Supplementary material - Impact of Na+ permeation on collective migration of pulmonary arterial endothelial cells (Xu et al)

Collective migration of endothelial cells is Endothelial migration is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na+ in collective cell migration remains unclear. Here, we examined the effect of Na+ permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by a scratch injury or barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. The first phase started with fast closure speed which then rapidly reduced within 5 hours after scratching. The second phase maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na+, we found that wound closure distance reduced more than 50%, fewer cells at the leading edge protruded prominent lamellipodia, and some sustained interendothelial gaps progressively increased in size over time and fused with adjacent gaps. When scratch-associated cellular injury was also missing, PAEC monolayer migrated much more slowly, and interendothelial gaps increased in size obviously towards the end. Pharmacological inhibition of the epithelial Na+ channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na+/Ca2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca2+ release-activated Ca2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. GSK-7975A also inhibits smaller inward Ca2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. Nevertheless, the combined treatment appeared to also cause accumulation of floating particles. Orai1 gene silencing using shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na+ or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data revealed that Na+ permeation into cells contributed to PAEC monolayer collective migration by increasing lamellipodial formation, causing accumulation of floating particles, and impairing intercellular adhesion.