RESUMO
VEGF and TGF-beta1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF-beta1 induces apoptosis. We have previously shown that VEGF/VEGF receptor-2 (VEGFR2) signaling mediates TGF-beta1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF-mediated apoptosis is required for TGF-beta1 induction of angiogenesis. In vitro the apoptotic effect of TGF-beta1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF-beta1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord-like structures by TGF-beta1 with an effect comparable to that of z-VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF-beta1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF-beta1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF-beta1 induction of both apoptosis and angiogenesis, an effect similar to that of z-VAD. Thus, TGF-beta1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF-mediated apoptosis as a potential target to control angiogenesis.
Assuntos
Apoptose/fisiologia , Neovascularização Fisiológica , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Capilares/anatomia & histologia , Capilares/fisiologia , Bovinos , Células Cultivadas , Galinhas , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/metabolismo , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Vein graft arterialization results in activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases-1 and -2 (ERK1/2), which have been implicated in cell proliferation, migration, and apoptosis. The goal of our study was to characterize the effect of MAPK inhibition on intimal hyperplasia (IH) in arterialized vein grafts in hypercholesterolemic rabbits. METHODS: Reversed bilateral jugular vein to common carotid artery interposition grafts were constructed in 16 New Zealand White rabbits. The veins were incubated for 30 min prior to grafting with either the synthetic ERK1/2 activation inhibitor UO126 or the control vehicle. Vein graft and control jugular vein were harvested 3 h, 1 d, and 28 d after arterialization for histological and biochemical analyses. RESULTS: Treatment with UO126 was associated with 31% reduction in mean intimal area (1.68 +/- 0.78 mm(2)versus 2.44 +/- 1.65 mm(2); mean +/- SD; P = 0.036) relative to controls. The intima-to-media ratio of UO126-treated vein grafts decreased by 29% (0.53 +/- 0.04 versus 0.74 +/- 0.06; mean +/- SD; P < 0.01) compared to controls, vehicle-treated vein grafts. There was also significant increase in apoptosis in UO126-treated vein graft medial cell layer at 1 d. CONCLUSION: Topical administration of UO126 before vein grafting significantly decreases IH in arterialized vein grafts in hypercholesterolemic rabbits. These results may have significant implications for the development of strategies aimed at blocking or reducing IH in bypass grafts. Therefore, further evaluation of this simple strategy to improve vein graft patency following coronary artery or peripheral vascular bypass surgery is warranted.
Assuntos
Butadienos/uso terapêutico , Artérias Carótidas/transplante , Inibidores Enzimáticos/uso terapêutico , Hiperplasia/prevenção & controle , Veias Jugulares/cirurgia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/uso terapêutico , Túnica Íntima/patologia , Animais , Apoptose , Artéria Carótida Primitiva/cirurgia , Divisão Celular , Ativação Enzimática/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Coelhos , Túnica Íntima/efeitos dos fármacosRESUMO
VEGF and TGF-beta1 induce angiogenesis but have opposing effects on vascular endothelial cells: VEGF promotes survival; TGF-beta1 induces apoptosis. We have previously shown that TGF-beta1 induces endothelial cell apoptosis via up-regulation of VEGF expression and activation of signaling through VEGF receptor-2 (flk-1). In context with TGF-beta1, VEGF signaling is transiently converted from a survival into an apoptotic one. VEGF promotes cell survival in part via activation of PI3K/Akt by a mechanism dependent on the formation of a multi-protein complex that includes flk-1 and the adherens junction proteins VE-cadherin and beta-catenin. Here we report that TGF-beta1 induces rearrangement of the adherens junction complex by separating flk-1 from VE-cadherin and increasing beta-catenin association with both flk-1 and VE-cadherin. This rearrangement is caused neither by changes in adherens junction mRNA or protein expression nor by post-translational modification, and requires VEGF signaling through flk-1. These results show that the adherens junction is an important regulatory component of TGF-beta1-VEGF interaction in endothelial cells.
Assuntos
Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Imunofluorescência , Humanos , beta Catenina/análiseRESUMO
Cardiovascular interventions that restore blood circulation to ischemic areas are accompanied by significant tissue damage, which triggers a vascular remodeling response that may result in restenosis of blood conduits. Early endothelial dysfunction and/or impairment is the early event of a cascade that leads, through an inflammatory response and dedifferentiation of medial smooth muscle cells with abundant deposition of extracellular matrix, to intimal hyperplasia. Here we present the molecular and cellular mechanisms of intimal hyperplasia secondary to vascular injury and discuss the potential role of therapeutic modulation of the intracellular signaling pathways that differentially effect vascular endothelial and smooth muscle cells. The role of mitogen-activated protein kinases (MAPKs) and the outcome of their modulation in these processes are highlighted here as they provide a promising therapeutic target for prevention of restenosis.
