Identification of Cysteine-Rich Angiogenic Inducer 61 as a Potential Antifibrotic and Proangiogenic Mediator in Scleroderma

Abstract

Objective: We previously identified CYR61 as a histone deacetylase 5 (HDAC-5)–repressed gene in systemic sclerosis (SSc; scleroderma) endothelial cells (ECs). When overexpressed, cysteine-rich angiogenic inducer 61 (CYR-61) promoted angiogenesis in SSc ECs. This study was undertaken to examine the role of CYR-61 in fibrosis and determine the mechanisms involved in CYR-61–mediated angiogenesis in SSc. Methods: Dermal ECs and fibroblasts were isolated from biopsy specimens from healthy subjects and patients with SSc. CYR-61 level was determined by quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. CYR-61 was overexpressed using a CYR61 vector or knocked down using small interfering RNA, and functional and mechanistic studies were then conducted in fibroblasts and ECs. Results: Lower CYR61 messenger RNA levels were observed in dermal fibroblasts and ECs from SSc patients than in those from healthy controls. In SSc fibroblasts, overexpression of CYR-61 led to significant reduction in the expression of profibrotic genes, including COL1A1 (P = 0.002) and ACTA2 (P = 0.04), and an increase in the expression of matrix-degrading genes, including MMP1 (P = 0.002) and MMP3 (P =0.004), and proangiogenic VEGF (P = 0.03). The antifibrotic effect of CYR-61 was further demonstrated by delay in wound healing, inhibition of gel contraction, inactivation of the transforming growth factor β pathway, and early superoxide production associated with senescence in SSc fibroblasts. In SSc ECs, overexpression of CYR-61 led to increased production of vascular endothelial cell growth factor. The proangiogenic effects of CYR-61 were mediated by signaling through αvβ3 receptors and downstream activation of AMP-activated protein kinase, AKT, and the endothelial cell nitric oxide synthase/nitric oxide pathway system. Conclusion: CYR-61, which is epigenetically regulated by HDAC-5, is a potent antifibrotic and proangiogenic mediator in SSc. Therapeutic intervention to promote CYR-61 activity or increase CYR-61 levels might be of benefit in SSc.