RESUMO
Peroxisome proliferator-activated receptor x03B3; agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor x03B3; agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Macrófagos/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Sequência de Bases , Sítios de Ligação , Cloreto de Cálcio , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/deficiência , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Dados de Sequência Molecular , PPAR gama/agonistas , PPAR gama/metabolismo , Pioglitazona , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Elevation of plasma high-density lipoprotein (HDL) cholesterol concentration reduces cardiovascular mortality and morbidity. HDLs have been shown to possess acute anti-inflammatory, antioxidant, and antithrombotic properties. We hypothesize that HDL therapy can acutely alter local and systemic manifestations of plaque instability. METHODS: Forty patients with early symptomatic carotid disease were randomized to either receive reconstituted HDL (rHDL) 40 mg/kg (n = 20) or placebo (n = 20). Carotid endarterectomies were performed 24 hr later. Plaques were obtained intraoperatively and used for measurement of thrombomodulatory genes expression. Plasma samples were collected before the infusion, 24 and 48 hr later to measure changes in systemic markers of plaque instability. RESULTS: No significant differences were noted in thrombomodulatory genes expression between the 2 groups. Systemic levels of tissue factor, matrix metalloproteinase 9 (MMP-9), and monocyte chemotactic factor-1 (MCP-1) were significantly reduced in the rHDL group. However, the effects on MMP-9 and MCP-1 were abolished in the immediate postoperative period. Although rHDL did not affect plasma interleukin-6 levels 24 hr following the infusion, it prevented the significant postoperative elevation seen in the placebo group. CONCLUSIONS: A single infusion of rHDL can acutely alter plasma biomarkers associated with plaque instability and cardiovascular morbidity.
Assuntos
Artéria Carótida Interna/cirurgia , Estenose das Carótidas/terapia , Endarterectomia das Carótidas , Lipoproteínas HDL/administração & dosagem , Placa Aterosclerótica , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Artéria Carótida Interna/metabolismo , Artéria Carótida Interna/patologia , Estenose das Carótidas/sangue , Estenose das Carótidas/complicações , Estenose das Carótidas/diagnóstico , Estenose das Carótidas/genética , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/sangue , Infusões Intravenosas , Lipoproteínas HDL/sangue , Londres , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. METHODS AND RESULTS: Using the angiotensin II-induced hypercholesterolemic and the CaCl(2)-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II-induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. CONCLUSIONS: Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Lipoproteínas HDL/sangue , Angiotensina II , Animais , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Ruptura Aórtica/sangue , Ruptura Aórtica/etiologia , Ruptura Aórtica/prevenção & controle , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Autofagia , Cloreto de Cálcio , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Injeções Subcutâneas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipoproteínas HDL/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3 structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals. METHODS AND RESULTS: Precontracted thoracic aorta and mesenteric artery segments from normotensive rats were relaxed by all 3 Kv7 activators, with potencies of BMS-204352=S-1>retigabine. We also tested these agents in the coronary circulation using the Langendorff heart preparation. BMS-204352 and S-1 dose dependently increased coronary perfusion at concentrations between 0.1 and 10 µmol/L, whereas retigabine was effective at 1 to 10 µmol/L. In addition, S-1 increased K(+) currents in isolated mesenteric artery myocytes. The ability of these agents to relax precontracted vessels, increase coronary flow, or augment K(+) currents was impaired considerably in tissues isolated from spontaneously hypertensive rats (SHRs). Of the 5 KCNQ genes, only the expression of KCNQ4 was reduced (≈3.7 fold) in SHRs aorta. Kv7.4 protein levels were ≈50% lower in aortas and mesenteric arteries from spontaneously hypertensive rats compared with normotensive vessels. A similar attenuated response to S-1 and decreased Kv7.4 were observed in mesenteric arteries from mice made hypertensive by angiotensin II infusion compared with normotensive controls. CONCLUSIONS: In 2 different rat and mouse models of hypertension, the functional impact of Kv7 channels was dramatically downregulated.
