RESUMO
BACKGROUND: Restenosis after percutaneous coronary intervention in coronary heart disease remains an unsolved problem. Clusterin (CLU) (or Apolipoprotein [Apo] J) levels have been reported to be elevated during the progression of postangioplasty restenosis and atherosclerosis. However, its role in neointimal hyperplasia is still controversial. OBJECTIVE: To elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin administration. METHODS: Male Wistar rats were randomly divided into three groups: the control group (n = 20), the model group (n = 20) and the statin intervention group (n = 32). The rats in the intervention group were given 10mg /kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury. Neointima formation was analyzed 1, 2, 3 and 4 weeks after balloon injury. The level of Apo J was measured by real-time PCR, immunohistochemistry and western blotting. RESULTS: Intimal/medial area ratio (intimal/medial, I/M) was increased after balloon-injury and reached the maximum value at 4weeks in the model group; I/M was slightly increased at 2 weeks and stopped increasing after rosuvastatin administration. The mRNA and protein levels of Apo J in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached maximum values at 2 weeks, which was earlier than in the model group (3 weeks). CONCLUSION: Apo J served as an acute phase reactant after balloon injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through up-regulation of Apo J. Our results suggest that Apo J exerts a protective role in the restenosis after balloon-injury in rats.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Anticolesterolemiantes/farmacologia , Lesões das Artérias Carótidas/tratamento farmacológico , Clusterina/efeitos dos fármacos , Reestenose Coronária/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Rosuvastatina Cálcica/farmacologia , Animais , Western Blotting , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Clusterina/análise , Reestenose Coronária/etiologia , Reestenose Coronária/patologia , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Masculino , Substâncias Protetoras/farmacologia , Distribuição Aleatória , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Túnica Média/efeitos dos fármacos , Túnica Média/patologiaRESUMO
Abstract Background: Restenosis after percutaneous coronary intervention in coronary heart disease remains an unsolved problem. Clusterin (CLU) (or Apolipoprotein [Apo] J) levels have been reported to be elevated during the progression of postangioplasty restenosis and atherosclerosis. However, its role in neointimal hyperplasia is still controversial. Objective: To elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin administration. Methods: Male Wistar rats were randomly divided into three groups: the control group (n = 20), the model group (n = 20) and the statin intervention group (n = 32). The rats in the intervention group were given 10mg /kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury. Neointima formation was analyzed 1, 2, 3 and 4 weeks after balloon injury. The level of Apo J was measured by real-time PCR, immunohistochemistry and western blotting. Results: Intimal/medial area ratio (intimal/medial, I/M) was increased after balloon-injury and reached the maximum value at 4weeks in the model group; I/M was slightly increased at 2 weeks and stopped increasing after rosuvastatin administration. The mRNA and protein levels of Apo J in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached maximum values at 2 weeks, which was earlier than in the model group (3 weeks). Conclusion: Apo J served as an acute phase reactant after balloon injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through up-regulation of Apo J. Our results suggest that Apo J exerts a protective role in the restenosis after balloon-injury in rats.
Resumo Fundamento: A reestenose após intervenção coronária percutânea (ICP) após doença coronariana continua um problema não solucionado. Estudos relataram que os níveis de clusterina (CLU), também chamada de apolipoproteína (Apo) J, encontram-se elevados na progressão da reestenose pós-angioplastia e na aterosclerose. Contudo, seu papel na hihperplasia neointimal ainda é controverso. Objetivo: Elucidar o papel da Apo J na hiperplasia neointimal na artéria carótida utilizando um modelo experimental com ratos in vivo, com e sem intervenção com rosuvastatina. Métodos: ratos Wistar machos foram divididos aleatoriamente em três grupos - grupo controle (n = 20), grupo modelo (n = 20), e grupo intervenção com estatina (n = 32). Os ratos no grupo intervenção receberam 10 mg/kg de rosuvastatina. Um cateter Fogarty 2 F foi introduzido para induzir lesão vascular. A formação de neoíntima foi analisada 1, 2, 3 e 4 semanas após lesão com balão. Concentrações de Apo J foram medidas por PCR em tempo real, imuno-histoquímica e western blotting. Resultados: A razão área íntima/média (I/M) aumentou após a lesão com balão e atingiu o valor máximo 4 semanas pós-lesão no grupo modelo; observou-se um pequeno aumento na I/M na semana 2, que cessou após a administração de rosuvastatina. Os níveis de mRNA e proteína da Apo J nas artérias carótidas aumentaram significativamente após administração de rosuvastatina em comparação ao grupo modelo, atingindo o máximo na semana 2, mais cedo em comparação ao grupo modelo (semana 3). Conclusão: A Apo J atuou como reagente de fase aguda após lesão com balão nas artérias carótidas de ratos. A rosuvastatina pode reduzir a formação de neoíntoma por aumento de Apo J. Nossos resultados sugerem que a Apo J exerce um papel protetor na reestenose após lesão com balão em ratos.
