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2.
J Immunol ; 177(11): 8266-72, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17114505

RESUMO

IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2(c) and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G(2)-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by approximately 50% as measured by [(3)H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.


Assuntos
Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucinas/metabolismo , Proteína Oncogênica v-akt/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Camundongos , Fosforilação , RNA Mensageiro/análise , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Interleucina 22
3.
Am J Physiol Heart Circ Physiol ; 290(6): H2178-86, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16473961

RESUMO

Induction of apoptosis represents a potential reaction of endothelial cells (ECs) after injury of the vascular endothelium. Beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in vascular diseases are widely recognized although the responsible mechanisms are not fully understood. Because it is not known whether PUFAs modulate EC apoptosis, we investigated the effects of n-3 and n-6 PUFAs on 4-hydroxynonenal (HNE)-induced EC apoptosis by annexin V staining and caspase-3 activation assays. Pretreatment with the n-3 fatty acid docosahexaenoic acid (DHA) reduced HNE-induced EC apoptosis. DHA-treated cells did not show the pronounced drop in intracellular GSH after HNE exposure seen in vehicle- or n-6 arachidonic acid-treated cells. This is most likely due to increased GSH levels in DHA-treated cells. Furthermore, DHA pretreatment increased ciap1 mRNA levels and transfection of cIAP1 small interfering RNA abolished the protective effect of DHA in HNE-induced apoptosis in HUVECs. Thus pretreatment of HUVECs with DHA reduces HNE-induced oxidative stress and apoptosis, and the protective effects of DHA seem to be dependent on cIAP1. The results provide a possible new mechanism for the atheroprotective effects of n-3 fatty acids in vascular disease.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/biossíntese , Aldeídos/antagonistas & inibidores , Aldeídos/toxicidade , Anexina A5/metabolismo , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Citometria de Fluxo , Glutationa/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico/patologia , Superóxidos/metabolismo , Transfecção
4.
FASEB J ; 19(10): 1293-5, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15939736

RESUMO

Nuclear factor-kappaB (NF-kappaB)-mediated vascular inflammation is a prominent characteristic of atherogenesis and restenosis. We noted that angioplastic injury to carotid artery elicited two phases of NF-kappaB activation characterized by an early activation in the arterial media and a late activation coupled with high levels of inhibitor of IkappaB kinase (IKK) activity in intima. These findings prompted us to elucidate the role for the different phases of NF-kappaB activation and IKK in the progress of vascular repair. Our results show that blockade of the early NF-kappaB activation by perivascular administration of pyrrolidine dithiocarbamate transiently attenuates the expression of proinflammatory genes in the injured vessels but does not affect intimal formation. Interruption of IKKbeta by overexpressing a dominant-negative IKKbeta in the injured artery effectively inhibited the late phase of NF-kappaB activation, resulting in down-regulation of inducible nitric oxide synthase, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 expression in conjunction with a 36% reduction in intima size, albeit with a lack of inhibitory effect on the early NF-kappaB activation. Collectively, these findings show that the IKKbeta-mediated late-phase NF-kappaB activation contributes to intimal hyperplasia and the accompanied vascular inflammatory responses.


Assuntos
Quinase I-kappa B/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Túnica Íntima/patologia , Vasculite/etiologia , Angioplastia/efeitos adversos , Animais , Apoptose , Artérias Carótidas/patologia , Proliferação de Células , Hiperplasia , Masculino , Músculo Liso Vascular/patologia , Ratos , Ratos Sprague-Dawley
5.
Artigo em Inglês | MEDLINE | ID: mdl-15853755

RESUMO

Growing evidence suggests that statins are more than simple lipid-lowering drugs. The so called pleiotropic effects of statins include multiple actions on cells of the vasculature. A large number of studies have confirmed that these compounds exert beneficial effects by mechanisms unrelated to cholesterol metabolism. For example, statins have been shown to inhibit the migration and proliferation of vascular smooth muscle cells (VSMC), and to induce apoptosis in this cell type. It is not yet clear if the induction of apoptosis in VSMC by statins is beneficial or detrimental. In the context of post-angioplasty restenosis, recurrent plaque growth after intervention, the inhibition of neointimal proliferation as well as a reduction of neointimal cell numbers by apoptosis is appealing. Multiple animal studies and clinical trials have therefore been undertaken to investigate effects of statin treatment on the development of restenosis, with very controversial results. Conversely, in advanced atherosclerotic lesions VSMC in the intima may stabilize the plaque and prevent plaque rupture by synthesizing collagen. VSMC in media adjacent to plaque areas or restenotic lesions should not be exposed to apoptosis promoting agents. In this context, recent evidence suggests that pravastatin protects such lesions by inhibiting inflammation and macrophage activation Our recent findings together with observations from other groups suggest that neointima cells are more sensitive to the induction of apoptosis than media VSMC. Importantly, statins were found to preferentially induce apoptosis in neointimal VSMC in our study. The purpose of the present review is to summarize statin effects on proliferation and apoptosis in VSMC in vitro and in vivo. Furthermore, the development of drug-coated stents may help to deliver high local doses of statins to enhance their effectiveness in the treatment of post-angioplasty restenosis.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Reestenose Coronária/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Humanos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia
6.
J Cell Physiol ; 204(3): 881-8, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15795939