Assuntos
Hipertensão/fisiopatologia , Canais de Potássio KCNQ/fisiologia , Angiotensina II/farmacologia , Animais , Aorta Torácica/fisiologia , Pressão Sanguínea/fisiologia , Carbamatos/farmacologia , Regulação para Baixo/fisiologia , Hipertensão/induzido quimicamente , Técnicas In Vitro , Indóis/farmacologia , Canais de Potássio KCNQ/agonistas , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/fisiologia , Camundongos , Fenilenodiaminas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Vasoconstritores/farmacologiaRESUMO
INTRODUCTION: Abdominal aortic aneurysms (AAA) are associated with inflammation, apoptosis, and matrix degradation. AAA tissue represents the end stage of disease, limiting its utility in identification of factors culpable for initiation of aneurysm development. Recent evidence suggests that AAAs are a local representation of a systemic disease of the vasculature. Morphologic and molecular changes, comparable to those found in the aneurysm wall, have been demonstrated in veins from patients with AAAs. Changes in the vascular tissue proteome of patients with AAAs were investigated, using inferior mesenteric vein (IMV), to gain insight into early molecular changes contributing to AAA development. METHODS: IMV was harvested from 16 patients with AAA and 16 matched controls. Whole IMV lysates were subjected to 2-D difference in gel electrophoresis (2D-DIGE) with quantitative densitometry. Protein spots differentially expressed in AAA were identified using mass spectrometry. Differential protein expression was validated by Western blotting and localized to cell type by immunohistochemistry (IHC). RESULTS: Decreased levels of prohibitin (AAA, 2.00 ± 1.37; controls, 3.81 ± 1.39; 1.9-fold change; P = .02) AAA (7.33 ± 3.9; controls, 14.5 ± 5.6; 2-fold change; P = .001), along with relative increases in a cleaved fragment of vimentin (AAA, 12.9 ± 9; controls, 6.9 ± 4.7; 2-fold change; P = .11) were identified in AAA patients. All proteins were localized to the vascular smooth muscle cells. CONCLUSIONS: Proteins important in combating the injurious effects of oxidative stress and modulating the response to inflammation appear reduced in the vasculature of patients with AAA. These changes may represent early events in AAA formation. Enhancing expression of these proteins might offer a novel therapeutic avenue to inhibit AAA development.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Músculo Liso Vascular/química , Miócitos de Músculo Liso/química , Proteínas/análise , Proteômica , Idoso , Anexina A1/análise , Aorta Abdominal/química , Western Blotting , Estudos de Casos e Controles , Densitometria , Eletroforese em Gel Bidimensional , Feminino , Humanos , Imuno-Histoquímica , Londres , Masculino , Espectrometria de Massas , Veias Mesentéricas/química , Pessoa de Meia-Idade , Proibitinas , Proteômica/métodos , Proteínas Repressoras/análise , Reprodutibilidade dos Testes , Vimentina/análiseRESUMO
BACKGROUND: Development and rupture of aortic aneurysms involve a combination of complex biological processes. Rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, has been shown to have a broad spectrum of effects in vivo. The hypothesis that rosiglitazone would reduce aneurysm expansion or rupture was tested in the angiotensin II (Ang II)-induced hypercholesterolemic mouse model. METHODS AND RESULTS: Apolipoprotein E-deficient mice, 12 months of age, were allocated to 4 groups. Three groups were infused with Ang II (1 microg . min(-1) . kg(-1)), and the fourth was infused with saline. Rosiglitazone was given 1 week before infusion and 1 week after infusion. At day 28, aortic size was measured, and tissues were collected for analyses. Both pretreatment and posttreatment with rosiglitazone inhibited the occurrence of fatal rupture (11 of 30 versus 0 of 30 versus 0 of 15; P=0.0013) and reduced maximal dilatation of the aorta (4.6+/-0.13 versus 2.4+/-0.48 versus 2.15+/-0.46 mm2; P<0.0001). Blood glucose, total cholesterol, body weight, and atherosclerosis did not differ between groups. Pretreatment with rosiglitazone inhibited the Ang II-induced expression of angiotensin type 1a Ang II receptor while having no effect on the angiotensin type 2 Ang II receptor, in addition to reducing Ang II-induced expression of E-selectin, tumor necrosis factor-alpha, and interleukin-6. CONCLUSIONS: Pretreatment or posttreatment with RGZ reduced aortic expansion and rupture in this mouse model. Reduction of lesions in animals pretreated with rosiglitazone is concomitant with decreased expression of inflammatory mediators. Further studies are needed to elucidate the precise mechanism.