Capacidad antifibrótica de las secreciones de las células madre derivadas de tejido adiposo equino en un modelo de endometrosis
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Date
2024
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Universidad de Concepción
Abstract
La endometrosis en yeguas es una enfermedad degenerativa, de gran impacto a nivel productivo. Es una patología multifactorial pero con un común denominador, se presenta en yeguas incapaces de resolver una inflamación post-monta en el endometrio, la cual conlleva a la perpetuación de esta condición y generar un ambiente fibrótico, caracterizado por una excesiva deposición de matriz extracelular, que compromete la arquitectura del tejido y funcionalidad de las glándulas endometriales, lo que a su vez genera un ambiente no favorable para la implantación embrionaria. Diversos tratamientos han sido probados pero con resultados poco favorables, es en este punto donde las terapias regenerativas aparecen como una alternativa. Las células madre presentan una probada actividad regenerativa y además han demostrado también capacidad antifibrótica en otros órganos. Esta acción terapéutica no está basada solo en su interacción directa con el tejido, sino también a partir de una acción paracrina de su secretoma, el cual puede contener diversas moléculas como factores de crecimiento, prostanoides y vesículas extracelulares.
A partir de esto, se plantea la siguiente hipótesis: Las secreciones de las células madre derivadas de tejido adiposo equino presentan actividad antifibrótica en un modelo de endometrosis. Para ello, primero se buscó establecer un modelo in vitro de endometrosis en células endometriales estromales, evaluándose el efecto de citoquinas proinflamatorias (IL1-β, IL-6, TNF-α) y TGF-β en células estromales provenientes de diferentes etapas del estro (Folicular y Luteal media). En la fase folicular, las citoquinas en combinación con TGF-β potencian la transcripción de genes relacionados a la fibrosis (CTGF, COL1A1, COL3A1 y TIMP1/MMP9) a partir un aumento de la fosforilación y posterior translocación a nivel nuclear del factor de transcripción SMAD2, aumento de expresión de miARNs profibróticos (mir-17, mir-21 y mir-433) en las células y en las vesículas extracelulares. Por otro lado, en la fase luteal media, se observó un aumento de la síntesis de PGE2 (prostanoide con actividad antifibrótica) y a su vez un aumento de miARNsantifibróticos (mir-26a, mir-29b, mir-29c, mir-145, mir-378 y mir-488), sin observar cambios en la expresión de los miARNs profibróticos. Una vez establecido este modelo in vitro, se procedió a evaluar la actividad antifibrótica de las secreciones de las células madre derivadas de tejido adiposo equino en el modelo de endometrosis anterior. Para ello, se pre-condicionó con TGF-β en 3 tiempos de exposición, 0, 4 y 24 horas. La exposición a TGF-β cambió las propiedades de las células madre, aumentando significativamente su capacidad de migración y favoreciendo la diferenciación a linaje condrocitario. Sin embargo, se observó una disminución de la diferenciación a linaje osteo y adipogénico con respecto al control (0 horas). Por otro lado, se demostró una notoria contracción de las células en la monocapa, así como la formación de nódulos, evidenciando los cambios a nivel de citoesqueleto y síntesis de matriz extracelular. A nivel molecular, 24 horas de exposición propició un aumento significativo de la expresión de los marcadores génicos de miofibroblastos αSMA, COL1A1 y TGF-β. A las 4 horas se observó en menor grado el aumento de marcadores miofibroblasticos y el alza de la expresión de los genes implicados en la síntesis y función de la PGE2, COX2, PTGES y el receptor de la PGE2, EP4. El aumento de secreción de PGE2 en el medio fue confirmado mediante ELISA. Estos hallazgos permitieron establecer una estrategia potencial del uso de secreciones de las