Assuntos
Animais , Masculino , Angioplastia Coronária com Balão/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lesões das Artérias Carótidas/tratamento farmacológico , Reestenose Coronária/tratamento farmacológico , Clusterina/efeitos dos fármacos , Anticolesterolemiantes/farmacologia , Fatores de Tempo , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Distribuição Aleatória , Western Blotting , Reprodutibilidade dos Testes , Resultado do Tratamento , Túnica Média/efeitos dos fármacos , Túnica Média/patologia , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Ratos Wistar , Substâncias Protetoras/farmacologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Reestenose Coronária/etiologia , Reestenose Coronária/patologia , Clusterina/análise , Reação em Cadeia da Polimerase em Tempo Real , Rosuvastatina Cálcica/farmacologiaRESUMO
OBJECTIVE: Intimal hyperplasia (IH) contributes to the failure of vascular interventions. While many investigational therapies inhibit the development of IH in animal models, few of these potential therapies can reverse established lesions. Inhaled carbon monoxide (CO) dramatically inhibits IH in both rats and pigs when given perioperatively. It also prevented the development of pulmonary arterial hypertension in rodents. Interestingly, CO could reverse pulmonary artery structural changes and right heart hemodynamic changes when administered after the establishment of pulmonary hypertension. Thus, we hypothesize that inhaled CO may mediate the regression of established neointimal lesions. METHODS: Rats underwent carotid artery balloon angioplasty injury. Carotid arteries were collected at 2 and 4 weeks after injury for morphometric analysis of the neointima. Another group was treated with inhaled CO (250 parts per million) for 1 hour daily from week 2 until week 4. Additional rats were sacrificed 3 days after initiating CO treatment, and the carotid arteries were examined for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end-labeling, proliferation by Ki67 staining, and autophagy by microtubule-associated protein light chain 3 I/II staining. RESULTS: At 2 weeks following injury, sizable neointimal lesions had developed (intimal/media = 0.92 ± 0.22). By 4 weeks, lesion size remained stable (0.80 ± 0.09). Delayed inhaled CO treatment greatly reduced neointimal lesion size vs the 2- and 4-week control mice (0.38 ± 0.05; P < .05). Arteries from the CO-treated rats exhibited significantly reduced apoptosis compared with control vessels (3.18% ± 1.94% vs 16.26% ± 5.91%; P = .036). Proliferation was also dramatically reduced in the CO-treated animals (2.98 ± 1.55 vs 10.37 ± 2.80; P = .036). No difference in autophagy between control and CO-treated rats was detected. CONCLUSIONS: Delayed administration of inhaled CO reduced established neointimal lesion size. This effect was mediated by the antiproliferative effect of CO on medial and intimal smooth muscle cells without increases in arterial wall apoptosis or autophagy. Future studies will examine additional time points to determine if there is temporal variation in the rates of apoptosis and autophagy.
Assuntos
Monóxido de Carbono/administração & dosagem , Lesões das Artérias Carótidas/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Neointima , Administração por Inalação , Angioplastia com Balão , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Artéria Carótida Externa/efeitos dos fármacos , Artéria Carótida Externa/patologia , Modelos Animais de Doenças , Esquema de Medicação , Hiperplasia , Masculino , Músculo Liso Vascular/patologia , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Hypoxia-ischemia on 3-day-old rats (HIP3) allows the investigation of HI damage in the immature brain. HIP3 is characterized for neurological disabilities caused by white matter injury. This study investigates the relationship between animals' sex and injured hemisphere on HIP3 consequences. Male and female Wistar rats had their right or left common carotid artery occluded under halotane anesthesia and exposed to 8% O2 for 1.5 h. Control rats received sham surgery and exposure to 1.5 h of room air in isolation of their mothers. Sex and injured hemisphere influence in Na+/K+ -ATPase activity 24h after lesion: females and the right brain hemispheres showed decreased enzymatic activity after HIP3. Cognitive impairment was observed in step-down inhibitory avoidance, in which females HIP3 left injured were the most damaged. Histological analysis showed a trend to white matter damage in females left injured without hemispherical nor hippocampal volume decrease in HIP3 rats at postnatal day 21. However, at PND90, hemisphere and sex effects were noted in hemispherical volume and myelination: left brain hemisphere and the females evidenced higher histological damage. Our results points to an increased resistance of male rats and right brain hemisphere to support the impairment caused in Na+/K+ -ATPase activity early after HIP3, and evidencing more discrete behavioral impairments and histological damage at adulthood. Present data adds new evidence of distinct effects of brain lateralization and sex vulnerability on biochemical, behavioral and histological parameters after hypoxia-ischemia.
Assuntos
Encéfalo/patologia , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/psicologia , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/fisiologia , Encéfalo/enzimologia , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/psicologia , Feminino , Lateralidade Funcional/fisiologia , Hipóxia-Isquemia Encefálica/enzimologia , Masculino , Atividade Motora/fisiologia , Fibras Nervosas Mielinizadas/patologia , Ratos , Ratos Wistar , Fatores Sexuais , ATPase Trocadora de Sódio-Potássio/análiseRESUMO
The purpose from this study was to investigate the consequences of sensory neurocompensation to carotid balloon injury in diabetic rats on angiotensin II-induced contraction and basal blood flow in contralateral carotid. Concentration-response curves for angiotensin II and blood flow were obtained in contralateral carotid from non-treated or capsaicin-treated streptozotocin-induced diabetic rats that underwent carotid balloon injury. Diabetes increased angiotensin II-induced contraction and impaired the blood flow in non-operated rat carotid. In diabetic rats, balloon injury led to neointima formation, which reduced the blood flow in ipsilateral carotid. Carotid balloon injury in diabetic rats reduced angiotensin II-induced contraction and restored the blood flow in contralateral carotid when compared to diabetic non-operated rat carotid. Capsaicin inhibited the effects evoked by carotid balloon injury on diabetic rat contralateral carotid. Endothelium removal, PEG-catalase (hydrogen peroxide scavenger) or l-NPA (neuronal nitric oxide synthase, nNOS, inhibitor) increased angiotensin II-induced contraction in contralateral carotid from diabetic operated rats to the levels observed in diabetic non-operated rat carotid. Our findings suggest that carotid balloon injury in diabetic rats elicits a neurocompensation that attenuates the diabetic hyperreactivity to angiotensin II in contralateral carotid by a sensory nerves-dependent mechanism mediated by hydrogen peroxide derived from endothelial nNOS. This sensory mechanism also restored the blood flow in this vessel, compensating the impaired blood flow in diabetic rat ipsilateral carotid. Thus, our major conclusions are that Diabetes confers a vasoprotective significance to the neurocompensation to carotid balloon injury in preventing further damage at carotid cerebral irrigation after angioplasty in diabetic subjects.
