for 6 h. The internalized RBITC-BSA was imaged by microscopy and the intensity of the internalized RBITC-BSA was quantitated using ImageJ (red, RBITC-BSA; blue, DAPI). Scale bar, 20 μ m. (H) Schematic illustration of the experimental procedure for assessing HRMEC monolayer permeability and the corresponding quantitative results. Data are presented as the mean ± SD. n=5 per group, except for western blot experiments (n=4). All statistical comparisons in this figure were performed between the C group and each of the other groups using one-way ANOVA followed by Dunnett's multiple comparisons test. * P<0.05 vs. C. C, control; GFP, green fluorescent protein; HRMEC, human retinal microvascular endothelial cell; ns, not significant; OD, optical density; PLVAP, plasmalemma vesicle-associated protein; RBITC, rhodamine B isothiocyanate; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; ZO-1, zonula occludens 1.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Fluorescence, Microscopy, Transfection, Labeling, shRNA, Stable Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Staining, Incubation, Permeability, Control

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B specifically regulates endothelium-pericyte interactions with no significant effect on pericyte function. HRMECs were transfected with lentiviral shUNC5B or shC and puromycin was used to select stably transfected HRMECs. (A) Following transduction, HRMECs were co-cultured with HRMVPCs for 6 h and then stained with NG2 (HRMVPCs) and IB4 (HRMECs) to detect the recruitment of pericytes toward endothelial cells. Scale bar, 100 μ m. (B) For BRB model formation, HRMVPCs were seeded in the lower compartment of the Transwell insert 1 h prior to the addition of HRMECs. Co-cultures were maintained for 2 days in complete DMEM. (C) Barrier properties of the BRB model were assessed by permeability analysis. Subsequently, HRMVPCs were transfected with lentiviral shUNC5B or shC, and puromycin was used to select stably transfected HRMVPCs. UNC5B expression was examined by (D) reverse transcription-quantitative PCR and (E) western blotting. (F) Apoptosis (measured by PI incorporation), (G) cell proliferation (measured by EdU incorporation) and (H) migration were examined using the described assays, and the results were quantified. Scale bar, 100 μ m. Data are presented as the mean ± SD. n=5 per group, except for western blot experiments (n=4). All statistical comparisons in this figure were performed between the C group and each of the other groups using one-way ANOVA followed by Dunnett's multiple comparisons test. * P<0.05 vs. C. BRB, blood-retinal barrier; C, control; ECs, endothelial cells; EdU, 5-ethynyl-2'-deoxyuridine; HRMEC, human retinal microvascular endothelial cell; HRMVPC, human retinal microvascular pericyte; IB4, isolectin B4; NG2, neuron-glial antigen 2; ns, not significant; PCs, pericytes; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Transfection, Stable Transfection, Transduction, Cell Culture, Staining, Permeability, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Migration, Control, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B silencing in endothelial cells disrupts blood-retinal barrier homeostasis in DR model mice and abrogates the protective effect of high-concentration netrin-1. At week 4 after successful DR model induction, different concentrations of netrin-1 (5, 50, 500, 1,000 and 5,000 ng/ml) were injected intravitreally. Retinal tissues were collected after ≥12 weeks of DR model induction for subsequent analysis. (A) EB staining was used to observe retinal vascular leakage in the different groups. Scale bar, 500 μ m. At week 4 after DR model induction, control shRNA-AAV carrying an endothelial cell-specific promoter sequence (DR + shC), 1,000 ng/ml netrin-1 (DR + Netrin-1), UNC5B shRNA-AAV carrying an endothelial cell-specific promoter sequence (DR + shUNC5B) or a combination of 1,000 ng/ml netrin-1 and UNC5B shRNA-AAV (DR + shUNC5B + Netrin-1) were injected. After ≥12 weeks of DR model induction, retinal tissues were collected for analysis. (B) EB staining showed retinal vascular leakage in the different groups. Scale bar, 500 μ m. (C) Periodic acid-Schiff staining revealed the formation of acellular capillaries (arrows) and the number of pericytes ('P') in the retinal tissues. Scale bar, 50 or 20 μ m. (D) Whole-mount retinal immunofluorescence staining showed the pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μ m. The data are presented as the mean ± SD. n=5 per group. All statistical comparisons in this figure were performed among all groups using one-way ANOVA followed by Tukey's multiple comparisons test. * P<0.05 vs. WT. # P<0.05 vs. DR/DR + shC. AAV, adeno-associated virus; DR, diabetic retinopathy; EB, Evans Blue; IB4, isolectin B4; NG2, neuron-glial antigen 2; ns, not significant; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B; WT, wild-type.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Concentration Assay, Injection, Staining, Control, shRNA, Sequencing, Immunofluorescence, Virus

