Epithelial tight junction regulation by endothelial paracrine signaling on a normal or high glucose environment

Background: Microvascular beds have been recognized as important sources of paracrine signals that maintain the homeostasis of surrounding tissues. This is of extreme relevance in diabetes, a microvascular disease. However, the consequences that microvascular alterations during diabetes have in the paracrine microenvironment and surrounding tissues is poorly understood. We have previously showed that endothelial-epithelial crosstalk is disrupted by diabetes and high glucose. We have also showed that secreted semaphorins regulate epithelial polarity. Understanding the paracrine role of endothelial semaphorins in maintaining epithelial barriers will shed light in processes affected by microvascular dysfunction during diabetes. In this study, we aimed to determine whether the semaphorin isoform Sema3F, secreted by endothelial cells, maintains the epithelial barrier integrity of tight junctions by promoting the trafficking of claudin-2 to the cell surface. Methods: We assessed claudin-2 surface expression using surface biotinylation in either cultured Calu-3 epithelial cells or native renal tubule epithelia from diabetic Akita mice. To study cell-cell communication, we co-cultured HUVEC endothelial cells with Calu-3 cells using transwell permeable supports under normal (5.5 mM) or high glucose (25 mM). Sema3F was silenced in HUVEC cells using a lentiviral vector carrying shRNA. Results: Surface expression of claudin-2 in renal epithelia from diabetic Akita mice was reduced by 76 ± 11% (p < 0.01), indicating dysregulated tight junction protein trafficking. In vitro, treatment with recombinant Sema3F (200 ng/mL) decreased surface claudin-2 in Calu-3 cells by 55 ± 1% (p < 0.01), but not under high glucose. These changes paralleled tight junction maturation over two weeks, as measured by transepithelial electrical resistance. To investigate the role of endogenous Sema3F, we silenced its expression in HUVEC cells and co-cultured them with Calu-3 cells. The presence of HUVEC cells stimulated claudin-2 surface expression in Calu-3 two-fold and accelerated tight junction maturation (p< 0.01). However, this stimulation was significantly diminished when Sema3F was silenced, indicating that endogenous Sema3F stimulated tight junctions in adjacent epithelial cells. Interestingly, under high glucose, Sema3F did not abolish the stimulatory effect of co-cultures. Conclusion: Semaphorin 3F secreted by endothelial cells stimulate tight junction maturation and increases claudin-2 surface expression in neighboring epithelial cells. On high glucose, this paracrine effect persists despite Sema3F silencing, suggesting the involvement of additional secreted factors. Funding. NIH: R35GM150570; R21DK136122 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.