RESUMO
Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Flavonoides/farmacologia , Hipertensão/prevenção & controle , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Angiotensina II , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Fosforilação , Ratos WistarRESUMO
BACKGROUND: Previous studies have indicated that the JAK/STAT signaling pathway is involved in modulating arterial adventitia inflammation response. In this study, we designed experiments to further investigate the effect of JAK2/STAT3/SOCS3 signaling in rabbit atherosclerosis process. METHODS: Atherosclerosis was induced in the abdominal arteries of rabbits by balloon injury of the aorta supplemented by the atherogenic diet. Simultaneously, in the process of atherosclerosis, animals underwent either ruxolitinib treatment or not for 12 weeks. At the end of the experimental period, all rabbits were sacrificed. The plaque areas in abdominal artery, the lipid burden of plaque and the calcium burden of plaque were detected by H&E staining, Oil Red O staining and Alizarin Red staining, respectively. In addition, rabbit plasma lipids and inflammatory cytokines were measured by biochemical test kits or ELISA kits. Finally, the expression and phosphorylation levels of JAK2/STAT3/SOCS3 pathway-related proteins were detected by RT-qPCR, western blot and immunohistochemistry assays. RESULTS: H&E staining and CT scan analysis showed that rabbit atherosclerosis model was constructed successfully. Ruxolitinib, an inhibitor of the Janus kinase 2 (JAK2), substantially reduced the area of atherosclerotic plaques in rabbits treated with high fat diet and balloon injury of the aorta. Moreover, ruxolitinib significantly decreased IL-6, IL-1ß, IFN-γ and TNF-α, but increased IL-10 and IL-17 levels in plasma of atherosclerotic rabbits. Additionally, ruxolitinib reduced plasma TC, TG and LDL-C contents and AIP value, while enhanced HDL-C level in atherosclerotic rabbits. Furthermore, we found that JAK2 and STAT3 phosphorylation were up-regulated in rabbits with atherosclerosis when compared with those of the control group, followed by the expression of SOCS3 was also increased due to the activation of JAK2 and STAT3. Interestingly, ruxolitinib could inactivate JAK2 and STAT3 pathway and decrease SOCS3 expression. CONCLUSION: Taken together, the inhibition of JAK2/STAT3/SOCS3 signaling pathway may be a novel method for the clinical treatment of artery atherosclerosis.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Janus Quinase 2/antagonistas & inibidores , Inibidores de Janus Quinases/farmacologia , Placa Aterosclerótica , Pirazóis/farmacologia , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Doenças da Aorta/sangue , Doenças da Aorta/enzimologia , Doenças da Aorta/patologia , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/patologia , Citocinas/sangue , Modelos Animais de Doenças , Mediadores da Inflamação/sangue , Janus Quinase 2/metabolismo , Lipídeos/sangue , Masculino , Nitrilas , Fosforilação , Pirimidinas , Coelhos , Transdução de SinaisRESUMO
Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.