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B overexpression in endothelial cells maintains blood-retinal barrier homeostasis in DR model mice. At week 4 after successful DR model induction, DR mice received a single retro-orbital injection of adeno-associated virus carrying an endothelial cell-specific promoter. Mice were assigned to two groups: One with overexpression of UNC5B (DR + oeUNC5B) and the other receiving an empty vector as a control (DR + NC). Retinal tissues were collected after ≥12 weeks of DR model induction for subsequent analysis. (A) EB staining showed retinal vascular leakage in the different groups. Scale bar, 500 μ m. (B) Periodic acid-Schiff staining revealed acellular capillary formation (arrows) and pericyte numbers ('P') in the retinas of the different groups. Scale bar, 50 or 20 μ m. (C) Whole-mount retinal immunofluorescence staining showed pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μ m. The data are presented as the mean ± SD. n=5 per group. All statistical comparisons in this figure were performed among all groups using one-way ANOVA followed by Tukey's multiple comparisons test. * P<0.05 vs. WT. # P<0.05 vs. DR + NC. DR, diabetic retinopathy; EB, Evans Blue; IB4, isolectin B4; NC, negative control; NG2, neuron-glial antigen 2; oe, overexpression; UNC5B, unc-5 netrin receptor B; WT, wild-type.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Over Expression, Injection, Virus, Plasmid Preparation, Control, Staining, Immunofluorescence, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B affects neurodegeneration and glial activation in the DR mouse model. After ≥14 weeks of DR model induction, retinal tissues were collected for analysis. Mice were divided into groups, including a WT group and four DR groups that received different AAV treatments: DR + shC, DR + shUNC5B, DR + NC and DR + oeUNC5B. Retinal cryosection immunofluorescence staining showed (A) the number of NeuN-positive cells (red, NeuN; blue, DAPI) and (B) TUBB3 fluorescence intensity (red, TUBB3; blue, DAPI) in different groups. Scale bar, 100 μ m. (C) Retinal flat-mount TUBB3 immunofluorescence staining showed the number of ganglion cells per field in different groups. Scale bar, 100 μ m. (D) Retinal cryosection vimentin staining demonstrated glial reactivity in different groups (red, vimentin; blue, DAPI). Scale bar, 100 μ m. The data are presented as the mean ± SD. n=5 per group. All statistical comparisons in this figure were performed among all groups using one-way ANOVA followed by Tukey's multiple comparisons test. * P<0.05 vs. WT. # P<0.05 vs. DR + shC. † P<0.05 vs. DR + NC. DR, diabetic retinopathy; GCL, ganglion cell layer; NC, negative control; NeuN, neuron-specific nuclear protein; oe, overexpression; RGC, retinal ganglion cell; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; TUBB3, β-III tubulin; UNC5B, unc-5 netrin receptor B; WT, wild-type.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Negative Control, Over Expression, shRNA, Control

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: UNC5B silencing in endothelial cells aggravates retinal edema in RVO model mice. In 6-8 week-old wild-type mice, UNC5B shRNA-AAV (shUNC5B) or control shRNA-AAV (shC), both carrying an endothelial cell-specific promoter sequence, were administered via posterior orbital vein injection. Following viral delivery, the mice were maintained for ≥4 weeks to allow sufficient gene knockdown before subsequent procedures. (A) Rose Bengal was injected via the tail vein, and retinal vein occlusion was induced by laser treatment within 20 min post-injection (created in BioRender; https://BioRender.com/g5aw0dv ). (B) Optical coherence tomography scans were performed 1 h, 1 day and 8 days after laser induction to observe retinal edema and atrophy. The data are presented as the mean ± SD. n=5 per group. Statistical comparisons between the UNC5B knockdown and control groups at each time point were performed using two-tailed unpaired Student's t-tests. * P<0.05 vs. shC. AAV, adeno-associated virus; RVO, retinal vein occlusion; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; UNC5B, unc-5 netrin receptor B.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: shRNA, Control, Sequencing, Injection, Knockdown, Tomography, Two Tailed Test, Virus