Assuntos
Angioplastia com Balão/efeitos adversos , Lesões das Artérias Carótidas/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Angiotensina II , Animais , Aorta/fisiopatologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Circulação Cerebrovascular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Peróxido de Hidrogênio/metabolismo , Masculino , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Wistar , Superóxidos/metabolismo , Vasoconstrição/fisiologiaRESUMO
BACKGROUND: Previous studies from our laboratory demonstrated that gremlin significantly increases vascular smooth muscle cell (VSMC) proliferation and migration. The present study investigates gremlin expression in the initial stages of rat carotid balloon injury and its effects on VSMC apoptosis. METHODS: Gremlin mRNA expression was evaluated in rat carotids and cultured VSMCs by quantitative PCR. Apoptosis was analyzed in A7r5 cells and rabbit primary VSMCs following gremlin gene overexpression or silencing by chromatin morphology and caspase-3 activity. RESULTS: Vascular injury promoted a significant decrease in gremlin mRNA levels. In addition, platelet-derived growth factor, angiotensin II and transforming growth factor (TGF)-beta1 promoted coordinated regulation of gremlin and bone morphogenetic protein (BMP)-4 expression in opposite directions according to the confluence status of VSMC culture. In A7r5 cells, gremlin overexpression was able to increase apoptosis, as demonstrated by chromatin morphology and caspase-3 activity, while BMP administration promoted opposite effects. Finally, in agreement with our results, gremlin gene silencing effectively suppressed apoptosis in A7r5 cells and rabbit VSMCs. CONCLUSION: Gremlin is regulated by growth factors and vascular injury and is involved in modulation of VSMC apoptosis. Modifications of gremlin expression during vascular injury may contribute to the apoptosis resistance of VSMCs.
Assuntos
Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Angioplastia com Balão/efeitos adversos , Angiotensina II/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Montagem e Desmontagem da Cromatina , Citocinas , Modelos Animais de Doenças , Masculino , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
INTRODUCTION: p27(Kip1) is a cyclin kinase inhibitor that induces cell cycle arrest. In this study, the efficacy of fusion protein TAT- p27(Kip1) to inhibit cell proliferation in rat perivascular injured carotid arteries was tested. METHODS: The cDNA of p27(Kip1) and GFP (green fluorescein protein) fused to the TAT epitope, which allows cell penetration, yielded TAT-p27 (Kip1) and TAT-GFP fusion proteins. In vitro biological activity on cell proliferation was evaluated by [(3)H] thymidine DNA incorporation in rabbit aortic endothelial cells (REC). An in vivo model used a silicone collar filled with saline positioned around the carotid vessel for 14 days to produce an increased adventitia cross-sectional area. RESULTS: TAT-p27(Kip1) inhibited REC proliferation in vitro using either 100, 200, and 500 nM compared to control (88.2 +/- 4.4, 81.3 +/- 7, 71.9 +/- 4.2 vs. 100 +/- 6.7%, N = 3, respectively, p < 0.05). This response was stable for purified proteins stored at -20*C for at least 23 days. In vivo , TAT-p27(Kip1) solution reduced adventitia cross-sectional area in a dose-dependent manner compared to TAT-GFP (area in mm(2) - TAT-p27(Kip1): 200 nM, 0.160 +/- 0.018; 500 nM, 0.050 +/- 0.005 vs. TAT-GFP: 500 nM, 0.595 +/- 0.066 vs. the contralateral: 0.047 +/- 0.005, N = 7, p < 0.01). CONCLUSION: Taken together, these results provide evidence that TAT-p27(Kip1) can inhibit vascular cells proliferation. It is the first successful demonstration that the cell permeable TAT-p27(Kip1) has potential as a vascular anti-proliferative agent.
Assuntos
Artérias Carótidas/citologia , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Produtos do Gene tat , Animais , Aorta/citologia , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/citologia , Epitopos , Masculino , Ratos , Ratos WistarRESUMO
PURPOSE: To compare through digital morphometry, the intimal thickening of the common ilíac arteries (CIA) in pigs, submitted to angioplasty and angioplasty followed by stent implantation. METHODS: Angioplasty was performed in 10 pigs in both CIA, followed by a stent implantation in the left CIA. After four weeks, the aorto-iliac segment was dissected. Histologic slices where divided in three groups: left CIA in the area of the proximal (group 1) and distal (group 2) implantation site of the stent and the region of angioplasty in the right CIA (group 3). The histological slices were digitalized to be analyzed by morphometry with calculation of the luminal, intimal and media layers areas. Descriptive statistics analysis was performed through average and standard deviation of areas on each group, using ANOVA, with LSD Post-Hoc test (p<0.05). RESULTS: A significant statistic difference was observed when the median area of intimal layer of the groups 1 and 2 where compared with group 3 and in the media layer area of the groups 1 and 2 when compared with group 3. No difference was observed in luminal areas among the three groups. CONCLUSION: Angioplasty followed by stent insertion produces an intimal thickening larger than that observed after simple angioplasty. However, the area of the media layer is smaller in the "angioplasty plus stent" group and there is no significant change in the luminal area among the three groups.