células madre como terapia libre de células en la patología fibrótica endometrial. Con los resultados obtenidos se definió un perfil secretor antifibrótico basado en un preacondicionamiento con TGF-β en las MSC, y se procedió a determinar los posibles blancos asociados a la actividad antifibrótica de las secreciones de dichas células derivadas de tejido adiposo equino en el modelo de endometrosis. Para ello, se analizaron los cambios en la expresión de miARNs relacionados con procesos fibróticos en MSC expuestas a diferentes tiempos de preacondicionamiento, la presencia y expresión de tales miARNs en vesículas extracelulares y los efectos de vesículasextracelulares derivadas de MSC pre-condicionadas, en la reversión de la fibrosis en el modelo in vitro. El perfil de expresión de miARNs tanto en las células como en las vesículas extracelulares fue congruente con lo encontrado en el anterior experimento, observándose una regulación a la alza de miARNs antifibróticos a las 4 horas (mir-29c, mir-145 y mir-200) y en contraposición un aumento de miARNs profibróticos a las 24 horas (mir-192,mir-199 y mir-433). Las vesículas extracelulares fueron añadidas por 48 horas al modelo fibrótico estromal y se evaluaron los marcadores génicos relacionados con la fibrosis, observando una baja significativa de los marcadores miofibroblásticos (αSMA, CTGF, COL1A1) en las células tratadas con las vesículas extracelulares derivadas de 4 horas de preacondicionamiento. Los cambios génicos globales fueron analizados mediante secuenciación de nueva generación. Los resultados evidenciaron 1526 genes sobreexpresados con respecto a las células estromales fibróticas. Mediante análisis de ontología génica se pudo evidenciar una prevalencia mayor de genes involucrados en la respuesta celular a interferones tipo I. Este mecanismo ha sido observado como antagonista a la acción de TGF-β ya que estimula directamente al represor del complejo SMAD2/3, SMAD7. Los resultados obtenidos no solo respaldan la acción antifibrótica de las vesículas derivadas del preacondicionamiento de 4 horas, sino que también identifican una diana molecular que podría ser relevante en el tratamiento de la endometrosis y enfermedades relacionadas.
Endometrosis in mares is a degenerative disease of great impact at the productive level. It is a multifactorial pathology but with a common denominator, it occurs in mares unable to resolve a post-breeding inflammation in the endometrium, which leads to perpetuate this condition and generate a fibrotic environment, characterized by an excessive deposition of extracellular matrix, which compromises the tissue architecture and functionality of the endometrial glands, generating an unfavorable environment for embryo implantation. Several treatments have been tried but with unfavorable results, it is at this point where regenerative therapies appear as an alternative. Stem cells have a proven regenerative activity and have also proven to have antifibrotic activity in other organs. This therapeutic action is not only based on their direct interaction with the tissue but also on the paracrine action of their secretome, which may contain various molecules such as growth factors, prostanoids and extracellular vesicles. From this, the following hypothesis is proposed: The secretions of stem cells derived from equine adipose tissue present anti-fibrotic activity in a model of endometrosis. For this purpose, we first sought to establish an in vitro model of endometrosis in endometrial stromal cells, evaluating the effect of proinflammatory cytokines (IL1-β, IL-6, TNF-α) and TGF-β in stromal cells from different stages of estrus (follicular and mid-luteal). It was obtained that in the follicular phase, cytokines in combination with TGF-β potentiate the transcription of genes related to fibrosis (CTGF, COL1A1, COL3A1 and TIMP1/MMP9) from increased phosphorylation and subsequent