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. 2019 Sep 24;10:1182. doi: 10.3389/fphys.2019.01182

FIGURE 4.

FIGURE 4

Low dose SDF-1α is protective against both mitochondrial reactive oxygen species (ROS) AND nitrosative stress. (A) Representative immunofluorescence images of human coronary artery endothelial cells (HCAECs) incubated with the mitochondrial O2– – sensitive fluorescent dye MitoSOX Red (10 um), and DAPI (blue) after treatment with SDF-1α recombinant (20 ng; n = 4), allogeneic MSC conditioned medium (n = 4) or autologous MSC conditioned medium (n = 5) and stimulated with LPS (1 ug/mL). (B) Quantification of MitoSOX Red fluorescence showed that LPS significantly increased superoxide production compared to control (301676 ± 7843 vs. 112664 ± 22307, p < 0.0001∗), whereas SDF-1α, allogeneic MSC conditioned medium, and autologous MSC conditioned medium prevented superoxide production induced by LPS (58674 ± 8145, 52665 ± 33625, 39535 ± 13687 AU, p < 0.0001∗, respectively. (C) Representative immunofluorescence images via confocal microscopy of HCAECs incubated with H2-DCF-DA (Green) that detects intracellular peroxides including H2O2 and DAPI (blue). (D) Quantification of the average of DCF fluorescence showing LPS induced significantly higher rates of hydrogen peroxide production compared to control (22491 ± 16371 vs. 4464 ± 2047, p < 0.0001∗), while SDF-1α, allogeneic MSC conditioned medium, and autologous conditioned medium attenuated cell hydrogen peroxide production (6172 ± 3920 AU, p < 0.01∗∗∗, 9753 ± 4751 AU, p < 0.05#, 46941 ± 89798, p < 0.05#, respectively). (E) Representative pictures of nitrotyrosine (3NT) expression in HCAECs stimulated with LPS followed by SDF-1α, allogeneic MSC conditioned medium, and autologous conditioned medium. (F) Quantification of 3NT fluorescence demonstrating that LPS significantly increased nitrotyrosine expression (75894 ± 13874 vs. 43546 ± 10020 AU, p < 0.001∗∗, and only SDF-1α and allogeneic MSC conditioned medium reduced nitrotyrosine expression (52132 ± 4395 AU, p < 0.05#, 34761 ± 4898 AU, p < 0.0001∗∗∗). Autologous MSC conditioned medium did not block LPS induced nitrotyrosine expression (56949 ± 21648 vs. 75894 ± 13874, respectively, p = NS).