Assuntos
Angioplastia com Balão/efeitos adversos , Lesões das Artérias Carótidas/patologia , Artéria Ilíaca/patologia , Stents/efeitos adversos , Túnica Íntima/patologia , Análise de Variância , Animais , Artéria Carótida Primitiva/patologia , Modelos Animais de Doenças , SuínosRESUMO
OBJETIVO: Analisar, por meio da morfometria digital, o espessamento intimal das artérias ilíacas comuns (AIC) de suínos, submetidas à angioplastia e à angioplastia seguida do implante de stent. MÉTODOS: Em dez suínos, foi realizada a angioplastia da AIC bilateral, seguida do implante do stent na AIC esquerda. Após quatro semanas, o segmento aorto-ilíaco foi retirado. As lâminas histológicas foram divididas em três grupos: segmento proximal (grupo 1) e distal (grupo 2) do local de implante do stent na AIC esquerda e a área da angioplastia da AIC direita (grupo 3). As imagens das lâminas foram digitalizadas e analisadas por programa de morfometria com cálculo das áreas luminal, da camada íntima e da camada média dos cortes histológicos. A análise estatística foi realizada através de média e desvio padrão das áreas em cada grupo, utilizando ANOVA, com teste Post-Hoc LSD (p<0,05). RESULTADOS: Na análise das médias das áreas obtidas, foi encontrada diferença estatisticamente significativa quanto à camada íntima dos grupos 1 e 2, quando comparados ao grupo 3 e em relação à camada média dos grupos 1 e 2 quando comparados ao grupo 3 e não se observou diferença significativa nas médias das áreas luminais dos três grupos. CONCLUSÃO: A angioplastia seguida do implante do stent gerou um espessamento intimal maior do que aquele produzido apenas pela angioplastia, porém, a área da camada média apresentou-se diminuída nos grupos "angioplastia + stent"; a luz arterial não apresentou diferença entre estes grupos.
Assuntos
Animais , Angioplastia com Balão/efeitos adversos , Lesões das Artérias Carótidas/patologia , Artéria Ilíaca/patologia , Stents/efeitos adversos , Túnica Íntima/patologia , Análise de Variância , Artéria Carótida Primitiva/patologia , Modelos Animais de Doenças , SuínosRESUMO
The rabbit carotid artery, injured by silicone collar, presents a perivascular inflammatory response and alterations in vascular responsiveness. Considering that angiotensin II (Ang II) plays an important role in cardiovascular physiology and pathology and that cardiovascular disease increases in postmenopausal women, the aim of this study was to investigate whether the Ang II contractile response in ovariectomized rat carotid artery is modified after a vascular injury by silicone collar. The positioning of the silicone collar around the common carotid artery for 14 days leads to an increased cross-sectional area of adventitial layer with inflammatory cells and an extensive angiogenesis. The Ang II-induced contraction was significantly decreased in collared arteries when compared with contralateral arteries. The reduction in the constrictor effect of Ang II in collared arteries was not modified by the presence of indomethacin (a non-selective inhibitor of cyclooxygenase) or PD 123,319 (a selective antagonist of the Ang II AT2 receptor). Moreover, while endothelium removal induced an increase in the Ang II responsiveness of both arteries (collared and contralateral), the Emax induced by Ang II was still lower in collared arteries. However, the "in vitro" pretreatment of the arteries with an inhibitor of nitric oxide synthase enzyme (L-NAME) significantly enhanced the maximal contractions response to Ang II only in injured arteries. Furthermore, the expression of iNOS (inducible nitric oxide synthase) was observed in the adventitial layer of collared arteries, indicating that the NO formed in the adventitial layer has an important role in injured arteries. Moreover, our data show impairment of extracellular calcium mobilization, mediated by Ang II, in the collared artery, although the intracellular calcium mobilization was not modified by the injury. In conclusion, the increased production of NO and a decrease in the calcium influx displayed by Ang II in the collared artery appears to counteract and reduce the biologic effect of Ang II.