Alterations of SS lead to the formation of membrane wounds in HUVEC that undergo repair by Ca²⁺-dependent early endosomal exocytosis. (A) Schematic depiction of the protocol for the pump flow assay used to induce alterations in laminar SS profiles to study the effect on endothelial membrane integrity in HUVEC. The x-axis shows the duration of the assay (in minutes), with the different phases of SS coupled to the flow experiment noted above vertically next to the black curve, and the y-axis shows the flow-induced SS values (in dynes cm^–2). Fluorescent dyes used at each stage of flow are highlighted on top of the graph in the representative colors. The black curve depicts the magnitude of the SS profiles applied during the course of the highest shear alteration experiment. (B) Representative live-cell microscopy images before and after shear alterations (shown here for 3 dynes cm^–2 to 14.1 dynes cm^–2). The endothelium is visualized with WGA 405S (glycoprotein binding dye that labels the endothelial PM; shown in blue) and wounded cells tracked using FM1-43FX (green) and PI (red) in the flow buffer. Hoechst nuclear stain is also used to identify all endothelial cells for analysis, and the direction of the laminar flow is indicated (left). The point of SSA is marked on the image with the red dashed arrow. Note that cells were wounded only upon shear alteration and not in unaltered low or high shear. (C) Quantification of wounded cells based on the influx of FM1-43FX signal, upon varying shear alterations during flow, represented as percentages. Uniform laminar shear flow (3 dynes cm^–2) served as negative control for shear alteration effects, and shear flow with the highest possible alteration achieved with the pump flow system (from 3 dynes cm^–2 to 14.1 dynes cm^–2) in the presence of the Ca²⁺ chelator EGTA served as positive wounding control. Legend on top indicates the various samples, which is the same for all further graphs. (D) Zoomed-in snapshot of the endothelial monolayer after shear alteration (at 11 minutes after flow) from panel B (indicated by a blue dashed box) shows examples of an unwounded cell with no FM1-43FX staining (no green FM1-43FX signal all over the cell; outlined in white dashes), a wounded and repaired cell identified by FM1-43FX staining only (green signal in the cytoplasm; shown in orange dashes), and wounded but nonrepaired cell, with FM1-43FX and PI staining appearing as yellow (green signal in cytoplasm and red PI signal in the nucleus appearing as yellow; shown in pink dashes). The minor nuclear signal for all cells in the FM1-43FX channel is due to the spectral bleed-through of Hoechst nuclear stain, which, however, is distinct from the FM1-43FX cytoplasmic localization that is seen only in wounded cells (also see supplemental Figure 1F). (E) Quantification of resealed cell counts performed as in panel C for shear alteration–induced wounding, based on FM1-43FX–positive and PI-negative signal (“SSA” in the graph corresponds to “SSA 3” in panel C). (F) HUVEC were transfected with the early endosomal marker, green fluorescent protein (GFP)–2xFYVE (a protein construct binding the endosomal lipid phosphatidylinositol 3-phosphate marking EE; shown in green), and subjected to the SSA-based flow assay at the maximum shear alteration (SSA 3 above). Images before and after flow alteration are shown. The wounded cell, outlined in white dashes, is identifiable by the FM4-64FX wounding dye (red) and PI (gray) signal. The GFP-2xFYVE channel alone is shown again in the bottom panels for clarity. (G) Counts of GFP-2xFYVE punctae in cells subjected to the SSA protocol are plotted for altered SS sample in wounded and nonwounded cells, along with unaltered shear negative control (low shear control with no wounded cells). SS-altered wounded cells are labeled as “SSA W” in the graph (red circles), non-wounded cells in the SS-altered sample labeled “SSA NW” (blue triangles), and cells in uniform SS control are depicted as “USS” (purple diamonds). Note that after shear alteration, the wounded cells showed fewer 2xFYVE punctae than the nearby unwounded cells or cells under unaltered shear flow. (H-I) HUVEC were transfected with the exocytosis marker, TfR-SEP (the endosomal protein transferrin receptor coupled to pH sensitive pHluorin to mark early endosomal exocytosis events; displayed in green), which shows low fluorescence at the internal acidic pH of EE but increased fluorescence after cell surface exposure due to the resulting neutralization. Still images before and after SSA are shown (H) and quantified (I) for the counts of TfR-SEP punctae in the cells after flow alteration. The wounded cell in panel H (shown in white dashes) displayed a punctate appearance of TfR-SEP all over the cell surface after flow alteration (indicated with white arrows), as opposed to the neighboring unwounded cell, which shows weakly fluorescent punctate TfR-SEP signals indicating the pre-existing TfR pool in EE. The increased counts of highly fluorescent cell surface punctae of TfR-SEP in wounded cells under shear alteration are quantified in panel I (graph legend same as panel G). Scale bars represent 50 μm in panel B; 20 μm in panel D; and 10 μm in panels F and H. Mean ± standard deviation (SD) shown for all graphs, with individual points indicating the distribution of the technical replicates for panels C and E and the cells measured for panels G and I (n = 3900-12 300 total cells in panel C; 0-404 wounded cells in panel E; 76-102 wounded cells in panel G; and 39-63 wounded cells in panel I, pooled from 3 to 4 independent experiments). Statistical analyses were performed using 1-way analysis of variance (ANOVA) with the Kruskal-Wallis test for panels C, G, and I and the Mann-Whitney U test for panel E. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.