Supplemental data from: Extracellular Na+ and Orai1 contribute to endothelial wound healing

Endothelial migration is necessary for wound healing and angiogenesis. Na⁺ has been incriminated as an important cellular signal mediating migration, although the ion channels regulating Na⁺ permeation that contribute to migration remain poorly understood. We have shown that Orai1 suppresses both constitutive Na⁺ leak and Na⁺ entry through store-operated calcium entry channels. Here, we examine the independent and combined contributions of extracellular Na⁺ and Orai1 to pulmonary artery endothelial cell (PAEC) wound healing. We found that PAEC monolayers migrated into wound areas and achieved confluence within 24 hours post-scratch. There was a fast healing phase within 5 hours, followed by a slow-healing phase between 5 and 24 hours. Na⁺ depletion reduced migration speed by at least 50%. Fewer cells at the migration front formed prominent lamellipodia. Some cells at the leading edge gradually detached from the monolayer. Reduction of Orai1 decreased wound closure speed by 25% within 6 hours. This Orai1-dependence was absent in Na⁺-depleted media and after 6 hours. In a non-scratch cell migration assay, the gap areas were partially sealed within 24 hours. Migration speed was reduced by 75% when extracellular Na⁺ was replaced by choline. Orai1 did not contribute to non-scratch cell migration, but extracellular Na⁺ did. Overall, extracellular Na⁺ and Orai1 together acutely regulate endothelial wound healing. Extracellular Na⁺ continuously contributes to wound healing between 6 and 24 hours. Extracellular Na⁺ but not Orai1 contributes to cell migration in the absence of injury. Orai1 may function as an injury sensor to acutely stimulate fast wound healing.