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Ruptura Aórtica/prevenção & controle , Hipoglicemiantes/farmacologia , Tiazolidinedionas/farmacologia , Angiotensina II/efeitos adversos , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Ruptura Aórtica/sangue , Ruptura Aórtica/induzido quimicamente , Ruptura Aórtica/genética , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Glicemia/análise , Peso Corporal/efeitos dos fármacos , Colesterol/sangue , Modelos Animais de Doenças , Selectina E/sangue , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/induzido quimicamente , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Hipercolesterolemia/prevenção & controle , Interleucina-6/sangue , Camundongos , Camundongos Knockout , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Receptores de Angiotensina/sangue , Rosiglitazona , Fator de Necrose Tumoral alfa/sangueRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) development is associated with increased angiogenesis and overexpression of vascular endothelial growth factor (VEGF). Inhibition of angiogenesis results in attenuation of experimental aneurysms. This study investigated the effects of recombinant human (rh)VEGF on experimental aneurysms. METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were assigned to one of four groups: (1) normal saline infusion (sham), (2) angiotensin-II (AngII) infusion, (3) AngII infusion plus 100 microg daily rhVEGF for 14 days (AngII+14dVEGF), or (4) AngII infusion plus 100 microg daily rhVEGF for 21 days (AngII+21dVEGF). Aortic maximum diameter and cross-sectional area were determined by magnetic resonance imaging and microscopy. All mice were sacrificed at day 28. RESULTS: Aneurysms developed in all mice in the AngII+14dVEGF and AngII+21dVEGF groups by day 21 compared with 40% in the AngII group. Treatment with rhVEGF increased maximum aortic diameter (P < .002) and cross-sectional area of aneurysms (P < .005) at day 21. This effect was maintained at day 28 (P < .0005). Decreasing rhVEGF treatment from 21 to 14 days did not attenuate aneurysm formation. Treatment with rhVEGF upregulated matrix metalloproteinase 2 gene expression within the aortic wall (P < .0009). CONCLUSIONS: Treatment with rhVEGF intensified the formation of AngII-induced aneurysms. Further studies are needed to investigate if antiangiogenic therapy may be a valid medical therapy against aneurysm expansion or rupture.
Assuntos
Angiotensina II , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Apolipoproteínas E/deficiência , Fator A de Crescimento do Endotélio Vascular , Angiotensina II/administração & dosagem , Animais , Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E/genética , Peso Corporal , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Humanos , Bombas de Infusão Implantáveis , Injeções Intraperitoneais , Angiografia por Ressonância Magnética , Masculino , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Knockout , Proteínas Recombinantes/administração & dosagem , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
OBJECTIVE: Matrix metalloproteinase (MMP) activity has been linked to abdominal aortic aneurysm (AAA) rupture. Medial neovascularization (MNV), a histopathologic characteristic of AAAs, involves proteolytic degradation of extracellular matrix by MMPs to facilitate endothelial cell migration. The role of MNV in aneurysm rupture is unknown. This study investigated whether MNV is increased in aneurysm rupture. METHODS AND RESULTS: Biopsy samples from aneurysm rupture edge were compared with control biopsy samples from aneurysm wall at the level of rupture and from anterior sac in 12 ruptured AAAs. Further controls were obtained from anterior sac of 10 nonruptured AAAs. MNV, microvessel diameter, maturity index, and inflammatory infiltrate were quantified using morphometric analyses following immunohistochemistry. Expression of proangiogenic mediators was quantified using quantitative real-time-polymerase chain reaction. Compared with anterior sac and aneurysm wall at level of rupture, MNV was increased (P<0.001) in rupture edge biopsy samples and consisted of smaller diameter (P<0.001) and more immature microvessels (P<0.001). mRNA expression of alpha(v)-integrin, vascular endothelial growth factor, vascular endothelial-cadherin, monocyte chemoattractant protein-1, and vimentin was increased (P<0.05) in rupture edge biopsy samples. CONCLUSIONS: This study demonstrated increased medial neovascularization and overexpression of proangiogenic cytokines at aneurysm rupture edge. Further investigations into whether this angiogenic response was a causative factor of aneurysm rupture are needed.
