Simvastatin and the Rho-kinase inhibitor Y-27632 prevent myofibroblast transformation in Peyronie's disease-derived fibroblasts via inhibition of YAP/TAZ nuclear translocation

Abstract

Objectives: To uncover the anti-myofibroblast (MFB) properties of Rho-kinase inhibitor (compound Y-27632) and simvastatin in an in vitro model of Peyronie's disease (PD), a sexually debilitating disease caused by an irreversible fibrotic plaque in the penile tunica albuginea (TA).

Materials and methods: Primary human fibroblasts (FBs) were isolated from surgically obtained TA tissue from patients with PD. To induce MFB status, cells were stimulated with 3 ng/mL transforming growth factor-β1 (TGF-β1). Increasing doses of Y-27632 and simvastatin were added. Real-time quantitative PCR was used to assess mRNA expression of α-smooth muscle actin (α-SMA), collagen III, elastin and connective tissue growth factor (CTGF) after 72 h. Western blot analysis was used to quantify α-SMA protein contents, and immunofluorescence (IF) was used to visualize MFB differentiation by staining for α-SMA after 72 h. A resazurin-based assay was used to assess cell viability to ensure the anti-MFB effect of the drugs. A mechanistic study was performed using IF staining for YAP/TAZ nuclear translocation.

Results: After 72 h of stimulation with TGF-β1, a six- to 10-fold upregulation of α-SMA could be observed. When treated with Y-27632 or simvastatin, the α-SMA, collagen III, elastin and CTGF mRNA expression was impeded. Additionally, TGF-β1 stimulation showed a twofold increase in α-SMA protein expression, which was reversed to non-stimulated levels after treatment with Y-27632 and simvastatin. Using IF, stimulated cells were identified as MFB (α-SMA+, Vim+) as opposed to the non-stimulated, Y-27632- and simvastatin-treated cells (α-SMA-, Vim+). The resazurin-based assay confirmed that the cell viability was not compromised by the administered drugs. On stimulation with TGF-β1, nuclear translocation of YAP/TAZ could be observed, which was prevented by adding the aforementioned compounds.

Conclusion: Transformation of FBs into the contractile and extracellular matrix-producing MFBs occurs after TGF-β1 stimulation. In our experiments, Rho-kinase inhibition and simvastatin treatment were shown to prevent this in TGF-β1-stimulated cells on an RNA and protein level through the inhibition of YAP/TAZ nuclear translocation. Y-27632 and simvastatin could become a novel treatment option in the early treatment of PD.

Keywords: #Andrology; #Peyronies; Peyronie's disease; Rho-kinase inhibition; statins; transforming growth factor-β.