Temporal gene signature of myofibroblast transformation in Peyronie's disease: first insights into the molecular mechanisms of irreversibility.

BACKGROUND: Transformation of resident fibroblasts to profibrotic myofibroblasts in the tunica albuginea is a critical step in the pathophysiology of Peyronie's disease (PD). We have previously shown that myofibroblasts do not revert to the fibroblast phenotype and we suggested that there is a point of no return at 36 hours after induction of the transformation. However, the molecular mechanisms that drive this proposed irreversibility are not known. AIM: Identify molecular pathways that drive the irreversibility of myofibroblast transformation by analyzing the expression of the genes involved in the process in a temporal fashion. METHODS: Human primary fibroblasts obtained from tunica albuginea of patients with Peyronie's disease were transformed to myofibroblasts using transforming growth factor beta 1 (TGF-ß1). The mRNA of the cells was collected at 0, 24, 36, 48, and 72 hours after stimulation with TGF-ß1 and then analyzed using a Nanostring nCounter Fibrosis panel. The gene expression results were analyzed using Reactome pathway analysis database and ANNi, a deep learning-based inference algorithm based on a swarm approach. OUTCOMES: The study outcome was the time course of changes in gene expression during transformation of PD-derived fibroblasts to myofibroblasts. RESULTS: The temporal analysis of the gene expression revealed that the majority of the changes at the gene expression level happened within the first 24 hours and remained so throughout the 72-hour period. At 36 hours, significant changes were observed in genes involved in MAPK-Hedgehog signaling pathways. CLINICAL TRANSLATION: This study highlights the importance of early intervention in clinical management of PD and the future potential of new drugs targeting the point of no return. STRENGTHS AND LIMITATIONS: The use of human primary cells and confirmation of results with further RNA analysis are the strengths of this study. The study was limited to 760 genes rather than the whole transcriptome. CONCLUSION: This study is to our knowledge the first analysis of temporal gene expression associated with the regulation of the transformation of resident fibroblasts to profibrotic myofibroblasts in PD. Further research is warranted to investigate the role of the MAPK-Hedgehog signaling pathways in reversibility of PD.

Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie's disease.

Drug repurposing has been shown to bring safe medications to new patient populations, as recently evidenced by the COVID-19 pandemic. We investigated whether we could use phenotypic screening to repurpose drugs for the treatment of Peyronie's disease (PD). PD is a fibrotic disease characterised by continued myofibroblast presence and activity leading to formation of a plaque in the penile tunica albuginea (TA) that can cause pain during erection, erectile dysfunction, and penile deformity. PD affects 3-9% of men with treatment options limited to surgery or injection of collagenase which can only be utilised at late stages after the plaque is formed. Currently there are no approved medications that can be offered to patients presenting with early disease before the formation of the plaque. Drug repurposing may therefore be the ideal strategy to identify medical treatments to address this unmet medical need in early PD. We used primary human fibroblasts from PD patients in a phenotypic screening assay that measures TGF-ß1-induced myofibroblast transformation which is the main cellular phenotype that drives the pathology in early PD. A library of FDA-approved 1,953 drugs was screened in duplicate wells at a single concentration (10 µM) in presence of TGF-ß1. The myofibroblast marker α-SMA was quantified after 72h incubation. A positive control of SB-505124 (TGF-ß1 receptor antagonist) was included on each plate. Hits were defined as showing >80% inhibition, whilst retaining >80% cell viability. 26 hits (1.3%) were identified which were divided into the following main groups: anti-cancer drugs, anti-inflammation, neurology, endocrinology, and imaging agents. Five of the top-ten drugs that increase myofibroblast-transformation appear to act on VEGFR. This is the first phenotypic screening of FDA-approved drugs for PD and our results suggest that it is a viable method to predict drugs with potential for repurposing to treat early PD.