Fig 1.
Contribution of extracellular Na+ to PAECs scratch wound closure.
(A) Phase-contrast images of PAEC monolayers. The top and bottom rows showed monolayers immediately after and 24 hours after scratching, respectively. The monolayers in the left and right columns were in a Na+- and choline-containing media, respectively. In the absence of extracellular Na+, wound closure was incomplete at 24 hours. (B) Bar plot of average wound closure distances over 24 hours in Na+- and choline-containing media. Over 24 hours, extracellular Na+ enabled PAECs to migrate farther. (C) Scatter plot of wound closure speeds in both Na+- and choline-containing media. The migration speed of PAECs was significantly slower in the absence of extracellular Na+ when compared to the Na+-containing counterpart. Both the bar plot and scatter plot showed mean ± SEM. Statistical significance was assessed using unpaired t-test with Welch’s correction (^—P < 0.0001).
Fig 2.
Contribution of extracellular Na+ to the 24-hour progression of PAEC wound closure.
(A) Sequential phase-contrast images of PAEC monolayers show wound closure in Na+- and choline-containing media. The wound closure was faster in Na+ than in choline condition. (B) The plot of wound closure distance as a function of time in a Na+- and choline-containing media. At each time point, wound closure distances were always quantified from time 0. In both conditions, wound closure occurred in two phases. These phases were analyzed using two linear regression fits, one from 0 to 190 minutes and other from 190 to 1390 minutes. The wound closure distance between 990 and 1390 minutes was significantly larger in Na+ condition than in choline condition. Bar plot of the slope of (C) the first linear regression fit (time 0 to 190 minutes), and (D) the second linear regression fit (time 190 and 1390 minutes) through the wound closure distances in Na+- and choline-containing media. During both phases, the wound closure distance in choline-containing media trailed that in the Na+-containing media. However, the difference was statistically significant only for the second phase. Scatter plots of wound closure distance in (E) 0–6 hours (or more accurate 0–390 minutes) and (F) 6–24 hours (**—p < 0.01, Wilcoxon matched paired t-test). (G) A plot of wound closure speed as a function of time quantified at a 100-minute interval. This plot also exhibited two linear phases. In the first phase, which spans 0–290 minutes, the wound closure speed decreased rapidly. In the second phase, which spans 290–1390 minutes, the wound closure speed changed minimally. Across the entire timespan, extracellular Na+ appeared to facilitate faster cell migration. (H) Distribution of wound closure speed displayed using Gaussian fit through the data. The distributions showed that extracellular Na+ increased variability in wound closure speed. Bar plot of the slope of (I) the first linear regression fit (time 0 to 290 minutes), and (J) the second linear regression fit (time 290 and 1390 minutes) through the wound closure speeds in Na+- and choline-containing media. During the first phase, the change in wound closure speed was similar in both conditions. However, during the second phase, the wound closure speed decreased in choline-containing media and increased in Na+-containing media. The data include at least 8 repeat experiments contributing to at least 8 images for each time point. Panels (B)-(G), (I), and (J) show mean values with standard errors. Statistical significance was assessed using two-way ANOVA followed by Bonferroni post hoc test and linear regression (ns—not significant, *—P < 0.05, **—P < 0.01, ***—P < 0.001, ^—P < 0.0001).
Fig 3.
Contribution of extracellular Na+ to PAEC protruding lamellipodia, sustaining and reinstating intercellular adhesion, and advancing persistently.
(A) Sequential phase-contrast images of a PAEC wound edge at 400-minute intervals in the presence and absence of extracellular Na+. PAECs at the leading edge protruded prominent and stable lamellipodia in Na+-containing media. In choline-containing media, fewer PAECs at the wound edge protruded lamellipodia, and most protruded lamellipodia were relatively smaller. Cells divided at the wound edge gradually formed interendothelial gaps and then detached from the monolayer. The detached individual cells thus lost lamellipodia and cell polarity, and eventually stopped migrating. (B) Sequential phase-contrast images of a confluent PAEC monolayer at 400-minute intervals in the presence and absence of extracellular Na+. In both conditions, transient gaps were both observed during cell divisions (arrowheads). Gaps were generally small and sealed quickly in Na+ condition (arrows). However, in choline condition, some sustained gaps were also observed. Some gaps formed even without cell division. The sustained gaps fused with adjacent newly formed gaps leading to gap size increase. Cell division instances are identified with arrowheads. Interendothelial gaps are identified with arrows.
