RESUMO
The paternally expressed gene 3 (Pw1/Peg3) is a mammalian-specific parentally imprinted gene expressed in stem/progenitor cells of the brain and endocrine tissues. Here, we compared phenotypic characteristics in Pw1/Peg3 deficient male and female mice. Our findings indicate that Pw1/Peg3 is a key player for the determination of sexual dimorphism in metabolism and behavior. Mice carrying a paternally inherited Pw1/Peg3 mutant allele manifested postnatal deficits in GH/IGF dependent growth before weaning, sex steroid dependent masculinization during puberty, and insulin dependent fat accumulation in adulthood. As a result, Pw1/Peg3 deficient mice develop a sex-dependent global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver. Furthermore, Pw1/Peg3 deficient males displayed reduced social dominance and competitiveness concomitant with alterations in the vasopressinergic architecture in the brain. This study demonstrates that Pw1/Peg3 provides an epigenetic context that promotes male-specific characteristics through sex steroid pathways during postnatal development.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Adiposidade , Animais , Tamanho Corporal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Impressão Genômica , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Herança Paterna , Fenótipo , Caracteres SexuaisRESUMO
BACKGROUND: Myocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery by promoting the development of noncontractile fibrotic areas. The mechanisms that lead to cardiac fibrosis are diverse and incompletely characterized. We explored whether the expansion of cardiac fibroblasts after MI can be regulated through a paracrine action of cardiac stromal cells. METHODS: We performed a bioinformatic secretome analysis of cardiac stromal PW1+ cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of candidates were subsequently analyzed in mouse and human hearts and plasmas. The relationship between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-segment-elevation MI and serial cardiac magnetic resonance imaging evaluations. RESULTS: Cardiac stromal PW1+ cells undergo a change in paracrine behavior after MI, and the conditioned media from these cells induced a significant increase in the proliferation of fibroblasts. We identified a total of 12 candidates as secreted proteins overexpressed by cardiac PW1+ cells after MI. Among these factors, GDF3 (growth differentiation factor 3), a member of the TGF-ß (transforming growth factor-ß) family, was markedly upregulated in the ischemic hearts. Conditioned media specifically enriched with GDF3 induced fibroblast proliferation at a high level by stimulation of activin-receptor-like kinases. In line with the secretory nature of this protein, we next found that GDF3 can be detected in mice and human plasma samples, with a significant increase in the days after MI. In humans, higher GDF3 circulating levels (measured in the plasma at day 4 after MI) were significantly associated with an increased risk of adverse remodeling 6 months after MI (adjusted odds ratio, 1.76 [1.03-3.00]; P=0.037), including lower left ventricular ejection fraction and a higher proportion of akinetic segments. CONCLUSIONS: Our findings define a mechanism for the profibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling after MI. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT01113268.
Assuntos
Infarto do Miocárdio , Miocárdio , Animais , Humanos , Camundongos , Cicatriz/patologia , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Fibrose , Fator 3 de Diferenciação de Crescimento/metabolismo , Miocárdio/metabolismo , Volume Sistólico , Fator de Crescimento Transformador beta/metabolismo , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Parental imprinting is a mammalian-specific form of epigenetic regulation in which one allele of a gene is silenced depending on its parental origin. Parentally imprinted genes have been shown to play a role in growth, metabolism, cancer, and behavior. Although the molecular mechanisms underlying parental imprinting have been largely elucidated, the selective advantage of silencing one allele remains unclear. The mutant phenotype of the imprinted gene, Pw1/Peg3, provides a key example to illustrate the hypothesis on a coadaptation between mother and offspring, in which Pw1/Peg3 is required for a set of essential maternal behaviors, such as nursing, nest building, and postnatal care. We have generated a novel Pw1/Peg3 mutant allele that targets the last exon for the PW1 protein that contains >90% of the coding sequence resulting in a loss of Pw1/Peg3 expression. In contrast to previous reports that have targeted upstream exons, we observe that maternal behavior and lactation are not disrupted upon loss of Pw1/Peg3. Both paternal and homozygous Pw1/Peg3 mutant females nurse and feed their pups properly and no differences are detected in either oxytocin neuron number or oxytocin plasma levels. In addition, suckling capacities are normal in mutant pups. Consistent with previous reports, we observe a reduction of postnatal growth. These results support a general role for Pw1/Peg3 in the regulation of body growth but not maternal care and lactation.
