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1.
J Vasc Res ; 45(2): 103-10, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17934321

RESUMO

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. METHODS AND RESULTS: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. CONCLUSIONS: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.


Assuntos
Aorta/metabolismo , Tecido Elástico/metabolismo , Elastina/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Aorta/enzimologia , Aorta/ultraestrutura , Proteína Morfogenética Óssea 1 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Tecido Elástico/enzimologia , Tecido Elástico/ultraestrutura , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , RNA Mensageiro/metabolismo , Tropoelastina/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 26(3): 604-10, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16373606

RESUMO

BACKGROUND: LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mphis) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS AND RESULTS: Pravastatin downregulated LOX-1 expression in cultured human Mphis and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg(-1) d(-1) pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mphis and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. CONCLUSIONS: The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hiperlipidemias/tratamento farmacológico , Pravastatina/farmacologia , Receptores Depuradores Classe E/genética , Animais , Aorta/patologia , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Células Cultivadas , Colesterol/sangue , Regulação para Baixo/efeitos dos fármacos , Feminino , Homozigoto , Humanos , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , NF-kappa B/metabolismo , Coelhos
3.
Cardiovasc Res ; 67(1): 142-50, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15949478

RESUMO

OBJECTIVE: Basic fibroblast growth factor (bFGF)-stimulated proliferation of coronary smooth muscle cells (cSMC) contributes to the pathogenesis of arteriosclerosis and restenosis. However, the molecular mechanisms involved are not fully understood. We have shown previously that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are required for the bFGF-stimulated mitogenic process in bovine cSMC. In this study, we determined the PKC isoform(s) involved and investigated their functional role in the bFGF-stimulated signaling and cell cycle progression in human and bovine cSMC. METHODS AND RESULTS: Downregulation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis, the activation of MAPK, and the expression of c-myc, demonstrating the involvement of PMA-sensitive PKC isoforms in growth factor-induced proliferation and the MAPK pathway. The PMA-sensitive classical PKC isoforms alpha, beta, gamma and novel PKC isoforms delta and epsilon were found in human cSMC. Whereas blocking of the classical PKC isoforms had no influence, the suppression of PKC delta by genetic and pharmacological approaches inhibited the bFGF-stimulated c-Raf1-MEK-MAPK-c-myc signaling and DNA synthesis in cSMC. In contrast to PKC epsilon, our results showed that bFGF activated PKC delta by phosphorylation in a time-dependent manner. In addition, inhibition of PKC delta induced a hypophosphorylation of the retinoblastoma protein and suppression of the cyclins D1 and A, demonstrating the importance of PKC delta for bFGF-induced cell cycle progression through the G1 phase in cSMC. CONCLUSIONS: Our results show that PKC delta is required for the bFGF-stimulated c-Raf1-MEK-MAPK-c-myc signaling pathway involved in the proliferation of cSMC. Therefore, it may be an interesting therapeutic target for preventing proliferative vascular disorders.


Assuntos
Vasos Coronários , Fator 2 de Crescimento de Fibroblastos/farmacologia , Sistema de Sinalização das MAP Quinases , Músculo Liso Vascular/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Northern Blotting/métodos , Western Blotting/métodos , Carbazóis/farmacologia , Linhagem Celular , Proliferação de Células , DNA/biossíntese , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Músculo Liso Vascular/citologia , Oligonucleotídeos Antissenso/farmacologia
4.
Arterioscler Thromb Vasc Biol ; 24(10): 1789-95, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15271788

RESUMO

OBJECTIVE: Atherogenesis represents a type of chronic inflammation and involves elements of the immune response, eg, the expression of proinflammatory cytokines. In advanced atherosclerotic lesions, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs). In vitro, the expression of LOX-1 is induced by inflammatory cytokines like TNF-alpha and transforming growth factor (TGF)-beta. Therefore, LOX-1 is thought to be upregulated locally in response to cytokines in vivo. METHODS AND RESULTS: We determined by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis whether the mediators of the acute phase response in inflammation, IL-1alpha, IL-1beta, and TNF-alpha, regulate LOX-1 expression in cultured SMC, and whether this regulation is influenced by peroxisome proliferator-activated receptor gamma (PPARgamma). We studied by immunohistochemistry whether these cytokines are spatially correlated with LOX-1 expression in advanced atherosclerotic lesions. We found upregulation of LOX-1 expression in SMC in a dose- and time-dependent manner after incubation with IL-1alpha, IL-1beta, and TNF-alpha. Simultaneous incubation with these cytokines at saturated concentrations had an additive effect on LOX-1 expression. The PPARgamma activator, 15d-PGJ(2), however, inhibited IL-1beta-induced upregulation of LOX-1. In the intima of atherosclerotic lesions regions of IL-1alpha, IL-1beta, and TNF-alpha expression corresponded to regions of LOX-1 expression. CONCLUSIONS: We suppose that upregulated LOX-1 expression in SMC of advanced atherosclerotic lesions is a response to these proinflammatory cytokines. Moreover, the proinflammatory effects of these cytokines can be decreased by the antiinflammatory effect of PPARgamma.


Assuntos
Citocinas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Prostaglandina D2/análogos & derivados , Receptores de LDL/biossíntese , Aorta/citologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Células Cultivadas , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-1/farmacologia , Lasers , Microdissecção/métodos , PPAR gama/metabolismo , Prostaglandina D2/farmacologia , RNA Mensageiro/biossíntese , Receptores de LDL Oxidado , Receptores Depuradores Classe E , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Túnica Íntima/citologia , Túnica Média/citologia
5.
Basic Res Cardiol ; 99(4): 272-8, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15221345

RESUMO

Autocrine stimulation and paracrine interaction between coronary smooth muscle cells (cSMC) and endothelial cells (EC) act as regulators of the vascular angiogenesis. Basic fibroblast growth factor (bFGF), its receptor FGF-R1, and coreceptor heparansulfate proteoglycan (HSPG) are important components involved in this angiogenic process. We investigated the influence of angiotensin (Ang) II on this trimolecular bFGF complex, the underlying signaling and the proliferative process in human cSMC. Ang II induces an AT1 receptor-dependent expression of bFGF and also upregulates the FGF-R1 and HSPG expression which is suppressed by losartan, the AT1 receptor blocker. AT1 receptor signaling which is characterized by phosphorylation of p42-mitogen-activated protein kinase (MAPK) is involved in Ang II-induced bFGF, FGF-R1 and HSPG upregulation and DNA synthesis in human cSMC. In contrast, inhibition of the AT2 receptor by PD123,319 has no influence on these Ang II-stimulated and via the MAPK cascade-mediated proangiogenic effects. Finally, our data show that the Ang II-induced DNA synthesis in cSMC is mediated via the bFGF expression. In conclusion, our results suggest that the Ang II-induced angiogenic effects in the vessel wall are supported by the AT1 receptor-stimulated and MAPK pathway-mediated upregulation of the autocrine/paracrine trimolecular bFGF complex in cSMC.


Assuntos
Angiotensina II/metabolismo , Vasos Coronários , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Indutores da Angiogênese/metabolismo , Comunicação Autócrina , Northern Blotting , Western Blotting , Células Cultivadas , Vasos Coronários/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Comunicação Parácrina , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Regulação para Cima
6.
Eur J Cell Biol ; 83(10): 521-30, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15679098

RESUMO

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.


Assuntos
Estenose da Valva Aórtica/metabolismo , Arteriosclerose/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Miócitos de Músculo Liso/metabolismo , Túnica Íntima/metabolismo , Animais , Aorta Abdominal/lesões , Aorta Abdominal/ultraestrutura , Estenose da Valva Aórtica/patologia , Arteriosclerose/patologia , Colesterol na Dieta/administração & dosagem , Conexina 43/genética , Conexina 43/ultraestrutura , Modelos Animais de Doenças , Progressão da Doença , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/ultraestrutura , Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Microscopia Confocal , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Túnica Íntima/patologia , Regulação para Cima , Grau de Desobstrução Vascular
8.
FASEB J ; 17(11): 1451-7, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12890699

RESUMO

GM-CSF takes part in the cytokine network regulating the metabolism of extracellular matrix (ECM) during atherogenesis. Since data also point to an effect of GM-CSF on the vascular ECM in general, the vascular collagenous matrix was studied in wild-type and GM-CSF-deficient mice. Histological examination revealed a disorganized vascular ECM in GM-CSF-deficient mice involving the collagenous matrix and elastic fiber system. As shown by electron microscopy, collagen bundles were disrupted and reduced. The diameter of fibrils varied widely. mRNA expression of collagens and related molecules was studied. Fibrillar collagens were markedly reduced, alpha1(I)procollagen to 16.5% of control levels alpha1(III)procollagen was abolished whereas the expression level of network-forming alpha1(VIII)procollagen was not altered. As shown by in situ hybridization, the number of collagen-expressing cells was reduced. Matrix metalloproteinases and their inhibitor 1 were not affected by GM-CSF deficiency. Our studies demonstrate that GM-CSF plays a major role in the cytokine network regulating the metabolism of vascular collagens. GM-CSF deficiency leads to an altered composition of the vascular collagenous matrix, i.e., reduced amount of fibrillar collagen, altered ratio of fibrillar and network-forming collagen, and failures in the fibrillogenesis. We suggest that GM-CSF is a basic requirement for the maintenance of vessel wall integrity and resilience.


Assuntos
Artérias/anatomia & histologia , Matriz Extracelular/ultraestrutura , Colágenos Fibrilares/biossíntese , Colágenos Fibrilares/ultraestrutura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Animais , Aorta/anatomia & histologia , Aorta/metabolismo , Aorta/ultraestrutura , Feminino , Colágenos Fibrilares/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Camundongos Knockout , RNA Mensageiro/biossíntese , Transcrição Gênica
9.
J Am Coll Cardiol ; 39(9): 1508-12, 2002 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11985915

RESUMO

OBJECTIVES: To assess the potential of the donor heart to respond to interleukin-6 (IL6), the present study investigated the expression of IL6 receptor components in the myocardium of donor hearts before transplantation. BACKGROUND: Donor heart dysfunction early after transplantation has been associated with the cytokine storm after donor brain death. Proinflammatory cytokines are thought to play a central role in this process. Interleukin-6 is of specific interest because it has been associated with cardiac allograft dysfunction and is related to an impaired prognosis. Its action requires expression of the specific IL6 receptor (IL6R), and the common signal transducer of the IL6 family glycoprotein 130 (gp130) in the donor heart. METHODS: The activation of IL6, IL6R and gp130 messenger ribonucleic acid (mRNA) and protein was studied via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology in donor hearts (n = 6) and compared with patients undergoing evaluation of ventricular arrhythmias (control, n = 9) or with advanced heart failure (n = 20). RESULTS: Messenger RNA of IL6, IL6R and gp130 was strongly expressed in all chambers of donor hearts, whereas right ventricles of control patients did not show any expression (donor vs. control: p < 0.005). Right ventricles of failing hearts showed IL6, IL6R and gp130 mRNA levels comparable with those found in donor hearts. Immunohistochemistry paralleled the RT-PCR data on the protein level. While IL6 was mainly expressed by myocytes, both receptor components were preferentially found mainly on interstitial cells. CONCLUSIONS: The expression of the IL6 receptor components in the donor heart before transplantation establishes the condition sine qua non for the response of the donor heart to circulating IL6. This mechanism may explain the close association of elevated IL6 serum levels to acute cardiac allograft dysfunction in the early perioperative period.


Assuntos
Antígenos CD/metabolismo , Transplante de Coração/imunologia , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocárdio/imunologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-6/metabolismo , Disfunção Ventricular/diagnóstico , Antígenos CD/genética , Biomarcadores/sangue , Receptor gp130 de Citocina , Coração , Humanos , Imuno-Histoquímica , Interleucina-6/sangue , Interleucina-6/genética , Glicoproteínas de Membrana/genética , Miocárdio/metabolismo , Miocárdio/patologia , Receptores de Interleucina-6/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doadores de Tecidos , Disfunção Ventricular/sangue
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