GRB2 serves as a viable target against skin fibrosis in systemic sclerosis by regulating endothelial cell apoptosis

Abstract

Background: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune system dysfunction, along with tissue fibrosis. Our previous study found GRB2 was downregulated by salvianolic acid B, a small molecule drug that attenuated skin fibrosis of SSc.

Objectives: Here we aim to investigate the role of GRB2 in SSc.

Methods: The microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc and healthy controls. Bleomycin (BLM)-induced skin fibrosis mice were used to explore how GRB2 downregulation affected fibrosis. The apoptosis of EA.hy926 endothelial cells was induced by H₂O₂ and apoptosis ratio was measured by flow cytometric. Transcriptome and phosphoproteomic analyses were performed to explore the regulated pathway.

Results: The expression of GRB2 was significantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-fold in Chinese. Double immunofluorescence staining showed the endothelial cells of SSc patient's skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skin fibrosis and apoptosis of endothelial cells in BLM mouse skin. The in vitro study showed that GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from H₂O₂-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis suggested the focal adhesion pathway was enriched in GRB2 siRNA transfected endothelial cells.

Conclusions: Our results demonstrated GRB2 highly expressed in endothelial cells of SSc skin, and inhibiting GRB2 could effectively attenuate BLM-induced skin fibrosis and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for SSc.

Keywords: Apoptosis; Endothelial cell; GRB2; Systemic sclerosis; Vascular dysfunction.