Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1–2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24–48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

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