Role of mesenchymal fibro/adipogenic progenitors in homeostasis and disease : from signaling to fibrosis

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2019
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Fibrosis, the dysregulated accumulation of connective tissue (CT), is a hallmark of multiple pathologies where inflammation is not fully resolved. The main components of the CT are the extracellular matrix (ECM) and its tissue-resident mesenchymal stromal cells (MSCs), which actively produce and remodel ECM during development, homeostasis, and disease. Fibrosis and/or fibro-fatty replacement of the functional tissue is a common feature of Duchenne Muscular Dystrophy (DMD), but it is also observed in neuromuscular diseases. It is generally accepted that these mesenchymal progenitor cells (MPCs) [hereafter referred to as fibro/adipogenic progenitors (FAPs)] are the progenitors of pathological myofibroblasts and adipocytes during muscular dystrophy and chronic muscle damage, but also important players for proper tissue regeneration. Their dysregulated activity during pathology impairs proper tissue repair. Our overall hypothesis is that efficient regeneration depends on a complex network of signaling interactions between multiple cell types leading to the proper coordination of their function. The best-known marker for FAPs is the receptor tyrosine kinase (RTK) plateletderived growth factor receptor alpha (PDGFRa). Several attempts to block the dysregulated RTK activity of PDGFRa in pathology have been successfully done, therefore, PDGFRa signaling emerges as a potential target against fibrosis. TGF-bmediated signaling pathways are commonly accepted to be fibrotic, to induce the proliferation of FAPs and myofibroblast differentiation from these precursors. TGF-b signaling and its ligands are augmented during DMD, and fibrosis in several tissues. Hence, TGF-b is a known driver of the fibrotic process. Here, we investigated the role of TGF-b signaling on PDGFRa expression and signaling, along with studying a functional cross-talk between PDGFRa and TGF-b in FAPs and MPCs. Canonical Wnt Tcf/Lef transcription factors are important players during development and controlling stem cell fate. Recently, the canonical Wnt signaling cascade emerged as a new system to study fibrosis in several tissues. Importantly, the role of Tcf/lef proteins in MPCs has not been studied yet. Additionally, whether TGF-b has an impact on Wnt signaling and/or may crosstalk through the regulation of these Wnt transcription factors has not been explored yet. In the present Ph.D. thesis, we focused on the TGF-b signaling pathway and its role in FAPs and MPCs. The main findings are; 1. MPCs are present in skeletal muscle under normal conditions and are increased in several models where muscle fibrosis and TGF-b are a hallmark. These Tcf7l2+ progenitors lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. 2. We suggest that the historically named muscle CT fibroblasts isolated via pre-plating correspond to a phenotypically and biochemically FAPs-like population of adherent MPCs. These adherent cells respond in the same fashion to TGF-b signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. 3. The role and/or cross-talk of TGF-b signaling in the regulation of PDGFRa expression and signaling in mesenchymal progenitors is described: i) PDGFRa expression is strongly reduced by TGF-b signaling in FAPs, Hic1+ MPs, skin fibroblasts, and two different MPs cell lines. ii) PDGFRa-mediated gene expression is altered during muscle regeneration in FAPs and after TGF-b1 treatment. iii) Pharmacological inhibition of PDGFRa impaired the TGF-b-mediated ECM remodeling and blocked the TGF-b1-induced migration of MPs. 4. the ubiquitin-proteasome system (UPS). Via Tcf7l2 pharmacological inhibition we suggest that Tcf7l2 plays a role in regulating FAPs survival and fate. Therefore, in this Ph.D. Thesis we studied and proposed that FAPs modulators and signaling governing pathways might be suitable for a novel therapeutic target for the treatment of MD, neurodegenerative diseases, non-malignant fibroproliferative disorders, and aging.Fibrosis, the dysregulated accumulation of connective tissue (CT), is a hallmark of multiple pathologies where inflammation is not fully resolved. The main components of the CT are the extracellular matrix (ECM) and its tissue-resident mesenchymal stromal cells (MSCs), which actively produce and remodel ECM during development, homeostasis, and disease. Fibrosis and/or fibro-fatty replacement of the functional tissue is a common feature of Duchenne Muscular Dystrophy (DMD), but it is also observed in neuromuscular diseases. It is generally accepted that these mesenchymal progenitor cells (MPCs) [hereafter referred to as fibro/adipogenic progenitors (FAPs)] are the progenitors of pathological myofibroblasts and adipocytes during muscular dystrophy and chronic muscle damage, but also important players for proper tissue regeneration. Their dysregulated activity during pathology