RESUMO

n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert beneficial effects in the prevention of cardiovascular disease, inflammation, and on tumor growth. To investigate effects of PUFAs on proliferation and apoptosis in endothelial cells, we tested the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA) in human umbilical vein endothelial cells (HUVEC). The mitochondrial membrane potential (MMP) and the production of reactive oxygen species were examined by flow cytometry. Phosphorylation of p53 or p38 MAP kinase, and total levels of p53 were measured by Western blot. DNA binding activity of p53 was analyzed with a TransAM transcription factor assay kit. Tube formation was assessed on Matrigel. In proliferating HUVEC, but not in confluent cells, DHA reduced cell viability and induced apoptosis, as demonstrated by increases in membrane leakage (propidium iodide (PI) staining), Annexin-V binding, sub G(1) phase in the cell cycle, and TUNEL-positive cells. AA had no effect on these parameters. In addition to a reduced MMP and increased reactive oxygen species, phosphorylation of p38 and p53 (serine 15) and impaired DNA binding of p53 were observed. There was no change in total levels of p53. The p38 inhibitor SB203580 had no effect on Annexin V binding. DHA also attenuated HUVEC tube formation. Taken together, DHA induces apoptosis in proliferating, but not in resting HUVEC, potentially via the phosphorylation of p53, resulting in decreased p53 DNA binding. The results suggest that anti-angiogenic effects of DHA may be due to induction of apoptosis in proliferating endothelial cells.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Superóxidos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Arterioscler Thromb Vasc Biol ; 25(3): 546-52, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15618544

RESUMO

OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. METHODS AND RESULTS: All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. CONCLUSIONS: Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.


Assuntos
Endotélio Vascular/fisiologia , Biologia Molecular/métodos , RNA Interferente Pequeno/genética , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Antígenos CD , Caderinas/genética , Linhagem Celular , Endotélio Vascular/citologia , Inativação Gênica , Hemangiossarcoma , Humanos , Microcirculação , Neovascularização Fisiológica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Circulação Pulmonar , RNA Mensageiro/genética , Sistemas do Segundo Mensageiro/fisiologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Neoplasias Vasculares , Vasculite/genética , Vasculite/imunologia , Vasculite/fisiopatologia
8.
Blood ; 104(9): 2761-6, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15242875

RESUMO

Endothelial progenitor cells (EPCs) play a role in the repair of ischemic or injured tissue. Because endothelial injury can be associated with apoptosis, we have investigated whether apoptotic bodies from mature endothelial cells (ECs) may affect growth and differentiation of EPCs in vitro. A 24-hour incubation of isolated human EPCs with apoptotic bodies-rich medium (ABRM) from ECs led to a significant increase in the number of spindle-shaped attached cells. EPCs were characterized by DiI-Ac-LDL/lectin staining and measurement of CD34 and kinase insert domain receptor (KDR) expression. The treatment with ABRM resulted in a 2-fold increase of DiI-Ac-LDL/lectin-positive cells and up-regulation of CD34 (22% +/- 2% versus 13% +/- 3%, P < .05 and KDR (49% +/- 12% versus 19% +/- 7%, P < .05). Fluorescence and confocal laser microscopy demonstrated the uptake of apoptotic bodies by the EPCs. Apoptotic bodies-depleted medium had no effect, whereas the incubation with suspension of apoptotic bodies induced effects similar to those of ABRM. Our results suggest that apoptotic bodies from ECs are taken up by EPCs, increasing their number and differentiation state. Such a mechanism may facilitate the repair of injured endothelium and may represent a new signaling pathway between progenitor and damaged somatic cells.


Assuntos
Apoptose , Endotélio Vascular/citologia , Células-Tronco/citologia , Adesão Celular , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Meios de Cultivo Condicionados/química , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Fagocitose , Veias Umbilicais
9.
Eur J Immunol ; 34(1): 241-50, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14971050

RESUMO

Cyclopentenone prostaglandins (CP-PG), such as prostaglandin A1 (PGA1) or 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), induce apoptosis in different cell types. PGJ2 is also a potent activator of the peroxisome proliferator-activated receptor-gamma (PPARgamma). We investigated whether PPARgamma regulates CP-PG-induced apoptosis in endothelial cells (EC). We show that CP-PG induce apoptosis in human umbilical vein EC (HUVEC). Incubation with PGA1 or PGJ2 for 24 h reduced HUVEC number and viability, while the synthetic activators Wy14643 or rosiglitazone had no effect. Flow cytometry and cell cycle analysis revealed externalized phosphatidylserine, caspase-3 activation, and an increased percentage of cells with a reduced DNA content by CP-PG treatment. EMSA demonstrated an activation of PPARgamma by PGJ2 and rosiglitazone. Immunohistochemistry of HUVEC and immunoblot analyses of protein extracts showed that PPARgamma was localized in the nuclei of HUVEC, and that CP-PG treatment decreased the amount of PPARgamma protein. This degradation was prevented by a pan-caspase inhibitor. Treatment of differentiated, endothelial-like PPARgamma-deficient stem cells, or of HUVEC transfected with dominant-negative PPARgamma with CP-PG, induced cell death and apoptosis. Our findings show that PGA1 and PGJ2 induce apoptosis in endothelial cells independent of PPARgamma. As the synthesis of PGJ2 is increased at sites of inflammation, our results may suggest a possible mechanism for endothelial damage.


Assuntos
Apoptose/efeitos dos fármacos , Prostaglandinas A/farmacologia , Veias Umbilicais/efeitos dos fármacos , Caspase 3 , Caspases/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo
10.
Circ Res ; 94(6): 776-84, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-14963004

RESUMO

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.5-fold higher arrest of monocytes and "memory" T lymphocytes than medial SMCs, which was dependent on both P-selectin and VLA-4, as demonstrated by blocking antibodies. The increase in monocyte arrest on niSMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, whereas "memory" T cell arrest was mediated by stromal cell-derived factor (SDF)-1alpha. This functional phenotype was paralleled by a constitutively increased mRNA and surface expression of P-selectin and of relevant chemokines in niSMCs, as assessed by real-time PCR and flow cytometry. The increased expression of P-selectin in niSMCs versus medial SMCs was associated with enhanced NF-kappaB activity, as revealed by immunofluorescence staining for nuclear p65 in vitro. Inhibition of NF-kappaB by adenoviral IkappaBalpha in niSMCs resulted in a marked reduction of increased leukocyte arrest in flow. Furthermore, P-selectin expression by niSMCs in vivo was confirmed in a hypercholesterolemic mouse model of vascular injury by double immunofluorescence and by RT-PCR after laser microdissection. In conclusion, we have identified a NF-kappaB-mediated proinflammatory phenotype of niSMCs that is characterized by increased P-selectin and chemokine expression and thereby effectively supports leukocyte recruitment.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Inflamação/patologia , Integrina alfa4beta1/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/fisiologia , Selectina-P/fisiologia , Animais , Aorta Torácica/lesões , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Adesão Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Quimiocina CX3CL1 , Quimiocinas CX3C/farmacologia , Constrição Patológica , Citocinas/biossíntese , Citocinas/genética , Citocinas/fisiologia , Endotélio Vascular/lesões , Regulação da Expressão Gênica , Hipercolesterolemia/metabolismo , Proteínas I-kappa B/fisiologia , Inflamação/metabolismo , Integrina alfa4beta1/biossíntese , Integrina alfa4beta1/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Músculo Liso Vascular/citologia , Inibidor de NF-kappaB alfa , Selectina-P/biossíntese , Selectina-P/genética , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-8B/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Recidiva , Reologia , Subpopulações de Linfócitos T/citologia
11.
Blood ; 103(7): 2585-92, 2004 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-14645014

RESUMO

Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0). LPA directly induced platelet shape change in blood and platelet-rich plasma obtained from all blood donors. However, LPA-stimulated platelet aggregation in blood was donor dependent. It could be completely blocked by apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors P2Y1 and P2Y12. These substances also inhibited LPA-induced aggregation of platelet-rich plasma and aggregation and serotonin secretion of washed platelets. These results indicate a central role for ADP-mediated P2Y1 and P2Y12 receptor activation in supporting LPA-induced platelet aggregation. Platelet aggregation and platelet-monocyte aggregate formation stimulated by LPA was insensitive to inhibition by aspirin. We conclude that LPA at concentrations approaching those found in vivo can induce platelet shape change, aggregation, and platelet-monocyte aggregate formation in whole blood and suggest that antagonists of platelet P2Y1 and P2Y12 receptors might be useful preventing LPA-elicited thrombus formation in patients with cardiovascular diseases.


Assuntos
Arteriopatias Oclusivas/sangue , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/fisiologia , Proteínas de Membrana/fisiologia , Ativação Plaquetária/fisiologia , Receptores Purinérgicos P2/fisiologia , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Tamanho Celular/efeitos dos fármacos , Humanos , Cinética , Ativação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Transdução de Sinais
12.
Atherosclerosis ; 169(2): 251-8, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12921976

RESUMO

In the context of atherogenesis and restenosis, vascular smooth muscle cell (SMC) proliferation and apoptosis play a crucial role. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) have been shown to inhibit the migration and proliferation of SMC, and to induce apoptosis in different cell types including SMC. However, it is not known whether these agents induce apoptosis in neointimal SMC. We investigated the effects of statin treatment on neointimal SMC as compared to medial cells by using trypan blue counting, MTT test, Annexin V staining, cell cycle analysis and a co-culture model. The incubation of neointimal or medial SMC with lovastatin reduced the MTT activity as well as the total cell number, and increased the amount of trypan blue positive cells, indicative of cell death. We tested by staining with Annexin V/propidium iodide, specific antibodies to active caspase-3, TUNEL reaction, and by the appearance of a sub-G1 peak, whether the observed increase in cell death was due to apoptosis. After treatment with lovastatin, programmed cell death was slightly increased in medial SMC, while neointimal cells showed a pronounced rate of apoptosis. In an attempt to mimic early phases of restenosis in vitro by seeding low density neointimal cells onto high density medial cells, we found that statin treatment induced cell death preferentially in the neointimal SMC. Our results suggest that statins enhance the rate of apoptosis in neointimal SMC, which may be an interesting feature to reduce restenosis after successful angioplasty.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Marcação In Situ das Extremidades Cortadas , Lovastatina/farmacologia , Masculino , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Músculo Liso Vascular/citologia , Ratos , Ratos Sprague-Dawley , Túnica Íntima/citologia
13.
Biochem Biophys Res Commun ; 307(2): 322-6, 2003 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12859958

RESUMO

Prostaglandins are a family of structurally related molecules formed by many cells in response to extrinsic stimuli. A member of this family, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), shows unique biological properties including anti-inflammatory, anti-viral, and anti-tumour activity, and has attracted much attention as a high affinity ligand for the peroxisome proliferator-activated receptor gamma. Increasing evidence points to additional effects. We investigated several structurally related prostaglandins in comparison to 15d-PGJ(2) with respect to their apoptosis-inducing capacity in human umbilical endothelial cells (HUVEC). Cell viability was tested with a modified MTT assay and apoptosis was detected by Annexin V staining and cell cycle analysis by flow cytometry. Incubation of confluent HUVECs with 15d-PGJ(2) markedly reduced endothelial cell viability which was due to apoptosis. In contrast, none of the other PGs tested affected cell viability. Interestingly, the cyclopentenone ring alone dose-dependently reduced cell viability and significantly induced apoptosis in HUVECs with as low a concentration as 0.25 microM. In conclusion, we report that the cyclopentenone moiety of cyPGs is an essential component for the apoptosis-inducing properties of 15d-PGJ(2). For 15d-PGJ(2) the position of the cyclopentenone ring in conjunction with the side chains yields a molecule with unique biological properties.


Assuntos
Apoptose , Ciclopentanos/química , Ciclopentanos/farmacologia , Endotélio Vascular/citologia , Prostaglandinas/química , Prostaglandinas/farmacologia , Linhagem Celular , Humanos , Estrutura Molecular , Prostaglandina D2/análogos & derivados , Prostaglandina D2/química , Prostaglandina D2/farmacologia
14.
Trends Cardiovasc Med ; 13(5): 201-6, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12837583

RESUMO

Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs). Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation. Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist. Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.


Assuntos
Endotélio/citologia , Neovascularização Fisiológica , Células-Tronco/citologia , Separação Celular , Humanos
15.
Arterioscler Thromb Vasc Biol ; 23(7): 1185-9, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12714439

RESUMO

Postnatal bone marrow contains a subtype of progenitor cells that have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells. Therefore, these cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. In general, EPCs are characterized by the expression of 3 markers, CD133, CD34, and the vascular endothelial growth factor receptor-2. During differentiation, EPCs obviously lose CD133 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor. EPCs seem to participate in endothelial repair and neovascularization of ischemic organs. Clinical studies using EPCs for neovascularization have just been started; however, the mechanisms stimulating or inhibiting the differentiation of EPC in vivo and the signals causing their migration and homing to sites of injured endothelium or extravascular tissue are largely unknown at present. Thus, future studies will help to explore areas of potential basic research and clinical application of EPCs.


Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Antígeno AC133 , Angioplastia com Balão , Antígenos CD , Antígenos CD34/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Células Endoteliais/transplante , Endotélio Vascular/lesões , Endotélio Vascular/fisiologia , Glicoproteínas/metabolismo , Humanos , Peptídeos/metabolismo , Regeneração , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo
16.
FASEB J ; 17(9): 1099-101, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12692090

RESUMO

The cyclopentenone prostaglandins (cPG) 15-deoxy-Delta12,14-prostaglandin J2 (dPGJ2) and PGA1 can inhibit multiple steps in nuclear factor (NF)-kappaB signaling and can induce cell death. Here we characterized the effects of dPGJ2 and PGA1 on the inflammatory induction of endothelial cell adhesion molecules (CAM). Pretreatment of endothelial cells with dPGJ2 or PGA1 at low concentrations dose dependently inhibited the up-regulation of CAM expression and monocyte arrest by tumor necrosis factor (TNF)-alpha but not expression of inhibitor of apoptosis proteins. Only at high concentrations, cPG enhanced TNF-alpha-induced cell death and inhibited TNF-alpha-induced IkappaB-alpha kinase (IKK) activation, IkappaB-alpha degradation, and NF-kappaB/p65 translocation, while promoting AP-1/c-jun phosphorylation. Expression of an IKK-beta mutant (C179A) resistant to interaction with cPG impaired cell death induction but not inhibition of CAM up-regulation by cPG. Gel shift and reporter gene analysis revealed that cPG at low concentrations directly impaired DNA binding of NF-kappaB and NF-kappaB-dependent transactivation. The synthetic analogs dPGA1 or dPGA2 were ineffective, indicating structural specificity of cPG. Thus, the suppression of endothelial CAM up-regulation with cPG is dissociated from cell death sensitization and IKK inhibition above threshold concentrations and related to interference with NF-kappaB binding. Our findings define distinct mechanisms for anti-inflammatory and proapoptotic effects of cPG in endothelial cells.


Assuntos
Moléculas de Adesão Celular/biossíntese , Endotélio Vascular/metabolismo , Prostaglandina D2/farmacologia , Prostaglandinas A/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Humanos , Quinase I-kappa B , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Modelos Biológicos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Prostaglandina D2/análogos & derivados , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/biossíntese
17.
Clin Cancer Res ; 8(3): 863-9, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11895920

RESUMO

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analogue of vitamin E, is a strong inducer of apoptosis, whereas alpha-tocopherol (alpha-TOH) lacks apoptogenic activity (J. Neuzil et al., FASEB J., 15: 403-415, 2001). Here we investigated the possible antineoplastic activities of alpha-TOH and alpha-TOS and further explored the potential of alpha-TOS as an antitumor agent. Using nude mice with colon cancer xenografts, we found that alpha-TOH exerted modest antitumor activity and acted by inhibiting tumor cell proliferation. In contrast, alpha-TOS showed a more profound antitumor effect, at both the level of inhibition of proliferation and induction of tumor cell apoptosis. alpha-TOS was nontoxic to normal cells and tissues, triggered apoptosis in p53(-/-) and p21(Waf1/Cip1(-/-)) cancer cells, and exerted a cooperative proapoptotic activity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) due to differences in proapoptotic signaling. Finally, alpha-TOS cooperated with tumor necrosis factor-related apoptosis-inducing ligand in suppression of tumor growth in vivo. Vitamin E succinate is thus a potent and highly specific anticancer agent and/or adjuvant of considerable therapeutic potential.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Animais , Proteínas Reguladoras de Apoptose , Caspases/metabolismo , Ensaio de Unidades Formadoras de Colônias , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Sinergismo Farmacológico , Humanos , Camundongos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miocárdio/citologia , Invasividade Neoplásica , Ligante Indutor de Apoptose Relacionado a TNF , Tocoferóis , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Tocoferol/farmacologia
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