Assuntos
Aneurisma Roto/complicações , Aneurisma da Aorta Abdominal/complicações , Citocinas/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Túnica Média/patologia , Idoso , Idoso de 80 Anos ou mais , Citocinas/genética , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/patologia , Integrina alfaV/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/etiologia , Neovascularização Patológica/genética , Distribuição TecidualRESUMO
The mechanisms of hypoxia-mediated aneurysm wall weakening and rupture are unknown. During hypoxia, strategies to maintain cellular ATP levels include increasing glycolysis (glycolytic strategy) or decreasing ATP consumption (metabolic depression). This study demonstrated that compared to anterior aneurysm sac, rupture edge overexpressed hypoxia-inducible factor-1-alpha (marker of hypoxia) and showed no significant difference in levels of combined ADP and ATP or lactate (glycolytic end product). Further studies are needed to confirm whether hypoxic AAA cells adapt through metabolic depression rather than glycolysis. The downregulation of protein synthesis during such metabolic depression may be a factor in hypoxia-mediated wall weakening.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Ruptura Aórtica/metabolismo , Hipóxia/metabolismo , Lactatos/metabolismo , Biomarcadores , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
To search for novel transcriptional pathways that are activated in abdominal aortic aneurysm rupture, cDNA microarrays were used to compare global mRNA expression at the aneurysm rupture edge to anterior sac, and selected results were confirmed using quantitative real-time-polymerase chain reaction (QRT-PCR). This study identified apoptosis, angiogenesis, and inflammation as potentially important participants during the process of aneurysm rupture.
Assuntos
Aneurisma da Aorta Abdominal/genética , Ruptura Aórtica/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genéticaRESUMO
Abdominal aortic aneurysm (AAA) rupture is associated with elevated levels of matrix metalloproteinase (MMP). Medial neovascularization is a known characteristic of established AAAs and involves proteolytic degradation of extracellular matrix by MMPs to facilitate endothelial cell proliferation and migration. This study evaluated the extent of neovascularization in abdominal aortic aneurysm rupture. Results indicated upregulation of proangiogenic cytokines and increased medial neovascularization at the aneurysm rupture edge compared with paired aneurysm anterior sac. Further investigations into the role of angiogenesis in aneurysm rupture may open novel therapeutic avenues to prevent aneurysm rupture.
Assuntos
Aneurisma da Aorta Abdominal/patologia , Ruptura Aórtica/patologia , Neovascularização Patológica/patologia , Aneurisma da Aorta Abdominal/genética , Ruptura Aórtica/genética , Biópsia , Citocinas/genética , Regulação da Expressão Gênica , Humanos , Integrina alfa5/genética , Neovascularização Patológica/genéticaRESUMO
Reduction of the remaining residual cardiovascular risk is a clinical unmet need currently being addressed through a combination of further reduction of plasma concentrations of low-density lipoproteins (LDLs) and increasing plasma concentrations of high-density lipoproteins (HDLs). This brief review sets out the so-called HDL hypothesis and summarises the clinical results of the family of drugs, which function to raise plasma HDL concentration through inhibition of cholesteryl ester transfer proteins (CEPT).
RESUMO
BACKGROUND: Since aortic diameter is the most -significant risk factor for rupture, we sought to identify stress-dependent changes in gene expression to illuminate novel molecular processes in aneurysm rupture. MATERIALS AND METHODS: We constructed finite element maps of abdominal computerized tomography scans (CTs) of seven abdominal aortic aneurysm (AAA) patients to map wall stress. Paired biopsies from high- and low-stress areas were collected at surgery using vascular landmarks as coordinates. Differential gene expression was evaluated by Illumina Array analysis, using the whole genome DNA-mediated, annealing, selection, extension, and ligation (DASL) gene chip (n = 3 paired samples). RESULTS: The sole significant candidate from this analysis, Lamin A/C, was validated at the protein level, using western blotting. Lamin A/C expression in the inferior mesenteric vein (IMV) of AAA patients was compared to a control group and in aortic smooth muscle cells in culture in response to physiological pulsatile stretch. -Areas of high wall stress (n = 7) correlate to those -regions which have the thinnest walls [778 µm (585-1120 µm)] in comparison to areas of lowest wall stress [1620 µm (962-2919 µm)]. Induced expression of Lamin A/C -correlated with areas of high wall stress from AAAs but was not significantly induced in the IMV from AAA patients compared to controls (n = 16). Stress-induced expression of Lamin A/C was mimicked by exposing aortic smooth muscle cells to prolonged pulsatile stretch. CONCLUSION: Lamin A/C protein is specifically increased in areas of high wall stress in AAA from patients, but is not increased on other vascular beds of aneurysm patients, suggesting that its elevation may be a compensatory response to the pathobiology leading to aneurysms.
RESUMO
The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.
