Figure 4
Figure 4. Rapid calcium mobilization and PDI expression after laser injury in vitro and in vivo. Fluo-4-AM (3μM) was incubated with cultured HUVECs before laser injury and observed by fluorescence microscopy. (A) The images show a representative field of cells before and after a direct laser pulse to the point indicated by X. Cell imaging was initiated before laser injury to obtain a baseline image, and the laser was fired during this capture. Increased green signal in subsequent time-lapse images represents increased intracellular calcium monitored by Fluo-4 fluorescence, shown in representative images. The Western blot on the right depicts an immunoblot with polyclonal anti-PDI antibody to detect PDI in conditioned medium from unactivated (lane 2) or laser-activated (lane 3) HUVECs. Lane 1: 2 ng recombinant human PDI. (B) Representative images of fixed and immunostained cells that have been activated by laser injury in the presence of plasma and calcium with (right) or without (left) a function blocking PDI antibody RL90. The cells were fixed after laser activation and stained for fibrin (red). Fluorescein isothiocyanate–phalloidin (green) and DAPI (4,6 diamidino-2-phenylindole; blue). (C) Quantification of fibrin signal detected on cultured endothelial: lane 1, unactivated cells; lane 2, laser-activated cells; lane 3, laser-activated cells in the presence of the PDI-inhibitory antibody RL90; lane 4, laser-activated cells in the presence of an isotype control antibody. The P values between the different groups were obtained using the unpaired t test. (D) Representative images of rapid activation of arteriolar endothelium and PDI expression after laser injury in confocal intravital microscopy. Rhod-2 (6μM) and Alexa 647–labeled polyclonal anti-PDI antibody (0.3 μg/g body weight) were infused 5 minutes before the first injury. Site of injury is indicated by an X. Platelet aggregation was blocked with the GPIIbIIIa antagonist, eptifibatide. Vessel imaging was initiated before laser injury, and the laser was fired after one z-stack of 30 planes was obtained. Each z-stack of 30 images was 8.7 seconds. The grid size was 10 μm. Calcium elevation was monitored by excitation of Rhod-2 at 561 nm (pseudocolored green), while the PDI signal was observed simultaneously at 647 nm (pseudocolored red).

Rapid calcium mobilization and PDI expression after laser injury in vitro and in vivo. Fluo-4-AM (3μM) was incubated with cultured HUVECs before laser injury and observed by fluorescence microscopy. (A) The images show a representative field of cells before and after a direct laser pulse to the point indicated by X. Cell imaging was initiated before laser injury to obtain a baseline image, and the laser was fired during this capture. Increased green signal in subsequent time-lapse images represents increased intracellular calcium monitored by Fluo-4 fluorescence, shown in representative images. The Western blot on the right depicts an immunoblot with polyclonal anti-PDI antibody to detect PDI in conditioned medium from unactivated (lane 2) or laser-activated (lane 3) HUVECs. Lane 1: 2 ng recombinant human PDI. (B) Representative images of fixed and immunostained cells that have been activated by laser injury in the presence of plasma and calcium with (right) or without (left) a function blocking PDI antibody RL90. The cells were fixed after laser activation and stained for fibrin (red). Fluorescein isothiocyanate–phalloidin (green) and DAPI (4,6 diamidino-2-phenylindole; blue). (C) Quantification of fibrin signal detected on cultured endothelial: lane 1, unactivated cells; lane 2, laser-activated cells; lane 3, laser-activated cells in the presence of the PDI-inhibitory antibody RL90; lane 4, laser-activated cells in the presence of an isotype control antibody. The P values between the different groups were obtained using the unpaired t test. (D) Representative images of rapid activation of arteriolar endothelium and PDI expression after laser injury in confocal intravital microscopy. Rhod-2 (6μM) and Alexa 647–labeled polyclonal anti-PDI antibody (0.3 μg/g body weight) were infused 5 minutes before the first injury. Site of injury is indicated by an X. Platelet aggregation was blocked with the GPIIbIIIa antagonist, eptifibatide. Vessel imaging was initiated before laser injury, and the laser was fired after one z-stack of 30 planes was obtained. Each z-stack of 30 images was 8.7 seconds. The grid size was 10 μm. Calcium elevation was monitored by excitation of Rhod-2 at 561 nm (pseudocolored green), while the PDI signal was observed simultaneously at 647 nm (pseudocolored red).

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