Assuntos
Antioxidantes/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Taurina/farmacologia , Angiotensina II , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/genética , Cloreto de Cálcio , Modelos Animais de Doenças , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Neutrófilos/enzimologia , Elastase Pancreática , Peroxidase/deficiência , Peroxidase/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMO
Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients (n=225) compared with the control group (n=100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Galectina 3/antagonistas & inibidores , Galectina 3/sangue , Elastase Pancreática , Pectinas/farmacologia , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/patologia , Proteínas Sanguíneas , Estudos de Casos e Controles , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Galectina 3/genética , Galectina 3/metabolismo , Galectinas , Humanos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação , RNA Mensageiro/sangue , RNA Mensageiro/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para CimaRESUMO
OBJECTIVE: Dipeptidyl peptidase-4 (DPP-4) inhibitor, a novel antidiabetic drug, has a cardioprotective effect on ischemia-reperfusion injury through an antioxidant effect. However, the effect of DPP-4 inhibitor on aneurysm formation has not been investigated. We aimed to test the hypothesis that the DPP-4 inhibitor, alogliptin, attenuates vascular oxidative stress and thus inhibits abdominal aortic aneurysm (AAA) formation. METHODS: AAAs were created with intraluminal elastase and extraluminal calcium chloride in 36 male rats. Rats were divided into three groups: a low dose of alogliptin group (group LD; 1 mg/kg/d), a high-dose group (group HD; 3 mg/kg/d), and a control group (group C, water). Alogliptin was administered by gastric gavage once daily beginning 3 days before surgery. On day 7 after aneurysm preparation, reactive oxygen species (ROS) expression was semiquantified by dihydroethidium staining, and the oxidation product of DNA produced by ROS, 8-hydroxydeoxyguanosine (8-OHdG), was measured by immunohistochemical staining. Blood glucose concentrations were measured. Hematoxylin and eosin and elastica Van Gieson stainings were performed on day 28, and the AAA dilatation ratio was calculated. RESULTS: On day 7 (six in each group), dihydroethidium staining of the aneurysm wall showed a reduced level of ROS expression (4.6 ± 0.6 in group C, 2.7 ± 0.3 in group LD, and 1.7 ± 0.5 in group HD; P < .0001) and showed fewer 8-OHdG-positive cells in alogliptin-treated samples (138.1 ± 7.4 cells in group C, 102.5 ± 4.5 cells in group LD, and 66.1 ± 4.5 cells in group HD; P < .0001) The treatment significantly reduced messenger RNA expression of matrix metalloproteinases (MMPs) in aneurysm walls (relative expression: MMP-2: 2.1 ± 0.4 in group C, 1.3 ± 0.3 in group LD, and 0.9 ± 0.2 in group HD; P < .001; MMP-9: 2.0 ± 0.5 in group C, 0.3 ± 0.3 in group LD, and 0.3 ± 0.2 in group HD; P < .001). On day 28 (six in each group), the aortic wall in groups LD and HD was less dilated (dilatation ratio: 199.2% ± 11.8% in group C, 159.6% ± 2.8% in group LD, and 147.1% ± 1.9% in group HD; P < .02 group C vs HD) and had higher elastin content than in group C. The difference in blood glucose levels among the three groups was not significant. CONCLUSIONS: The DPP-4 inhibitor, alogliptin, attenuates aneurysm formation and expansion dose-dependently in a rat AAA model via an antioxidative action.
Assuntos
Antioxidantes/administração & dosagem , Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Piperidinas/administração & dosagem , Uracila/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Administração Oral , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Cloreto de Cálcio , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Dilatação Patológica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Elastase Pancreática , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Uracila/administração & dosagemRESUMO
BACKGROUND: Hyperbaric oxygen (HBO) mitigates ischemia-reperfusion (IR) injury via a nitric oxide mechanism that is nitric oxide synthase (NOS) dependent. The purpose of this study was to investigate this NOS-dependent mechanism by examining isoform-specific, tissue-specific, and time-specific upregulation of NOS mRNA, protein, and enzymatic activity. METHODS: We raised a gracilis flap in Wistar rats that were separated into early and late phases. Treatment groups included nonischemic control, IR, HBO-treated ischemia-reperfusion (IR-HBO), and nonischemic HBO control. We harvested tissue-specific samples from gracilis, rectus femoris, aorta, and pulmonary tissues and processed them by reverse transcription polymerase chain reaction and Western blot to determine upregulation of isoform-specific NOS mRNA and protein. We also harvested tissue for NOS activity to investigate upregulation of enzymatic activity. Data are presented as mean ± standard error of the mean with statistics performed by analysis of variance. P ≤ 0.05 was considered significant. RESULTS: There was no increase in NOS mRNA in the early phase. In the late phase, there was a significant increase in endothelial-derived NOS (eNOS) mRNA in IR-HBO compared with IR in gracilis muscle (79.4 ± 22.3 versus 36.1 ± 4.5; P < 0.05) and pulmonary tissues (91.0 ± 31.2 versus 30.2 ± 3.1; P < 0.01). There was a significant increase in the late-phase eNOS pulmonary protein IR-HBO group compared with IR (235.5 ± 46.8 versus 125.2 ± 14.7; P < 0.05). Early-phase NOS activity was significantly increased in IR-HBO compared with IR in pulmonary tissue only (0.049 ± 0.009 versus 0.023 ± 0.003; P < 0.05). CONCLUSIONS: The NOS-dependent effects of HBO on IR injury may result from a systemic effect involving an early increase in eNOS enzymatic activity followed by a late-phase increase in eNOS protein expression within the pulmonary tissues.
