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Article Open Access

Endothelial UNC5B regulates blood‑retinal barrier homeostasis

  • Authors:
    • Yujia Yao
    • Suyu Wang
    • Jiajun Li
    • Qianzi Jin
    • Ziyi Chen
    • Qin Jiang
    • Keran Li

  • View Affiliations / Copyright

    Affiliations: Department of Ophthalmology, The Affiliated Eye Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
    Copyright: © Yao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 100
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    Published online on: February 20, 2026
       https://doi.org/10.3892/ijmm.2026.5771
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Abstract

The blood‑retinal barrier (BRB), a critical component of the retinal neurovascular unit (NVU), is essential for maintaining retinal homeostasis. Dysfunction of the BRB contributes to vascular leakage, neuronal degeneration and gliosis, which are the core pathological hallmarks of diabetic retinopathy (DR) and retinal vein occlusion (RVO). Despite the importance of the BRB, the molecular mechanisms underlying the preservation of BRB integrity under pathological conditions remain unclear. The present study identified the endothelial receptor unc‑5 netrin receptor B (UNC5B) as a critical regulator of BRB and NVU homeostasis and a potential therapeutic target for neurovascular protection. Analysis of a public Gene Expression Omnibus single‑cell transcriptomic dataset, cell and animal models, and clinical samples revealed reduced UNC5B expression in the aqueous humor of patients and in the retinas of DR and RVO models. In vitro, endothelial knockdown of UNC5B increased apoptosis (assessed by PI/calcein‑AM staining), impaired barrier function (evaluated by BSA uptake and permeability of cell monolayer) and reduced pericyte recruitment, whereas UNC5B knockdown in pericytes had no detectable effects on pericyte proliferation, apoptosis or migration. In vivo, endothelial‑specific UNC5B deficiency markedly exacerbated retinal vascular leakage and structural damage in the DR model, as evidenced by Evans blue leakage, Periodic acid‑Schiff staining and immunofluorescence analyses. Furthermore, UNC5B knockdown abolished the protective effects of high‑dose netrin‑1 administration in DR mice. Endothelial UNC5B modulation, including knockdown and overexpression, affected not only the vascular integrity but also the neural components within the NVU, as evidenced by altered retinal ganglion cell degeneration and glial activation in the DR model, assessed using NeuN, β‑III tubulin and vimentin staining. In the RVO model, endothelial UNC5B deficiency aggravated retinal edema and thinning, as revealed by in vivo retinal imaging. Mechanistically, transcriptomic and protein analyses revealed that UNC5B downregulation was associated with increased extracellular matrix protein deposition and reduced Hippo pathway activity. Collectively, these findings established UNC5B as a key mediator of BRB and NVU stability, and highlighted its therapeutic potential in maintaining vascular integrity and protecting neural elements in retinal vascular diseases.
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Figure 1

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.

Figure 2

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 μm. 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.

Figure 3

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 μm. 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 μm. (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 μm. (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.

Figure 4

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 μm. (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 μm. 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.

Figure 5

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 μm. 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 μm. (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 μm. (D) Whole-mount retinal immunofluorescence staining showed the pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μm. 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.

Figure 6

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 μm. (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 μm. (C) Whole-mount retinal immunofluorescence staining showed pericyte coverage in the retinas of the different groups (red, NG2; green, IB4). Scale bar, 200 μm. 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.

Figure 7

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 μm. (C) Retinal flat-mount TUBB3 immunofluorescence staining showed the number of ganglion cells per field in different groups. Scale bar, 100 μm. (D) Retinal cryosection vimentin staining demonstrated glial reactivity in different groups (red, vimentin; blue, DAPI). Scale bar, 100 μm. 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.

Figure 8

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.

Figure 9

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.

Figure 10

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.
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Copy and paste a formatted citation
Spandidos Publications style
Yao Y, Wang S, Li J, Jin Q, Chen Z, Jiang Q and Li K: Endothelial UNC5B regulates blood‑retinal barrier homeostasis. Int J Mol Med 57: 100, 2026.
APA
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., & Li, K. (2026). Endothelial UNC5B regulates blood‑retinal barrier homeostasis. International Journal of Molecular Medicine, 57, 100. https://doi.org/10.3892/ijmm.2026.5771
MLA
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., Li, K."Endothelial UNC5B regulates blood‑retinal barrier homeostasis". International Journal of Molecular Medicine 57.4 (2026): 100.
Chicago
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., Li, K."Endothelial UNC5B regulates blood‑retinal barrier homeostasis". International Journal of Molecular Medicine 57, no. 4 (2026): 100. https://doi.org/10.3892/ijmm.2026.5771
Copy and paste a formatted citation
x
Spandidos Publications style
Yao Y, Wang S, Li J, Jin Q, Chen Z, Jiang Q and Li K: Endothelial UNC5B regulates blood‑retinal barrier homeostasis. Int J Mol Med 57: 100, 2026.
APA
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., & Li, K. (2026). Endothelial UNC5B regulates blood‑retinal barrier homeostasis. International Journal of Molecular Medicine, 57, 100. https://doi.org/10.3892/ijmm.2026.5771
MLA
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., Li, K."Endothelial UNC5B regulates blood‑retinal barrier homeostasis". International Journal of Molecular Medicine 57.4 (2026): 100.
Chicago
Yao, Y., Wang, S., Li, J., Jin, Q., Chen, Z., Jiang, Q., Li, K."Endothelial UNC5B regulates blood‑retinal barrier homeostasis". International Journal of Molecular Medicine 57, no. 4 (2026): 100. https://doi.org/10.3892/ijmm.2026.5771
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