Fig 4.
Inhibition of Na+ permeation through ENaC and NCX channels slowed PAEC scratch wound closure.
(A) Brightfield images of scratched PAEC monolayers at 0, 6, and 24-hours in vehicle control, 90 μM amiloride, and 10 μM benzamil conditions. Benzamil treatment caused accumulation of floating particles over 24 hours. Studies were all conducted in Na+-containing media. Scatter plots of wound closure distance in (B) 0–6, (C) 6–24, and (D) 0–24 hours. Benzamil was more potent than amiloride to slow PAEC monolayer wound closure. Data were reported as mean ± SEM. (E) Magnified view of the marginal regions of the control, amiloride-treated, and benzamil-treated cells after 24 hours of migration. An arrowhead points to a floating particle. Statistical significance was assessed using one-way ANOVA with Bonferroni post hoc test (ns—not significant, *—P < 0.05, **—P < 0.01, ***—P < 0.0001).
Fig 5.
Inhibition of Orai1 and TRPC1/4 did not affect overall wound closure.
(A) Brightfield images of scratched PAEC monolayers at 0, 6, and 24-hours in vehicle control, 10 μM GSK-7975A, 1 nM Pico145, and combined 10 μM GSK-7975A and 1 nM Pico145 conditions. Combined GSK-7975A and Pico145 treatment caused an accumulation of floating particles over 24 hours. Studies were all conducted in Na+-containing migration media. Scatter plots of wound closure distance in (B) 0–6, (C) 6–24, and (D) 0–24 hours. Data were reported as mean ± SEM. (E) Magnified view of the marginal regions of the control, GSK-7975A-treated, Pico145-treated, and both GSK-7975A- and Pico145-treated cells after 24 hours of wound closure. An arrowhead points to a floating particle. Statistical significance was assessed using one-way ANOVA with Bonferroni post hoc test (ns—not significant, *—P < 0.05).
Fig 6.
In the presence of extracellular Na+, Orai1 silencing slowed endothelial wound closure.
(A) Phase-contrast images of scratched Orai1-expressing and -silenced PA2879 endothelial monolayers at 0, 6 and 24 hours in the presence and absence of extracellular Na+. Scatter plots of wound closure distance in (B) 0–6 hours, (C) 6–24 hours, and (D) 0–24 hours. In the presence of extracellular Na+, wound closure distances in Orai1-silenced PA2979 monolayers were less than the distances in Orai1-expressing monolayers. However, in the absence of extracellular Na+, the contribution of Orai1 was not significant. Data were reported as mean ± SEM. (E) Magnified view of the marginal regions of the Orai1-expressing and -silenced cells at 24 hours of wound closure in Na+- and choline-containing media. Cells near the wound edge spread larger in Na+- than in choline-containing media. Intercellular gaps were observed in the absence of extracellular Na+. Arrows point to typical interendothelial gaps. Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test (ns—not significant, ^—P < 0.0001).
Fig 7.
Extracellular Na+ promoted PAEC monolayer unscratched gap closure.
The presence of both Orai1 and extracellular Na+ improved intercellular adhesion. (A) Phase-contrast images of Orai1-expressing and -silenced PA2879 monolayers at 0, 6 and 24 hours in the presence and absence of extracellular Na+. Scatter plots of gap closure distances in (B) 0–6 hours, (C) 6–24 hours, and (D) 0–24 hours. Replacing extracellular Na+ with choline significantly slowed PA2879 gap closure whereas silence of Orai1 had no effect on gap closure. (E) Magnified view of the marginal regions of Orai1-expressing and -silenced monolayers at 17 and 24 hours of migration in Na+- and choline-containing media. Without scratch-associated cellular injury, the marginal and submarginal cells were of similar size. Lack of extracellular Na+ and/or Orai1 compromised intercellular adhesion. Arrows point to typical interendothelial gaps. Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test (ns—not significant, ^—P < 0.0001).