Assuntos
Músculo Liso Vascular/química , Tropomiosina/química , Tropomiosina/genética , Sequência de Aminoácidos , Animais , Aorta/anatomia & histologia , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fases de Leitura Aberta , Isoformas de Proteínas , Coelhos , Sequências Repetitivas de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Tropomiosina/metabolismo , Regiões não TraduzidasRESUMO
High-density lipoproteins (HDLs) have been shown to reduce the organ injury and mortality in animal models of shock by reducing the expression of adhesion molecules and proinflammatory enzymes. However, there is limited evidence that HDL treatment reduces inflammation. As inflammation plays an important role in the development of colitis as well as ischemia/reperfusion (I/R) injury of the intestine, we have investigated the effects of HDL in animal models of associated with gut injury and inflammation (splanchnic artery occlusion [SAO] shock and dinitrobenzene sulfonic acid [DNBS]-induced colitis). We report here for the first time that the administration of reconstituted HDLs (recHDLs; 80 mg/kg i.v. bolus 30 min prior to ischemia in the SAO-shock model or 40 mg/kg i.v. every 24 h in the colitis model) exerts potent anti-inflammatory effects (e.g., reduced inflammatory cell infiltration and histological injury, and delayed the development of the clinical signs) in vivo. Furthermore, recHDL reduced the staining for nitrotyrosine and poly(ADP-ribose) (immunohistochemistry) and the expression of intercellular adhesion molecule-1 in the ileum of SAO-shocked rats and in the colon from DNBS-treated rats. Thus, recHDL reduces the inflammation caused by intestinal I/R and colitis. HDLs may represent a novel therapeutic approach for the therapy of inflammation of the gut.
Assuntos
Colite/complicações , Colite/patologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Lipoproteínas HDL/farmacologia , Traumatismo por Reperfusão , Animais , Colite/induzido quimicamente , Dinitrofluorbenzeno/análogos & derivados , Dinitrofluorbenzeno/farmacologia , Imuno-Histoquímica , Inflamação/tratamento farmacológico , Inflamação/patologia , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/análise , Lipoproteínas HDL/administração & dosagem , Masculino , Ratos , Ratos Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
The salutary effects of high-density lipoproteins (HDLs) in animal and human models of endotoxic shock have in the past been attributed to the ability of this lipoprotein to bind to lipopolysaccharide. However, the precise mechanisms for the protective effect of HDL are unclear. The first objective of this study was to determine the effects of HDLs on the organ injury and dysfunction associated with acute severe endotoxemia. Second, to gain insight into the mechanism of action of HDL, we also investigated the effect of HDLs on 1) the expression of P-selectin and intercellular adhesion molecule-1 in the kidneys of rats treated with endotoxin and 2) the rise in the plasma levels of tumor necrosis factor-alpha (TNF-alpha). Rats were given Escherichia coli lipopolysaccharide (6 mg/kg i.v.), pretreated with either vehicle (n = 9) or reconstituted HDL (rHDL; apolipoprotein A-I/phosphatidylcholine proteoliposomes, n = 10), and were monitored for 6 h. Here we report that rHDL attenuates the renal injury and dysfunction caused by endotoxin in the rat. In addition, rHDL reduced the degree of histological tissue injury in the lung, liver and intestine and attenuated the expression of P-selectin and intercellular adhesion molecule-1 in the renal glomerulus. Interestingly, pretreatment of rats with rHDL did not prevent the hypotension nor the rise in plasma levels of TNF-alpha (at 90 min) caused by endotoxin. Thus, rHDL reduces the organ injury/dysfunction, but does not affect the circulatory failure, nor the rise in plasma levels of TNF-alpha caused by endotoxin in the rat. We propose that the mechanisms of these beneficial effects of HDL may be related to direct inhibition of adhesion molecule expression.
