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.

内皮细胞迁移对于伤口愈合与血管生成至关重要。钠离子（Na+）已被证实是介导细胞迁移的关键细胞信号，然而调控迁移相关钠离子渗透的离子通道机制仍不甚明确。本团队此前已证实，Orai1可同时抑制组成型钠离子漏流以及通过钙池操纵性钙通道介导的钠离子内流。本研究探讨了细胞外钠离子与Orai1各自及联合对肺动脉内皮细胞（pulmonary artery endothelial cell, PAEC）伤口愈合的影响。实验结果显示，PAEC细胞单层可迁移至划痕区域，并在划痕后24小时内达到细胞汇合。划痕后的愈合过程分为两个阶段：5小时内为快速愈合期，5至24小时为缓慢愈合期。去除细胞外钠离子可使迁移速率降低至少50%，且迁移前沿的细胞形成显著片状伪足的数量减少，部分前沿细胞逐渐从细胞单层脱离。下调Orai1的表达可在6小时内使伤口闭合速率降低25%，但该依赖Orai1的效应在钠离子缺失培养基中以及6小时后均不复存在。在非划痕细胞迁移实验中，间隙区域可在24小时内部分闭合。当细胞外钠离子被胆碱替代时，迁移速率降低75%。Orai1对非划痕细胞迁移无调控作用，但细胞外钠离子仍发挥关键作用。综上，细胞外钠离子与Orai1可联合急性调控内皮细胞伤口愈合：细胞外钠离子在6至24小时内持续参与伤口愈合过程；在无损伤的情况下，仅细胞外钠离子（而非Orai1）参与细胞迁移。Orai1或可作为损伤感受器，急性刺激快速伤口愈合。