Abstract 331: Hdac5 Represses The Angiogenic Growth Factor Fgf2 And Guidance Molecules And Thereby Blocks Endothelial Cell Function
Histone deacetylases (HDAC) modulate gene expression for cardiac hypertrophy and angiogenesis. HDAC7 is essentially required for vascular development and vessel integrity. We used specific siRNA to investigate the role of other HDAC isoenzymes for angiogenic endothelial cell activity. Consistent with previous reports, HDAC7 suppression inhibited migration and capillary-like sprouting of endothelial cells, whereas, in contrast, siRNA against HDAC5 surprisingly increased migration to 121 ± 4% (P < 0.005) and sprout formation to 216 ± 35% of scrambled siRNA (P < 0.05). HDAC5 is a negative regulator of angiogenic endothelial cell activity, since overexpression of a constitutively nuclear localized HDAC5 construct, S259A/S498A, inhibited sprout formation (62 ± 17% of empty vector, P < 0.005). By mRNA profiling, HDAC5-siRNA induced the expression of secreted angiogenic factors including fibroblast growth factor 2 (FGF2, bFGF). Conditioned medium from HDAC5-siRNA-transfected endothelial cells stimulated migration to 248 ± 34% (P < 0.05) and sprout formation to 163 ± 18% (P < 0.005), suggesting that secreted gene products contribute to the proangiogenic effect of HDAC5-siRNA. Vice versa, HDAC5 overexpression inhibited the FGF2 promoter activity in reporter gene assays, and chromatin immunoprecipitation showed binding of HDAC5 to the FGF2 promoter, suggesting that HDAC5 is a repressor at the FGF2 gene. A neutralizing FGF2 antibody attenuated HDAC5-siRNA-induced sprouting from 331 ± 50 to 203 ± 22% of scrambled siRNA (P < 0.01 vs. HDAC5-siRNA alone and vs. scrambled siRNA + FGF2 ab), suggesting that both FGF2-dependent and -independent signals mediate the effect of HDAC5 suppression. As further candidate target genes, HDAC5-siRNA induced the expression of axon guidance molecules SLIT2 and EphB4, which also regulate vessel pathfinding and angiogenic network formation. The combination of FGF2 and EphB4 neutralizing antibodies with inhibition of the SLIT2 receptor Robo1 additionally reduced sprout induction by HDAC5-siRNA (P < 0.05). In summary, HDAC5-siRNA induces angiogenic genes, among those the secreted gene products FGF2 and SLIT2 as well as membrane-bound EphB4 causally contribute to increased capillary-like sprouting of endothelial cells.