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1.
Biochem Biophys Res Commun ; 432(1): 105-10, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23376071

RESUMO

Much recent work has highlighted the key role of adipose tissue as an endocrine organ that secretes a number of adipocytokines, linking adiposity, especially intra-abdominal visceral fat, and the pathogenesis of cardiovascular and metabolic diseases. However, the role of epicardial adipose tissue (EAT), another important visceral fat depot situated in close proximity to epicardial coronary arteries and myocardium, has been less well studied. In this study, we sought to characterize EAT by comparing gene expression profiles of EAT, omental adipose tissue (OAT), and subcutaneous adipose tissue (SCAT) in patients who underwent elective coronary artery bypass graft surgery for critical coronary artery disease (CAD) and identify molecules involved in inflammation. A total of 15,304 probes were detected in all depots, and 231 probes were differentially expressed. Significantly higher expression of pro-inflammatory genes such as interleukin-1ß, -6, and -8, and chemokine receptor 2 was observed in EAT, even when compared with OAT. Among them, serglycin was one of the most abundantly expressed genes in EAT. Serglycin expression was induced during adipocytic differentiation of 3T3L1 cells. Serglycin was secreted from adipocytes, and tumor necrosis factor-α stimulated its expression and secretion in adipocytes. Serglycin was also present in human serum samples. These results suggest that human EAT has strong inflammatory properties in patients with CAD and provide novel evidence that serglycin is an adipocytokine highly expressed in EAT.


Assuntos
Adipocinas/biossíntese , Tecido Adiposo/metabolismo , Doença da Artéria Coronariana/metabolismo , Pericárdio/metabolismo , Proteoglicanas/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Adipocinas/genética , Animais , Doença da Artéria Coronariana/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Proteoglicanas/genética , Transcriptoma , Fator de Necrose Tumoral alfa/farmacologia , Proteínas de Transporte Vesicular/genética
2.
Biochem Biophys Res Commun ; 428(4): 500-5, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23123625

RESUMO

PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.


Assuntos
Proteína de Ligação a Androgênios/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Coração/embriologia , Desenvolvimento Muscular , Mioblastos Cardíacos/citologia , Miócitos Cardíacos/citologia , Proteínas Smad/metabolismo , Proteína de Ligação a Androgênios/genética , Animais , Linhagem Celular Tumoral , Camundongos , Mioblastos Cardíacos/metabolismo , Transdução de Sinais
3.
Biochem Biophys Res Commun ; 426(3): 317-23, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22935419

RESUMO

While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2'Z, 3'E]-6-bromoindirubin-3'-oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Coração/embriologia , Miócitos Cardíacos/citologia , Organogênese , Fatores de Transcrição/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Citocinas , Regulação para Baixo , Células-Tronco Embrionárias/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
4.
Am J Physiol Heart Circ Physiol ; 300(1): H154-61, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20952669

RESUMO

Members of the fibroblast growth factor (FGF) family have been clinically applied to the treatment of ischemic diseases because of their strong angiogenic actions. Although tissue ischemia is predominantly caused by atherosclerosis, the roles of endothelial FGF receptors (FGF-Rs) in atherosclerosis remain obscure. We generated endothelial cell (EC)-targeted constitutively active FGF-R2-overexpressing mice, using the Tie2 promoter (Tie2-FGF-R2-Tg), and crossed them with apolipoprotein E (ApoE)-deficient mice (ApoE-KO) to generate Tie2-FGF-R2-Tg/ApoE-deficient mice (Tie2-FGF-R2-Tg/ApoE-KO). After being fed a Western diet for 8 wk, the Tie2-FGF-R2-Tg/ApoE-KO demonstrated 2.0-fold greater atherosclerotic lesion area on the luminal surfaces of the aortas than the ApoE-KO (P < 0.01). The level of p21(Cip1) protein, a cell cycle inhibitor, in the FGF-R2-overexpressing EC was 2.5-fold greater than that in the wild-type (WT) EC at the baseline (P < 0.01). FGF-R2 overexpression in the EC resulted in increased expression of VCAM-1 and ICAM-1, acceleration of apoptosis, and decreased proliferative activity, all of which were normalized by small interfering RNA (siRNA)-mediated knockdown of p21(Cip1) (75% reduction in protein level, P < 0.01). Furthermore, the expression of PDGF-B and Egr-1, a PDGF/p21(Cip1)-inducible transcription factor, in the aortic endothelium of Tie2-FGF-R2-Tg/ApoE-KO was significantly greater than that in ApoE-KO. The proliferation of vascular smooth muscle cells in the aortic media of Tie2-FGF-R2-Tg/ApoE-KO was 2.0-fold higher than that in ApoE-KO (P < 0.01). Thus our study reveals that endothelial FGF-R2 signaling aggravates atherosclerosis by promoting p21(Cip1)-mediated EC dysfunction and cautions against the use of FGF for therapeutic angiogenesis in the setting of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Apoptose , Aterosclerose/fisiopatologia , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dieta , Endotélio Vascular/fisiopatologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Arterioscler Thromb Vasc Biol ; 30(10): 1908-15, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20651281

RESUMO

OBJECTIVE: Vascular calcification is an important risk factor for cardiovascular diseases. Here, we investigated a role of dedifferentiated vascular smooth muscle cells (VSMCs) in the atherosclerotic intimal calcification. METHODS AND RESULTS: We prepared human cultured VSMCs in either redifferentiatiated or dedifferentiated state and analyzed the gene expressions of bone-calcification regulatory factors. Expression of bone morphogenetic protein-2 (BMP-2), a potent initiator for osteoblast differentiation, was significantly enhanced in dedifferentiated VSMCs. Furthermore, endogenous BMP-2 antagonists, such as noggin, chordin, and matrix gamma-carboxyglutamic acid protein, were all downregulated in the dedifferentiated VSMCs. Conditioned medium from dedifferentiated VSMCs, but not from redifferentiated VSMCs, stimulated the osteoblastic differentiation of the mesenchymal progenitor C2C12 cells, which was abolished by BMP-2 knockdown. In atherosclerotic intima from apolipoprotein (apo)E-deficient mice, αSM-actin-positive cells, presumably dedifferentiated VSMCs, expressed BMP-2. We generated BMP-2-transgenic mice using αSM-actin promoter and crossed them with apoE-deficient mice (BMP-2-transgenic/apoE-knockout). Significantly accelerated atherosclerotic intimal calcification was detected in BMP-2-transgenic/apoE-knockout mice, although serum lipid concentration and atherosclerotic plaque size were not different from those in apoE-knockout mice. Enhanced calcification appeared to be associated with the frequent emergence of osteoblast-like cells in atherosclerotic intima in BMP-2-transgenic/apoE-knockout mice. CONCLUSIONS: Our findings collectively demonstrate an important role of dedifferentiated VSMCs in the pathophysiology of atherosclerotic calcification through activating paracrine BMP-2 osteogenic signals.


Assuntos
Aterosclerose/etiologia , Proteína Morfogenética Óssea 2/fisiologia , Calcinose/etiologia , Osteogênese/fisiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Proteína Morfogenética Óssea 2/genética , Calcinose/genética , Calcinose/patologia , Calcinose/fisiopatologia , Desdiferenciação Celular , Diferenciação Celular , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Osteoblastos/patologia , Osteoblastos/fisiologia , Osteogênese/genética , Comunicação Parácrina , Túnica Íntima/patologia , Túnica Íntima/fisiopatologia
6.
Arterioscler Thromb Vasc Biol ; 30(1): 60-7, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19834109

RESUMO

OBJECTIVE: Bone marrow (BM)-derived endothelial progenitor cells (EPCs) and vascular smooth muscle progenitor cells (VPCs) contribute to neointima formation, whereas the angiotensin II (Ang II) type 1 receptor (AT(1))-mediated action on BM-derived progenitors remains undefined. METHODS AND RESULTS: A wire-induced vascular injury was performed in the femoral artery of BM-chimeric mice whose BM was repopulated with AT(1)-deficient (BM-Agtr1(-/-)) or wild-type (BM-Agtr1(+/+)) cells. Neointima formation was profoundly reduced by 38% in BM-Agtr1(-/-) mice. Although the number of circulating EPCs (Sca-1(+)Flk-1(+)) and extent of reendothelialization did not differ between the 2 groups, the numbers of both circulating VPCs (c-Kit(-)Sca-1(+)Lin(-)) and tissue VPCs (Sca-1(+)CD31(-)) incorporated into neointima were markedly decreased in BM-Agtr1(-/-) mice. The accumulation of aggregated platelets and their content of stromal cell-derived factor-1alpha (SDF-1alpha) were significantly reduced in BM-Agtr1(-/-) mice, accompanied by a decrease in the serum level of SDF-1alpha. Thrombin-induced platelets aggregation was dose-dependently inhibited (45% at 0.1 IU/mL, P<0.05) in Agtr1(-/-) platelets compared with Agtr1(+/+) platelets, accompanied by the reduced expression and release of SDF-1alpha. CONCLUSIONS: The BM-AT(1) receptor promotes neointima formation by regulating the mobilization and homing of BM-derived VPCs in a platelet-derived SDF-1alpha-dependent manner without affecting EPC-mediated reendothelialization.


Assuntos
Medula Óssea/fisiologia , Quimiocina CXCL12/metabolismo , Células-Tronco Hematopoéticas/citologia , Músculo Liso Vascular/citologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Anticorpos/farmacologia , Plaquetas/metabolismo , Linhagem da Célula/fisiologia , Quimiocina CXCL12/imunologia , Quimiocina CXCL12/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Liso Vascular/metabolismo , Agregação Plaquetária/fisiologia , Receptor Tipo 1 de Angiotensina/genética , Túnica Íntima/citologia , Túnica Íntima/metabolismo
7.
Biochem Biophys Res Commun ; 390(4): 1202-7, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19879243

RESUMO

Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin-angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion. MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.


Assuntos
Endotélio Vascular/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Neovascularização Patológica/enzimologia , Receptores de Superfície Celular/fisiologia , Renina/fisiologia , Animais , Movimento Celular , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/biossíntese , Renina/genética , Sistema Renina-Angiotensina/fisiologia , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor de Pró-Renina
8.
Am J Physiol Heart Circ Physiol ; 297(5): H1673-84, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19749165

RESUMO

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klotho(-/-) aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting P(i)-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.


Assuntos
Calcinose/patologia , Proliferação de Células , Transdiferenciação Celular , Senescência Celular , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteoblastos/patologia , Fatores Etários , Envelhecimento/metabolismo , Envelhecimento/patologia , Fosfatase Alcalina/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose , Calcinose/genética , Calcinose/metabolismo , Calcinose/prevenção & controle , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Glucuronidase/deficiência , Glucuronidase/genética , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteínas Klotho , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Túnica Média/metabolismo , Túnica Média/patologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína de Matriz Gla
9.
Arterioscler Thromb Vasc Biol ; 29(10): 1529-36, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19628784

RESUMO

BACKGROUND: The angiotensin II (Ang II) type 1 (AT(1)) receptor is expressed in bone marrow (BM) cells, whereas it remains poorly defined how Ang II regulates differentiation/proliferation of monocyte-lineage cells to exert proatherogenic actions. METHODS AND RESULTS: We generated BM chimeric apoE(-/-) mice repopulated with AT(1)-deficient (Agtr1(-/-)) or wild-type (Agtr1(+/+)) BM cells. The atherosclerotic development was significantly reduced in apoE(-/-)/BM-Agtr1(-/-) mice compared with apoE(-/-)/BM-Agtr1(+/+) mice, accompanied by decreased numbers of BM granulocyte/macrophage progenitors (GMP:c-Kit(+)Sca-1(-)Lin(-)CD34(+)CD16/32(+)) and peripheral blood monocytes. Macrophage-colony-stimulating factor (M-CSF)-induced differentiation from hematopoietic stem cells (HSCs:c-Kit(+)Sca-1(+)Lin(-)) to promonocytes (CD11b(high)Ly-6G(low)) was markedly reduced in HSCs from Agtr1(-/-) mice. The expression of M-CSF receptor c-Fms was decreased in HSCs/promonocytes from Agtr1(-/-) mice, accompanied by a marked inhibition in M-CSF-induced phosphorylation of PKC-delta and JAK2. c-Fms expression in HSCs/promonocytes was mainly regulated by TNF-alpha derived from BM CD45(-)CD34(-) stromal cells, and Ang II specifically regulated the TNF-alpha synthesis and release from BM stromal cells. CONCLUSIONS: Ang II regulates the expression of c-Fms in HSCs and monocyte-lineage cells through BM stromal cell-derived TNF-alpha to promote M-CSF-induced differentiation/proliferation of monocyte-lineage cells and contributes to the proatherogenic action.


