ObjectivesTo uncover the anti-myofibroblast (MFB) properties of Rhokinase 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). ConclusionTransformation of FBs into the contractile and extracellular matrix-producing MFBs occurs after TGF-b1 stimulation. In our experiments, Rho-kinase inhibition and simvastatin treatment were shown to prevent this in TGF-b1-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.

word count: 328 Text body word count: 3100 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). PD is a sexual debilitating disease caused by an irreversible fibrotic plaque in the penile tunica albuginea (TA). Treatment is limited to surgically restoring anatomical shape. Evidence for effective medical treatment is lacking.Material and Methods: Primary human fibroblasts were isolated from surgically obtained TA tissue from PD patients. To induce MFB status, cells where stimulated with 3 ng/mL TGF-β1. Increasing doses of Y-27632 and simvastatin were added. RT-qPCR was used to assess mRNA expression of alpha-smooth muscle actin (α-SMA), collagen III, elastin and CTGF after 72 h. WB was used to quantify α-SMA protein contents and IF visualized MFB differentiation by staining for α-SMA after 72 h. Resazurin-based assay was performed to assess cell viability to ensure 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 6-to10-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 two-fold 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. Upon stimulation with TGF-β1, nuclear translocation of YAP/TAZ could be observed, which was prevented by adding the aforementioned compounds.Conclusion: Transformation of fibroblasts into the contractile and extracellular matrix-producing myofibroblasts 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.

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