RESUMO
Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.
Assuntos
Artérias/crescimento & desenvolvimento , Sistema Hematopoético/citologia , Interleucina-3/fisiologia , Antígenos Comuns de Leucócito , Neovascularização Fisiológica , Fator de Transcrição STAT5/metabolismo , Animais , Células da Medula Óssea , Proliferação de Células , Humanos , Camundongos , Células-TroncoRESUMO
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Proteínas do Leite/metabolismo , Receptores de Interleucina-3/metabolismo , Transativadores/metabolismo , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibronectinas/metabolismo , Humanos , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Janus Quinase 2 , Proteínas do Leite/genética , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S/fisiologia , Fator de Transcrição STAT5 , Transdução de Sinais/fisiologia , Transativadores/genética , Ativação Transcricional/fisiologia , Proteínas Supressoras de TumorRESUMO
OBJECTIVE: Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPARgamma in regulating CAC expansion in normal and diabetic settings. METHODS AND RESULTS: Normal and diabetic CACs were used. siRNA technology, EMSA, and chromatin immunoprecipitation (ChIP) assay as well as site-directed mutagenesis of the STAT5 response element in the PPARgamma promoter enabled us to demonstrate that STAT5 transcriptional activity controls PPARgamma expression. Moreover, FACS analysis, coimmunoprecipitation experiments, and ChIP assay revealed that a STAT5/PPARgamma transcriptional complex controls cyclin D1 expression and CAC progression into the cell-cycle. Conversely, PPARgamma agonists, by preventing the expression of STAT5 and the formation of the STAT5/PPARgamma heterodimeric complex failed to promote CAC expansion. Finally, we demonstrated that diabetic CAC functional capability can be recovered by molecules able to activate the STAT5/PPARgamma transcriptional complex. CONCLUSIONS: Our data identify the STAT5/PPARgamma heterodimers as landmark of CAC expansion and provide evidences for a mechanism that partially rescues CAC bioavailability in diabetic setting.
Assuntos
Diabetes Mellitus Tipo 2/genética , Angiopatias Diabéticas/genética , Neovascularização Patológica/genética , PPAR gama/fisiologia , Fator de Transcrição STAT5/genética , Transcrição Gênica , Animais , Células da Medula Óssea/fisiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Feminino , Genes Reporter , Humanos , Luciferases/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , PPAR gama/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Fator de Transcrição STAT5/fisiologiaRESUMO
Modified LDL is a major cause of injury to the endothelium in diabetes. In the present study, we analyzed the effects on endothelial cells of LDL recovered from type 2 diabetic patients (dm-LDL) or from nondiabetic subjects (n-LDL). Treatment of human umbilical vein endothelial cells with dm-LDL, but not n-LDL, led to the accumulation of cells in G1. To dissect the molecular mechanisms of this effect, we analyzed the expression and function of the cyclin-dependent kinase inhibitor p21(waf), a cell cycle regulator known to be a target of the signal transducers and activators of transcription (STATs). dm-LDL led to transient STAT5 phosphorylation and the formation of a STAT5-containing complex and activated p21(waf) expression at the transcriptional level. Expression of the dominant-negative form of STAT5B, but not of STAT5A, significantly decreased both p21(waf) expression and the fraction of cells in G1. Finally, immunofluorescence analysis demonstrated that activated STAT5 is expressed in newly formed intraplaque vessels and in endothelial cells lining the luminal side of the plaque. Similarly, p21(waf) immunoreactivity was found in the neointimal vasculature. Our results suggest a role of STAT5B as a regulator of gene expression in diabetes-associated vascular disease.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/efeitos dos fármacos , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/sangue , Lipoproteínas LDL/sangue , Lipoproteínas LDL/farmacologia , Proteínas do Leite , Transativadores/metabolismo , Idoso , Arteriosclerose/sangue , Arteriosclerose/etiologia , Arteriosclerose/patologia , Estudos de Casos e Controles , Ciclo Celular/fisiologia , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Proteínas de Ligação a DNA/genética , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas LDL/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT5 , Transativadores/genética , Proteínas Supressoras de TumorRESUMO
As vascular endothelial growth factor (VEGF), interleukin-3 (IL-3), released into the tumor microenvironment stimulates motogenic and mitogenic activity of normal and transformed cells. In the present study, we investigate the effects of IL-3 and VEGF on neoplastic vascular growth. Engagement of IL-3 receptor beta common (IL-3R beta c) contributes to both IL-3- and VEGF-induced Rac1 activation, cell migration and in vitro tube-like structure formation as shown by the expression of the dominant-negative IL-3R beta c construct (Delta455). In normal and transformed endothelial cells (EC) as well as in HEK 293 cells expressing KDR and IL-3R, VEGF and IL-3 treatment induces the formation of a KDR/IL-3R beta c complex. Moreover, as shown by the IL-3R Delta455 mutant or by the kinase dead KDR, functional receptors are required for this interaction. Consistent with the contribution of IL-3R beta c in both IL-3- and VEGF-mediated angiogenic signal, a reduced number of vessels inside tumors are found in mice injected with cells expressing the IL-3R Delta455 mutant. Thus, these findings provide a novel mechanism through which IL-3 and VEGF support cell survival and tumor neovascularization.
Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica , Receptores de Interleucina-3/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Humanos , Imunoprecipitação , Interleucina-3/fisiologia , Invasividade Neoplásica , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Interleukin-3 (IL-3) expression by tumor-infiltrating lymphocytes (TILs) and its effects on vessel assembly were evaluated. TILs from 'in situ' human breast cancers expressed CD4/CD25 antigens and IL-3. An injection of Matrigel containing SMC and IL-3 or basic-fibroblast growth factor (bFGF) into SCID mice confirmed the neoangiogenetic effect of both factors. However, in response to IL-3, but not to bFGF, only few SMC became incorporated into the nascent vessels. To evaluate the possibility that signals emanated by the nascent vasculature in the presence of IL-3 may negatively regulate SMC recruitment, conditioned media (CM) from IL-3-treated endothelial cells (EC) or SMC were tested for their biological effects on SMC and EC. CM from IL-3-treated SMC stimulated the migration of EC. In contrast, the migration of SMC was not affected by CM from IL-3-stimulated EC; however, it was greatly enhanced by blocking transforming growth factor beta (TGF beta) activity. TGF beta immunoenzymatic assay demonstrated the following: (i) the absence of TGF beta activity in CM from IL-3-stimulated EC; (ii) a barely detectable TGF beta activity in CM from IL-3-stimulated SMC; and (iii) the presence of TGF beta activity in the supernatants of SMC stimulated with CM from IL-3-, but not from bFGF-stimulated EC. Increased TGF beta mRNA expression was only detected in SMC stimulated with CM from IL-3-treated EC. Finally, the inhibitory signals induced by IL-3 in vivo were abrogated by the addition of the neutralizing TGF beta antibody. Thus, the positive immunostaining for IL-3 by TILs in 'in situ' breast cancers sustains the possibility that early in tumor development, IL-3 can contribute to the chronic immaturity of these vessels.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Interleucina-3/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Neovascularização Patológica , Fator de Crescimento Transformador beta/metabolismo , Animais , Neoplasias da Mama/imunologia , Células Cultivadas , Colágeno , Meios de Cultivo Condicionados/farmacologia , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Interleucina-3/metabolismo , Laminina , Linfócitos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos SCID , Mitógenos/farmacologia , Modelos Biológicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteoglicanas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Advanced glycation end products (AGEs) have been implicated in the accelerated vascular injury occurring in diabetes. We recently reported that LDL prepared from type 2 diabetic patients (dm-LDL), but not normal LDL (n-LDL) triggered signal transducers and activators of transcription STAT5 activation and p21(waf) expression in endothelial cells (ECs). The aims of the present study were to investigate the role of LDL glycation in dm-LDL- mediated signals and to analyze the molecular mechanisms leading to STAT5 activation. We found that glycated LDL (gly-LDL) triggered STAT5 activation, the formation of a prolactin inducible element (PIE)-binding complex containing STAT5, and increased p21(waf) expression through the activation of the receptor for AGE (RAGE). We also demonstrated that dm-LDL and gly-LDL, but not n-LDL treatment induced the formation of a stable complex containing the activated STAT5 and RAGE. Moreover, gly-LDL triggered src but not JAK2 kinase activity. Pretreatment with the src kinase inhibitor PP1 abrogated both STAT5 activation and the expression of p21(waf) induced by gly-LDL. Consistently, gly-LDL failed to activate STAT5 in src(-/-) fibroblasts. Collectively, our results provide evidence for the role of glycation in dm-LDL-mediated effects and for a specific role of src kinase in STAT5-dependent p21(waf) expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/sangue , Produtos Finais de Glicação Avançada/metabolismo , Lipoproteínas LDL/sangue , Proteínas do Leite , Transativadores/metabolismo , Quinases da Família src/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Glicosilação , Humanos , Proteínas Recombinantes/metabolismo , Valores de Referência , Fator de Transcrição STAT5 , Transfecção , Veias UmbilicaisRESUMO
Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are characterized by mucosal immune cell activation that is driven by a cytokine imbalance. Several cytokines involved in IBD act through the activation of the signal transducers and activators of transcription (STAT) family. We investigated the activation of STAT3 in the mucosa of CD and UC patients, and evaluated whether this event is specific for IBD patients. Using immunofluorescence and immunoblotting, total and phosphorylated STAT3 levels were assessed in biopsy specimens, isolated lamina propria mononuclear cells, and peripheral blood mononuclear cells from patients with CD, UC, other forms of intestinal inflammation, and control subjects. Immunoblotting revealed phosphorylated STAT3 in mucosal biopsy specimens from patients with CD, UC, celiac disease, and acute self-limited colitis, but not in the normal mucosa of control subjects. In IBD patients, STAT3 activation was confined to actively inflamed areas. Accordingly, activated STAT3 was detected in isolated lamina propria mononuclear cells from inflamed IBD tissues, but not in peripheral blood mononuclear cells from control subjects or IBD patients. Immunofluorescence demonstrated that the sources of activated STAT3 were macrophages and T lymphocytes, but not neutrophils. STAT3 activation also was detected in T cells infiltrating the duodenal mucosa of celiac disease patients. We conclude that STAT3 signaling occurs in both CD and UC, where it is strictly confined to areas of active inflammation and is limited to infiltrating macrophages and T cells. The occurrence of STAT3 signaling in other acute and chronic intestinal inflammatory conditions suggests that, rather than a specific feature of IBD, it represents a fundamental signaling pathway that is shared by multiple forms of gut inflammation.
Assuntos
Doença de Crohn/fisiopatologia , Doenças Inflamatórias Intestinais/fisiopatologia , Transdução de Sinais , Adulto , Estudos de Casos e Controles , Proteínas de Ligação a DNA , Feminino , Humanos , Inflamação , Mucosa Intestinal , Macrófagos , Masculino , Pessoa de Meia-Idade , Fator de Transcrição STAT3 , Linfócitos T , Transativadores , Ativação TranscricionalRESUMO
The molecular events associated with acute and chronic exposure of mesangial cells (MC) to hyperglycemia were evaluated. We found that, unlike high glucose (HG) and Amadori adducts, advanced glycation end products (AGE) and transforming growth factor-beta (TGF-beta) induced p21waf expression and accumulation of MC in G0/G1. TGF-beta1 blockade inhibited AGE-mediated collagen production but only partially affected AGE-induced p21waf expression and cell-cycle events, indicating that AGE by binding to AGE receptor (RAGE) per se could control MC growth. Moreover, AGE and TGF-beta treatment led to the activation of the signal transduction and activators of transcription (STAT)5 and the formation of a STAT5/p21SIE2 complex. The role of STAT5 in AGE- and TGF-beta-mediated p21waf expression and growth arrest, but not collagen production, was confirmed by the expression of the dominant negative STAT5 (DeltaSTAT5) or the constitutively activated STAT5 (1*6-STAT5) constructs. Finally, in p21waf-/- fibroblasts both AGE and TGF-beta failed to inhibit cell-cycle progression. A potential in vivo role of these mechanisms was sustained by the increasing immunoreactivity for the activated STAT5 and p21(waf) in kidney biopsies from early to advanced stage of diabetic nephropathy. Our data indicate that AGE- and TGF-beta-mediated signals, by converging on STAT5 activation and p21waf expression, may regulate MC growth.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Nefropatias Diabéticas/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transativadores/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Albuminúria/metabolismo , Albuminúria/patologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Colágeno/biossíntese , Colágeno/genética , Inibidor de Quinase Dependente de Ciclina p21 , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Nefropatias Diabéticas/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Mesângio Glomerular/citologia , Humanos , Hipertrofia , Proteínas do Leite/genética , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Proteinúria/metabolismo , Proteinúria/patologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/efeitos dos fármacos , Fator de Transcrição STAT5 , Transativadores/deficiência , Transativadores/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE: To characterize the molecules and the mechanisms regulating the neoangiogenetic process in advanced atherosclerotic plaques. METHODS AND RESULTS: Western blot and immunofluorescence analysis of atherosclerotic specimens demonstrated that unlike neovessels from early lesions that expressed vascular endothelial growth factor (VEGF) and angiopoietin1 (Angio1), vessels from advanced lesions expressed VEGF and angiopoietin 2 (Angio2). Moreover, only few neovessels from advanced lesions showed a positive immunostaining for proliferating cell nuclear antigen. Angio1-elicited and Angio2-elicited intracellular events in endothelial cells (EC) demonstrated that while Angio1 triggered Erk1/Erk2 mitogen activated protein kinases (MAPK) and Akt activation, Angio2 (50 ng/mL) induced STAT5 activation and p21waf expression and increased the fraction of cells in G1. Both Angio2-mediated events were abrogated by expressing a dominant negative STAT5 construct (DeltaSTAT5). Consistent with the expression of Angio2 in neovessels of advanced lesions a transcriptionally active STAT5 was detected. Moreover, co-immunoprecipitation experiments revealed the presence of a STAT5/Tie2 molecular complex in neointima vessels from advanced, but not from early, lesions. CONCLUSIONS: In advanced lesions, the activation of the Tie2-mediated STAT5 signaling pathway may negatively regulate vessel growth.
