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

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.

Simvastatin and ROCK Inhibitor Y-27632 Inhibit Myofibroblast Differentiation of Graves’ Ophthalmopathy-Derived Orbital Fibroblasts via RhoA-Mediated ERK and p38 Signaling Pathways

“…Some studies have demonstrated the inhibitory effects of simvastatin and Y-27632 on TGF-β-induced myofibroblast differentiation from fibroblasts derived from different disease specimens, such as nasal polyps ( 24 ), keloids ( 20 ), and penile tunica albuginea from Peyronie’s disease ( 21 ). To the best of our knowledge, this is the first study to investigate the antifibrotic effects of simvastatin and Y-27632 in GO-derived fibroblasts.…”

“…Statins inhibit the synthesis of intermediates of cholesterol biosynthesis such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are essential for the post-translational modification of the Rho family proteins ( 8 ). Therefore, statins have a potential therapeutic role in fibrotic diseases owing to their inhibitory effects on RhoA/ROCK signaling ( 20 , 21 ). Simvastatin, a type of statin, can suppress TGF-β-induced myofibroblast transformation of fibroblasts through RhoA/ROCK inhibition ( 20 , 21 ).…”

“…Therefore, statins have a potential therapeutic role in fibrotic diseases owing to their inhibitory effects on RhoA/ROCK signaling ( 20 , 21 ). Simvastatin, a type of statin, can suppress TGF-β-induced myofibroblast transformation of fibroblasts through RhoA/ROCK inhibition ( 20 , 21 ).…”

The Natural History of Peyronie's Disease

“…In the last few years, we acknowledged a growing interest in basic and translational research on PD pathogenesis, including the developing of the first in vitro cell culture models [10,11]. Even though, little progresses have been made towards further elucidating the real etiology of the disease.…”

New insights into the pathogenesis of Peyronie's disease: A narrative review

“…The ROCK inhibitor, Y-27632, and simvastatin prevent fibrotic changes in TA by inhibiting YAP/TAZ nuclear translocation and downregulating CTGF mRNA expression. 124 Fasudil, a RhoA/Rho kinase (ROCK) inhibitor significantly induces the apoptosis of human fibroblasts derived from urethral scar tissues and decreases their proliferative activity in the absence or presence of TGF-β1 stimulation in a dose and time-dependent manner. Fasudil also suppresses actin polymerization and collagen synthesis, and induces apoptosis in human urethral scar fibroblasts stimulated with or without TGF-β1 via the Rho/ROCK signaling pathway.…”