Assuntos
Oxigenoterapia Hiperbárica , Óxido Nítrico Sintase/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Aorta Abdominal/enzimologia , Pulmão/enzimologia , Masculino , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologiaRESUMO
BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is thought to play a central role in abdominal aortic aneurysm (AAA) initiation. Doxycycline, a tetracycline analogue, has direct MMP-9-inhibiting properties in vitro, and it effectively suppresses AAA development in rodents. Observed inhibition of AAA progression, and contradictory findings in human studies evaluating the effect of doxycycline therapy on aortic wall MMP-9, suggest that the effects of doxycycline extend beyond MMP-9 inhibition and that the effect may be dose-dependent. METHODS: This clinical trial evaluated the effect of 2 weeks of low- (50 mg/d), medium- (100 mg/d), or high-dose (300 mg/d) doxycycline vs no medication in four groups of 15 patients undergoing elective AAA repair. The effect of doxycycline treatment on MMP and cysteine proteases, and their respective inhibitors, was evaluated by quantitative polymerase chain reaction, Western blot analysis, immunocapture protease activity assays, and immunohistochemistry. RESULTS: Doxycycline was well tolerated and no participants dropped out. Doxycycline treatment reduced aortic wall MMP-3 and MMP-25 messenger RNA expression (P < .045 and P < .014, respectively), selectively suppressed neutrophil collagenase and gelatinase (MMP-8 and MMP-9) protein levels (P < .013 and <.004, respectively), and increased protein levels of the protease inhibitors tissue inhibitor of metalloproteinase 1 and cystatin C (P < .029). As for the apparent selective effect on neutrophil-associated proteases, we sought for a reducing effect on aortic wall neutrophil content that was indeed confirmed by immunohistochemical analysis that revealed a 75% reduction in aneurysm wall neutrophil content (P < .001). CONCLUSIONS: Independent of its dose, short-term preoperative doxycycline therapy improves the proteolytic balance in AAA, presumably through an effect on aortic wall neutrophil content. This study provides a rationale for doxycycline treatment in patients with an AAA as well as in other (vascular) conditions involving neutrophil influx such as Kawasaki disease and Behçet disease.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/tratamento farmacológico , Fármacos Cardiovasculares/uso terapêutico , Doxiciclina/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Inibidores de Proteases/uso terapêutico , Procedimentos Cirúrgicos Vasculares , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal/enzimologia , Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/cirurgia , Cistatina C/metabolismo , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Estudos Prospectivos , RNA Mensageiro/metabolismo , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development. METHODS AND RESULTS: Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl2-mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells. CONCLUSIONS: Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs.
Assuntos
Aneurisma da Aorta Abdominal/tratamento farmacológico , Elastina/efeitos dos fármacos , Taninos Hidrolisáveis/uso terapêutico , Administração Tópica , Animais , Aorta Abdominal/química , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/patologia , Calcinose/induzido quimicamente , Calcinose/etiologia , Cloreto de Cálcio/toxicidade , Células Cultivadas/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/efeitos dos fármacos , Taninos Hidrolisáveis/administração & dosagem , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The ability to modify the enzymatic processes involved in promoting atherosclerotic plaque disruption and to serially monitor atherosclerotic evolution could provide novel information in the management of patients with atherosclerosis. We studied the effects of a statin (atorvastatin) and its combination with an acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor (avasimibe) on atherosclerotic regression and plaque stability as measured by matrix metalloproteinase 1 and 3 (MMP-1 and MMP-3) levels. Watanabe Heritable Hyperlipidemic (WHHL) rabbits treated with atorvastatin alone experienced an attenuated increase in atherosclerotic burden versus controls as determined by MR imaging. The mean vessel wall area (VWA) prior to drug therapy was 5.57 +/- 0.01 mm2. The VWA increased to 6.71 +/- 0.03 and 7.16 +/- 0.03 mm2, respectively, in atorvastatin-treated and control groups (p < 0.0001 for both). The combination of atorvastatin and avasimibe induced a significant regression of the previously established atherosclerotic lesions, with the VWA decreasing to 4.54 +/- 0.04 mm2 (p = 0.009). Atorvastatin alone induced a nonsignificant reduction in the percent staining of MMP-1 in atherosclerotic lesions, but the combination treatment with avasimibe led to a significant reduction versus controls (p = 0.005). However, a reduction in MMP-3 staining was significant for rabbits treated with both atorvastatin alone (p = 0.007) and in combination with avasimibe (p = 0.04) versus controls. In this animal model, the addition of avasimibe to atorvastatin has beneficial effects on both atherosclerotic plaque regression and stabilization.
