Fig. 4

Endothelial cell migration depends on a Cx43-formed channel-mediated increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). a, Representative images (left) and fluorescence intensity analysis (right) of the increase in [Ca²⁺]_i observed in endothelial cells of the migration front 15 min after scratching the monolayer in control conditions and in the presence of 50 µM 18-β-Glycyrrhetenic acid (ß-GA), a general Cx-formed channel blocker, or 200 µM ^37,43Gap27, a peptide designed to block channels formed by Cx37 or Cx43. Variations in the levels of [Ca²⁺]_i were assessed with the fluorescent Ca²⁺ indicator Fluo-4. b, Representative images of the changes in [Ca²⁺]_i of endothelial cells (left) in which the differences in the subcellular distribution of the Ca²⁺ signal attained in a cell of the migration front (Cell 2) and a cell within the monolayer (Cell 1) are highlighted in a 3D analysis (middle and right). c, Fluorescence intensity analysis of the Fluo-4 signal measured along the endothelial cells length (from back to front) in the migration front and the monolayer. d, Analysis of the changes in [Ca²⁺]_i levels attained in the rear and anterior edge of endothelial cells of the migration front in control conditions and after the treatment with ß-GA or ^37,43Gap27. Changes in Fluo-4 signal are expressed as the area under the curve (AUC). Note that cells were treated with the Cx blocking peptide ^37,43Gap27 for only 10 min to inhibit Cx hemichannels, without affecting gap junction channels. Dot red lines depict the edge of the migration front. Numbers inside the bars or in parentheses indicate the n value. Values are means ± SEM. ***, P < 0.001 vs. Control by one-way ANOVA plus Bonferroni post hoc test. †††, P < 0.001 vs. Anterior by paired Student’s t-test