Abstract 143: Enrichment of Dimethylation of Lysine 4 on Histone 3 and Resident Vascular Stem Cell Transition to Vascular Smooth Muscle Cells

Data support a role for stem cell-derived vascular smooth muscle cells (SMC) in arteriosclerosis. Epigenetics play a critical role in SMC differentiation where histone proteins associated with the myosin heavy chain (Myh11) promoter are post-translationally modified by dimethylation of lysine 4 on histone 3 (H3K4me2). Studies report that ‘de-differentiated’ SMC do not exhibit loss of H3K4me2 at the Myh11 promoter even when mRNA levels decrease thereby allowing this modification to be used to track differentiated SMC. Our aim was to determine the level of H3K4me2 and H3K27me3 methylation of differentiated SMCs and compare to resident vascular stem cells and stem cell-derived SMC. Murine adventitial Sca1 + stem cells (APCs), rat medial Sox10 + multipotent vascular stem cells (MVSCs) and CD44 + bone-marrow derived mesenchymal stem cells (MSCs) were all examined for methylation of lysine 4 (H3K4me2) and lysine 27 (H3K27me3) on histone 3 associated with the Myh11 promoter, before and after SMC differentiation and compared to fresh aortic differentiated SMC and sub-cultured de-differentiated SMC in vitro by Chromatin Immunoprecipitation (ChIP) assay. Cells were also examined for Myh11 expression, stemness (telomerase activity) and multipotentcy. Differentiated rat and murine SMC were enriched for H3K4Me2 at the Myh11 promoter, compared to H3K27me3. Phenotypically de-differentiated sub-cutured rat and murine SMC were enriched for H3K4Me2 when compared to H3K27me3, but to a much lesser extent when compared to differentiated aortic SMC. In contrast, resident APCs and MVSCs, and bone-marrow derived MSCs, were all enriched for H3K27me3, concomitant with significant telomerase activity and multipotent differentiation capacity. The levels of enrichment of H3K27me3 dropped significantly after SMC differentiation with TGF-βeta1 concomitant with a significant enrichment of H3K4me2 to levels that mimicked the level of enrichment in de-differentiated SMC when compared to aortic differentiated SMC. De-differentiated SMC exhibit reduced enrichment of H3K4me2 at the Myh11 promoter region when compared to differentiated aortic SMCs, but mimic the level of enrichment of H3K4me2 observed following resident vascular stem cell differentiation to SMCs.