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
Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.
Assuntos
Transcrição Reversa , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e EspecificidadeRESUMO
The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics. However, the accuracy, reliability and reproducibility of qPCR data face challenges arising from various factors associated with experimental design, execution, data analysis and inadequate reporting details. Addressing these concerns, the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have emerged as a cohesive framework offering a standardised set of recommendations that describe the essential information required for assessing qPCR experiments. By emphasising the importance of methodological rigour, the MIQE guidelines have made a major contribution to improving the trustworthiness, consistency, and transparency of many published qPCR results. However, major challenges related to awareness, resources, and publication pressures continue to affect their consistent application.
Assuntos
Reprodutibilidade dos Testes , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
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.