Assuntos
Reestenose Coronária/enzimologia , Reestenose Coronária/terapia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Substâncias de Crescimento/metabolismo , Humanos , Hiperplasia , Túnica Íntima/patologiaRESUMO
Vein graft failure following bypass surgery is a frequent and important clinical problem. The vascular injury caused by arterialization is responsible for vein graft intimal hyperplasia, a lesion generated by medial smooth muscle cell proliferation and migration into the intima, increased extracellular matrix deposition, and formation of a thick neointima. Development of the neointima into a typical atherosclerotic lesion and consequent stenosis ultimately result in vein graft failure. Endothelial damage, inflammation, and intracellular signaling through mitogen-activated protein kinases (MAPKs) have been implicated in the early stages of this process. We therefore investigated the effects of topical inhibition of ERK-1/2 MAPK activation on vascular cell proliferation and apoptosis, and on the inflammatory response in a canine model of vein graft arterialization. For this purpose, vein grafts were incubated with the MEK-1/2 inhibitor, UO126, ex vivo for 30 min before grafting. This treatment effectively abolished arterialization-induced ERK-1/2 activation, decreased medial cell proliferation, and increased apoptosis. UO126 treatment also inhibited the vein graft infiltration by myeloperoxidase-positive inflammatory cells that follows vein graft arterialization. Thus, topical ex vivo administration of MAPK inhibitors can provide a pharmacological tool to prevent or reduce the vascular cell responses that lead to vein graft intimal hyperplasia and graft failure.
Assuntos
Anti-Inflamatórios/administração & dosagem , Apoptose/efeitos dos fármacos , Butadienos/farmacologia , Artérias Carótidas/cirurgia , Inflamação/tratamento farmacológico , Veias Jugulares/transplante , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Administração Tópica , Animais , Anti-Inflamatórios/farmacologia , Butadienos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Cães , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Animais , Nitrilas/administração & dosagemRESUMO
Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis. HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27(kip1) cyclin-cdk inhibitor. HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin. Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells. TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain. Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration. The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation. Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration. These results predicted a migration defect in p27-deficient cells. Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect. These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/metabolismo , Actinas/ultraestrutura , Sequência de Aminoácidos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Movimento Celular , Células Cultivadas , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina , Proteínas Inibidoras de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Fúngicas/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: C-reactive protein (CRP), an acute phase reactant, is an independent predictor of coronary artery syndromes and a mediator of the vascular response to injury. CRP has been found in arterialized vein grafts and has been linked to atherogenesis; however, its involvement in vein graft early failure or intimal hyperplasia has not been assessed. This study was designed to investigate the mechanism(s) of CRP up-regulation in arterialized vein grafts. METHODS: Carotid artery bypass with arterialized jugular vein grafts (AVG) was performed in 18 dogs. AVG were harvested at 3, 8, and 24 hours and 4, 14, and 28 days, using the femoral vein obtained at the time of AVG harvest as a control. Serum CRP levels were characterized by enzyme-linked immunosorbent assay; AVG expression of CRP was studied by immunofluorescence, Western blotting, in situ hybridization, Northern blotting, and quantitative RT-PCR. RESULTS: CRP levels peaked at 24 hours in serum and AVG but remained at baseline in control veins. By double immunofluorescence, CRP was associated with the media and adventitia of AVG. However, Northern blotting analysis showed no CRP mRNA expression in AVG. Reverse transcriptase polymerase chain reaction analysis confirmed the lack of up-regulation of CRP in AVG. CONCLUSION: CRP levels are increased in AVG, peaking 24 hours after arterialization. However, no significant production of CRP was detected in AVG. Therefore, increased CRP levels within AVG appear to originate mostly from CRP diffusion from the systemic circulation. These results have significant implications for the development of strategies aimed at blocking CRP up-regulation in bypass grafts.