Assuntos
Células-Tronco Hematopoéticas/citologia , Monócitos/citologia , Receptor Tipo 1 de Angiotensina/fisiologia , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Aterosclerose/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Janus Quinase 2/metabolismo , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Quinase C-delta/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/análise , Receptores de LDL/fisiologia , Fator de Necrose Tumoral alfa/fisiologia
10.
Heart Vessels ; 24(1): 54-62, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19165570

RESUMO

Intracellular Na(+) ([Na(+)](i)) regulation plays a crucial role in the structural, mechanical, and electrical properties of myocardium. It is assumed that the [Na(+)](i) handling system may differ not only between normal and diseased hearts but also regionally within a heart. To gain new insight concerning disease- and region-dependent differences in the [Na(+)](i)-regulatory system, we investigated mRNA expression of Na+ transporters, the principal determinants of [Na(+)](i). Nonischemic pressure-overloaded hypertrophy was created by suprarenal abdominal aortic constriction of 50% for 7 weeks. mRNA abundances of Na(+)-Ca(2+) exchanger (NCX1), Na(+)-H(+) exchanger (NHE1), Na(+)-K(+)-2Cl(-) exchanger (NKCC1) and Na(+), K(+)-ATPase multigene family(alpha(1), alpha(2), alpha(3), and beta(1) isoforms) were measured by the real-time quantitative polymerase chain reaction method. mRNA abundance of all transporters mediating Na(+) influx (NCX1, NHE1, and NKCC1) was significantly upregulated as compared to normal. In contrast, Na(+)-efflux-mediating transporter (Na(+), K(+)-ATPase) mRNA expression was unaltered between normal and hypertrophic hearts. Losartan, an angiotensin II AT1 receptor antagonist, significantly attenuated upregulation of Na(+)-influx-mediating transporters induced by aortic constriction. The onset of Na(+)-influx-mediating transporter upregulation occurred within 5 days following constriction. In normal and hypertrophied hearts, mRNA of all Na(+)-influx-mediating transporters was expressed in order of abundance as: apex > septum approximately free wall of left ventricles. A transmural gradient in expression was also evident in normal hearts (midcardium > endo- and epicardium), which was attenuated under hypertrophic development. Myocardial hypertrophy is associated with significant changes in the spatial distribution and expression levels of Na(+) transporters. The upregulation of Na influx transporters during hypertrophy may contribute to the remodeling process, modulate contractility and promote arrhythmias.


Assuntos
Cardiomiopatia Hipertrófica/genética , Regulação da Expressão Gênica , Miocárdio/metabolismo , RNA Mensageiro/genética , ATPase Trocadora de Sódio-Potássio/genética , Animais , Cardiomiopatia Hipertrófica/metabolismo , Modelos Animais de Doenças , Predisposição Genética para Doença , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/biossíntese
11.
J Am Coll Cardiol ; 52(23): 1858-1865, 2008 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19038683

RESUMO

OBJECTIVES: This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction. BACKGROUND: Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. METHODS: We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion. RESULTS: Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size. CONCLUSIONS: Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.


Assuntos
Transplante de Células/métodos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Coração/fisiologia , Infarto do Miocárdio/terapia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Humanos , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Reperfusão Miocárdica , Placebos , Distribuição Aleatória , Reperfusão , Suínos
12.
Am J Physiol Heart Circ Physiol ; 295(6): H2512-21, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18978195

RESUMO

Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by Nomega-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression.


Assuntos
Apoptose , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Células Endoteliais/enzimologia , Soro/metabolismo , Caspase 3/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , RNA Helicases DEAD-box/genética , Regulação para Baixo , Endorribonucleases/genética , Células Endoteliais/patologia , Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , MicroRNAs/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
J Biol Chem ; 283(39): 26705-13, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18662983

RESUMO

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.


Assuntos
Antígenos de Diferenciação/biossíntese , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Musculares/biossíntese , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Animais , Antígenos de Diferenciação/genética , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Endoderma/citologia , Endoderma/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Proteínas Musculares/genética , Miócitos Cardíacos/citologia , Organogênese/fisiologia , Fator de Crescimento Transformador beta/genética
14.
Am J Physiol Cell Physiol ; 295(2): C490-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18508909

RESUMO

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.