Assuntos
Epigênese Genética , Fatores de Transcrição Kruppel-Like/genética , Comportamento Materno , Proteínas Mutantes/genética , Alelos , Animais , Éxons , Feminino , Regulação da Expressão Gênica , Impressão Genômica/genética , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Lactação/genética , Camundongos , Proteínas Mutantes/biossíntese , Neurônios/metabolismoRESUMO
Pw1/Peg3 is a parentally imprinted gene expressed in adult stem cells in every tissue thus far examined including the stem cells of the hair follicle. Using a Pw1/Peg3 reporter mouse, we carried out a detailed dissection of the stem cells in the bulge, which is a major stem cell compartment of the hair follicle in mammalian skin. We observed that PW1/Peg3 expression initiates upon placode formation during fetal development, coincident with the establishment of the bulge stem cells. In the adult, we observed that PW1/Peg3 expression is found in both CD34+ and CD34- populations of bulge stem cells. We demonstrate that both populations can give rise to new hair follicles, reconstitute their niche, and self-renew. These results demonstrate that PW1/Peg3 is a reliable marker of the full population of follicle stem cells and reveal a novel CD34- bulge stem-cell population. Stem Cells 2017;35:1015-1027.
Assuntos
Antígenos CD34/metabolismo , Folículo Piloso/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Autorrenovação Celular , Separação Celular , Expressão Gênica , Genes Reporter , Camundongos Endogâmicos C57BL , Coloração e Rotulagem , Nicho de Células-TroncoRESUMO
Vascular associated endothelial cell (ECs) progenitors are still poorly studied and their role in the newly forming vasculature at embryonic or postnatal stage remains elusive. In the present work, we first defined a set of genes highly expressed during embryo development and strongly downregulated in the adult mouse. In this group, we then concentrated on the progenitor cell marker Peg3/PW1. By in vivo staining of the vasculature we found that only a subset of cells coexpressed endothelial markers and PW1. These cells were quite abundant in the embryo vasculature but declined in number at postnatal and adult stages. Using a reporter mouse for PW1 expression, we have been able to isolate PW1-positive (PW1posECs) and negative endothelial cells (PW1negECs). PW1-positive cells were highly proliferative in comparison to PW1negECs and were able to form colonies when seeded at clonal dilution. Furthermore, by RNAseq analysis, PW1posECs expressed endothelial cell markers together with mesenchymal and stem cell markers. When challenged by endothelial growth factors in vitro, PW1posECs were able to proliferate more than PW1negECs and to efficiently form new vessels in vivo. Taken together these data identify a subset of vessel associated endothelial cells with characteristics of progenitor cells. Considering their high proliferative potential these cells may be of particular importance to design therapies to improve the perfusion of ischemic tissues or to promote vascular repair. Stem Cells 2017;35:1328-1340.
Assuntos
Vasos Sanguíneos/citologia , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Biomarcadores/metabolismo , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proliferação de Células/efeitos dos fármacos , Separação Celular , Desenvolvimento Embrionário/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , FenótipoRESUMO
RATIONALE: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1(+) progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. OBJECTIVE: To determine the role of pulmonary PW1(+) progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. METHODS AND RESULTS: We investigated their contribution during chronic hypoxia-induced vascular remodeling in Pw1(nLacZ+/-) mouse expressing ß-galactosidase in PW1(+) cells and in differentiated cells derived from PW1(+) cells. PW1(+) progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1(+) cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1(+) cells and the proportion of ß-gal(+) vascular SMC were increased, indicating a recruitment of PW1(+) cells and their differentiation into vascular SMC during early chronic hypoxia-induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1(+) cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed ß-gal(+) SMC were not derived from circulating bone marrow-derived PW1(+) progenitor cells, confirming a resident origin of the recruited PW1(+) cells. The number of pulmonary PW1(+) cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. CONCLUSIONS: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension-associated vascular remodeling.
Assuntos
Hipertensão Pulmonar/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , Músculo Liso Vascular/metabolismo , Células-Tronco/metabolismo , Remodelação Vascular/fisiologia , Animais , Células Cultivadas , Humanos , Hipertensão Pulmonar/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/patologia , Ratos , Células-Tronco/patologiaRESUMO
Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.
Assuntos
Antígenos Ly/genética , Epigênese Genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Células Musculares/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Compostos de Vanádio/farmacologia , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Camundongos Nus , Camundongos Transgênicos , Microscopia de Fluorescência , Células Musculares/citologia , Células Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/lesões , Músculo Esquelético/fisiopatologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Regeneração/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS/HYPOTHESIS: Pw1 or paternally-expressed gene 3 (Peg3) encodes a zinc finger transcription factor that is widely expressed during mouse embryonic development and later restricted to multiple somatic stem cell lineages in the adult. The aim of the present study was to define Pw1 expression in the embryonic and adult pancreas and investigate its role in the beta cell cycle in Pw1 wild-type and mutant mice. METHODS: We analysed PW1 expression by immunohistochemistry in pancreas of nonpregant and pregnant mice and following injury by partial duct ligation. Its role in the beta cell cycle was studied in vivo using a novel conditional knockout mouse and in vitro by lentivirus-mediated gene knockdown. RESULTS: We showed that PW1 is expressed in early pancreatic progenitors at E9.5 but becomes progressively restricted to fully differentiated beta cells as they become established after birth and withdraw from the cell cycle. Notably, PW1 expression declines when beta cells are induced to proliferate and loss of PW1 function activates the beta cell cycle. CONCLUSIONS/INTERPRETATION: These results indicate that PW1 is a co-regulator of the beta cell cycle and can thus be considered a novel therapeutic target in diabetes.