Assuntos
Proteína C-Reativa/biossíntese , Ponte de Artéria Coronária , Veias Jugulares/transplante , Animais , Aterosclerose/fisiopatologia , Proteína C-Reativa/fisiologia , Cães , Ensaio de Imunoadsorção Enzimática , Sobrevivência de Enxerto , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
The formation of blood capillaries from preexisting vessels (angiogenesis) and vascular remodeling secondary to atherosclerosis or vessel injury are characterized by endothelial cell migration and proliferation. Numerous growth factors control these cell functions. Basic fibroblast growth factor (FGF-2), a potent angiogenesis inducer, stimulates endothelial cell proliferation, migration, and proteinase production in vitro and in vivo. However, mice genetically deficient in FGF-2 have no apparent vascular defects. We have observed that endothelial cell migration in response to mechanical damage in vitro is accompanied by activation of the extracellular signal-regulated kinase (ERK) pathway, which can be blocked by neutralizing anti-FGF-2 antibodies. Endothelial cells from mice that are genetically deficient in FGF-2 neither migrate nor activate ERK in response to mechanical wounding. Addition of exogenous FGF-2 restores a normal cell response, which shows that impaired migration results from the genetic deficiency of this growth factor. Injury-induced ERK activation in endothelial cells occurs only at the edge of the wound. In addition, FGF-2-induced ERK activation mediates endothelial cell migration in response to wounding without a significant effect on proliferation. These data show that FGF-2 is a key regulator of endothelial cell migration during wound repair.
Assuntos
Movimento Celular , Endotélio/fisiologia , Fator 2 de Crescimento de Fibroblastos/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Divisão Celular , Células Cultivadas , Endotélio/citologia , Endotélio/enzimologia , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Deleção de Genes , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Estresse MecânicoRESUMO
In addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two platelet-associated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releases of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium.
Assuntos
Plaquetas/fisiologia , Comunicação Celular , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Linfocinas/fisiologia , Plaquetas/metabolismo , Divisão Celular , Sobrevivência Celular , Técnicas de Cocultura , Fatores de Crescimento Endotelial/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: Long-term durability of saphenous vein grafts used for coronary artery bypass grafting is limited by neointimal formation. Arterial vascular injury is known to activate intracellular mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-jun N-terminal kinases, that affect cell differentiation, proliferation, migration, and apoptosis. This study tests the hypothesis that these mitogen-activated protein kinases are activated in saphenous veins during preparation for coronary artery bypass grafting. METHODS: Saphenous veins were harvested from 10 patients undergoing coronary artery bypass grafting. A specimen from each vein was placed in ice-cold lysis buffer immediately after harvesting (t = 0). The remaining tissue was incubated at room temperature in normal saline, 0.1% dimethylsulfoxide (vehicle), or 50 mmol/L PD98059 (mitogen-activated protein kinase kinase-1/2 inhibitor) until the vein was grafted (mean 50 minutes). To study kinetics of intracellular signaling pathways, canine saphenous veins were harvested, and mitogen-activated protein kinases and PI-3 kinase pathways were studied after different incubation time intervals. Extracted proteins were analyzed by Western blotting or in vitro kinase assay. RESULTS: The human saphenous veins showed elevated levels of active extracellular signal-regulated kinase after harvesting (t = 0) and prior to implant (t = 1). Incubation with PD98059 resulted in decreased activation of extracellular signal-regulated kinase. Kinetics of canine saphenous veins showed extracellular signal-regulated kinase and c-jun N-terminal kinase activation, in a time-dependent manner, along with activation of the growth factor-regulated PI3 kinase pathway. CONCLUSIONS: This study characterizes activation of extracellular signal-regulated kinases and c-jun N-terminal kinases during vein graft preparation and demonstrates the ability to inhibit extracellular signal-regulated kinase activation by simple incubation with a specific inhibitor. Further studies are needed to evaluate the significance of these findings with respect to graft durability.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Veias/efeitos dos fármacos , Veias/transplante , Animais , Cães , Humanos , Coleta de Tecidos e Órgãos , Veias/enzimologiaRESUMO
OBJECTIVE: Vascular injury results in activation of the mitogen-activated protein kinases-extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK)-which have been implicated in cell proliferation, migration, and apoptosis. The goal of this study was to characterize mitogen-activated protein kinase activation in arterialized vein grafts. METHODS: Carotid artery bypass using reversed external jugular vein was performed in 29 dogs. Vein grafts were harvested after 30 minutes and 3, 8, and 24 hours, and 4, 7, 14, and 28 days. Contralateral external jugular vein and external jugular vein interposition vein-to-vein grafts were used as controls. Vein graft extracts were analyzed for extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK) activation. Proliferating cell nuclear antigen expression was investigated as a parameter of cell proliferation. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining and intimal hyperplasia by morphometric examination of tissue sections. RESULTS: Significant intimal hyperplasia was observed at 28 days. Over the time points studied, vein graft arterialization resulted in bimodal activation of both extracellular-signal regulated kinase and p38(MAPK) (30 minutes through 3 hours; 4 days) but did not induce activation of c-jun N-terminal kinase. Proliferating cell nuclear antigen expression increased from days 1 through 28, and apoptosis increased between 8 and 24 hours. CONCLUSION: Vein graft arterialization induces bimodal activation of extracellular-signal regulated kinase and p38(MAPK); however, in contrast with what is described in arterial injury, it does not induce c-jun N-terminal kinase activation. These results provide the first comprehensive characterization of the mitogen-activated protein kinase signaling pathways activated in vein graft arterialization and identify mitogen-activated protein kinases as potential mediators of vein graft remodeling and subsequent intimal hyperplasia.
