Background/Purpose: Vascular abnormalities represent a fundamental event in the pathogenesis of scleroderma (SSc) in that endothelial cell (EC) damage triggers a self-fueling process ending in pathological tissue fibrosis. In our previous study we identified CYR61 as a histone deacetylase 5-target gene in SSc ECs, and further confirmed its role in impaired angiogenesis in SSc. As CYR61 is also involved in fibrosis, we hypothesize that CYR61 is beneficial for SSc through its anti-fibrotic and pro-angiogenic properties. In this study, we examined the anti-fibrotic role of CYR61 in SSc fibroblasts, and also dissected the mechanism of its pro-angiogenic property in SSc ECs.

Methods: Dermal ECs and fibroblasts were isolated from biopsies from healthy subjects or patients with diffuse cutaneous SSc. CYR61 was determined by qPCR and ELISA. CYR61 was overexpressed using a CYR61 vector. Angiogenesis was assessed by an in vitro Matrigel tube formation assay. The scratch wound assay and gel contraction assay were used to evaluate fibroblast function. Cell proliferation was assessed by ki67 immunofluorescence, while superoxide production was measured using dihydroethidium. Cell senescence was analyzed by measuring p21. A t-test was used to compare differences between groups, and a p-value of <0.05 was considered significant.

Results: In both SSc ECs and fibroblasts, CYR61 levels were significantly lower compared to control cells. In SSc ECs, overexpression of CYR61 was accompanied with elevation of NOS3 as well as increased secretion of CYR61 and VEGF. The cell surface receptor for CYR61 on ECs, integrin αvβ3, was significantly elevated on SSc ECs. The proangiogenic properties of CYR61 was blocked by neutralizing antibodies for αvβ3, as well as inhibitors for AMPK or AKT/NO signaling pathways. In SSc fibroblasts, overexpression of CYR61 led to significant decrease in profibrotic genes including COL1A1 and ACTA2 while increasing anti-fibrotic PPARG. CYR61 overexpression also resulted in increased secretion of matrix degrading MMP1 and 3, as well as pro-angiogenic VEGF. The anti-fibrotic effect of CYR61 was further demonstrated by delay in wound healing and inhibition of gel contraction. CYR61 led to early superoxide production followed by decrease in cell proliferation and cell senescence in SSc fibroblasts.

Conclusion:

In this study we showed that deficiency in CYR61 potentially contributes to impaired angiogenesis and enhanced fibrosis in SSc ECs and fibroblasts. CYR61 overexpression in SSc ECs led to increase excretion of CYR61, which through binding to αvβ3, activates the AMPK/AKT pathways to promote angiogenesis. CYR61 also induced pro-angiogenic VEGF expression to further increase its pro-angiogenic potential. In addition, CYR61-overexpressed SSc fibroblasts were converted from extracellular matrix-producing myofibroblasts into extracellular matrix-degrading senescent cells. Moreover, the ability of CYR61 to increase VEGF secretion in fibroblasts may modulate angiogenesis in SSc ECs. Taken all together, our data supports the beneficial role of CYR61 in SSc and warrants the use of CYR61 mimetics as a therapeutic option.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identifying-matricellular-protein-cyr61-as-a-potential-anti-fibrotic-and-pro-angiogenic-mediator-in-scleroderma/