Background/Purpose: Scleroderma (SSc) is a complex disease that involves activation of the immune system, vascular complications, and tissue fibrosis. Although the pathogenesis of this disease is largely unknown, epigenetic dysregulation has been implicated. In this study, we focused on the histone methyltransferase enhancer of zeste homolog 2 (EZH2), which is the catalytic component of the polycomb repressor complex 2 and mediates trimethylation of lysine 27 of histone 3 (H3K27me3). It has been reported that EZH2 regulates angiogenesis, and it is also involved in fibrosis. We hypothesize that EZH2 contributes to impaired angiogenesis and enhanced fibrosis in SSc, and inhibition of EZH2 improves these key events in SSc.

Methods: Dermal endothelial cells (ECs) and fibroblasts were isolated from biopsies from healthy subjects or patients with diffuse cutaneous SSc. EZH2 was overexpressed using an EZH2 vector. Inhibition of EZH2 was achieved by using EZH2 inhibitor DZNep or EZH2 siRNA. Angiogenesis was assessed by an in vitro Matrigel tube formation assay. The scratch wound assay was used to evaluate fibroblast migration. The effect of DZNep (50mg/kg/day for 14 days) in vivo was assessed in a bleomycin-induced skin fibrosis model. A t-test was used to compare differences between groups, and a p-value of <0.05 was considered significant. Genome-wide DNA methylation was evaluated using the Illumina Infinium Methylation EPIC BeadChip Array.

Results: EZH2 and H3K27me3 were significantly elevated in SSc ECs and fibroblasts compared to healthy controls. Overexpression of EZH2 in normal ECs led to significant decrease in tube formation, while decrease in EZH2 in SSc ECs, achieved by siRNA or DZNep treatment, restored normal angiogenesis. In SSc fibroblasts, DZNep treatment dose-dependently reduced EZH2, as well as pro-fibrotic COL1A1, TGFB1, and FRA2. In addition, DZNep significantly reduced pro-angiogenic genes VEGF and FGF2, as well as genes involved in DNA methylation, including DNMT1, DNMT3A, and MECP2. EZH2 inhibition in SSc fibroblasts also led to genome-wide changes in the methylome, and the expression of differentially methylated genes was confirmed by qPCR, including anti-fibrotic IL7 and migration-involved LRRC16A, both hypermethylated and downregulated. In the scratch wound assay, DZNep-treated SSc fibroblasts showed wider wound width at 48 hours post-injury compared to untreated SSc fibroblasts. The gene expression profiles and scratch wound results were further confirmed in EZH2-overexpressing normal fibroblasts, as these cells showed a pro-fibrotic phenotype, mimicking what was seen in SSc fibroblasts. In a bleomycin prevention model, DZNep successfully prevented significant skin fibrosis to occur, assessed by both skin thickness and hydoxyproline analysis.

Conclusion: EZH2 is overexpressed in SSc ECs and fibroblasts, and this overexpression is profibrotic and results in impaired angiogenesis in this disease. Inhibition of EZH2 restored normal angiogenesis in SSc ECs, and normalized the pro-fibrotic phenotype in SSc fibroblasts as well as in an animal model. Targeting EZH2 or EZH2-regulated genes may open new therapeutic avenues for patients with SSc.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-ezh2-stops-fibrosis-and-improves-angiogenesis-in-scleroderma/