Anti-fibrotic properties of RhoA kinase-inhibition in a cell culture model of Peyronie’s Disease
Milenkovic, U1; Ilg, MM2; van Renterghem, K3; De Ridder, D1; Albersen, M1
1: KU Leuven, Belgium; 2: Anglia Ruskin University, UK; 3: University of Hasselt, Belgium
Objective: Peyronie’s disease (PD) is defined as the formation of a fibrotic plaque in the penile tunica albuginea (TA). Most common symptoms are pain, erectile dysfunction and irreversible penile curvature. Currently, evidence for reliable medical treatment is lacking. This study aimed to uncover the anti-myofibroblast (MFB) properties of RhoA kinase-inhibitor (Ri) (compound Y27632) in an in vitro setting.
Materials and Methods: Human primary fibroblasts were isolated from TA of patients with PD (N=4). To confirm fibroblast (FB) identity, cells were stained for vimentin (Vim) using immunofluorescence (IF). To induce MFB status, cells where stimulated with 3ng/mL TGF- β1 (TBS). Increasing doses of Y27632 where added (1-10-30mM). RT-qPCR was used to assess mRNA expression of alpha-smooth muscle actin (α-SMA) after 24h. WB was used to quantify α-SMA protein contents and IF visualized MFB differentiation by staining for α-SMA after 72h. Resazurin-based assay was performed to assess cell viability to ensure anti-MFB effect of the drugs.
Results: After 24h of TBS a 6-fold upregulation of α-SMA compared to non-stimulated cells could be observed. When treated with Y27632, the α-SMA mRNA expression returned to non-stimulated levels. 72h of TBS showed a 50% increase in α-SMA protein expression on WB, which was reversed to non-stimulated levels after treatment with Y27632. Using IF, TBS cells were identified as MFB (α-SMA+, Vim+) as opposed to the non-stimulated and Y27632-treated cells (α-SMA-, Vim+). The resazurin-based assay confirmed that the cell viability was not compromised when administering increasing doses of Y27632.
Conclusion: TGF- β1 stimulation causes a transformation of fibroblasts into the contractile and extracellular matrix-producing myofibroblasts, with α-SMA as its hallmark. Rho A-kinase inhibition has proven its anti-myofibroblast activity in TGF- β1 stimulated cells on an RNA and protein level. Y27632 has the potential to become a new weapon in the waging battle against PD.
Acknowledgement: Funded by European Society for Sexual Medicine, Functional Urology Fund of UZ Leuven & Fund for Peyronie’s Disease Research of UZ Leuven.
Work supported by industry: no.Go Back