Figure 4.

Conditioned medium from HDAC1‐depleted CMCs exhibit altered paracrine signaling potency in vitro. A and B, HAEC transwell migration assays in response to CM from shNT, shHDAC1, or untransduced CMCs. HAEC medium (−Ctrl) and HAEC medium+FBS (+Ctrl) are shown. Values are mean±SEM (n=3). Representative images of HAEC transwell migration assays (scale=1 mm) are shown in (B). Data were analyzed by unpaired, 1‐way ANOVA and P values determined using the post hoc Tukey multiple comparison test. C, Oxidative stress assays were performed on HAECs pretreated with CM (shNT, shHDAC1, or untransduced CMCs) by their incubation with increasing concentrations of H₂O₂. Relative cell viability is reported. Values are mean±SEM (n=6). Data were analyzed by 2‐way ANOVA and subject to post hoc analysis using the Tukey multiple comparison test. D and E, HAEC growth was assessed following their propagation in CM from shHDAC1, shNT, or untransduced CMCs. Representative images of CM‐treated HAECs incubated with PrestoBlue metabolizable fluorometric reagent are depicted in (D). Values are mean±SEM (n=6). Growth data were analyzed by the Kruskal–Wallis test and P values determined using a post hoc uncorrected Dunn's test. F, Representative fluorescent microscopy images (scale=1000 μm) and (G) graph enumerating HUVEC tube formation in response to incubation with CM. HUVEC base (−Ctrl), CMC base (−Ctrl), and HUVEC base+inducer (low serum growth supplement [LSGS]; +Ctrl) controls are shown (n=4 each). Values are mean±SEM (n=6 for experimental groups). Resultant tube formation data were analyzed by unpaired, 1‐way ANOVA and P values determined using the post hoc Tukey multiple comparison test. All experiments utilized 1:3 shRNA viral titer dilutions. CM indicates conditioned medium; CMC, cardiac mesenchymal stromal cell; HAEC, human aortic endothelial cell; HDAC1, histone deacetylase 1; HUVEC, human umbilical vein endothelial cell; shHDAC1, short hairpin RNA‐histone deacetylase 1; shNT, short hairpin RNA‐non target; UT, untransduced.