impairs proper tissue repair. Our overall hypothesis is that efficient regeneration depends on a complex network of signaling interactions between multiple cell types leading to the proper coordination of their function. The best-known marker for FAPs is the receptor tyrosine kinase (RTK) plateletderived growth factor receptor alpha (PDGFRa). Several attempts to block the dysregulated RTK activity of PDGFRa in pathology have been successfully done, therefore, PDGFRa signaling emerges as a potential target against fibrosis. TGF-bmediated signaling pathways are commonly accepted to be fibrotic, to induce the proliferation of FAPs and myofibroblast differentiation from these precursors. TGF-b signaling and its ligands are augmented during DMD, and fibrosis in several tissues. Hence, TGF-b is a known driver of the fibrotic process. Here, we investigated the role of TGF-b signaling on PDGFRa expression and signaling, along with studying a functional cross-talk between PDGFRa and TGF-b in FAPs and MPCs. Canonical Wnt Tcf/Lef transcription factors are important players during development and controlling stem cell fate. Recently, the canonical Wnt signaling cascade emerged as a new system to study fibrosis in several tissues. Importantly, the role of Tcf/lef proteins in MPCs has not been studied yet. Additionally, whether TGF-b has an impact on Wnt signaling and/or may crosstalk through the regulation of these Wnt transcription factors has not been explored yet. In the present Ph.D. thesis, we focused on the TGF-b signaling pathway and its role in FAPs and MPCs. The main findings are; 1. MPCs are present in skeletal muscle under normal conditions and are increased in several models where muscle fibrosis and TGF-b are a hallmark. These Tcf7l2+ progenitors lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. 2. We suggest that the historically named muscle CT fibroblasts isolated via pre-plating correspond to a phenotypically and biochemically FAPs-like population of adherent MPCs. These adherent cells respond in the same fashion to TGF-b signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. 3. The role and/or cross-talk of TGF-b signaling in the regulation of PDGFRa expression and signaling in mesenchymal progenitors is described: i) PDGFRa expression is strongly reduced by TGF-b signaling in FAPs, Hic1+ MPs, skin fibroblasts, and two different MPs cell lines. ii) PDGFRa-mediated gene expression is altered during muscle regeneration in FAPs and after TGF-b1 treatment. iii) Pharmacological inhibition of PDGFRa impaired the TGF-b-mediated ECM remodeling and blocked the TGF-b1-induced migration of MPs. 4. the ubiquitin-proteasome system (UPS). Via Tcf7l2 pharmacological inhibition we suggest that Tcf7l2 plays a role in regulating FAPs survival and fate. Therefore, in this Ph.D. Thesis we studied and proposed that FAPs modulators and signaling governing pathways might be suitable for a novel therapeutic target for the treatment of MD, neurodegenerative diseases, non-malignant fibroproliferative disorders, and aging.Fibrosis, the dysregulated accumulation of connective tissue (CT), is a hallmark of multiple pathologies where inflammation is not fully resolved. The main components of the CT are the extracellular matrix (ECM) and its tissue-resident mesenchymal stromal cells (MSCs), which actively produce and remodel ECM during development, homeostasis, and disease. Fibrosis and/or fibro-fatty replacement of the functional tissue is a common feature of Duchenne Muscular Dystrophy (DMD), but it is also observed in neuromuscular diseases. It is generally accepted that these mesenchymal progenitor cells (MPCs) [hereafter referred to as fibro/adipogenic progenitors (FAPs)] are the progenitors of pathological myofibroblasts and adipocytes during muscular dystrophy and chronic muscle damage, but also important players for proper tissue regeneration. Their dysregulated activity during pathology impairs proper tissue repair. Our overall hypothesis is that efficient regeneration depends on a complex network of signaling interactions between multiple cell types leading to the proper coordination of their function. The best-known marker for FAPs is the receptor tyrosine kinase (RTK) plateletderived growth factor receptor alpha (PDGFRa). Several attempts to block the dysregulated RTK activity of PDGFRa in pathology have been successfully done, therefore, PDGFRa signaling emerges as a potential target against fibrosis. TGF-bmediated signaling pathways are commonly accepted to be fibrotic, to induce the proliferation of FAPs and myofibroblast differentiation from these precursors. TGF-b signaling and its ligands are augmented during DMD, and fibrosis in several tissues. Hence, TGF-b is a known driver of the fibrotic process. Here, we investigated the role of TGF-b signaling on PDGFRa expression and signaling, along with studying a functional cross-talk between PDGFRa and TGF-b in FAPs and MPCs. Canonical Wnt Tcf/Lef transcription factors are important players during development and controlling stem cell fate. Recently, the canonical Wnt signaling cascade emerged as a new system to study fibrosis in several tissues. Importantly, the role of Tcf/lef proteins in MPCs has not been studied yet. Additionally, whether TGF-b has an impact on Wnt signaling and/or may crosstalk through the regulation of these Wnt transcription factors has not been explored yet. In the present Ph.D. thesis, we focused on the TGF-b signaling pathway and its role in FAPs and MPCs. The main findings are; 1. MPCs are present in skeletal muscle under normal conditions and are increased in several models where muscle fibrosis and TGF-b are a hallmark. These Tcf7l2+ progenitors lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. 2. We suggest that the historically named muscle CT fibroblasts isolated via pre-plating correspond to a phenotypically and biochemically FAPs-like population of adherent MPCs. These adherent cells respond in the same fashion to TGF-b signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. 3. The role and/or cross-talk of TGF-b signaling in the regulation of PDGFRa expression and signaling in mesenchymal progenitors is described: i) PDGFRa expression is strongly reduced by TGF-b signaling in FAPs, Hic1+ MPs, skin fibroblasts, and two different MPs cell lines. ii) PDGFRa-mediated gene expression is altered during muscle regeneration in FAPs and after TGF-b1 treatment. iii) Pharmacological inhibition of PDGFRa impaired the TGF-b-mediated ECM remodeling and blocked the TGF-b1-induced migration of MPs. 4. the ubiquitin-proteasome system (UPS). Via Tcf7l2 pharmacological inhibition we suggest that Tcf7l2 plays a role in regulating FAPs survival and fate. Therefore, in this Ph.D. Thesis we studied and proposed that FAPs modulators and signaling governing pathways might be suitable for a novel therapeutic target for the treatment of MD, neurodegenerative diseases, non-malignant fibroproliferative disorders, and aging.Fibrosis, the dysregulated accumulation of connective tissue (CT), is a hallmark of multiple pathologies where inflammation is not fully resolved. The main components of the CT are the extracellular matrix (ECM) and its tissue-resident mesenchymal stromal cells (MSCs), which actively produce and remodel ECM during development, homeostasis, and disease. Fibrosis and/or fibro-fatty replacement of the functional tissue is a common feature of Duchenne Muscular Dystrophy (DMD), but it is also observed in neuromuscular diseases. It is generally accepted that these mesenchymal progenitor cells (MPCs) [hereafter referred to as fibro/adipogenic progenitors (FAPs)] are the progenitors of pathological myofibroblasts and adipocytes during muscular dystrophy and chronic muscle damage, but also important players for proper tissue regeneration. Their dysregulated activity during pathology impairs proper tissue repair. Our overall hypothesis is that efficient regeneration depends on a complex network of signaling interactions between multiple cell types leading to the proper coordination of their function. The best-known marker for FAPs is the receptor tyrosine kinase (RTK) plateletderived growth factor receptor alpha (PDGFRa). Several attempts to block the dysregulated RTK activity of PDGFRa in pathology have been successfully done, therefore, PDGFRa signaling emerges as a potential target against fibrosis. TGF-bmediated signaling pathways are commonly accepted to be fibrotic, to induce the proliferation of FAPs and myofibroblast differentiation from these precursors. TGF-b signaling and its ligands are augmented during DMD, and fibrosis in several tissues. Hence, TGF-b is a known driver of the fibrotic process. Here, we investigated the role of TGF-b signaling on PDGFRa expression and signaling, along with studying a functional cross-talk between PDGFRa and TGF-b in FAPs and MPCs. Canonical Wnt Tcf/Lef transcription factors are important players during development and controlling stem cell fate. Recently, the canonical Wnt signaling cascade emerged as a new system to study fibrosis in several tissues. Importantly, the role of Tcf/lef proteins in MPCs has not been studied yet. Additionally, whether TGF-b has an impact on Wnt signaling and/or may crosstalk through the regulation of these Wnt transcription factors has not been explored yet. In the present Ph.D. thesis, we focused on the TGF-b signaling pathway and its role in FAPs and MPCs. The main findings are; 1. MPCs are present in skeletal muscle under normal conditions and are increased in several models where muscle fibrosis and TGF-b are a hallmark. These Tcf7l2+ progenitors lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. 2. We suggest that the historically named muscle CT fibroblasts isolated via pre-plating correspond to a phenotypically and biochemically FAPs-like population of adherent MPCs. These adherent cells respond in the same fashion to TGF-b signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. 