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 to play a role. 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. It mediates trimethylation of lysine 27 of histone 3, which acts as a repressive epigenetic mark. 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.

Methods:  Dermal endothelial cells (ECs) and fibroblasts were isolated from biopsies from healthy subjects or patients with diffuse cutaneous SSc. EZH2, collagen 1a1 (Col1a1), α-smooth muscle actin (ACTA2), fos-related antigen 2 (Fra2), and peroxisome proliferator-activated receptor gamma (PPARG) expression were determined by qPCR. EZH2 expression was also determined by Western blotting. EZH2 was overexpressed using an EZH2 vector. Inhibition of EZH2 was achieved by using EZH2 inhibitor DZNep (0.2μM-5μM) or EZH2 siRNA. Angiogenesis was assessed by an in vitro Matrigel tube formation assay. The scratch wound assay was used to evaluate fibroblast migration. A paired t-test was used to compare differences between groups, and a p-value of <0.05 was considered significant. Genome-wide DNA methylation status was evaluated using the Illumina Infinium Methylation EPIC BeadChip Array and the Illumina GenomeStudio platform was used to analyze the methylation data.

Results:   The expression of EZH2 was significantly elevated in both SSc ECs and fibroblasts compared to healthy controls. To evaluate the effect of EZH2 on EC angiogenesis, EZH2 was overexpressed in normal ECs. This led to a significant decrease in tube formation on Matrigel compared to sham-transfected cells. In contrast, silencing of EZH2 in SSc ECs restored normal angiogenesis. In normal fibroblasts, overexpression of EZH2 led to increase in Col1a1 and Fra2 mRNA expression. In contrast, SSc fibroblasts treated with DZNep showed a dose-dependent reduction in Cola1a and Fra2 mRNA expression, suggesting that inhibiting EZH2 attenuates profibrotic potential in SSc fibroblasts. In addition, we observed a dose-dependent reduction in mRNA expression of the myofibroblast marker ACTA2 in SSc fibroblasts treated with DZNep, but this did not reach statistical significance. The antifibrotic PPARG decreased at lower doses of DZNep but increased significantly at the highest dose (5μM) compared to untreated SSc fibroblasts. In the scratch wound assay, EZH2-overexpressing normal fibroblasts showed an increase in cell migration while DZNep-treated SSc fibroblasts showed wider wound width at 48 hours post-injury compared to untreated SSc fibroblasts. Genome-wide DNA methylation changes after manipulation of EZH2 expression were used to identify additional EZH2-regulated targets, which will help to further mechanistically understand the angiogenic and fibrotic effects of EZH2 in SSc.

Conclusion:  Our results uncovered an important role for EZH2 in SSc. EZH2 is overexpressed in SSc ECs and fibroblasts, and this overexpression is profibrotic and results in impaired angiogenesis in this diseases. Targeting EZH2 or EZH2-regulated genes may open new therapeutic avenues for patients with SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ezh2-modulates-angiogenesis-and-fibrosis-in-scleroderma/