nuclear translocation of the transcription factor SMAD2, increased expression of profibrotic miRNA (mir-17, mir-21 and mir-433) in the cells and in the extracellular vesicles. On the other hand, in the mid-luteal phase, we observed an increase in the synthesis of PGE2 (prostanoid with antifibrotic activity) and in turn an increase in antifibrotic miRNAs (miR-26a, miR-29b, miR-29c, miR-145, miR-378 and mir-488) was observed, without observing changes in the expression of profibrotic miRNAs. Once this in vitro model was established, we proceeded to evaluate the antifibrotic activity of equine adipose tissue-derived stem cell secretions in the previous endometrosis model. For this purpose, they were preconditioned with TGF-β at 3 exposure times, 0, 4 and 24 hours. Exposure to TGF-β changed the properties of the stem cells, significantly increasing their migration capacity and favoring differentiation to chondrocyte lineage. However, a decrease in differentiation to osteo- and adipogenic lineage was observed concerning the control (0 hours). On the other hand, a notorious contraction of the cells in the monolayer was demonstrated, as well as the formation of nodules, evidencing changes at the cytoskeleton and extracellular matrix synthesis levels. At the molecular level, 24 hours of exposure led to a significant increase in the expression of myofibroblast gene markers αSMA, COL1A1 and TGF-β. At 4 hours, the increase in myofibroblast markers and the upregulation of the expression of genes involved in the synthesis and function of PGE2, COX2, PTGES and the PGE2 receptor, EP4, were observed to a lesser degree. The increased secretion of PGE2 in the medium was confirmed by ELISA. These findings allowed establishing a potential strategy of using stem cell secretions as a cell-free therapy in endometrial fibrotic pathology. With the results obtained, an antifibrotic secretory profile was defined based on preconditioning with TGF-β in MSCs, and we proceeded to determine the possible targets associated with the antifibrotic activity of the secretions of these cells derived from equine adipose tissue in the endometrial endometrosis model. To this end, we analyzed the changes in the expression of miRNAs related to fibrotic processes in MSCs exposed to different pre-conditioning times, the presence and expression of such miRNAs in extracellular vesicles, and the effects of extracellular vesicles derived from pre-conditioned MSCs on the reversal of fibrosis in the in vitro model. The expression profile of miRNAs in both cells and extracellular vesicles was congruent with that found in the previous experiment, observing an up-regulation of antifibrotic miRNAs at 4 hours (mir-29c, mir-145 and mir-200) and in contrast an increase of profibrotic miRNAs at 24 hours (mir-192, mir-199 and mir-433). Extracellular vesicles were added for 48 hours to the stromal fibrotic model and fibrosis-related gene markers were assessed, observing a significant downregulation of myofibroblastic markers (αSMA, CTGF, COL1A1) in cells treated with extracellular vesicles derived from 4 hours of preconditioning. Global gene changes were analyzed by next-generation sequencing. The results evidenced 1526 overexpressed genes concerning fibrotic stromal cells. Gene ontology analysis revealed a higher prevalence of genes involved in the cellular response to type I interferons. This mechanism has been observed as an antagonist to the action of TGF-β, since it stimulates the direction of the cellular response to type I interferons. This mechanism has been observed as an antagonist to the action of TGF-β as it directly stimulates the repressor of the SMAD2/3 complex, SMAD7. The results obtained not only support the antifibrotic action of vesicles derived from 4-hour preconditioning, but also identify a molecular target that could be relevant in the treatment of endometrosis and related diseases.