Assuntos
Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Pâncreas/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Imuno-Histoquímica , Células Secretoras de Insulina/citologia , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/embriologiaRESUMO
The satellite cell is the major tissue-resident stem cell underlying muscle regeneration; however, multiple non-satellite myogenic progenitors as well as non-myogenic populations that support the muscle regenerative process have been identified. PW1 is expressed in satellite cells as well as in a subset of interstitial cells with myogenic potential termed PICs (PW1+ interstitial cells). Microarray profiling revealed that PICs express a broad range of genes common to mesenchymal stem cells, whereas satellite cells express genes consistent with a committed myogenic progenitor. Isolated PICs from both young and adult muscles can differentiate into smooth and skeletal muscle and fat whereas satellite cells are restricted to a skeletal muscle fate. We demonstrate that the adipogenic potential of PICs corresponds to a subpopulation that expresses platelet derived growth factor receptor alpha (PDGFRα) and overlaps with the recently described interstitial adipogenic progenitors. By contrast, PICs with myogenic potential do not express PDGFRα. Moreover, we observe a discrete and transient population of juvenile PICs based upon SCA1 expression that disappears by 3 weeks of postnatal development coincident with a switch in the cellular and genetic mechanisms underlying postnatal muscle growth.
Assuntos
Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/citologia , Adipogenia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , TranscriptomaRESUMO
Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT-PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Interação Gene-Ambiente , Impressão Genômica , Desnutrição , Animais , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Desnutrição/genética , Desnutrição/metabolismo , Camundongos , Placenta/metabolismo , Placentação , GravidezRESUMO
A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Animais , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Marcadores Genéticos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , GravidezRESUMO
The epicardium is a reservoir of progenitors that give rise to coronary vasculature and stroma during development and mediates cardiac vascular repair. However, its role as a source of progenitors in the adult mammalian heart remains unclear due to lack of clear lineage markers and single-cell culture systems to elucidate epicardial progeny cell fate. We found that in vivo exposure of mice to physiological hypoxia induced adult epicardial cells to re-enter the cell cycle and to express a subset of developmental genes. Multiplex single cell transcriptional profiling revealed a lineage relationship between epicardial cells and smooth muscle, stromal cells, as well as cells with an endothelial-like fate. We found that physiological hypoxia promoted a perinatal-like progenitor state in the adult murine epicardium. In vitro clonal analyses of purified epicardial cells showed that cell growth and subsequent differentiation is dependent upon hypoxia, and that resident epicardial cells retain progenitor identity in the adult mammalian heart with self-renewal and multilineage differentiation potential. These results point to a source of progenitor cells in the adult heart that can be stimulated in vivo and provide an in vitro model for further studies.
Assuntos
Pericárdio , Células-Tronco , Animais , Diferenciação Celular/genética , Proliferação de Células , Hipóxia/metabolismo , Mamíferos , Camundongos , Pericárdio/metabolismoRESUMO
Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1+) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1+ cells expressing PDGFRα. PW1nLacZ reporter mice were used to follow the fate of pulmonary PW1+ progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1+ progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1+ progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1+ progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.
Assuntos
Hipertensão Pulmonar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Animais , Proliferação de Células , Células Cultivadas , Humanos , Hipertensão Pulmonar/metabolismo , Hipóxia , Pulmão , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Remodelação VascularRESUMO
There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVß1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFß at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.
Assuntos
Integrina alfaV/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Venenos de Serpentes/uso terapêutico , Células Estromais/efeitos dos fármacos , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Fibrose , Integrina alfaV/fisiologia , Fatores de Transcrição Kruppel-Like/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , RNA Mensageiro/biossíntese , Análise de Célula Única , Venenos de Serpentes/farmacologia , Células Estromais/química , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Pw1/Peg3 is an imprinted gene expressed from the paternally inherited allele. Several imprinted genes, including Pw1/Peg3, have been shown to regulate overall body size and play a role in adult stem cells. Pw1/Peg3 is expressed in muscle stem cells (satellite cells) as well as a progenitor subset of muscle interstitial cells (PICs) in adult skeletal muscle. We therefore examined the impact of loss-of-function of Pw1/Peg3 during skeletal muscle growth and in muscle stem cell behavior. We found that constitutive loss of Pw1/Peg3 function leads to a reduced muscle mass and myofiber number. In newborn mice, the reduction in fiber number is increased in homozygous mutants as compared to the deletion of only the paternal Pw1/Peg3 allele, indicating that the maternal allele is developmentally functional. Constitutive and a satellite cell-specific deletion of Pw1/Peg3, revealed impaired muscle regeneration and a reduced capacity of satellite cells for self-renewal. RNA sequencing analyses revealed a deregulation of genes that control mitochondrial function. Consistent with these observations, Pw1/Peg3 mutant satellite cells displayed increased mitochondrial activity coupled with accelerated proliferation and differentiation. Our data show that Pw1/Peg3 regulates muscle fiber number determination during fetal development in a gene-dosage manner and regulates satellite cell metabolism in the adult.
Assuntos
Impressão Genômica , Fatores de Transcrição Kruppel-Like/fisiologia , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/fisiologia , Regeneração/genética , Animais , Animais Recém-Nascidos , Autorrenovação Celular/genética , Células Cultivadas , Desenvolvimento Fetal/genética , Dosagem de Genes/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Fibras Musculares Esqueléticas/citologia , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70-80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.
RESUMO
Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Esquelético/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Cistos , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica , Glucosidases/genética , Glucosidases/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hepatopatias , Músculo Esquelético/citologia , Fatores de TranscriçãoRESUMO
Skeletal muscle-derived PW1pos/Pax7neg interstitial cells (PICs) express and secrete a multitude of proregenerative growth factors and cytokines. Utilizing a porcine preclinical skeletal muscle injury model, delivery of allogeneic porcine PICs (pPICs) significantly improved and accelerated myofiber regeneration and neocapillarization, compared with saline vehicle control-treated muscles. Allogeneic pPICs did not contribute to new myofibers or capillaries and were eliminated by the host immune system. In conclusion, allogeneic pPIC transplantation stimulated the endogenous stem cell pool to bring about enhanced autologous skeletal muscle repair and regeneration. This allogeneic cell approach is considered a cost-effective, easy to apply, and readily available regenerative therapeutic strategy.
RESUMO
BACKGROUND: Pw1 gene expression is a marker of adult stem cells in a wide range of tissues. PW1-expressing cells are detected in the heart but are not well characterized. OBJECTIVES: The authors characterized cardiac PW1-expressing cells and their cell fate potentials in normal hearts and during cardiac remodeling following myocardial infarction (MI). METHODS: A human cardiac sample was obtained from a patient presenting with reduced left ventricular (LV) function following a recent MI. The authors used the PW1nLacZ+/- reporter mouse to identify, track, isolate, and characterize PW1-expressing cells in the LV myocardium in normal and ischemic conditions 7 days after complete ligature of the left anterior descending coronary artery. RESULTS: In both human and mouse ischemic hearts, PW1 expression was found in cells that were mainly located in the infarct and border zones. Isolated cardiac resident PW1+ cells form colonies and have the potential to differentiate into multiple cardiac and mesenchymal lineages, with preferential differentiation into fibroblast-like cells but not into cardiomyocytes. Lineage-tracing experiments revealed that PW1+ cells differentiated into fibroblasts post-MI. Although the expression of c-Kit and PW1 showed little overlap in normal hearts, a marked increase in cells coexpressing both markers was observed in ischemic hearts (0.1 ± 0.0% in control vs. 5.7 ± 1.2% in MI; p < 0.001). In contrast to the small proportion of c-Kit+/PW1- cells that showed cardiogenic potential, c-Kit+/PW1+ cells were fibrogenic. CONCLUSIONS: This study demonstrated the existence of a novel population of resident adult cardiac stem cells expressing PW1+ and their involvement in fibrotic remodeling after MI.
Assuntos
Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/genética , Miocárdio/metabolismo , RNA/genética , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/genética , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismoRESUMO
Fibro-adipogenic progenitors (FAPs) are an interstitial cell population in adult skeletal muscle that support muscle regeneration. During development, interstitial muscle connective tissue (MCT) cells support proper muscle patterning, however the underlying molecular mechanisms are not well understood and it remains unclear whether adult FAPs and embryonic MCT cells share a common lineage. We show here that mouse embryonic limb MCT cells expressing the transcription factor Osr1, differentiate into fibrogenic and adipogenic cells in vivo and in vitro defining an embryonic FAP-like population. Genetic lineage tracing shows that developmental Osr1+ cells give rise to a subset of adult FAPs. Loss of Osr1 function leads to a reduction of myogenic progenitor proliferation and survival resulting in limb muscle patterning defects. Transcriptome and functional analyses reveal that Osr1+ cells provide a critical pro-myogenic niche via the production of MCT specific extracellular matrix components and secreted signaling factors.