Regions of DSS in mice are prone to endothelial membrane wounds, which undergo Ca²⁺-mediated repair using EE. (A) C57BL/6J mice were perfused immediately after euthanasia with a buffer containing the wounding dye FM4-64FX (displayed in green), washed, and fixed. The aorta was prepared, stained for endothelial cells by anti–PECAM-1 antibodies (red) and for all cells by DAPI (4′,6-diamidino-2-phenylindole) nuclear stain (blue), and imaged by confocal microscopy. FM4-64FX was perfused either in the presence of Ca²⁺ or the Ca²⁺ chelator, EGTA, to study the role of Ca²⁺-dependent membrane repair, and perfusions without FM4-64FX served as the no dye negative control. Representative images of regions of laminar SS (LSS; top) and DSS in the aorta (bottom) are shown, also marked in the schematic of the mouse aorta on the left. The schematic of the aorta shows the regions of LSS (blue line) and DSS (orange line) examined, with the potential flow profiles in these regions depicted with gray arrows. The FM4-64FX signal was considered to indicate wounded cells, only if it correlated to PECAM-1–stained endothelial cells and a corresponding DAPI signal. Note that enhanced FM4-64FX signal (labeled with white arrows) was specifically observed in regions of DSS around the branches of arteries, and no staining was seen in the no dye control. High magnification images of LSS (blue dashed box) and DSS regions (orange dashed box) are shown on the right. The FM4-64FX localizations around the branches are clearly distinct from the background punctae observed in all cells, and some are indicated with white arrows (also see supplemental Figure 3). (B) Quantifications of FM4-64FX positive endothelial cells in DSS and LSS regions of the aorta are represented as cell counts per 10⁴ μm² of aorta area. Wounded endothelial cells were only observed in regions of DSS (separated from LSS regions by a dashed black line in the graph). Note the wounded endothelial cell counts are significantly higher in perfusions with EGTA (red circles) than Ca²⁺ (blue triangles) or no dye controls (purple diamonds). Legend on the right indicates the various perfusion conditions, also for all further graphs. (C) Wound dye perfusions into mice were performed as above, and along with the aorta, the superior vena cava, pulmonary artery, and pulmonary vein were examined (marked on the images and in the schematic on the left as “1” indicating the aorta, “2” marking vena cava, and “3” and “4” labeling the pulmonary artery and vein, respectively). No FM4-64FX signal corresponding to PECAM-1–stained cells could be seen in vessels other than aorta (white arrows) in all conditions, indicating no inherent membrane damage in these other vessels. (D) Quantifications of the wounded cell counts in panel C. (E) Mice were perfused with FM4-64FX wound dye (shown in green), washed, and then perfused again with a second dye, Live-or-Dye Fix 488 (red), washed, fixed and aorta sections were stained for PECAM-1 to label endothelial cells (dark blue) and with DAPI to label all cells (light blue), as above. Wound-tracking dyes were perfused either in the presence of Ca²⁺ or the Ca²⁺ chelator, EGTA, to study the population of cells undergoing Ca²⁺-dependent membrane repair, and perfusion without both wound dyes served as the no wound dye negative control. High magnification images of DSS regions are shown here (also see supplemental Figure 4A for a fluorescence overview). Along with the FM4-64FX signal around branches (white arrows marking wounded endothelial cells), Live-or-Dye signal was also noted (yellow arrows) in PECAM-1–labeled cells, indicative of wounded and non-repaired endothelial cells. This Live-or-Dye signal is significantly higher in EGTA conditions, suggesting an active Ca²⁺ dependent repair mechanism. (F) Percentages of the non-repaired endothelial cells (Live-or-Dye positive) as in panel E are represented relative to the total number of wounded (FM4-64FX–positive) endothelial cells across the perfusion conditions. Note that cells under EGTA perfusion show a major defect in membrane repair, as opposed to those cells in the presence of Ca²⁺ (see supplemental Figure 4D for the repaired cell analyses). (G) FM4-64FX wound dye perfusions into mice were performed as before, and after fixation and dissection, the aorta was stained for EE with anti–EEA-1 (early endosome antigen 1) antibodies (red), with anti–PECAM-1 antibodies to identify endothelial cells (dark blue), and with DAPI to label all cells (light blue). Zoomed-in regions of DSS are displayed (see supplemental Figure 5A for low zooms). Punctate EEA-1 staining can be observed in all PECAM-1–stained endothelial cells. FM4-64FX–positive wounded cells around branches (indicated with white arrows in zooms) showed reduced EEA-1 staining as compared to unwounded PECAM-1–positive cells without FM4-64FX stain or PECAM-1 cells far from the curvature (both indicated with yellow arrows). (H) EEA-1 fluorescence intensities were measured in endothelial cells around the DSS exposed branches (as in panel G; labeled “Proximal to DSS” on the x-axis) and in cells far away from the branches (“Distal to DSS”), normalized to the corresponding PECAM-1 endothelial cell signals, and plotted. Note that the cells around the bifurcation show significantly reduced EEA-1 staining (separated from distal region measurements in the graph by a dashed black line), indicating early endosomal involvement in membrane wound repair. (I) Model showing the membrane damaging effect of SS of blood flow on the endothelial lining of a blood vessel, which occurs upon mechanically challenging alterations or disturbances in SS of blood flow. Scale bars represent 20 μm in panels A and C and 10 μm in zoom-ins in panels A, E, and G. Mean ± SD with the distribution of all samples shown for the graphs (n = 36-103 fields of view pooled from 14 mice in panel B; 13-73 regions from 6 mice in panel D; 10-20 regions each combined from 3 mice in panel F; and 133-191 cells from 3 mice in panel H). Statistical comparisons using 1-way ANOVA with the Kruskal-Wallis test were performed for all analyses. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.