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: Silencing of UNC5B promotes ECM-related protein synthesis and inhibits the Hippo signaling pathway. HRMECs were transfected with lentiviral UNC5B shRNA, and stable UNC5B-silenced cell lines (shUNC5B) were constructed using puromycin selection. A control group was established by transfection with negative control shRNA (shC). A total of three samples from each group were used for transcriptome sequencing, and differentially expressed genes were analyzed by Gene Ontology and KEGG functional enrichment analysis. The lollipop plots displayed the enrichment features of the two groups in terms of (A) biological processes and (B) KEGG pathways. (C) Western blotting was used to detect the expression of ECM-related proteins in HRMECs after silencing of UNC5B. (D) Western blotting was also used to assess the activation of the Hippo signaling pathway in HRMECs after silencing of UNC5B. The data are presented as the mean ± SD. n=4 per group. All statistical comparisons in this figure were performed between the C group and each of the other groups using one-way ANOVA followed by Dunnett's multiple comparisons test. * P<0.05 vs. C. C, control; ECM, extracellular matrix; HRMEC, human retinal microvascular endothelial cell; KEGG, Kyoto Encyclopedia of Genes and Genomes; MST, mammalian Ste20-like kinase; p-, phosphorylated; padj, adjusted P-value; shRNA, short hairpin RNA; shC, scramble control shRNA; shUNC5B, UNC5B shRNA; TAZ, transcriptional co-activator with PDZ-binding motif; UNC5B, unc-5 netrin receptor B; YAP, yes-associated protein.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Transfection, shRNA, Construct, Selection, Control, Negative Control, Sequencing, Functional Assay, Western Blot, Expressing, Activation Assay, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Endothelial UNC5B regulates blood-retinal barrier homeostasis

doi: 10.3892/ijmm.2026.5771

Figure Lengend Snippet: Role of endothelial UNC5B in maintaining BRB and NVU integrity under normal and pathological conditions. Normal UNC5B expression sustains BRB and NVU stability, whereas its downregulation in diabetic retinopathy and retinal vein occlusion is linked to Hippo signaling suppression, and impairment of both BRB and NVU integrity. Created in BioRender; https://BioRender.com/xp1oos1 . BRB, blood-retinal barrier; ECM, extracellular matrix; MST, mammalian Ste20-like kinase; NVU, neurovascular unit; P, phosphorylated; TAZ, transcriptional co-activator with PDZ-binding motif; UNC5B, unc-5 netrin receptor B; YAP, yes-associated protein.

Article Snippet: The lentiviral expression vectors for human UNC5B shRNA were developed and produced by Shanghai GeneChem Co., Ltd.

Techniques: Expressing, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Restoring compromised blood-retina-barrier integrity with Netrin-1 overexpression

doi: 10.1007/s00018-025-05903-6

Figure Lengend Snippet: Effects of global and endothelial Unc5b deletion on retinal angiogenesis and the BRB. ( A ) Unc5b expression in retinal scRNA-seq samples. ( B ) Survival curve after neonatal global Unc5b gene deletion, Mantel-cox test. ( C ) Gene deletion strategy. ( D ) Whole-mount P5 retinas of indicated genotypes stained with IB4 and ( E ) Quantification of vascular outgrowth and density. ( F ). Whole-mount P5 retinas of indicated genotypes stained with Ib4 and ( G ) Quantification of vascular outgrowth and density. ( H ) Gene deletion strategy. ( I ) IB4 staining and ( J ) quantification of superficial, intermediate, and deep layers at P12. (K) Unc5b gene deletion and tracer injection strategy. ( L ) Whole-mount P12 retinas after i.p. injection with sulfo-NHS-biotin for 1 h. (M) Unc5b gene deletion strategy. ( N ) Whole-mount P12 retinas stained with the indicated antibodies. A: Artery, V: Vein. Data are shown as mean ± SEM. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups, ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Each dot represents one eye. At least 4 mice were used per genotype for each experiment

Article Snippet: Unc5b fl/fl mice [ ] were bred with Cdh5Cre ERT2 (endothelial deletion) (Taconic, Biosciences), or with RosaCre ERT2 (global deletion).

Techniques: Expressing, Staining, Injection, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Restoring compromised blood-retina-barrier integrity with Netrin-1 overexpression

doi: 10.1007/s00018-025-05903-6

Figure Lengend Snippet: Working model. NTN1iGOF mice display BRB tightening and rescue of vascular leakage in two mouse models of pathological ocular neovascularization, but only mild transient effects on retinal angiogenesis. As global Unc5b deletion prevents BRB tightening, reinforcing Netrin-1 Unc5b signaling may be a therapeutic strategy to prevent vision loss. Figure was created using BioRender and SMART

Article Snippet: Unc5b fl/fl mice [ ] were bred with Cdh5Cre ERT2 (endothelial deletion) (Taconic, Biosciences), or with RosaCre ERT2 (global deletion).

Techniques: