High Glucose Undermines Epithelial-Endothelial Crosstalk as Determinant of Epithelial Barrier Properties and Angiogenesis

Epithelia are selective barriers composed by the polarized epithelial layer, the basement membrane and the underlying vasculature. The epithelial-endothelial unit should be considered a functional entity regulated by epithelial physiology and angiogenesis. Diseases like diabetes are known to impair the homeostasis of vascular and epithelial cells. However, the consequences in epithelial-endothelial crosstalk remain insufficiently studied. We previously identified a potential cell-cell crosstalk pathway between epithelial and endothelial cells mediated by Semaphorins (ligands) and Plexins (receptors). Here we studied whether high glucose and diabetes affect cell-cell crosstalk as mediator of angiogenesis and epithelial tight junctions. Our objectives are to determine whether high glucose and diabetes impair epithelial-endothelial crosstalk, and to establish Semaphorin-Plexins as mediators of barrier properties in the epithelial-endothelial unit. We developed primary cultures of kidney endothelial cells from diabetic Akita mice to measure angiogenesis in matrigel. We studied epithelial tight junction maturation as trans-epithelial electrical resistance (TEER) in Calu-3 cells in transwells. We measured expression at the plasma membrane of angiogenesis receptors and tight junction claudins via surface biotinylation. We first determined that kidney endothelial primary cultures expressed endothelial markers Kdr, Cdh5 (qRT-PCR), underwent angiogenesis (Matrigel), and were stimulated by VEGF (pro-angiogenic). We observed reduced angiogenesis in endothelial cells from diabetic mice, but this did not correlate with protein expression of angiogenesis receptors VEGFR1/ R2, Plexin-D1, Nrp1/ 2 (Western blot). However, surface expression of Plexin-D1 (anti-angiogenic) was 305 ± 63% higher in cells from diabetic mice (t-test p<0.05), highlighting stimulation of an anti-angiogenic pathway. Similarly, we found 332 ±105% higher Plexin-D1 surface expression in endothelial cells on high (25 mM) vs. normal (5.5 mM) glucose but not on mannitol osmolality control (p<0.05, ANOVA). A semaphorin isoform, Sema3G that we found is secreted by epithelial cells, prevented the VEGF-stimulation in matrigel angiogenesis (p<0.05, ANOVA). To study the reciprocal crosstalk, we measured development of tight junctions in Calu-3 epithelial cells. Treatment with semaphorin Sema3F (produced by endothelial cells) stimulated TEER. However, this stimulation was absent on high glucose. To correlate TEER with tight junction proteins, we measured expression of claudins and found that high glucose (but not mannitol), decreased the pore-forming claudin-2. In contrast, claudins 3, 7, 16, 19, 20, 22, 24 were increased. Since these claudins are expected to strengthen tight junctions, we measured TEER and found it to be increased by high glucose (p<0.05, ANOVA). To explore the role of diabetes, we measured surface Claudin-2 in renal tubules of diabetic mice and found it to be decreased by 95 ± 3% (t-test p<0.01), consistent with a decrease in pore-forming claudins. We conclude that high glucose and diabetes impair epithelial barrier properties by undermining epithelial tight junctions and angiogenesis, possibly via Semaphorin-Plexin cell-cell communication. NIH, American Heart Association This abstract was presented at the American Physiology Summit 2025 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.