Assuntos
Artérias Carótidas/cirurgia , Veias Jugulares/enzimologia , Veias Jugulares/transplante , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Divisão Celular , Cães , Ativação Enzimática , Hiperplasia , Proteínas Quinases JNK Ativadas por Mitógeno , Veias Jugulares/metabolismo , Veias Jugulares/patologia , Antígeno Nuclear de Célula em Proliferação/análise , Túnica Íntima/patologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses.
Assuntos
Epiderme/efeitos da radiação , Proteínas Quinases JNK Ativadas por Mitógeno , Queratinócitos/efeitos da radiação , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo , Transdução de Sinais/efeitos da radiação , Células Cultivadas , Células HeLa/efeitos da radiação , Temperatura Alta , Humanos , MAP Quinase Quinase 4 , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/genética , Raios UltravioletaRESUMO
BACKGROUND: Vascular endothelial cell apoptosis is central in atherosclerosis and intimal hyperplasia. Transforming growth factor (TGF)-beta1 induces endothelial cell apoptosis through unidentified mechanism(s). Although TGF-beta1 signals through the Smad proteins, in some nonendothelial cell types it also activates the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK [p38(MAPK)]). p38(MAPK) relays apoptotic signals in several cell types. We hypothesized that TGF-beta1 activates endothelial cell MAPKs and induces apoptosis through p38(MAPK) activation. METHODS: Human umbilical vein or bovine capillary endothelial cells were incubated with TGF-beta1 for 0.5 to 12 hours. MAPK activation was characterized by Western blotting with antibodies to phosphorylated extracellular signal-regulated kinase 1/2, p38(MAPK), or c-Jun N-terminal kinases 1/2. To study apoptosis, extracts of cells incubated with TGF-beta1 for 6 hours with or without MAPK inhibitors were characterized by Western blotting analysis of poly (ADP-Ribose) polymerase degradation. RESULTS: TGF-beta1 induced p38(MAPK), extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase 1/2 activation and increased apoptosis. Inhibition of p38(MAPK) significantly reduced TGF-beta1-induced apoptosis. In contrast, inhibition of other signaling pathways was ineffective. CONCLUSIONS: TGF-beta1 induces endothelial cell apoptosis through p38(MAPK) activation. Because TGF-beta1 is upregulated in vascular remodeling, p38(MAPK) is a potential target to prevent endothelial cell apoptosis during this process.
Assuntos
Apoptose/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Western Blotting , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
TGF-ß1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-ß1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-ß1 induces endothelial cell expression of VEGF, which mediates TGF-ß1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-ß1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, ß, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-ß1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, ß, and γ. VEGF activates p38ß, whereas TGF-ß1 activates p38α. TGF-ß1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38ß is activated, and the surviving cells become refractory to TGF-ß1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-ß1 induction of apoptosis, whereas downregulation of p38ß or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38ß and p38γ transduce survival signaling. TGF-ß1 activation of p38α is mediated by VEGF, which in the absence of TGF-ß1 activates p38ß. Therefore, these results show that TGF-ß1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38ß to the proapoptotic p38α.
Assuntos
Apoptose/genética , Sobrevivência Celular/genética , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Animais , Bovinos , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Serine proteases are a key component of the inflammatory response as they are discharged from activated leukocytes and mast cells or generated through the coagulation cascade. Their enzymatic activity plays a major role in the body's defense mechanisms but it has also an impact on vascular homeostasis and tissue remodeling. Here we focus on the biological role of serine proteases in the context of cardiovascular disease and their mechanism(s) of action in determining specific vascular and tissue phenotypes. Protease-activated receptors (PARs) mediate serine protease effects; however, these proteases also exert a number of biological activities independent of PARs as they target specific protein substrates implicated in vascular remodeling and the development of cardiovascular disease thus controlling their activities. In this review both PAR-dependent and -independent mechanisms of action of serine proteases are discussed for their relevance to vascular homeostasis and structural/functional alterations of the cardiovascular system. The elucidation of these mechanisms will lead to a better understanding of the molecular forces that control vascular and tissue homeostasis and to effective preventative and therapeutic approaches.
