Estudio del efecto antifibrótico del secretoma de células madre mesenquimales equinas acondicionadas con PGE2 en modelos in vitro de fibrosis endometrial
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Date
2025
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Universidad de Concepción
Abstract
El objetivo de esta tesis fue evaluar el potencial terapéutico de células madre mesenquimales derivadas del endometrio equino (ET-eMSCs), preacondicionadas con prostaglandina E₂ (PGE₂), y sus vesículas extracelulares (VEs), en la reversión del fenotipo fibrótico de miofibroblastos endometriales y explantes, condición asociada a la endometrosis, una patología reproductiva común en yeguas. Esta investigación propuso abordar si la interacción entre ET-eMSCs y miofibroblastos puede modular la secreción de factores antifibróticos y generar cambios funcionales en las células involucradas, mediante tres objetivos específicos: (1) caracterizar las propiedades antifibróticas de las ET-eMSCs preacondicionadas con PGE₂ y su secretoma, (2) determinar si las VEs derivadas de estas células son suficientes para inducir cambios en el fenotipo de miofibroblastos, y (3) identificar, mediante análisis proteómico cuantitativo, los mecanismos moleculares involucrados en dicha interacción.
Los resultados demostraron que las ET-eMSCs preacondicionadas con PGE₂, así como su medio condicionado libre de detritos y sus VEs aisladas, modulan la función biológica de miofibroblastos in vitro, reduciendo la expresión de marcadores fibróticos como α-SMA y CTGF, e inhibiendo la deposición de colágeno tipo I. Adicionalmente, el análisis cuantitativo proteómico mediante LCMS/MS y SILAC reveló que el co-cultivo de ET-eMSCs con miofibroblastos genera un cambio significativo en el perfil secretor de ambas poblaciones celulares. Se detectaron proteínas asociadas a la remodelación de la matriz extracelular, respuesta al estrés oxidativo, plegamiento de proteínas, organización del citoesqueleto y regulación inmune. En particular, la interacción bidireccional activó rutas que promueven la reorganización tisular y la resolución del daño, indicando que el efecto terapéutico no se limita a una acción unidireccional, sino que ambos tipos celulares contribuyen activamente al ambiente antifibrótico.
Esto tiene importantes implicancias para el desarrollo de terapias regenerativas en medicina veterinaria y humana, ya que se demostró que las ET-eMSCs pretratadas con PGE₂ pueden ser programadas para secretar un perfil bioactivo más eficaz, capaz de revertir cambios fibróticos preexistentes. Además, la utilización de VEs como herramientas terapéuticas no invasivas refuerza la posibilidad de aplicar este enfoque de forma segura y específica. El sistema de co-cultivo y la integración de herramientas proteómicas avanzadas permitieron establecer un modelo robusto para el estudio de la fibrosis endometrial equina y aportan evidencia sólida sobre la plasticidad celular y la posibilidad de inducir regresión del fenotipo patológico mediante intervención molecular dirigida.
En términos prácticos, esta tecnología ofrece un marco prometedor para mejorar la eficiencia reproductiva en equinos mediante tratamientos personalizados que restauren la funcionalidad endometrial. A largo plazo, podría extenderse a otras especies y contextos clínicos donde las enfermedades fibróticas comprometan la función tisular.
The objective of this thesis was to evaluate the therapeutic potential of equine endometrial-derived mesenchymal stem cells (ET-eMSCs), preconditioned with prostaglandin E₂ (PGE₂), and their extracellular vesicles (VEs), in reversing the fibrotic phenotype of endometrial myofibroblasts and explants. This condition is associated with endometrosis, a common reproductive disorder in mares. The research aimed to determine whether the interaction between ET-eMSCs and myofibroblasts can influence the secretion of antifibrotic factors and cause functional changes in the cells involved, through three specific objectives: (1) to characterize the antifibrotic properties of ET-eMSCs preconditioned with PGE₂ and their secretome, (2) to assess if VEs derived from these cells can induce changes in the myofibroblast phenotype, and (3) to identify, via quantitative proteomic analysis, the molecular mechanisms underlying such interactions. The results showed that ET-eMSCs preconditioned with PGE₂, their detritus-free conditioned medium, and isolated VEs influence the biological function of myofibroblasts in vitro, decreasing the expression of fibrotic markers such as α-SMA and CTGF, and preventing the buildup of type I collagen. Additionally, quantitative proteomic analysis using LC-MS/MS and SILAC indicated that coculturing ET-eMSCs with myofibroblasts causes a significant change in the secretory profile of both groups. Proteins associated with extracellular matrix remodeling, oxidative stress response, protein folding, cytoskeleton organization, and immune regulation were detected. In particular, the bidirectional interaction activated pathways that promote tissue reorganization and damage resolution, indicating that the therapeutic effect is not limited to a unidirectional action, but that both cell types actively contribute to the antifibrotic environment. This has important implications for the development of regenerative therapies in veterinary and human medicine, as it was shown that ET-eMSCs pretreated with PGE₂ can be programmed to secrete a more effective bioactive profile, capable of reversing pre-existing fibrotic changes. Moreover, the use of VEs as noninvasive therapeutic tools reinforces the possibility of applying this approach in a safe and targeted manner. The co-culture system and the integration of advanced proteomic tools allowed the establishment of a robust model for the study of equine endometrial fibrosis and