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The paracellular pathway of an epithelial cellular sheet can be divided into two parts: one between two adjacent cells sealed by tight junctions (TJs) and one at tricellular contacts (TCs), where the corners of three cells meet. At TCs of epithelial cells, there is a specialized mode of TJs, namely tricellular TJs (tTJs), required for full barrier function of the cellular sheet. However, tTJs have not been described in endothelial cells to date. Here, we investigated whether tTJs occur in endothelial cells by analyzing the TC localizations of tTJ markers, tricellulin and angulin family proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2), by immunofluorescence staining of frozen sections of various tissues from adult mice. Endothelial TCs in most tissues revealed no detectable staining of tricellulin or angulins. However, tricellulin and angulin-1/LSR were specifically concentrated in TCs of brain and retinal endothelial cells, which form the blood-brain barrier (BBB) and inner blood-retinal barrier (BRB), respectively. Even in the brain, endothelial cells in the choroid plexus and the median eminence, one of the circumventricular organs, did not show concentration of tricellulin or angulins at TCs. These findings indicate the existence of tTJs in endothelial cells in vivo and suggest that tTJs impart important characteristics to the BBB and inner BRB.

Localization of Angulin-1/LSR and Tricellulin at Tricellular Contacts of Brain and Retinal Endothelial Cells <i>in vivo</i>