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.

Emerging Roles of Matricellular Proteins in Systemic Sclerosis

“…Protein levels of CCN1 from dermal fibroblasts of lesioned skin also reveal conflicting expression patterns. Comparable expression between healthy and SSc-derived fibroblasts was reported by Tsou et al, while fibroblasts from a small cohort of patients with early onset (<2 years) dcSSc found CCN1 to be overexpressed in comparison to healthy matched controls [ 63 , 65 ].…”

“…Post developmental expression of CCN1, like many MCPs is observed during tissue damage and disease; however, circulating levels of CCN1 in serum samples of SSc patients have been inconsistently reported in the literature. Lin et al report CCN1 to be significantly elevated in SSc from their analysis of several autoimmune diseases [ 62 ], while other reports found no difference in plasma CCN1 in both lcSSc and dcSSc patients versus healthy controls [ 63 , 64 ]. Protein levels of CCN1 from dermal fibroblasts of lesioned skin also reveal conflicting expression patterns.…”

“…Using an in vivo model of bleomycin-induced skin scleroderma in mice, targeted knockout of CCN1 in fibroblasts limited type 1 collagen accumulation with reduced skin thickness [ 65 ]. Alternatively, CCN1 overexpression in isolated dermal fibroblasts cultured from skin biopsies of dcSSc patients suggest an antifibrotic role due to the downregulated expression of fibrotic genes COL1A1 and ACTA2 , and consequently, slower cell migration and diminished gel contractile strength [ 63 ]. A mechanistic explanation by Tsou et al propose a twofold rationale for the attenuation of fibrogenesis by CCN1 overexpression in these dcSSc fibroblasts.…”

Inhibition of histone readers bromodomain extra-terminal proteins alleviates skin fibrosis in experimental models of scleroderma

“…Cells were lysed in RIPA buffer containing protease inhibitors. Western blotting was performed following the protocol as described previously (11). After blocking the blots for an hour, the blots were probed with antibodies against BRD2, BRD3, BRD4, collagen I, and Cell proliferation assays.…”

“…Gel contraction and cell migration assays. The Cell Contraction Assay kit (Cell Biolabs) was used for gel contraction and was conducted as previously described (11).…”