Assuntos
Acetatos/uso terapêutico , Doenças da Aorta/tratamento farmacológico , Aterosclerose/tratamento farmacológico , Ácidos Heptanoicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipidemias/tratamento farmacológico , Pirróis/uso terapêutico , Esterol O-Aciltransferase/antagonistas & inibidores , Ácidos Sulfônicos/uso terapêutico , Acetamidas , Acetatos/administração & dosagem , Animais , Aorta Abdominal/enzimologia , Aorta Abdominal/lesões , Aorta Abdominal/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Aterosclerose/enzimologia , Aterosclerose/etiologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Atorvastatina , Cateterismo/efeitos adversos , Colesterol/sangue , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Ácidos Heptanoicos/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hiperlipidemias/sangue , Hiperlipidemias/complicações , Hiperlipidemias/genética , Imageamento por Ressonância Magnética , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 3 da Matriz/análise , Pirróis/administração & dosagem , Coelhos , Distribuição Aleatória , Sulfonamidas , Ácidos Sulfônicos/administração & dosagemRESUMO
BACKGROUND AND OBJECTIVES: The vascular extracellular matrix is maintained by a dynamic balance between matrix synthesis and degradation. This equilibrium is disrupted in arterial pathologies such as abdominal aortic aneurysm. Low-level laser irradiation (LLLI) promotes wound healing. However, its effect on smooth muscle cells (SMCs), a central player in these responses, has not been established. The current study was designed to determine the effects of LLLI on arterial SMC proliferation, inflammatory markers, and matrix proteins. STUDY DESIGN/MATERIALS AND METHODS: Porcine primary aortic SMCs were irradiated with a 780 nm laser diode (1 and 2 J/cm(2)). Trypan blue exclusion assay, immunofluorescent staining for collagen I and III, Sircol assay, gelatin zymography, and RT-PCR were used to monitor proliferation; collagen trihelix formation; collagen synthesis; matrix metalloproteinase-2 (MMP-2) activity, and gene expression of MMP-1, MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and IL-1-beta, respectively. RESULTS: LLLI-increased SMC proliferation by 16 and 22% (1 and 2 J/cm(2), respectively) compared to non-irradiated cells (P<0.01 and P<0.0005). Immediately after LLLI, trihelices of collagen I and III appeared as perinuclear fluorescent rings. Collagen synthesis was increased twofold (2 days after LLLI: 14.3+/-3.5 microg, non-irradiated control: 6.6+/-0.7 microg, and TGF-beta stimulated control: 7.1+/-1.2 microg, P<0.05), MMP-2 activity after LLLI was augmented (over non-irradiated control) by 66+/-18% (2 J/cm(2); P<0.05), and MMP-1 gene expression upregulated. However, TIMP-2 was upregulated, and MMP-2 gene expression downregulated. IL-1-beta gene expression was reduced. CONCLUSIONS: LLLI stimulates SMC proliferation, stimulates collagen synthesis, modulates the equilibrium between regulatory matrix remodeling enzymes, and inhibits pro-inflammatory IL-1-beta gene expression. These findings may be of therapeutic relevance for arterial diseases such as aneurysm where SMC depletion, weakened extracellular matrix, and an increase in pro-inflammatory markers are major pathologic components.