Assuntos
Desenvolvimento Muscular/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Mioblastos/metabolismo , Animais , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular , Creatina Quinase Forma MM/genética , Citoplasma/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Proteína MyoD/genética , Mioblastos/citologia , Miogenina/genética , Miosinas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Regeneração/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacos
15.
Circulation ; 116(9): 1041-51, 2007 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-17698736

RESUMO

BACKGROUND: The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. METHODS AND RESULTS: Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2-/- mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)-mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor-dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor-induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor-mediated Src association with PLCgamma1 and phosphorylation of 783Tyr-PLCgamma1 also were abolished by PYK2 deficiency. CONCLUSION: These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLCgamma1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase-mediated vasoactive function and angiogenic response.


Assuntos
Quinase 2 de Adesão Focal/fisiologia , Coração/fisiologia , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína Oncogênica v-akt/fisiologia , Análise de Variância , Animais , Cálcio/fisiologia , Ativação Enzimática , Quinase 2 de Adesão Focal/deficiência , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Camundongos , Camundongos Knockout , Fosforilação , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/fisiologia , Vasodilatação
16.
Biochem Biophys Res Commun ; 359(2): 341-7, 2007 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-17537408

RESUMO

We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.


Assuntos
Regulação da Expressão Gênica , Neovascularização Fisiológica , Osteopontina/biossíntese , Osteopontina/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Fibronectinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , Osteopontina/metabolismo , RNA/metabolismo
17.
J Cell Sci ; 120(Pt 10): 1791-800, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17502484

RESUMO

Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERT(GFP)-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation.


Assuntos
Antígenos Ly/metabolismo , Células Clonais/metabolismo , Proteínas de Membrana/metabolismo , Mioblastos Cardíacos/metabolismo , Miócitos Cardíacos/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Animais , Antígenos Ly/genética , Apoptose/genética , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Clonais/citologia , Regulação para Baixo/genética , Sobrevivência de Enxerto/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mioblastos Cardíacos/citologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Comunicação Parácrina/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Telomerase/genética , Telomerase/metabolismo
18.
Am J Physiol Heart Circ Physiol ; 293(2): H1254-64, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17416604

RESUMO

The Na(+)-HCO(3)(-) cotransporter (NBC) plays a key role in intracellular pH (pH(i)) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH(i) was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J(NBC)) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na(+)/H(+) exchange was also increased in hypertrophied myocytes, the relative enhancement of J(NBC) was larger, 6) membrane depolarization markedly increased J(NBC) in hypertrophied myocytes, and 7) losartan, an ANG II AT(1) receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na(+) overload, which may render the ventricles more vulnerable to Ca(2+) overload during ischemia-reperfusion.


Assuntos
Hipertensão/complicações , Hipertrofia Ventricular Esquerda/metabolismo , Miócitos Cardíacos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Transcrição Gênica , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Aorta Abdominal/cirurgia , Modelos Animais de Doenças , Ventrículos do Coração/metabolismo , Concentração de Íons de Hidrogênio , Hipertensão/genética , Hipertensão/metabolismo , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/genética , Ligadura , Losartan/farmacologia , Masculino , Potenciais da Membrana , Miócitos Cardíacos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Simportadores de Sódio-Bicarbonato/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima
19.
Biochem Biophys Res Commun ; 352(3): 668-74, 2007 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-17150187

RESUMO

Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the delta-sarcoglycan (delta-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the delta-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated delta-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored delta-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat delta-sarcoglycan complex mutant cardiomyopathy.


Assuntos
Cardiomiopatias/patologia , Cardiomiopatias/cirurgia , Mioblastos/citologia , Mioblastos/transplante , Neovascularização Fisiológica/fisiologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Resultado do Tratamento
20.
Biochem Biophys Res Commun ; 352(3): 635-41, 2007 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-17150190

RESUMO

Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3beta may be crucial modulators for hCSC maintenance in human heart.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteína Oncogênica v-akt/metabolismo , Transdução de Sinais/fisiologia , Adulto , Idoso , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Criança , Pré-Escolar , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade
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