Assuntos
Angiopoietina-2/fisiologia , Arteriosclerose/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Células Endoteliais/efeitos dos fármacos , Receptor TIE-2/fisiologia , Transativadores/fisiologia , Angiopoietina-1/biossíntese , Angiopoietina-1/genética , Angiopoietina-1/fisiologia , Angiopoietina-2/biossíntese , Angiopoietina-2/genética , Angiopoietina-2/farmacologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/patologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Doença da Artéria Coronariana/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/genética , Progressão da Doença , Células Endoteliais/citologia , Endotélio Vascular/patologia , Fase G1/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Substâncias Macromoleculares , Proteínas do Leite/genética , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Fator de Transcrição STAT5 , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Transcrição Gênica , Transfecção , Túnica Íntima/patologiaRESUMO
OBJECTIVE: Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects. RESEARCH DESIGN AND METHODS: Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47(phox) and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model. RESULTS: Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG. CONCLUSIONS: These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Endotélio Vascular/fisiopatologia , Grelina/uso terapêutico , Células-Tronco/fisiologia , Acilação , Idoso , Animais , Doadores de Sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Lipídeos/sangue , Masculino , Camundongos , Camundongos Obesos , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Valores de Referência , Células-Tronco/efeitos dos fármacosRESUMO
Lipid abnormalities and oxidative stress, by stimulating mesangial cell (MC) proliferation, can contribute to the development of diabetes-associated renal disease. In this study we investigated the molecular events elicited by oxidized low density lipoproteins (ox-LDL) in MC. We demonstrate that in MC cultured in the presence of ox-LDL, survival and mitogenic signals on Akt and Erk1/2 MAPK pathways are induced, respectively. Moreover, as shown by the expression of the dominant negative Rac-1 construct, we first report that ox-LDL-mediated cell survival and cell cycle progression depend on Rac-1 GTPase-mediated reactive oxygen species production and on epidermal growth factor receptor transactivation. By silencing Akt and blocking Erk1/2 MAPK pathways, we also demonstrate that these signals are downstream to Rac-1/reactive oxygen species production and epidermal growth factor receptor activation. Finally, by endogenous depletion of beta4 integrin, expressed in MC, we provide evidence that the expression of this adhesion molecule is essential for ox-LDL-mediated MC dysfunction. Our data identify a novel signaling pathway involved in oxidative stress-induced diabetes-associated renal disease and provide the rationale for therapeutically targeting beta4 integrin.
Assuntos
Regulação da Expressão Gênica , Integrina beta4/biossíntese , Sistema de Sinalização das MAP Quinases , Mitose , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/terapia , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Mesangiais , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mitose/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K(+) channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the beta(1) integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/beta(1) complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1-dependent cell migration and invasion, and hence confer a greater malignancy.
Assuntos
Movimento Celular , Canais de Potássio Éter-A-Go-Go/metabolismo , Integrina beta1/metabolismo , Leucemia Mieloide/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Doença Aguda , Adulto , Idoso , Adesão Celular , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
We investigated the role of c-Kit and the membrane-bound ligand (mbKitL) in endothelial progenitor cell (EPC) recruitment by microvascular endothelial cells (ECs). We demonstrated that inflammatory activation induced the expression of the mbKitL on ECs both in vitro and in vivo, and that recruitment of EPCs depended on c-Kit/mbKitL interaction. Depletion of endogenous c-Kit or inhibition of c-Kit enzymatic activity by imatinib mesylate prevented adhesion of EPCs to activated ECs both in vitro and in vivo, indicating that a functional c-Kit on EPCs is essential. We also demonstrate that Akt was the downstream molecule regulating cell adhesion. A potential role of the c-Kit/mbKitL interaction in pathological settings is sustained by the expression of the mbKitL on ECs lining intraplaque neovessels. Thus, our results provide new insights into the mechanisms underlying EPC recruitment and the bases for novel strategies to hinder pathological angiogenesis.
Assuntos
Endotélio Vascular/metabolismo , Inflamação/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fator de Células-Tronco/metabolismo , Células-Tronco/metabolismo , Animais , Adesão Celular , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Neovascularização Patológica/metabolismo , Proteína Oncogênica v-akt/fisiologia , Transdução de SinaisRESUMO
Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes.