RESUMO
AIM: The biological and pharmacological properties of vessels used in coronary artery bypass graft (CABG) surgery are as important as their mechanical properties. The aim of this study was to investigate the possible role of protein kinase C (PKC)-dependent mechanisms in leptin-induced relaxation in the human internal mammary artery (IMA). METHODS: IMA rings, obtained from patients undergoing CABG surgery, were suspended in isolated tissue baths containing Krebs-Henseleit solution, which were continuously gassed with 95% O2 and 5% CO2 at 37(°)C. RESULTS: The IMA rings were pre-contracted with increasing concentrations of norepinephrine (NE 10(-9)-10(-4) mol/l) and the relaxation responses to sodium nitroprusside (SNP), a nitrosovasodilator, and leptin were studied in the presence and absence of a PKC inhibitor. Leptin (1 µM) caused a dose-dependent relaxation in NE pre-contracted IMA rings. Pre-treatment with a PKC inhibitor significantly attenuated this vasorelaxatory response to leptin in human isolated IMA. CONCLUSION: It was found that SNP and leptin caused significant relaxation of the NE pre-contracted human IMA rings, and PKC was probably the sub-cellular mediator for this effect. Our findings may have clinical or pharmacological importance as it could be hypothesised that obese subjects who have a left IMA bypass graft would have better myocardial perfusion.
Assuntos
Leptina/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Nitroprussiato/farmacologia , Norepinefrina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Anastomose de Artéria Torácica Interna-Coronária , Artéria Torácica Interna/enzimologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Coleta de Tecidos e ÓrgãosRESUMO
No-touch (NT) saphenous vein (SV) grafts are superior to SVs harvested by the conventional technique (CT), with a patency comparable with the internal thoracic artery (ITA). Preservation of the vasa vasorum is implicated in the success of NT harvesting. We compared the vasa vasorum and endothelial nitric oxide synthase (eNOS) in NT SV with ITA and radial artery (RA) grafts. Skeletonized SV (SSV) was also analyzed. The NT SV had a higher number and larger vasa vasorum compared with ITA (P = .0001) and RA (P = .0004) that correlated with eNOS protein. Activity of eNOS in SSV grafts was significantly lower than NT SV grafts (P = 004). Since a high proportion of the vasa vasorum are removed in SSV using the CT, we suggest that preservation of the vasa vasorum and eNOS-derived NO contributes to the high patency for NT as compared with SSV grafts.
Assuntos
Ponte de Artéria Coronária , Artéria Torácica Interna/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Radial/enzimologia , Veia Safena/enzimologia , Coleta de Tecidos e Órgãos/métodos , Vasa Vasorum/enzimologia , Idoso , Western Blotting , Ponte de Artéria Coronária/efeitos adversos , Oclusão de Enxerto Vascular/enzimologia , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Imuno-Histoquímica , Masculino , Artéria Torácica Interna/fisiopatologia , Artéria Torácica Interna/transplante , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Artéria Radial/fisiopatologia , Artéria Radial/transplante , Ensaios Clínicos Controlados Aleatórios como Assunto , Veia Safena/fisiopatologia , Veia Safena/transplante , Coleta de Tecidos e Órgãos/efeitos adversos , Vasa Vasorum/fisiopatologia , Vasa Vasorum/transplante , Grau de Desobstrução VascularRESUMO
BACKGROUND: After coronary artery bypass graft surgery, older women have less favorable clinical outcome and lower conduit patency compared with men. This less favorable outcome can be in part ascribed to impaired endothelium-derived nitric oxide (eNOS) production. This study evaluated endothelial nitric oxide synthase expression in internal mammary artery from postmenopausal women undergoing coronary artery bypass graft surgery. METHODS: Internal mammary artery segments were obtained from 20 postmenopausal woman and 20 matched male patients. Twenty more segments from younger patients were used as controls. Expression of eNOS messenger RNA in internal mammary artery endothelial cells were evaluated by polymerase chain reaction and real-time quantitative reverse transcription polymerase chain reaction. The eNOS protein level was assayed by Western blot. Vascular dynamics of specimens were evaluated by organ chamber methodology. RESULTS: In postmenopausal women, the band of messenger RNA for eNOS was reduced by 37.4% and by 25.2%, respectively, compared with matched men and the control group (62.6%±4.8% versus 74.8%±5.3%, p<0.001). In comparison with the control group lane, the eNOS protein immunoreactive band was 44.2% decreased in postmenopausal women and 34.5% decreased in matched men, and was significantly decreased in postmenopausal women as compared with matched men (55.8%±4.6% versus 65.5%±5.2%, p<0.001). Nitric oxide-mediated vasomotor dynamics were consistent with reduced eNOS production. CONCLUSIONS: Internal mammary artery endothelial cells from women after menopause undergoing coronary surgery have impaired expression of messenger RNA for eNOS and reduced eNOS levels. Reduced bioactivity of nitric oxide translates into impaired endothelial metabolism that could contribute to worse surgical outcome.
Assuntos
Doença da Artéria Coronariana/genética , Endotélio Vascular/enzimologia , Regulação da Expressão Gênica , Artéria Torácica Interna/enzimologia , Óxido Nítrico Sintase Tipo III/genética , Pós-Menopausa/sangue , RNA Mensageiro/genética , Adulto , Fatores Etários , Western Blotting , Angiografia Coronária , Ponte de Artéria Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/cirurgia , Endotélio Vascular/fisiopatologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Artéria Torácica Interna/fisiopatologia , Artéria Torácica Interna/transplante , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/biossíntese , Complicações Pós-Operatórias , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Vasodilatação/genéticaRESUMO
The resistance of internal mammary artery (IMA) toward atherosclerosis is not well understood. In plasma, homocysteine (Hcy) occurs in reduced, oxidized, homocysteine thiolactone and a component of proteins as a result of N- or S-homocysteinylation. We evaluated S/N-homocysteinylated protein levels in IMA fragments of patients undergoing coronary artery bypass grafting, and whether they were affected by genetic common variants. We tested whether tHcy, Hcy-S-protein levels, genotypes or Hcy-induced methylation modifications were related to differences in iNOS, Ddah2, and eNOS gene expression between territories. A small percentage of Hcy-S-proteins were found in IMA fragments. The Mthfr C677T (rs1801133) and Pon-1 Leu55Met (rs854560) variants were associated with Hcy-S-proteins. We observed a gradual difference according to Hcy-S-protein levels in the methylation degree of the Ddah2 gene promoter in aortic, but not in IMA, fragments. No correlation between the degree of methylation and the Ddah2 gene expression levels was found in both types of analyzed fragments. Total Hcy but not Hcy-S-proteins correlated with iNOS promoter methylation. Analyzed variants seem to contribute to the in vivo Hcy binding properties to IMA. The contribution of the Hcy-derived methylation modifications to Ddah2 and eNOS gene expression seems to be tissue-specific and independent of the Ddah2/ADMA/eNOS pathway. Hcy-derived methylation modifications to the iNOS gene promoter contribute to a lesser extent to iNOS gene expression.
Assuntos
Aorta/enzimologia , Aterosclerose , Homocisteína , Artéria Torácica Interna/enzimologia , Idoso , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aterosclerose/enzimologia , Aterosclerose/genética , Ponte de Artéria Coronária , Feminino , Expressão Gênica , Genótipo , Homocisteína/genética , Homocisteína/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Polimorfismo Genético , Regiões Promotoras GenéticasRESUMO
OBJECTIVES: Endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine is a cardiovascular risk factor that is elevated in patients with coronary artery disease. We hypothesized that novel endothelial nitric oxide synthase enhancer AVE3085 might improve the endothelial function altered by asymmetric dimethylarginine in the human internal thoracic artery. METHODS: Cumulative concentration-relaxation curves to acetylcholine (-11 to -5 log mol/L) were established in left internal thoracic artery rings (n = 65) from 27 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log mol/L) in the absence or presence of asymmetric dimethylarginine (100 µmol/L) or AVE3085 (30 µmol/L). Protein expressions of endothelial nitric oxide synthase and levels of superoxide anion production were detected. RESULTS: Maximal relaxation induced by acetylcholine was significantly attenuated by asymmetric dimethylarginine (12.7% ± 2.3% vs 35.3% ± 5.0% in control; P < .05) and significantly restored by AVE3085 (23.4% ± 2.8%; P < .05). AVE3085 also markedly restored endothelial nitric oxide synthase expression (0.29 ± 0.008; P = .012) reduced by asymmetric dimethylarginine (0.05 ± 0.04 vs 0.36 ± 0.03 in control; P = .014). Increased superoxide anion production by asymmetric dimethylarginine (2.97 ± 0.25 vs 0.51 ± 0.10 relative light units/[s/mg] in control; P < .05) was inhibited by AVE3805 (0.62 ± 0.104 relative light units/[s/mg]; P < .05). CONCLUSIONS: AVE3085 may restore endothelium-dependent relaxation reduced by asymmetric dimethylarginine through upregulation of endothelial nitric oxide synthase expression and inhibition of production of superoxide anion in human internal thoracic artery. These findings provide new insights into endothelial protection of coronary bypass grafting vessels to improve long-term patency of grafts.
Assuntos
Arginina/análogos & derivados , Benzodioxóis/farmacologia , Ativadores de Enzimas/farmacologia , Indanos/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Idoso , Antioxidantes/farmacologia , Arginina/metabolismo , Western Blotting , Citoproteção , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Humanos , Técnicas In Vitro , Masculino , Artéria Torácica Interna/enzimologia , Artéria Torácica Interna/fisiopatologia , Pessoa de Meia-Idade , Miografia , Superóxidos/metabolismo , Grau de Desobstrução Vascular/efeitos dos fármacosRESUMO
BACKGROUND: The internal mammary artery (IMA) has a better long-term patency rate than the radial artery (RA), but the underlying molecular mechanisms are unclear. We compared endothelial nitric oxide synthase (eNOS) and related NO release in these two arteries. METHODS: Real-time polymerase chain reaction was used to quantify eNOS messenger RNA (mRNA) expression level in the endothelial cells of IMAs and RAs. eNOS protein localization was determined by immunohistochemistry. NO release from the endothelium of IMAs and RAs was directly measured by an electrochemical method using a membrane-type NO-sensitive electrode. RESULTS: Endothelial nitric oxide synthase mRNA expression level was significantly higher in the endothelial cells of IMAs than in RAs (1.03±0.19 vs 0.53±0.09, n=7, p<0.05), but was similar in the whole vascular tissue. eNOS protein immunoreactivity was higher in the endothelial cells of IMAs than in RAs. NO release at both levels in IMAs was significantly greater than in RAs (basal: 17.5±1.9 vs 10.2±0.7 nM, n=11 each, p=0.003; stimulated with bradykinin -7 log M: 31.5±3.6 vs 14.3±5.3 nM, n=6 each, p=0.02). CONCLUSIONS: Endothelial cells in the IMA express higher levels of eNOS mRNA and protein than those in the RA, which is linked with higher release of NO. These findings may be related to the superior long-term patency rate of the IMA vs the RA. This study also provides some basic genetic information for grafting arteries.
Assuntos
Doença das Coronárias/genética , Regulação da Expressão Gênica , Artéria Torácica Interna/enzimologia , Óxido Nítrico Sintase Tipo III/genética , RNA Mensageiro/genética , Artéria Radial/enzimologia , Grau de Desobstrução Vascular/genética , Ponte de Artéria Coronária , Doença das Coronárias/metabolismo , Doença das Coronárias/cirurgia , Humanos , Imuno-Histoquímica , Artéria Torácica Interna/transplante , Óxido Nítrico Sintase Tipo III/biossíntese , Reação em Cadeia da Polimerase , Artéria Radial/transplanteRESUMO
OBJECTIVE: Cilnidipine is a novel, long-action L/N-type dihydropyridine calcium channel blocker that has recently been used for antihypertensive therapy. We investigated the vasorelaxation effect of cilnidipine with regard to its calcium channel blockage and nitric oxide-cyclic guanosine monophosphate-dependent mechanism in human internal thoracic artery. METHODS: Fresh human internal thoracic arteries taken from discarded tissues of patients undergoing coronary artery bypass surgery were studied. Concentration-relaxation curves for cilnidipine in comparison with nifedipine were studied. The expression level of endothelial nitric oxide synthase mRNA was assayed by quantitative real-time polymerase chain reaction, and the phosphorylation of endothelial nitric oxide synthase at Ser(1177) was determined by Western blotting analysis. RESULTS: Cilnidipine and nifedipine caused nearly full relaxation in potassium-precontracted internal thoracic artery. Pretreatment with cilnidipine at the clinical plasma concentration significantly depressed the maximal contraction. Endothelium denudation (47.7% ± 7.0%, P < .05) and inhibition of endothelial nitric oxide synthase (48.6% ± 6.1%, P < .05) or guanylate cyclase (41.6% ± 3.8%, P < .01) significantly reduced the cilnidipine-induced endothelium-dependent relaxation (73.9% ± 6.4%). Cilnidipine increased the expression of endothelial nitric oxide synthase mRNA by 42.4% (P < .05) and enhanced phosphorylation level of endothelial nitric oxide synthase at Ser(1177) by 37.0% (P < .05). CONCLUSIONS: The new generation of calcium channel antagonist cilnidipine relaxes human arteries through calcium channel antagonism and increases production of nitric oxide by enhancement of endothelial nitric oxide synthase. The dual mechanisms of cilnidipine in human arteries demonstrated in this study may prove particularly important in vasorelaxing therapy in cardiovascular diseases.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Idoso , Western Blotting , Canais de Cálcio/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Artéria Torácica Interna/enzimologia , Pessoa de Meia-Idade , Nifedipino/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina , Vasoconstritores/farmacologiaRESUMO
This study was designed to determine whether K+ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10(-6)M). Incubation with indomethacin (3 x 10(-6)M), superoxide dismutase (150 U/ml) and bosentan (10(-5)M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using L-NOArg (10(-4)M) or ODQ (3 x 10(-6)M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO(2)) was reduced when rings were contracted with an enriched K+ solution (30 mM), by voltage-dependent K+ (K(v)) channel blockade with 4-amynopiridine (4-AP; 10(-4)M), by Ca(2+)-activated K+ (K(Ca)) channel blockade with tetraethylammonium (TEA; 10(-3)M), and by apamin (5 x 10(-7)M) plus charybdotoxin (ChTx; 10(-7)M) but not when these were added alone. In contrast, large conductance K(Ca) (BK(Ca)), ATP-sensitive K+ (K(ATP)) and inwardly rectifying K+ (K(ir)) channel blockade with iberiotoxin (IbTx; 10(-7)M), glibenclamide (10(-6)M) and BaCl(2) (3 x 10(-5)M), respectively, did not alter the concentration-response curves to acetylcholine and NaNO(2). Na+-K+ ATPase pump inhibition with ouabain (10(-5)M) practically abolished acetylcholine and NaNO(2) relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K+ and K(v) channels, and Na+-K+-ATPase pump activation.
Assuntos
Acetilcolina/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/fisiologia , Óxido Nítrico/metabolismo , Canais de Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Masculino , Artéria Torácica Interna/enzimologia , Artéria Torácica Interna/metabolismo , SuínosRESUMO
Haptides are a family of 19-21-mer cell-binding and permeating peptides homologous to sequences in the C termini on both fibrinogen ß- and γ-chain (Cß and preCγ, respectively). The effect of the Haptides on the cardiovascular system was studied by different assays, including the activity of isolated perfused rat heart and blood vessels in the organ bath. Haptides (50-80 µg/ml) decreased the hemodynamic functions of perfused rat hearts by up to 60% (p < 0.05) in a dose-dependent manner. Whole fibrinogen or a control nonrelated peptide (Cα) did not show such an effect. The NO donor, sodium nitroprusside, reversed the inhibitory effects of Haptides. L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, did not further augment the effect of the Haptides. Perfused (FITC)Haptides were attached to the coronary endothelium. In myocardial homogenates and HUVEC, Haptides significantly decreased eNOS activity, but had no effect on the contraction of isolated cultured adult cardiomyocytes. Haptides also significantly enhanced the contraction of rings of rat aorta and human mammary artery vessels ex vivo only when the endothelium was intact. Haptides seem to affect the coronary endothelium, but not the cardiomyocytes, by inhibiting eNOS activity, causing vasoconstriction, temporary ischemia and impaired myocardial function that seem to be related to the amino acid composition of the Haptides.
Assuntos
Vasos Coronários/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibrinogênio/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Peptídeos/farmacologia , Animais , Células Cultivadas , Vasos Coronários/enzimologia , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Fibrinogênio/química , Hemodinâmica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Artéria Torácica Interna/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Perfusão , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
OBJECTIVE: Aim of the study was to discuss a new mechanism underlying the poor graft patency of GSV from diabetic patients and provide a rationale for selecting suitable grafts in diabetic patients. MATERIALS AND METHODS: The discarded matched RA, IMA, and GSV from 7 diabetics and 7 nondiabetic patients undergoing CABG were collected and tested for their contractile response to phenylephrine (PE) and their relaxation response to fasudil (a inhibitor of Rho-kinase) and used for immunohistochemical and mRNA detection of RhoA/ROK. RESULTS: The relaxation response to fasudil of GSV taken from diabetic patients was markedly increased but the relaxation response to fasudil of IMA and RA from diabetic patients was not. Immunohistochemistry and mRNA expression of RhoA/ROK was significantly increased in GSV from diabetic patients compared to that of IMA and RA from diabetic patients. RhoA/ROK immunohistochemistry and mRNA expression were significantly increased in GSV from diabetic patients compared with GSV from nondiabetic controls. CONCLUSIONS: RhoA/ROK expression and function in GSV from diabetic patients is significantly increased compared with IMA and RA from diabetic patients and GSV from nondiabetic patients. This contributes to a higher incidence of atherosclerosis and a lower long-term patency of GSV from diabetic patients.
Assuntos
Ponte de Artéria Coronária , Doença da Artéria Coronariana/cirurgia , Diabetes Mellitus/enzimologia , RNA Mensageiro/análise , Veia Safena/enzimologia , Veia Safena/transplante , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Adulto , Idoso , Estudos de Casos e Controles , Ponte de Artéria Coronária/efeitos adversos , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/genética , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Feminino , Regulação Enzimológica da Expressão Gênica , Oclusão de Enxerto Vascular/enzimologia , Oclusão de Enxerto Vascular/etiologia , Humanos , Imuno-Histoquímica , Masculino , Artéria Torácica Interna/enzimologia , Pessoa de Meia-Idade , Fenilefrina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Artéria Radial/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veia Safena/efeitos dos fármacos , Veia Safena/fisiopatologia , Grau de Desobstrução Vascular , Vasoconstrição , Vasoconstritores/farmacologia , Vasodilatação , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries. METHODS: Human internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists. RESULTS: The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 microM bisindolylmaleimide I and 10 microM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 microM SB203580, 10 microM PD98059 and 10 microM SP600125, respectively). CONCLUSION: In conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.
Assuntos
Artéria Torácica Interna/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Receptor de Endotelina B/metabolismo , Transdução de Sinais , Vasoconstrição , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Relação Dose-Resposta a Droga , Endotelina-1/metabolismo , Feminino , Humanos , Masculino , Artéria Torácica Interna/efeitos dos fármacos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Regulação para Cima , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.
Assuntos
Doença da Artéria Coronariana/metabolismo , Endotélio Vascular/química , Artéria Torácica Interna/química , Músculo Liso Vascular/química , Peptidil Dipeptidase A/análise , Artéria Radial/química , Receptores de Angiotensina/análise , Actinas/análise , Enzima de Conversão de Angiotensina 2 , Ponte de Artéria Coronária , Doença da Artéria Coronariana/enzimologia , Endotélio Vascular/enzimologia , Humanos , Artéria Torácica Interna/citologia , Artéria Torácica Interna/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/enzimologia , Artéria Radial/citologia , Artéria Radial/enzimologiaRESUMO
Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.
Assuntos
Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/enzimologia , Peptidil Dipeptidase A/análise , Idoso , Enzima de Conversão de Angiotensina 2 , Cromatografia Líquida de Alta Pressão , Células Endoteliais/enzimologia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ/métodos , Macrófagos/enzimologia , Masculino , Artéria Torácica Interna/enzimologia , Miócitos de Músculo Liso/enzimologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , RNA Mensageiro/análise , Sistema Renina-Angiotensina/fisiologia , Estatísticas não ParamétricasRESUMO
Dysregulation of matrix metalloproteinase (MMP)-2 in the vasculature has been suggested to be associated with increased prevalence of cardiovascular disease and renal injury. In this descriptive study, we hypothesized that arterial MMP-2 activity is elevated in the presence of cardiovascular risk factors such as diabetes, hypertension, smoking and ageing, and that it correlates with the degree of kidney function. MMP-2 activity in internal mammary arteries (n = 37) was measured using gelatinolytic zymography, and cutoffs were determined using sample-derived medians. Patient demographics and clinical data were analyzed, and the estimated glomerular filtration rate (eGFR) was calculated. High MMP-2 activity (>60,000 units) was associated with age, hypertension and diabetes (p = 0.0034, 0.06 and 0.0034, respectively). Multivariate analysis showed that age and diabetes were independent predictors of high MMP-2 activity. There is a trend towards increased MMP-2 activity and reduced eGFR (p = 0.010). The current exploratory work describes that the activity of MMP-2 in the internal mammary artery is correlated with age, hypertension, diabetes and eGFR. It is the first report suggesting that MMP-2 in the arterial vasculature could be the possible mediator crucial in linking the progression of kidney function to cardiovascular disease.
Assuntos
Envelhecimento , Diabetes Mellitus Tipo 2 , Hipertensão , Nefropatias , Artéria Torácica Interna/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Taxa de Filtração Glomerular , Humanos , Pessoa de Meia-IdadeRESUMO
The CYBA gene variants have been inconsistently associated with coronary heart disease (CHD) risk. A case-control study was conducted genotyping 619 subjects to explore the contribution of C242T and A640G to CHD risk in the population. A significant risk was found associated with GG homozygosity (odds ratio (OR) 2.132, 95% confidence interval, 1.113-4.085). The C242T variant was associated with CHD risk in women. Bias due to population stratification was analysed. Phenotype changes linked to these polymorphisms were evaluated. Superoxide measurements revealed higher production as indicated by the presence of the G and T alleles. Differences in mRNA concentration in heterozygous A640G samples were analysed. Higher levels of G allele mRNA compared with A allele mRNA were found. NAD(P)H oxidase p22phox sub-unit expression was evaluated with Western blot. Experiments revealed a gradual relationship in p22phox protein expression according to genotypes of the analysed variants. Those GG TT double homozygous showed increased p22phox protein expressions regarding AA CC double homozygous. This study has demonstrated increased expression and activity of the NAD(P)H system components during atherogenesis and the results could help explain the relevance of the A640G variant as a CHD marker.
Assuntos
Doença das Coronárias/genética , Regulação Enzimológica da Expressão Gênica , NADPH Oxidases/genética , Polimorfismo Genético , Adulto , Estudos de Casos e Controles , Células Cultivadas , Doença das Coronárias/enzimologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Homozigoto , Humanos , Peróxidos Lipídicos/sangue , Macrófagos/enzimologia , Masculino , Artéria Torácica Interna/enzimologia , Pessoa de Meia-Idade , Monócitos/enzimologia , NADPH Oxidases/metabolismo , Razão de Chances , Fenótipo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Medição de Risco , Fatores de Risco , Fatores Sexuais , Espanha , Superóxidos/metabolismo , Regulação para CimaRESUMO
OBJECTIVES: We tested the hypothesis of whether an inhibition of the nitroglycerin (GTN) bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) contributes to GTN tolerance in human blood vessels. BACKGROUND: The hemodynamic effects of GTN are rapidly blunted by the development of tolerance, a phenomenon associated with increased formation of reactive oxygen species (ROS). Recent studies suggest that ROS-induced inhibition of ALDH-2 accounts for tolerance in animal models. METHODS: Segments of surgically removed arteria mammaria and vena saphena from patients undergoing coronary bypass surgery were used to examine the vascular responsiveness to GTN and the endothelium-dependent vasodilator acetylcholine. The ALDH-2 activity and expression in these segments were assessed by the conversion of a benzaldehyde or its derivative to the benzoic acid metabolite and by Western blotting technique. RESULTS: In contrast to patients not treated with nitrates (n = 36), patients treated with GTN for 48 h (n = 14) before surgery showed tolerance to GTN and endothelial dysfunction in arterial and venous vessels. In vivo GTN tolerance was mimicked in vitro by incubation of nontolerant vessels with the ALDH-2 inhibitor benomyl. In vivo GTN treatment decreased vascular aldehyde dehydrogenase activity compared with nontolerant vessels and decreased the expression of ALDH-2 in arterial tissue. Incubation of control venous vessels with GTN caused a significant attenuation of aldehyde dehydrogenase activity that was reversed by presence of the sulfhydryl group donor dithiothreitol. CONCLUSIONS: Long-term GTN treatment induces tolerance and endothelial dysfunction in human vessels, associated with an inhibition and down-regulation of vascular ALDH-2. Thus, these findings extend results of previous animal studies to humans.
Assuntos
Aldeído Desidrogenase/metabolismo , Tolerância a Medicamentos/fisiologia , Artéria Torácica Interna/efeitos dos fármacos , Infarto do Miocárdio/enzimologia , Nitroglicerina/farmacologia , Veia Safena/efeitos dos fármacos , Acetilcolina/farmacologia , Idoso , Aldeído-Desidrogenase Mitocondrial , Esquema de Medicação , Feminino , Humanos , Masculino , Artéria Torácica Interna/enzimologia , Artéria Torácica Interna/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroglicerina/administração & dosagem , Estresse Oxidativo/fisiologia , Veia Safena/enzimologia , Veia Safena/fisiopatologia , Técnicas de Cultura de Tecidos , Vasodilatadores/farmacologiaRESUMO
Risk factors for cardiovascular diseases (CVD) increase oxidative stress, and they are proposed to hasten endothelial cell (EC) damage and dysfunction. Our objective was to elucidate the impact of chronic exposure to risk factors for CVD on senescence of EC isolated and cultured from internal mammary arterial segments of patients with severe coronary artery disease. Senescence induced by serial passages resulted in progressive telomere shortening, and short initial telomeres predicted early appearance of senescence in culture. Neither time course of senescence nor telomere length was age-dependent, suggesting that biological age, rather than chronological age, determined the dynamics. Senescence appeared earlier in patients with longer history of risk factor for CVD, and multivariate analysis suggested that hypertension hastened the onset of senescence. Risk factors for CVD override the effects of chronological aging likely by generating stress-dependent damage: senescent EC exhibited oxidative stress (increase in lipid peroxydation and caveolin-1 gene expression) and cell damage markers (loss of eNOS expression and increase in Cox2 mRNA, lower TRF1 protein level). Thus, cell senescence was triggered both by telomere-dependent and -independent pathways. In conclusion, chronic exposure to risk factors for CVD accelerated the development of endothelial senescence that could contribute to the pathogenesis of CVD.
Assuntos
Doenças Cardiovasculares/etiologia , Senescência Celular , Doença da Artéria Coronariana/patologia , Células Endoteliais/patologia , Artéria Torácica Interna/patologia , Adulto , Fatores Etários , Idoso , Índice de Massa Corporal , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Caveolina 1/genética , Caveolina 1/metabolismo , Proliferação de Células , Células Cultivadas , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Feminino , Humanos , Hipertensão/complicações , Hipertensão/patologia , Peroxidação de Lipídeos , Masculino , Artéria Torácica Interna/enzimologia , Artéria Torácica Interna/metabolismo , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , RNA Mensageiro/metabolismo , Fatores de Risco , Índice de Gravidade de Doença , Fumar/efeitos adversos , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: Nitric oxide (NO) inhibits thrombus formation, vascular contraction, and smooth muscle cell proliferation. We investigated whether NO release is enhanced after endothelial NO synthase (eNOS) gene transfer in atherosclerotic human carotid artery ex vivo. METHODS AND RESULTS: Western blotting and immunohistochemistry revealed that transduction enhanced eNOS expression; however, neither nitrite production nor NO release measured by porphyrinic microsensor was altered. In contrast, transduction enhanced NO production in non-atherosclerotic rat aorta and human internal mammary artery. In transduced carotid artery, calcium-dependent eNOS activity was minimal and did not differ from control conditions. Vascular tetrahydrobiopterin concentrations did not differ between the experimental groups. Treatment of transduced carotid artery with FAD, FMN, NADPH, L-arginine, and either sepiapterin or tetrahydrobiopterin did not alter NO release. Superoxide formation was similar in transduced carotid artery and control. Treatment of transduced carotid artery with superoxide dismutase (SOD), PEG-SOD, PEG-catalase did not affect NO release. CONCLUSIONS: eNOS transduction in atherosclerotic human carotid artery results in high expression without any measurable activity of the recombinant protein. The defect in the atherosclerotic vessels is neither caused by cofactor deficiency nor enhanced NO breakdown. Since angioplasty is performed in atherosclerotic arteries,eNOS gene therapy is unlikely to provide clinical benefit.
Assuntos
Aterosclerose/metabolismo , Artérias Carótidas/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Adenoviridae/genética , Idoso , Animais , Aorta/enzimologia , Aorta/metabolismo , Aterosclerose/enzimologia , Aterosclerose/genética , Artérias Carótidas/enzimologia , Linhagem Celular , Feminino , Vetores Genéticos , Humanos , Masculino , Artéria Torácica Interna/enzimologia , Artéria Torácica Interna/metabolismo , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/genética , Ratos , Superóxidos/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transdução GenéticaRESUMO
OBJECTIVE: Recent reports have demonstrated that long-term patency of the gastroepiploic artery (GEA) in coronary artery bypass grafting (CABG) is less satisfactory compared with the internal thoracic artery (ITA). However, the reason has not been fully elucidated. Angiotensin II is known to play an important role in the development of intimal hyperplasia, we hypothesized that the GEA is different from the ITA with respect to angiotensin II-forming ability. Accordingly, we measured activities of angiotensin II-forming enzymes, angiotensin-converting enzyme (ACE) and chymase, in human GEA and ITA. METHODS: Remnant of the GEAs and ITAs were obtained from 24 patients who underwent CABG in which both conduits were used simultaneously. Activities of ACE and chymase were measured by using the extract form the GEA or ITA. Sections of the GEA or ITA were immunohistochemically stained with anti-human chymase antibody. RESULTS: The ACE activity of the GEA (0.28+/-0.16 mU/mg protein) was greater than that of the ITA (0.18+/-0.11, p < 0.001). The chymase activity of the GEA (11.11+/-7.15 mU/mg protein) was also greater than that in the ITA (7.13+/-4.89, p < 0.001). The density of chymase-positive cells in the GEA (3.8+/-4.2 cells/mm2) was greater than that in the ITA (1.1+/-1.2, p < 0.01). CONCLUSION: Activities of both ACE and chymase were significantly greater in the GEA compared with the ITA. The GEA may be different from the ITA with respect to potential ability of angiotensin II-formation.