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TGF?-dependent epithelial-to-mesenchymal transition is required to generate cardiospheres from human adult heart biopsies.

ABSTRACT: Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor ?1 (TGF?1) and drastically blocked by the type I TGF?-receptor inhibitor SB431452, indicating that TGF?-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGF? is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.

SUBMITTER: Forte E

PROVIDER: S-EPMC4146498 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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TGFβ-dependent epithelial-to-mesenchymal transition is required to generate cardiospheres from human adult heart biopsies.

Forte Elvira E Miraldi Fabio F Chimenti Isotta I Angelini Francesco F Zeuner Ann A Giacomello Alessandro A Mercola Mark M Messina Elisa E

Stem cells and development 20120807 17

Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in ...[more]

PMID: 22765842

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Project description:Background & aimsChronic failure of mechanisms that promote effective regeneration of dead hepatocytes causes replacement of functional hepatic parenchyma with fibrous scar tissue, ultimately resulting in cirrhosis. Therefore, defining and optimizing mechanisms that orchestrate effective regeneration might prevent cirrhosis. We hypothesized that effective regeneration of injured livers requires hepatocytes to evade the growth-inhibitory actions of TGFβ, since TGFβ signaling inhibits mature hepatocyte growth but drives cirrhosis pathogenesis.MethodsWild-type mice underwent 70% partial hepatectomy (PH); TGFβ expression and signaling were evaluated in intact tissue and primary hepatocytes before, during, and after the period of maximal hepatocyte proliferation that occurs from 24-72 h after PH. To determine the role of Yap1 in regulating TGFβ signaling in hepatocytes, studies were repeated after selectively deleting Yap1 from hepatocytes of Yap1flox/flox mice.ResultsTGFβ expression and hepatocyte nuclear accumulation of pSmad2 and Yap1 increased in parallel with hepatocyte proliferative activity after PH. Proliferative hepatocytes also upregulated Snai1, a pSmad2 target gene that promotes epithelial-to-mesenchymal transition (EMT), suppressed epithelial genes, induced myofibroblast markers, and produced collagen 1α1. Deleting Yap1 from hepatocytes blocked their nuclear accumulation of pSmad2 and EMT-like response, as well as their proliferation.ConclusionInteractions between the TGFβ and Hippo-Yap signaling pathways stimulate hepatocytes to undergo an EMT-like response that is necessary for them to grow in a TGFβ-enriched microenvironment and regenerate injured livers.Lay summaryThe adult liver has an extraordinary ability to regenerate after injury despite the accumulation of scar-forming factors that normally block the proliferation and reduce the survival of residual liver cells. We discovered that liver cells manage to escape these growth-inhibitory influences by transiently becoming more like fibroblasts themselves. They do this by reactivating programs that are known to drive tissue growth during fetal development and in many cancers. Understanding how the liver can control programs that are involved in scarring and cancer may help in the development of new treatments for cirrhosis and liver cancer.

Project description:Phenotype reprogramming during transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) is an extensive and dynamic process, orchestrated by the integration of biological signaling across multiple time scales. As part of the numerous transcriptional changes necessary for EMT, TGFβ-initiated Smad3 signaling results in remodeling of the redox environment and decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated activity through antioxidants, the possibility of a positive feedback loop exists, albeit the time scales on which these mechanisms operate are quite different. We hypothesized that the decreased nucleophilic tone acquired during EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Previous findings supporting such a mechanism were characterized independent of each other; we sought to investigate these relationships within a singular experimental context. In this study, we characterized multivariate representations of phenotype as they evolved over time, specifically measuring expression of epithelial/mesenchymal differentiation, redox regulators, and Smad transcription factors. In-cell Western (ICW) assays were developed to evaluate multivariate phenotype states as they developed during EMT. Principal component analysis (PCA) extracted anticorrelations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected a compensatory up-regulation of Smad2 and Smad4 following cessation of TGFβ signaling. Measuring transcript expression following EMT, we identified down-regulation of numerous antioxidant genes concomitant with up-regulation of NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGFβ treatment increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased glutathione redox potential. Our findings suggest that the decreased nucleophilic tone during EMT coincides with the acquisition of a mesenchymal phenotype over too long a time scale to enable enhanced Smad3 phosphorylation during initiation of EMT. We further challenged the mesenchymal phenotype following EMT through antioxidant and TGFβ inhibitor treatments, which failed to induce a mesenchymal-epithelial transition (MET). Our characterization of multivariate phenotype dynamics during EMT indicates that the decrease in nucleophilic tone occurs alongside EMT; however, maintenance of the mesenchymal phenotype following EMT is independent of both the nascent redox state and the continuous TGFβ signaling.

Dataset Information

TGF?-dependent epithelial-to-mesenchymal transition is required to generate cardiospheres from human adult heart biopsies.

Publications

TGFβ-dependent epithelial-to-mesenchymal transition is required to generate cardiospheres from human adult heart biopsies.

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