RESUMO
OBJECTIVE: The inflammatory process of aortic stenosis involves the differentiation of aortic valve myofibroblasts into osteoblasts. Osteopontin, a proinflammatory glycoprotein, both stimulates differentiation of myofibroblasts and regulates the deposition of calcium by osteoblasts. Osteopontin levels are increased in patients with such conditions as end-stage renal disease, ectopic calcification, and autoimmune disease. We hypothesized that increased plasma osteopontin levels might be associated with the presence of aortic valve calcification and stenosis. METHODS: Venous blood from volunteers older than 65 years undergoing routine echocardiographic analysis or aortic valve surgery for aortic stenosis was collected. Plasma osteopontin levels were measured by means of enzyme-linked immunosorbent assay. The presence of aortic stenosis was defined as an aortic valve area of less than 2.0 cm(2). Aortic valve calcification was assessed by using a validated echocardiographic grading system (1, none; 2, mild; 3, moderate; 4, severe). Comparisons were performed with nonpaired t tests. RESULTS: Aortic stenosis was present in 23 patients (mean age, 78 years) and was absent in 7 patients (mean age, 72 years). Aortic valve calcification scores were 3.5 +/- 0.6 and 1.3 +/- 0.5 in patients with and without aortic stenosis, respectively (P < .001). Patients with no or mild aortic valve calcification had lower osteopontin levels compared with patients with moderate or severe aortic valve calcification (406.1 +/- 165.8 vs 629.5 +/- 227.5 ng/mL, P = .01). Similarly, patients with aortic stenosis had higher osteopontin levels compared with patients without aortic stenosis (652.2 +/- 218.7 vs 379.7 +/- 159.9 ng/mL, P < .01). CONCLUSION: Increased levels of plasma osteopontin are associated with the presence of aortic valve calcification and stenosis. These findings suggest that osteopontin might play a functional role in the pathogenesis of calcific aortic stenosis.
Assuntos
Estenose da Valva Aórtica/sangue , Calcinose/sangue , Osteopontina/sangue , Idoso , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/fisiopatologia , Calcinose/diagnóstico por imagem , Calcinose/fisiopatologia , Ecocardiografia , Feminino , Humanos , MasculinoRESUMO
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.
Assuntos
Proliferação de Células , Metaloproteinase 14 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Alanina/metabolismo , Alanina/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Proteínas ras/metabolismoRESUMO
After over thirty years from its discovery, research on basic fibroblast growth factor (FGF-2) keeps revealing new aspects of the complexity of its gene expression as it evolved in the eukaryotic organisms. The discovery of multiple forms of FGF-2 generated by alternative translation from AUG and non-canonical CUG codons on the same mRNA transcript has led to the characterization of a low molecular weight (LMW) FGF-2 form and various high molecular weight (HMW) forms (four in humans). In this review, we discuss the biochemical features and biological activities of the different FGF-2 forms. In particular, we focus on the properties that are unique to the HMW forms and its biological functions.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica , Iniciação Traducional da Cadeia Peptídica , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Códon , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Neoplasias/irrigação sanguínea , Isoformas de ProteínasRESUMO
VEGF and TGF-beta1 are potent angiogenesis inducers with opposing effects on endothelial cells. TGF-beta1 induces apoptosis; VEGF protects endothelial cells from apoptosis. We found that TGF-beta1 promotes endothelial cell expression of FGF-2, which up-regulates VEGF synthesis. Inhibition of VEGF signaling through VEGF receptor 2 (flk-1) abrogates TGF-beta1-induced apoptosis and p38(MAPK) activation. Inhibition of p38(MAPK) blocks TGF-beta1-induced apoptosis, showing that VEGF/flk-1-mediated activation of p38(MAPK) is required for TGF-beta1 induction of apoptosis. In the absence of TGF-beta1, VEGF activates p38(MAPK) and promotes endothelial cell survival. However, in context with TGF-beta1, VEGF/flk-1-mediated activation of p38(MAPK) results in apoptosis. Thus, cross-talk between TGF-beta1 and VEGF signaling converts VEGF/flk-1-activated p38(MAPK) into a proapoptotic signal. This finding illustrates an unexpected role of VEGF and indicates that VEGF can be pharmacologically converted into an apoptotic factor, a novel approach to antiangiogenesis therapy.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/deficiência , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts.