RESUMO
Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.
Assuntos
Miócitos Cardíacos , Fator 2 Relacionado a NF-E2 , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/farmacologia , RNA-Seq , Diferenciação Celular/genéticaRESUMO
RATIONALE: The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. OBJECTIVE: A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. METHODS AND RESULTS: Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. CONCLUSIONS: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Modelos Cardiovasculares , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Humanos , Proteína de Matriz GlaRESUMO
Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Actinina/metabolismo , Adipócitos/citologia , Animais , Proliferação de Células , Células Cultivadas , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Troponina I/metabolismoRESUMO
RATIONALE: Endothelial cells have the ability to undergo endothelial-mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell-like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood. OBJECTIVE: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs. METHODS AND RESULTS: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein-deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein-deficient mice. CONCLUSIONS: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.
Assuntos
Calcinose , Endotélio Vascular/metabolismo , Mesoderma/metabolismo , Serina Endopeptidases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Ativação Enzimática , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Immunoblotting , Calicreínas/genética , Calicreínas/metabolismo , Mesoderma/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Serina Endopeptidases/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteína de Matriz GlaRESUMO
Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp(-/-)) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp(-/-) mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/deficiência , Guanilato Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Malformações Arteriovenosas Intracranianas/etiologia , Malformações Arteriovenosas Intracranianas/prevenção & controle , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Análise de Variância , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/fisiologia , Células Endoteliais/fisiologia , Efrina-B2/metabolismo , Proteínas da Matriz Extracelular/genética , Deleção de Genes , Immunoblotting , Proteína Jagged-1 , Proteína Jagged-2 , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Família Eph/metabolismo , Proteínas Serrate-Jagged , Microtomografia por Raio-X , Proteína de Matriz GlaRESUMO
Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Diferenciação Celular/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Tecido Adiposo , Animais , Animais Geneticamente Modificados , Proteína Morfogenética Óssea 4/genética , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/patologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/biossíntese , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Ratos , Proteína de Matriz GlaRESUMO
RATIONALE: Vascular calcification is a regulated process that involves osteoprogenitor cells and frequently complicates common vascular disease, such as atherosclerosis and diabetic vasculopathy. However, it is not clear whether the vascular endothelium has a role in contributing osteoprogenitor cells to the calcific lesions. OBJECTIVE: To determine whether the vascular endothelium contributes osteoprogenitor cells to vascular calcification. METHODS AND RESULTS: In this study, we use 2 mouse models of vascular calcification, mice with gene deletion of matrix Gla protein, a bone morphogenetic protein (BMP)-inhibitor, and Ins2Akita/+ mice, a diabetes model. We show that enhanced BMP signaling in both types of mice stimulates the vascular endothelium to contribute osteoprogenitor cells to the vascular calcification. The enhanced BMP signaling results in endothelial-mesenchymal transitions and the emergence of multipotent cells, followed by osteoinduction. Endothelial markers colocalize with multipotent and osteogenic markers in calcified arteries by immunostaining and fluorescence-activated cell sorting. Lineage tracing using Tie2-Gfp transgenic mice supports an endothelial origin of the osteogenic cells. Enhancement of matrix Gla protein expression in Ins2Akita/+ mice, as mediated by an Mgp transgene, limits the generation of multipotent cells. Moreover, matrix Gla protein-depleted human aortic endothelial cells in vitro acquire multipotency rendering the cells susceptible to osteoinduction by BMP and high glucose. CONCLUSIONS: Our data suggest that the endothelium is a source of osteoprogenitor cells in vascular calcification that occurs in disorders with high BMP activation, such as deficiency of BMP-inhibitors and diabetes mellitus.
Assuntos
Calcinose/fisiopatologia , Proteínas de Ligação ao Cálcio/fisiologia , Transdiferenciação Celular/fisiologia , Angiopatias Diabéticas/fisiopatologia , Células Endoteliais/patologia , Endotélio Vascular/fisiopatologia , Proteínas da Matriz Extracelular/fisiologia , Insulina/fisiologia , Células-Tronco Multipotentes/patologia , Doenças Vasculares/fisiopatologia , Animais , Aorta/citologia , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula , Células Cultivadas/efeitos dos fármacos , Diabetes Mellitus Tipo 2/genética , Angiopatias Diabéticas/genética , Modelos Animais de Doenças , Endotélio Vascular/patologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Glucose/farmacologia , Heterozigoto , Humanos , Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/fisiologia , Proteínas Musculares/fisiologia , RNA Interferente Pequeno/farmacologia , Receptor TIE-2/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Proteína de Matriz GlaRESUMO
The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.
Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/metabolismo , Fator 2 de Diferenciação de Crescimento/antagonistas & inibidores , Fator 2 de Diferenciação de Crescimento/metabolismo , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/antagonistas & inibidores , Receptores de Activinas Tipo II/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/efeitos dos fármacos , Especificidade por SubstratoRESUMO
The vasculature, an organ that penetrates every other organ, is ideally poised to be the site where pools of stem cells are placed, to be deployed and committed in response to feedback regulation, and to respond to demands for new vascular structures. These pools of multipotent cells are often under the regulation of various members of the transforming growth factor-ß superfamily, including the bone morphogenetic proteins and their antagonists. Regulation of stem cell populations affects their recruitment, differentiation, spatial organization, and their coordination with host tissue. Loss and dysregulation of feedback control cause a variety of diseases that involve ectopic tissue formation, including atherosclerotic lesion formation and calcification, diabetic vasculopathies, and arteriovenous malformations.
Assuntos
Vasos Sanguíneos/citologia , Neovascularização Fisiológica , Células-Tronco/fisiologia , Animais , Vasos Sanguíneos/fisiologia , Diferenciação Celular , Proliferação de Células , Retroalimentação Fisiológica , Humanos , Regeneração , Células-Tronco/metabolismoRESUMO
RATIONALE: Diabetes mellitus is frequently complicated by cardiovascular disease, such as vascular calcification and endothelial dysfunction, which have been associated with bone morphogenetic proteins (BMPs). OBJECTIVE: To determine whether hyperglycemia in vitro and diabetes in vivo promote vascular BMP activity and correlate with vascular calcification. METHODS AND RESULTS: Increased glucose augmented expression of BMP-2 and BMP-4; the BMP inhibitors matrix Gla protein (MGP) and Noggin; activin-like kinase receptor (ALK)1, -2, -3 and -6; the BMP type 2 receptor; and the vascular endothelial growth factor in human aortic endothelial cells (HAECs). Diabetes induced expression of the same factors in the aortic wall of 3 animal models of diabetes, Ins2(Akita/+) mice, db/db mice, and HIP rats (rats transgenic for human islet amyloid polypeptide), representative of types 1 and 2 diabetes. Conditioned media from glucose-treated HAECs increased angiogenesis in bovine aortic endothelial cells, as mediated by BMP-4, and osteogenesis in calcifying vascular cells, as mediated by BMP-2. BMP-4, MGP, ALK1, and ALK2 were predominantly expressed on the endothelial side of the aorta, and small interfering RNA experiments showed that these genes were regulated as a group. Diabetic mice and rats showed a dramatic increase in aortic BMP activity, as demonstrated by SMAD1/5/8 phosphorylation. This was associated with increased osteogenesis and calcium accumulation. These changes were prevented in the Ins2(Akita/+) mice by breeding them with MGP transgenic mice, which increased aortic BMP inhibition. CONCLUSIONS: Hyperglycemia and diabetes activate vascular BMP activity, which is instrumental in promoting vascular calcification and may be limited by increasing BMP inhibition.
Assuntos
Aorta/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Transdução de Sinais/fisiologia , Receptores de Ativinas/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Proteína Morfogenética Óssea 4/metabolismo , Calcinose/metabolismo , Calcinose/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Proteínas da Matriz Extracelular/metabolismo , Glucose/farmacologia , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Osteogênese/fisiologia , Ratos , Ratos Transgênicos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína de Matriz GlaRESUMO
White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.
Assuntos
Adipócitos Brancos/citologia , Desdiferenciação Celular/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Multipotentes/citologia , Adipócitos Brancos/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/farmacologia , Desdiferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Expressão Gênica , Fatores de Diferenciação de Crescimento/farmacologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/efeitos dos fármacos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Transplante de Células-TroncoRESUMO
Multipotent cells derived from white adipose tissue have been shown to differentiate into multiple lineages including neurogenic lineages. However, the high innervation of brown adipose tissue by the sympathetic nervous system suggest it might be a better source of neural precursor cells. To investigate potential differences between white and brown progenitors, we cultured white and brown dedifferentiated fat (wDFAT and brDFAT) cells from mouse and human adipose tissue and compared marker expression of neural precursors, and neuronal and glial cells, using fluorescence-activated cell sorting, bright-field imaging, immunofluorescence, and RNA analysis by qPCR. The results showed that both wDFAT and brDFAT cells had the capacity to generate neuronal and glial-like cells under neurogenic conditions. However, the brDFAT cells exhibited enhanced propensity for neurogenic differentiation. The neurogenic cells were at least in part derived from Adiponectin-expressing cells. TdTomato-expressing cells derived from Adiponectin (Adipoq) Cre ERT2 -tdTomato flox/flox mice gave rise to individual cells and cell clusters with neurogenic characteristics. Moreover, human brDFAT cells demonstrated a similar ability to undergo neurogenic differentiation after treatment with neurogenic medium, as assessed by immunofluorescence and qPCR. Together, our results support that brDFAT cells have ability to undergo neurogenic differentiation.
Assuntos
Tecido Adiposo Marrom , Células-Tronco Neurais , Tecido Adiposo Branco , Animais , Diferenciação Celular/fisiologia , Camundongos , NeurogêneseRESUMO
Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.
Assuntos
Adipócitos/citologia , Desdiferenciação Celular/fisiologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Adipócitos/metabolismo , Adipócitos/transplante , Animais , Transplante de Células , Células Cultivadas , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica/fisiologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: High-density lipoproteins (HDL) have antiinflammatory effects on the vascular endothelium. Because bone morphogenetic proteins (BMPs) are known to be inflammatory mediators, we examined the effect of HDL on BMP signaling. METHODS AND RESULTS: Increasing concentrations of HDL progressively enhanced expression of the activin-like kinase receptor (ALK)1 and ALK2 in human aortic endothelial cells as determined by real-time polymerase chain reaction and immunoblotting. Induction of ALK1 was a result of enhanced ALK2 expression as determined by siRNA interference, and was associated with increased levels of vascular endothelial growth factor (VEGF) and matrix Gla protein (MGP). The HDL-induction of ALK2 was dependent on BMP-signaling, and affected coregulation of the ALK2 gene by the homeodomain proteins MSX2, DLX3, and DLX5, as determined by reporter gene assays, siRNA interference, and chromatin immunoprecipitation. Apolipoprotein A-I transgenic mice, known to have high HDL and inhibition of atherogenesis, exhibited similar changes in aortic gene expression as seen in endothelial cells treated with HDL in vitro. CONCLUSIONS: We conclude that HDL benefits the arterial wall by allowing for enhanced ALK1 and ALK2 signaling, resulting in an increase of VEGF and MGP, essential for endothelial cell survival and prevention of vascular calcification, respectively.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Animais , Apolipoproteína A-I/genética , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Sequência de Bases , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Bovinos , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína de Matriz GlaRESUMO
The bone morphogenetic proteins (BMPs) belong to the same superfamily as related to transforming growth factor ß (TGFß), growth and differentiation factors (GDFs), and activins. They were initially described as inducers of bone formation but are now known to be involved in morphogenetic activities and cell differentiation throughout the body, including the development of adipose tissue and adipogenic differentiation. BMP4 and BMP7 are the most studied BMPs in adipose tissue, with major roles in white adipogenesis and brown adipogenesis, respectively, but other BMPs such as BMP2, BMP6, and BMP8b as well as some inhibitors and modulators have been shown to also affect adipogenesis. It has become ever more important to understand adipose regulation, including the BMP pathways, in light of the strong links between obesity and metabolic and cardiovascular disease. In this review, we summarize the available information on BMP signaling in adipose tissue using preferentially articles that have appeared in the last decade, which together demonstrate the importance of BMP signaling in adipose biology.
Assuntos
Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
Multipotent cells derived from white mature adipocytes, referred to as dedifferentiated fat (DFAT) cells have the capacity differentiate into endothelial cells. The objective of this study was to modify the isolation method for DFAT cells in order to optimize the endothelial lineage potential. The adipocytes were preincubated for 24 hours, washed, and then incubated for 5 days to allow the generated DFAT cells to remain in proximity to the adipocytes while the cells aggregated into cell clusters. The DFAT cells rapidly differentiated into adipocytes after which endothelial-like cells (ECs) emerged and formed tube-like structure closely associated with the newly differentiated adipocytes. The lipid-filled cells then gradually disappeared whereas the network of tube structure expanded over the course of 3 weeks. ECs accounted for 35-45% of the cells derived from the DFAT cells, as assessed by qPCR, immunofluorescence and fluorescence-activated cell sorting. The DFAT cell-derived ECs could also be further enriched by magnetic sorting, thereby serving as a mouse cell line for further research.
RESUMO
OBJECTIVE: Obesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis. METHODS: We generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the AdiponectinCre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency. RESULTS: Our studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO2, VCO2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice. CONCLUSIONS: BMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas de Transporte/genética , Modelos Animais de Doenças , Ingestão de Alimentos , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Estudos de Associação Genética , Produto da Acumulação Lipídica , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transdução de Sinais/genética , Termogênese/genética , Termogênese/fisiologia , Transcriptoma , Proteína Desacopladora 1/genéticaRESUMO
The vascular endothelium is critical for induction of appropriate lineage differentiation in organogenesis. In this study, we report that dysfunctional pulmonary endothelium, resulting from the loss of matrix Gla protein (MGP), causes ectopic hepatic differentiation in the pulmonary epithelium. We demonstrate uncontrolled induction of the hepatic growth factor (HGF) caused by dysregulated cross talk between pulmonary endothelium and epithelium in Mgp-null lungs. Elevated HGF induced hepatocyte nuclear factor 4 α (Hnf4a), which competed with NK2 homeobox 1 (Nkx2.1) for binding to forkhead box A2 (Foxa2) to drive hepatic differentiation in Mgp-null airway progenitor cells. Limiting endothelial HGF reduced Hnf4a, abolished interference of Hnf4a with Foxa2, and reduced hepatic differentiation in Mgp-null lungs. Together, our results suggest that endothelial-epithelial interactions, maintained by MGP, are essential in pulmonary cell differentiation.
Assuntos
Comunicação Celular , Diferenciação Celular , Endotélio Vascular/metabolismo , Células Epiteliais/metabolismo , Mucosa Respiratória/metabolismo , Animais , Endotélio Vascular/citologia , Células Epiteliais/citologia , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Camundongos , Camundongos Knockout , Proteínas/genética , Proteínas/metabolismo , Mucosa Respiratória/citologiaRESUMO
Mesenchymal stem cells (MSCs) have attracted the attention of researchers and clinicians for their ability to differentiate into a number of cell types, participate in tissue regeneration, and repair the damaged tissues by producing various growth factors and cytokines, as well as their unique immunoprivilege in alloreactive hosts. The immunomodulatory functions of exogenous MSCs have been widely investigated in immune-mediated inflammatory diseases and transplantation research. However, a harsh environment at the site of tissue injury/inflammation with insufficient oxygen supply, abundance of reactive oxygen species, and presence of other harmful molecules that damage the adoptively transferred cells collectively lead to low survival and engraftment of the transferred cells. Preconditioning of MSCs ex vivo by hypoxia, inflammatory stimulus, or other factors/conditions prior to their use in therapy is an adaptive strategy that prepares MSCs to survive in the harsh environment and to enhance their regulatory function of the local immune responses. This review focuses on a number of approaches in preconditioning human MSCs with the goal of augmenting their capacity to regulate both innate and adaptive immune responses.
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BACKGROUND: To study white coat (WC) hypertension in centenarians, a cross-sectional surveillance was carried out on Uygurs, a long-lived population in China. METHODS: Twenty-four-hour ambulatory blood pressure (BP) monitoring (ABPM) was performed in 33 centenarians (age range, 100 to 113 years) and compared with 100 elderly subjects (age range, 65 to 70 years). All subjects were clinically healthy and capable of self-care. Subjects had no history, signs, or symptoms of cardiovascular disease and were receiving no medical treatments. Office BP, 24-h mean, daytime and night-time BP, pulse pressure, heart rate, standard deviation (SD), and coefficient of variation (CV) of the same variables were extracted from ABPM. The WC effect was defined as the difference between mean office and daytime BP. RESULTS: Centenarians demonstrated higher prevalence of WC hypertension, compared to elderly group (15% vs. 5%). The WC effect was also greater in centenarians than in elderly subjects, and was more marked for systolic BP than for diastolic BP and heart rate. The WC effect for systolic BP was positively correlated with both SD (r = 0.45, P < .01) and CV (r = 0.55, P < .01) for 24-h systolic BP in centenarians, but not in elderly subjects. CONCLUSIONS: Prevalence of WC hypertension was greater in centenarians than in elderly subjects. The WC effect and BP variation may be increased in centenarians. Previously observed higher BPs seen in very elderly individuals might be explained by the greater impact of WC hypertension.