Antifibrotic effects of CCN1/CYR61 in primary portal myofibroblasts through induction of reactive oxygen species and resulting apoptosis

Cysteine-rich protein 61 (CCN1/CYR61) is a matricellular protein of the CCN family that comprises six secreted CCN proteins in mammals [1, 2]. In adults, CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies showed that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transform into an extracellular matrix-degrading phenotype [3]. We here investigated CCN1/CYR61 expression in primary profibrogenic liver cells (i.e. hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels in primary parenchymal liver cells (i.e. hepatocytes) were only marginal compared to that found in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly found during early periods of insult, while the expression declined during prolonged phases of fibrogenesis. We further generated an adenovirus type 5 encoding CCN1/CYR61 (i.e. Ad5-CMV-CCN1/CYR61) and transduced it into primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 inhibits production of collagen type I at both mRNA and protein levels in these cells as evidenced by quantitative real time PCR, Western blot and immunocytochemistry. Interestingly, CCN1/CYR61 further induces production or reactive oxygen species (ROS) leading to apoptosis as shown by ROS staining and TUNEL assay, while β-galactosidase staining of senescence phenotype showed no difference from respective controls. Therefore, we conclude that in line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in the process of liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells therefore might be beneficial in liver fibrotic therapy.