provided solid evidence on cellular plasticity and the possibility of inducing regression of the pathological phenotype by targeted molecular intervention. Proteins associated with extracellular matrix remodeling, oxidative stress response, protein folding, cytoskeleton organization, and immune regulation were detected. In particular, the bidirectional interaction activated pathways that promote tissue reorganization and damage resolution, indicating that the therapeutic effect is not limited to a unidirectional action, but that both cell types actively contribute to the antifibrotic environment. This has significant implications for the development of regenerative therapies in both veterinary and human medicine, as it was demonstrated that ET-eMSCs pretreated with PGE₂ can be programmed to secrete a more potent bioactive profile, capable of reversing existing fibrotic changes. Furthermore, using VEs as noninvasive therapeutic tools strengthens the potential for applying this approach safely and precisely. The co-culture system and the integration of advanced proteomic techniques enabled the creation of a robust model for studying equine endometrial fibrosis, providing strong evidence of cellular plasticity and the potential to induce regression of the pathological phenotype through targeted molecular intervention. In practical terms, this technology offers a promising platform for improving reproductive efficiency through personalized treatments that restore endometrial function over the long term. It could also be applied to other species and clinical situations where fibrotic diseases impair tissue.
The objective of this thesis was to evaluate the therapeutic potential of equine endometrial-derived mesenchymal stem cells (ET-eMSCs), preconditioned with prostaglandin E₂ (PGE₂), and their extracellular vesicles (VEs), in reversing the fibrotic phenotype of endometrial myofibroblasts and explants. This condition is associated with endometrosis, a common reproductive disorder in mares. The research aimed to determine whether the interaction between ET-eMSCs and myofibroblasts can influence the secretion of antifibrotic factors and cause functional changes in the cells involved, through three specific objectives: (1) to characterize the antifibrotic properties of ET-eMSCs preconditioned with PGE₂ and their secretome, (2) to assess if VEs derived from these cells can induce changes in the myofibroblast phenotype, and (3) to identify, via quantitative proteomic analysis, the molecular mechanisms underlying such interactions. The results showed that ET-eMSCs preconditioned with PGE₂, their detritus-free conditioned medium, and isolated VEs influence the biological function of myofibroblasts in vitro, decreasing the expression of fibrotic markers such as α-SMA and CTGF, and preventing the buildup of type I collagen. Additionally, quantitative proteomic analysis using LC-MS/MS and SILAC indicated that coculturing ET-eMSCs with myofibroblasts causes a significant change in the secretory profile of both groups. Proteins associated with extracellular matrix remodeling, oxidative stress response, protein folding, cytoskeleton organization, and immune regulation were detected. In particular, the bidirectional interaction activated pathways that promote tissue reorganization and damage resolution, indicating that the therapeutic effect is not limited to a unidirectional action, but that both cell types actively contribute to the antifibrotic environment. This has important implications for the development of regenerative therapies in veterinary and human medicine, as it was shown that ET-eMSCs pretreated with PGE₂ can be programmed to secrete a more effective bioactive profile, capable of reversing pre-existing fibrotic changes. Moreover, the use of VEs as noninvasive therapeutic tools reinforces the possibility of applying this approach in a safe and targeted manner. The co-culture system and the integration of advanced proteomic tools allowed the establishment of a robust model for the study of equine endometrial fibrosis and provided solid evidence on cellular plasticity and the possibility of inducing regression of the pathological phenotype by targeted molecular intervention. Proteins associated with extracellular matrix remodeling, oxidative stress response, protein folding, cytoskeleton organization, and immune regulation were detected. In particular, the bidirectional interaction activated pathways that promote tissue reorganization and damage resolution, indicating that the therapeutic effect is not limited to a unidirectional action, but that both cell types actively contribute to the antifibrotic environment. This has significant implications for the development of regenerative therapies in both veterinary and human medicine, as it was demonstrated that ET-eMSCs pretreated with PGE₂ can be programmed to secrete a more potent bioactive profile, capable of reversing existing fibrotic changes. Furthermore, using VEs as noninvasive therapeutic tools strengthens the potential for applying this approach safely and precisely. The co-culture system and the integration of advanced proteomic techniques enabled the creation of a robust model for studying equine endometrial fibrosis, providing strong evidence of cellular plasticity and the potential to induce regression of the pathological phenotype through targeted molecular intervention. In practical terms, this technology offers a promising platform for improving reproductive efficiency through personalized treatments that restore endometrial function over the long term. It could also be applied to other species and clinical situations where fibrotic diseases impair tissue.
Description
Tesis presentada para optar al grado académico de Doctora en Ciencias Veterinarias
Keywords
Células madre, Animales - Reproducción, Yeguas