Endometrosis in mares is a degenerative disease of great impact at the productive level. It is a multifactorial pathology but with a common denominator, it occurs in mares unable to resolve a post-breeding inflammation in the endometrium, which leads to perpetuate this condition and generate a fibrotic environment, characterized by an excessive deposition of extracellular matrix, which compromises the tissue architecture and functionality of the endometrial glands, generating an unfavorable environment for embryo implantation. Several treatments have been tried but with unfavorable results, it is at this point where regenerative therapies appear as an alternative. Stem cells have a proven regenerative activity and have also proven to have antifibrotic activity in other organs. This therapeutic action is not only based on their direct interaction with the tissue but also on the paracrine action of their secretome, which may contain various molecules such as growth factors, prostanoids and extracellular vesicles. From this, the following hypothesis is proposed: The secretions of stem cells derived from equine adipose tissue present anti-fibrotic activity in a model of endometrosis. For this purpose, we first sought to establish an in vitro model of endometrosis in endometrial stromal cells, evaluating the effect of proinflammatory cytokines (IL1-β, IL-6, TNF-α) and TGF-β in stromal cells from different stages of estrus (follicular and mid-luteal). It was obtained that in the follicular phase, cytokines in combination with TGF-β potentiate the transcription of genes related to fibrosis (CTGF, COL1A1, COL3A1 and TIMP1/MMP9) from increased phosphorylation and subsequent nuclear translocation of the transcription factor SMAD2, increased expression of profibrotic miRNA (mir-17, mir-21 and mir-433) in the cells and in the extracellular vesicles. On the other hand, in the mid-luteal phase, we observed an increase in the synthesis of PGE2 (prostanoid with antifibrotic activity) and in turn an increase in antifibrotic miRNAs (miR-26a, miR-29b, miR-29c, miR-145, miR-378 and mir-488) was observed, without observing changes in the expression of profibrotic miRNAs. Once this in vitro model was established, we proceeded to evaluate the antifibrotic activity of equine adipose tissue-derived stem cell secretions in the previous endometrosis model. For this purpose, they were preconditioned with TGF-β at 3 exposure times, 0, 4 and 24 hours. Exposure to TGF-β changed the properties of the stem cells, significantly increasing their migration capacity and favoring differentiation to chondrocyte lineage. However, a decrease in differentiation to osteo- and adipogenic lineage was observed concerning the control (0 hours). On the other hand, a notorious contraction of the cells in the monolayer was demonstrated, as well as the formation of nodules, evidencing changes at the cytoskeleton and extracellular matrix synthesis levels. At the molecular level, 24 hours of exposure led to a significant increase in the expression of myofibroblast gene markers αSMA, COL1A1 and TGF-β. At 4 hours, the increase in myofibroblast markers and the upregulation of the expression of genes involved in the synthesis and function of PGE2, COX2, PTGES and the PGE2 receptor, EP4, were observed to a lesser degree. The increased secretion of PGE2 in the medium was confirmed by ELISA. These findings allowed establishing a potential strategy of using stem cell secretions as a cell-free therapy in endometrial fibrotic pathology. With the results obtained, an antifibrotic secretory profile was defined based on preconditioning with TGF-β in MSCs, and we proceeded to determine the possible targets associated with the antifibrotic activity of the secretions of these cells derived from equine adipose tissue in the endometrial endometrosis model. To this end, we analyzed the changes in the expression of miRNAs related to fibrotic processes in MSCs exposed to different pre-conditioning times, the presence and expression of such miRNAs in extracellular vesicles, and the effects of extracellular vesicles derived from pre-conditioned MSCs on the reversal of fibrosis in the in vitro model. The expression profile of miRNAs in both cells and extracellular vesicles was congruent with that found in the previous experiment, observing an up-regulation of antifibrotic miRNAs at 4 hours (mir-29c, mir-145 and mir-200) and in contrast an increase of profibrotic miRNAs at 24 hours (mir-192, mir-199 and mir-433). Extracellular vesicles were added for 48 hours to the stromal fibrotic model and fibrosis-related gene markers were assessed, observing a significant downregulation of myofibroblastic markers (αSMA, CTGF, COL1A1) in cells treated with extracellular vesicles derived from 4 hours of preconditioning. Global gene changes were analyzed by next-generation sequencing. The results evidenced 1526 overexpressed genes concerning fibrotic stromal cells. Gene ontology analysis revealed a higher prevalence of genes involved in the cellular response to type I interferons. This mechanism has been observed as an antagonist to the action of TGF-β, since it stimulates the direction of the cellular response to type I interferons. This mechanism has been observed as an antagonist to the action of TGF-β as it directly stimulates the repressor of the SMAD2/3 complex, SMAD7. The results obtained not only support the antifibrotic action of vesicles derived from 4-hour preconditioning, but also identify a molecular target that could be relevant in the treatment of endometrosis and related diseases.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Veterinarias
Keywords
Yeguas - Chile, Endometriosis, Marcadores genéticos