Assuntos
Aorta Abdominal/efeitos da radiação , Expressão Gênica/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Metaloproteinases da Matriz/efeitos da radiação , Músculo Liso Vascular/efeitos da radiação , Miócitos de Músculo Liso/efeitos da radiação , Análise de Variância , Animais , Aorta Abdominal/enzimologia , Colágeno/biossíntese , Colágeno/efeitos da radiação , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , SuínosRESUMO
OBJECTIVE: Collagen plays a major role in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. Smokers often have weakened arterial walls associating with aneurysm and thinned atherosclerotic plaque caps leading to rupture and acute coronary syndromes. We hypothesize that these detrimental effects on arterial wall by tobacco are partially mediated by disturbed collagen metabolism. METHODS AND RESULTS: We first investigated the effect of cigarette smoke extracts (CSE) on prolyl-4-hydroxylase (P4H) expression and collagen production in human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCSMCs). After exposure to 0.01-U CSE for 24 h, expression of P4Halpha-a rate limiting subunit of P4H enzyme responsible for the formation of 4-hydroxyproline in mature functional collagen, was significantly down-regulated according to Western blotting and quantitative RT-PCR (HAEC p < 0.01 and HCSMC p < 0.001) when treated by CSE. The decreased P4Halpha expression was corresponded with reduced cellular collagen levels (HAEC p < 0.001 and HCSMC p < 0.001). We also found that one of the cigarette components benzo(a)pyrene exerted similar effect as CSE, but not nicotine or acrolein. We further examined P4H expression in a few human atherosclerotic abdominal aortas. These in vivo data demonstrated that smokers had thinner atherosclerotic cap thickness and lower levels of P4Halpha and collagen. CONCLUSIONS: Our study suggests that cigarette may interfere with one of the key enzymes in arterial wall collagen metabolism, which may be responsible for thin fibrous cap in atherosclerotic lesion, impaired arterial wall extensibility, and increased likelihood of aneurysm in smokers.
Assuntos
Benzo(a)pireno/toxicidade , Colágeno/biossíntese , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Fumar/metabolismo , Acroleína/toxicidade , Aorta Abdominal/enzimologia , Aorta Abdominal/patologia , Células Cultivadas , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Nicotina/toxicidade , Extratos Vegetais/farmacologia , RNA Mensageiro/metabolismo , Fumaça/análise , Fumar/efeitos adversos , Nicotiana/químicaRESUMO
Among components of oxidized low density lipoproteins, cholesterol derivatives oxidized in position 7 inhibit endothelium-dependent arterial relaxation by decreasing the release of the main endothelium-derived relaxing factor, nitric oxide (NO). The aim of the present study was to bring new insights into the molecular mechanism by which 7-ketocholesterol can block the endothelium-dependent arterial relaxation. Superoxide dismutase did not prevent the inhibitory effect of 7-ketocholesterol on endothelium-dependent relaxation, and consistent observations were made whether superoxide dismutase was conjugated or not to polyethylene glycol. In addition, neither glutathione supplementation, nor oxypurinol, i.e. a xanthine oxidase inhibitor could reverse the effect of 7-ketocholesterol, indicating that NO was not inactivated by superoxide anion. A direct alteration of the activity of the calcium-dependent NO synthase could also be ruled out, since identical relaxing effects of the calcium ionophore A23187 were observed whether arterial rings were treated or not with 7-ketocholesterol. 4 Whereas the above observations come in support of an early, inhibitory action of 7-ketocholesterol, the specific blockade of one given subtype of membrane receptors could be discarded, and similar inhibitions were observed when either muscarinic or purinergic receptors were stimulated. Finally, the blockade of protein kinase C activity by chelerythrine arose as the sole relevant tool in preventing the effect of 7-ketocholesterol on the endothelium-dependent relaxation of rabbit aortic rings. In addition, complementary studies on cultured bovine aortic endothelial cells came in direct support of the ability of 7-ketocholesterol to activate PKC. In conclusion, 7-ketocholesterol that is present in human hypercholesterolaemic plasma, in atherosclerotic arteries, and in many processed foods can block the release of NO by vascular endothelial cells through its ability to activate PKC.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Cetocolesteróis/farmacologia , Proteína Quinase C/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Aorta Abdominal/enzimologia , Relação Dose-Resposta a Droga , Endotélio Vascular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Técnicas In Vitro , Proteína Quinase C/antagonistas & inibidores , Coelhos , Vasodilatação/fisiologiaRESUMO
