Potential Role of Diabetes in Impaired Semaphorin-Mediated Maintenance of Epithelial Barrier Properties

Abstract

Microvasculature-secreted factors create a local niche for tissue specialization and homeostasis. Diseases like diabetes are considered a microvascular disease. However, the consequences of microvascular alteration in surrounding tissues like epithelial barriers have not been suffciently explored. The objective of this study is to determine whether a cell-cell communication protein, Semaphorin-3E (Sema3E), which we have previously found increased in diabetic mice and in endothelial cells cultured on high glucose, impairs the barrier properties of epithelial cells. We determined by ELISA that HUVEC endothelial cells secreted Sema3E (7.8 ±1.8 pg/sq.cm/day) and while mannitol osmolality control 15mM had no effect, high glucose (25mM) increased Sema3E secretion 6-fold (ANOVA, p<0.01). To test the effect of Sema3E in the tight junction barrier properties of epithelial cells, we measured trans-epithelial electrical resistance (TEER) in Calu-3 epithelial cells cultured in trans-well permeable support. We found that 200ng/ml of recombinant Sema3E inhibited the acquisition of TEER over the course of 2 weeks (vehicle: 803 ± 31 Ω/cm^2 vs. Sema3E: 465 ± 15 Ω/cm^2; t-test p<0.01) The weakening of tight junctions was due to decreased presence of Claudin-2 at the cell surface as measured by surface biotinylation (Surface/intracellular ratio= vehicle: 0.356 ± 0.043 vs. Sema3E: 0.020 ± 0.003; t-test p<0.01). Consequently, the polarity of Calu-3 cells was also impaired as we determined by measuring the polarized distribution of the basolateral protein E-cadherin. We observed a reduction of basolateral E-cadherin from 88 ± 3% (vehicle) to 65 ± 6% (Sema3E), t-test p<0.05. Finally, to determine whether surface Claudin-2 expression was decreased in epithelial cells of diabetic mice, we performed surface biotinylation in freshly isolated kidney renal tubules. We observed that the surface/intracellular ratio of Claudin-2 was decreased by 76% in kidney tubules from type-1 diabetic mice (Akita strain), compared to healthy control mice (control: 4.23 ± 1.44 ratio vs. Akita: 1.03 ± 0.16 ratio, t-test p<0.05). We conclude that glucose stimulates Sema3E secretion from endothelial cells, and this signal protein impairs the epithelial barrier properties of epithelial cells by reducing Claudin-2 presence at tight junctions. Reduced surface Claudin-2 may be a feature of diabetic epithelial cells. American Heart Association NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.