Voltage-gated sodium channels play a central role in the Ca²⁺signaling that mediates the endothelial cell migration observed during angiogenesis

Angiogenesis is a complex process that involves proliferation, migration, and formation of tubular vascular structures by endothelial cells. The coordination of this process depends on the control of endothelial migration through the increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ), which is triggered by the release of Ca 2+ from intracellular stores and maintained along the time by the subsequent Ca 2+ entry from the extracellular space. It has been observed that the increase in [Ca 2+ ] i is more pronounced in the rear part of endothelial cells (ECs), which is associated with the redistribution of caveolin-1, a caveolae structural protein. Interestingly, we recently demonstrated that the activation of Cx43 hemichannels contributes to the Ca 2+ increase observed in the rear part of migrating ECs. ECs also exhibit an increase in intracellular Na + concentration and membrane depolarization during migration, which may contribute to the Ca 2+ signaling by the activation of the reverse mode of Na + -Ca 2+ exchanger (NCX). ECs express voltage-gated sodium channels (Na v ), but the participation of these channels in endothelial cell migration remains to be demonstrated. Therefore, in the present study, we evaluated the involvement of Na v channels in the Cx43 hemichannel-mediated signaling associated with the increase of [Ca 2+ ] i in EC migration. Proliferation and EC migration were evaluated in primary cultures of rat mesenteric ECs by the analysis of bromodeoxyuridine incorporation in the wound healing assay. Changes in [Ca 2+ ] i were recorded using the fluorescent Ca 2+ indicator Fluo-4 and hemichannel opening was assessed by measuring ethidium uptake. The distribution of Na v channels and caveolin-1 was analyzed by immunofluorescence, and their colocalization was examined using proximity ligation assay (PLA). The results show that treatment with 1 µM tetrodotoxin (TTX) or with selective inhibitors of Na v 1.2 and Na v 1.6, lamotrigine and 4,9-anhydro-TTX, respectively, reduced EC migration and the Ca 2+ signaling observed in the migration front, without affecting proliferation. Consistent with this, TTX inhibited the tubular-like structure formation in ECs cultured in Matrigel. The increase in [Ca 2+ ] i was also inhibited by the disruption of caveolae with methyl-β-cyclodextrin or the inhibition of NCX reverse mode with SEA0400. Interestingly, endothelial cell migration was associated with the redistribution of Na v 1.2 and Na v 1.6 channels and caveolin-1 to the posterior part of the cells, but only Na v 1.2 channels increased its spatial association with caveolin-1. In addition, blockade of Na v channels reduced ethidium uptake in ECs of the migration front, suggesting that Na v channel activation is required for the opening of Cx hemichannels. These results indicate that the functional association of Na v 1.2 and Na v 1.6 channels with the activation of the reverse mode of NCX plays an important role in the Ca 2+ signaling that mediates endothelial cell migration and angiogenesis. Anillo ANID/ACT210057 and CONICYT 21170977 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.