Rabbit abdominal aortic smooth muscle cells (SMC) were stably transfected with the cDNA of porcine phospholipid hydroperoxide glutathione peroxidase (PHGPx) by means of a retroviral gene transfer technique, to create a model for studying cellular processes relevant to atherogenesis. The transfected cells (SMC/PHGPx) had approximately 4-fold higher PHGPx activity when cultured in the presence of selenite whereas the parental cells did not show any significant increase in PHGPx or total GPx activity upon selenium supplementation. In situ functionality of PHGPx was validated by inhibition of linoleic acid hydroperoxide-induced toxicity, dihydrorhodamine oxidation, NFkappaB activation and apoptosis. SMC grown in 1% FCS responded to oxidized LDL (oxLDL) with a marked proliferation, as measured by [3H]thymidine incorporation, irrespective of selenium supplementation. In SMC/PHGPx grown with or without selenite under control conditions or exposed to native LDL, thymidine incorporation was generally depressed. Also, oxLDL-induced proliferation was lower in SMC/PHGPx compared to untransfected SMC up to 24 h of incubation. After 40 h, however, selenite supplementation restored maximum proliferation response to oxLDL in SMC/PHGPx. The results suggest a proliferative effect of endogenous hydroperoxides in SMC. They further reveal that hydroperoxy lipids of oxLDL contribute to the induction of proliferation, but also suggest involvement of hydroxy lipids in the response to oxLDL.
Assuntos
Aorta Abdominal/citologia , Aorta Abdominal/enzimologia , Glutationa Peroxidase/biossíntese , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , NF-kappa B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Transferência de Genes , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Oxirredução , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Coelhos , Selênio/farmacologiaRESUMO
Skeletal muscle and myocardium express microNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, microNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/microNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and microNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor N(G)-nitro-L-arginine. These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.
Assuntos
Aorta Abdominal/enzimologia , Aorta Torácica/enzimologia , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Animais , Elementos Antissenso (Genética) , Western Blotting , Encéfalo/enzimologia , Cálcio/farmacologia , DNA Complementar , Feminino , Técnicas Imunoenzimáticas , Potenciais da Membrana/fisiologia , Músculo Esquelético/enzimologia , Músculo Liso Vascular/enzimologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo III , Nitroarginina/farmacologia , Norepinefrina/farmacologia , Cloreto de Potássio , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Túnica Média/enzimologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasoconstritores/farmacologiaRESUMO
The effect of dietary supplementation with vitamin A on the healing of rat aortas was studied. Rats were divided into two groups: rats fed a standard chow and rats fed the chow supplemented with 150 IU vitamin A palmitate/g diet. Seven rats from each group were fed with the above diets for 7 days, killed with ether, and the abdominal aorta excised and assayed for hydroxyproline content and collagenase activity. Ten rats from each group were fed for 7 days on the above diets and then underwent a longitudinal aortotomy which was sutured with prolene sutures under pentobarbital anesthesia. The rats were maintained on their respective diets for 7 days and then killed with ether, their abdominal aorta was excised and both the segment with the arteriotomy and the adjacent distal normal segment were analyzed for hydroxyproline content and collagenase activity. Seven rats from each group were fed with the above diets and then underwent transverse division of the abdominal aorta and reanastomosis using nylon sutures under pentobarbital anesthesia. Rats were maintained on their respective diets throughout the postoperative period. Seven days later, all rats were killed with ether and the bursting strength of the aortic anastomoses was measured. The results showed that vitamin A supplementation in non-operated animals had no significant effect on aortic hydroxyproline content or collagenase activity. In rats undergoing longitudinal aortotomy and suture, there was a significant increase in hydroxyproline content both at the healing arteriotomy and at the adjacent non-wounded aorta in the vitamin A-supplemented group. There was also a significant increase in bursting strength of the healing aortic anastomosis in the vitamin A-supplemented rats.(ABSTRACT TRUNCATED AT 250 WORDS)