3. The role and/or cross-talk of TGF-b signaling in the regulation of PDGFRa expression and signaling in mesenchymal progenitors is described: i) PDGFRa expression is strongly reduced by TGF-b signaling in FAPs, Hic1+ MPs, skin fibroblasts, and two different MPs cell lines. ii) PDGFRa-mediated gene expression is altered during muscle regeneration in FAPs and after TGF-b1 treatment. iii) Pharmacological inhibition of PDGFRa impaired the TGF-b-mediated ECM remodeling and blocked the TGF-b1-induced migration of MPs. 4. the ubiquitin-proteasome system (UPS). Via Tcf7l2 pharmacological inhibition we suggest that Tcf7l2 plays a role in regulating FAPs survival and fate. Therefore, in this Ph.D. Thesis we studied and proposed that FAPs modulators and signaling governing pathways might be suitable for a novel therapeutic target for the treatment of MD, neurodegenerative diseases, non-malignant fibroproliferative disorders, and aging.Fibrosis, the dysregulated accumulation of connective tissue (CT), is a hallmark of multiple pathologies where inflammation is not fully resolved. The main components of the CT are the extracellular matrix (ECM) and its tissue-resident mesenchymal stromal cells (MSCs), which actively produce and remodel ECM during development, homeostasis, and disease. Fibrosis and/or fibro-fatty replacement of the functional tissue is a common feature of Duchenne Muscular Dystrophy (DMD), but it is also observed in neuromuscular diseases. It is generally accepted that these mesenchymal progenitor cells (MPCs) [hereafter referred to as fibro/adipogenic progenitors (FAPs)] are the progenitors of pathological myofibroblasts and adipocytes during muscular dystrophy and chronic muscle damage, but also important players for proper tissue regeneration. Their dysregulated activity during pathology impairs proper tissue repair. Our overall hypothesis is that efficient regeneration depends on a complex network of signaling interactions between multiple cell types leading to the proper coordination of their function. The best-known marker for FAPs is the receptor tyrosine kinase (RTK) plateletderived growth factor receptor alpha (PDGFRa). Several attempts to block the dysregulated RTK activity of PDGFRa in pathology have been successfully done, therefore, PDGFRa signaling emerges as a potential target against fibrosis. TGF-bmediated signaling pathways are commonly accepted to be fibrotic, to induce the proliferation of FAPs and myofibroblast differentiation from these precursors. TGF-b signaling and its ligands are augmented during DMD, and fibrosis in several tissues. Hence, TGF-b is a known driver of the fibrotic process. Here, we investigated the role of TGF-b signaling on PDGFRa expression and signaling, along with studying a functional cross-talk between PDGFRa and TGF-b in FAPs and MPCs. Canonical Wnt Tcf/Lef transcription factors are important players during development and controlling stem cell fate. Recently, the canonical Wnt signaling cascade emerged as a new system to study fibrosis in several tissues. Importantly, the role of Tcf/lef proteins in MPCs has not been studied yet. Additionally, whether TGF-b has an impact on Wnt signaling and/or may crosstalk through the regulation of these Wnt transcription factors has not been explored yet. In the present Ph.D. thesis, we focused on the TGF-b signaling pathway and its role in FAPs and MPCs. The main findings are; 1. MPCs are present in skeletal muscle under normal conditions and are increased in several models where muscle fibrosis and TGF-b are a hallmark. These Tcf7l2+ progenitors lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. 2. We suggest that the historically named muscle CT fibroblasts isolated via pre-plating correspond to a phenotypically and biochemically FAPs-like population of adherent MPCs. These adherent cells respond in the same fashion to TGF-b signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. 3. The role and/or cross-talk of TGF-b signaling in the regulation of PDGFRa expression and signaling in mesenchymal progenitors is described: i) PDGFRa expression is strongly reduced by TGF-b signaling in FAPs, Hic1+ MPs, skin fibroblasts, and two different MPs cell lines. ii) PDGFRa-mediated gene expression is altered during muscle regeneration in FAPs and after TGF-b1 treatment. iii) Pharmacological inhibition of PDGFRa impaired the TGF-b-mediated ECM remodeling and blocked the TGF-b1-induced migration of MPs. 4. the ubiquitin-proteasome system (UPS). Via Tcf7l2 pharmacological inhibition we suggest that Tcf7l2 plays a role in regulating FAPs survival and fate. Therefore, in this Ph.D. Thesis we studied and proposed that FAPs modulators and signaling governing pathways might be suitable for a novel therapeutic target for the treatment of MD, neurodegenerative diseases, non-malignant fibroproliferative disorders, and aging.
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Tesis (Doctor of Philosophy in Biological Sciences with mention in Cell and Molecular Biology)--Pontificia Universidad CatĆ³lica de Chile, 2019
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