RESUMO
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.
Assuntos
Induração Peniana , Masculino , Humanos , Induração Peniana/genética , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Hedgehog/metabolismo , Pênis , Células Cultivadas , Fibroblastos/metabolismoRESUMO
Extracellular vesicles have emerged as important mediators of cell-to-cell communication in the pathophysiology of fibrotic diseases. One such disease is Peyronie's disease (PD), a fibrotic disorder of the penis caused by uncontrolled transformation of resident fibroblasts to alpha-smooth muscle actin positive myofibroblasts. These cells produce large amounts of extracellular matrix, leading to formation of a plaque in the penile tunica albuginea (TA), causing pain, penile curvature, and erectile dysfunction. We have used primary fibroblasts derived from the TA of PD patients to explore the role of transforming growth factor beta 1 (TGF-ß1), a key signalling factor in this process. TGF-ß1 treatment elicited a range of responses from the myofibroblasts: (i) they secreted extracellular vesicles (EVs) that were more numerous and differed in size and shape from those secreted by fibroblasts, (ii) these EVs prevented TGF-ß1-induced transformation of fibroblasts in a manner that was dependent on vesicle uptake and (iii) they prevented phosphorylation of Erk1/2, a critical component in modulating fibrogenic phenotypic responses, but did not affect TGF-ß1-induced Smad-signalling. We posit that this effect could be linked to enrichment of TSG-6 in myofibroblast-derived EVs. The ability of myofibroblast-derived vesicles to prevent further myofibroblast transformation may establish them as part of an anti-fibrotic negative feedback loop, with potential to be exploited for future therapeutic approaches.
Assuntos
Vesículas Extracelulares , Fibroblastos , Miofibroblastos , Fator de Crescimento Transformador beta1 , Vesículas Extracelulares/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Humanos , Miofibroblastos/metabolismo , Fosforilação , Masculino , Fibroblastos/metabolismo , Moléculas de Adesão Celular/metabolismo , Sistema de Sinalização das MAP Quinases , Induração Peniana/metabolismo , Induração Peniana/patologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células Cultivadas , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Transdução de SinaisRESUMO
Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-ß1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03-300 µM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis. When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts. This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.