Thomas Eschenhagen, Roberto Bolli, Thomas Braun, Loren J. Field, Bernd K. Fleischmann, Jonas Frisén, Mauro Giacca, Joshua M. Hare, Steven R. Houser, Richard T. Lee, Eduardo Marbán, James F. Martin, Jeffery D. Molkentin, Charles E. Murry, Paul R. Riley, Pilar Ruiz-Lozano, Hesham A. Sadek, Mark A. Sussman and Joseph A. Hill
Circulation. 2017;CIRCULATIONAHA.117.029343, originally published July 6, 2017
shRNA‐mediated depletion of HDAC1 in human CMCs. Representative (A) epifluorescence microscopy images (n=3; scale=400 μm) and (B and C) flow cytometric analysis (values are mean±SEM; n=3) of CMCs 72 hours after transduction with the MISSION pLKO.1‐puro‐CMV‐TurboGFP Positive Control Vector (1:3 or 1:10 viral titer dilutions). Flow cytometry data were arcsine‐transformed and analyzed by unpaired, 1‐way ANOVA. P values were calculated using the post hoc Bonferroni multiple comparison test. The efficacy of HDAC1 knockdown in CMCs assessed by (D) qPCR (values are mean±SEM; n=4) and (E) Western blot (representative image; n=4). F, Bar graph denoting densitometric quantification of resulting HDAC1 immunoblots. Values represent mean protein expression (relative to β‐actin)±SEM (n=4). qPCR and Western blot data were log base 10 (y=log10 y) transformed and analyzed by unpaired, 1‐way ANOVA. P values were calculated using the post hoc Bonferroni multiple comparison test. CMCs indicates cardiac mesenchymal stromal cells; FSC‐A, forward scatter pulse area; GFP, green fluorescent protein; HDAC1, histone deacetylase 1; shHDAC1, short hairpin RNA‐histone deacetylase 1; shNT, short hairpin RNA nontarget; UT, untransduced.
By Joseph B.Moore, JohnZhao, Annalara G.Fischer, Matthew C.L.Keith, DavidHagan, MarcinWysoczynski and RobertoBolli
HDAC1 depletion alters human CMC cytokine secretion patterns. A, Schematic detailing the isolation of conditioned medium (CM) from virally transduced...Show More
HDAC1 depletion alters human CMC cytokine secretion patterns. A, Schematic detailing the isolation of conditioned medium (CM) from virally transduced CMCs. CM was harvested from shRNA nontarget (shNT) or shRNA HDAC1 (shHDAC1) transduced CMCs 72 hours after lentiviral exposure. Lentivirus dilutions (1:3 or 1:10) were used. B, CM was incubated with cytokine array membranes (Proteome Profiler Human XL Cytokine Array Kit; R&D Systems, Minneapolis, MN) and detected using enhanced chemiluminescence. C, Proteome cytokine array heatmap illustrating post hoc densitometric quantification. Relative pixel density is shown (shHDAC1 relative to shNT controls). D, Cytokine expression changes stratified according to function. (+) denotes upregulation; (−) denotes downregulation. Angio. indicates angiogenesis; Apopt., apoptosis; CMCs, cardiac mesenchymal stromal cells; Diff., differentiation; HDAC1, histone deacetylase 1; shHDAC1, short hairpin RNA‐histone deacetylase 1; shNT, short hairpin RNA‐non target; Surv., survival; UT, untransduced.Show Less
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.
HDAC1‐depletion stimulates basic fibroblast growth factor (bFGF) expression in human CMCs. A, qPCR assays evaluating the expression of known trophic factors involved in cell‐mediated cardiac repair in shHDAC1, shNT, or untransduced CMCs. Values are mean±SEM (n=4). qPCR data were log base 10 (y=log10 y) transformed and analyzed by 2‐way ANOVA. P values were calculated using the post hoc Bonferroni multiple comparison test. B, Representative immunoblot evaluating bFGF expression in total protein isolates derived from shHDAC1, shNT, or untransduced CMCs (n=4). C, Densitometric quantification of bFGF immunoblots (expression relative to β‐actin). Values are mean±SEM (n=4). Western blot data were log base 10 (y=log10 y) transformed and analyzed by unpaired, 1‐way ANOVA. P values were calculated using the post hoc Bonferroni multiple comparison test. All experiments utilized 1:3 shRNA viral titer dilutions. HDAC1 indicates histone deacetylase 1; shHDAC1, short hairpin RNA‐histone deacetylase 1; shNT, short hairpin RNA‐non target; UT, untransduced.
Basic fibroblast growth factor (bFGF) knockdown inhibits paracrine signaling‐mediated activation of endothelial cell proliferation and tube assembly in HDAC1‐depleted CMCs. A, Immunoblots evaluating the expression of bFGF in total protein extracts derived from shRNA‐transduced CMCs (n=4). B, Densitometric analysis of bFGF immunoblots (expression relative to β‐actin). Values are mean±SEM (n=4). Western blot data were log base 10 (y=log10 y) transformed and analyzed by unpaired, 1‐way ANOVA. P values were calculated using the post hoc Holm–Sidak multiple comparison test. C, HAEC growth was assessed following their propagation in CM from all shRNA‐transduced CMC groups (untransduced, shNT, shHDAC1, shbFGF, and shHDAC1+shbFGF). Values are mean±SEM (n=11). Growth data were analyzed by 1‐way ANOVA and P values determined using the post hoc Dunnett multiple comparison test. D, Representative fluorescent microscopy images (scale=1000 μm) and (E) graph enumerating HUVEC tube formation in response to incubation with CM from untransduced, shNT, shHDAC1, shbFGF, or shHDAC1+shbFGF transduced CMCs. HUVEC base (−Ctrl), CMC base (−Ctrl), and HUVEC base+inducer (low serum growth supplement [LSGS]; +Ctrl) controls are included. Values are mean±SEM (n=8 for control and experimental groups). Tube formation data were analyzed using 1‐way ANOVA and P values determined using the post hoc Dunnett 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; shbFGF, short hairpin RNA basic fibroblast growth factor; shHDAC1, short hairpin RNA‐histone deacetylase 1; shNT, short hairpin RNA nontarget; UT, untransduced.
