We extended our work to examine the impact of epigenetics in scleroderma, focusing initially on DNA methylation changes in dermal fibroblasts. More recently, we have also explored the role of epigenetic dysregulation on the on the vascular aspect of the disease. We hypothesized that the anti-angiogenic histone deacetylase 5 (HDAC5) contributes to impaired angiogenesis in scleroderma by repressing pro-angiogenic factors in endothelial cells. We showed that HDAC5 is indeed overexpressed in endothelial cells isolated from patients with diffuse cutaneous scleroderma compared to healthy controls, and that silencing HDAC5 restored normal angiogenesis in scleroderma endothelial cells. We then took an unbiased approach to examine genome-wide changes in chromatin accessibility after HDAC5 knockdown using an assay for transposase-accessible chromatin using sequencing (ATAC-seq). HDAC5 knockdown showed increased chromatin accessibility compared to control cells, and a total 75 genes were located in these sites. To identify genes that were related to angiogenesis or fibrosis, the genes were subject to bioinformatics analysis. Using this approach, among those genes that were in the more open regions, at least 16 genes were involved in angiogenesis and a couple were involved in fibrosis. Through functional assays we identified 3 novel HDAC5-target genes, CYR61, PVRL2, and FSTL1 that were associated with impaired angiogenesis in scleroderma. In this study, we provided a link between epigenetic regulation and impaired angiogenesis in scleroderma, and presented a novel mechanism for the dysregulated angiogenesis that marks the initiation of this disease.
Summary of our approach to identify novel targets to help better understand and treat endothelial dysfunction in scleroderma
(Experimental design used in Tsou et al. Arthritis and Rheumatology 2016)