Assuntos
Lipoproteínas HDL/uso terapêutico , Choque Séptico/terapia , Choque/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Endotoxemia , Endotoxinas/metabolismo , Endotoxinas/farmacologia , Escherichia coli/metabolismo , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/metabolismo , Intestino Delgado/patologia , Rim/metabolismo , Lipase/sangue , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Lesão Pulmonar , Masculino , Selectina-P/biossíntese , Selectina-P/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
There is strong epidemiological evidence that patients with diabetes have a lower incidence of abdominal aortic aneurysm. The precise mechanism of this negative association is unknown. Whilst a number of studies have supported the hypothesis that protection is a function of diabetes-mediated changes in the vascular extracellular matrix biology, there is also support for the idea that the treatment regimens used in diabetes may afford protection against AAA. In particular the pleiotropic drug family, the thiazolidinediones have been examined as candidates to ameliorate aneurysm formation. Both the thiazolidinediones, and the structurally related family, fibrates, have been shown to have anti-inflammatory and antioxidative effects, in addition to ability to modulatate glucose and lipid homeostasis. In this brief review we present the current data exploring the use of thiazolidinediones in experimental aneurysm development. Despite the fact that both thiazolidinediones Rosiglitazone and Pioglitazone are no longer prescribed in Europe and the US, they have provided important insights into the mechanism of action, and the application of other pleiotropic drugs in the treatment of AAA. One such pleiotropic drug is high-density lipoproteins (HDLs), which have been shown to have a broad spectrum of effects, including activation of PPARs, which may favour their use as a new drug target for protection against AAA development.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Diabetes Mellitus/epidemiologia , Tiazolidinedionas/farmacologia , Animais , Aneurisma da Aorta Abdominal/epidemiologia , Diabetes Mellitus/tratamento farmacológico , Humanos , Hipoglicemiantes/farmacologia , Lipoproteínas HDL/metabolismo , Terapia de Alvo Molecular , Receptores Ativados por Proliferador de Peroxissomo/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Pioglitazona , Fatores de Risco , RosiglitazonaRESUMO
OBJECTIVE: Development and rupture of aortic aneurysms (AA) is a complex process involving inflammation, cell death, tissue and matrix remodelling. The thiazolidinediones (TZDs) including Rosiglitazone (RGZ) are a family of drugs which act as agonists of the nuclear peroxisome proliferator-activated receptors and have a broad spectrum of effects on a number of biological processes in the cardiovascular system. In our previous study we have demonstrated that RGZ has a marked effect on both aneurysm rupture and development, however, the precise mechanism of this is unknown. METHODS AND RESULTS: In the present study, we examined possible targets of RGZ action in the early stages of Angiotensin II-induced AA in apolipoprotein E-deficient mice. For this purpose we employed immunoblotting, ELISA and antibody array approaches. We found that RGZ significantly inhibited c-Jun N-terminal kinase (JNK) phosphorylation and down-regulated toll-like receptor 4 (TLR4) expression at the site of lesion formation in response to Angiotensin II infusion in the initiation stage (6-72 h) of experimental AA development. Importantly, this effect was also associated with a decrease of CD4 antigen and reduction in production of TLR4/JNK-dependant proinflammatory chemokines MCP-1 and MIP-1α. CONCLUSION: These data suggest that RGZ can modulate inflammatory processes by blocking TLR4/JNK signalling in initiation stages of AA development.
Assuntos
Aneurisma Aórtico/induzido quimicamente , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Angiotensina II , Animais , Aneurisma Aórtico/prevenção & controle , Ruptura Aórtica/prevenção & controle , Regulação para Baixo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Rosiglitazona , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: Circulating plasma interleukin-6 (IL-6) concentrations are elevated in patients with abdominal aortic aneurysms (AAAs) compared with controls. In vitro studies suggest that the aneurysm is the source of the IL-6. Because IL-6 is an independent risk factor for cardiovascular mortality, elevation of this cytokine may be significant in these patients, who represent a group at increased risk from cardiovascular death. The aim of this study was to directly measure in vivo aortic IL-6 concentrations, testing the hypothesis that aneurysms secrete IL-6 into the circulation. METHODS: Before endovascular aneurysm repair took place, blood was sampled from the entire length of the aorta in 27 patients with AAA and nine with thoracic aneurysms (TAs). A control group consisted of 15 patients without aneurysms undergoing angiography. Plasma IL-6 was determined using enzyme-linked immunosorbent assay, and high-sensitivity C-reactive protein (hs-CRP) was measured turbidimetrically. Aneurysm surface area was calculated from axial computed tomography scans. RESULTS: Mean IL-6 concentrations (pg/mL) were higher in the TA and AAA groups compared with controls (10.4 +/- 3.7 and 4.9 +/- 0.5 vs 2.7 +/- 0.5, P = .002). There was a significant difference in plasma IL-6 concentration corresponding to aneurysm position in the AAA (P = .002) and TA (P = .008) groups, with both patterns conforming to a linear trend. This pattern was not observed in the control group, in which no significant difference in IL-6 concentrations was found throughout the aorta. Peak IL-6 occurred earlier in TAs compared with AAAs (descending aorta vs iliac artery) corresponding to aneurysm position (P = .0007). Linear regression revealed a positive correlation between aneurysm surface area and mean plasma IL-6 (Spearman's correlation, P = .003). The mean surface areas of the TAs, at 0.07 m2 (interquartile range [IQR], 0.06 to 0.09), were higher than those of the AAAs at 0.03 m2 (IQR, 0.02 to 0.04; P = .002). High-sensitivity CRP was within normal limits, and no significant differences were found between the AAA group and the controls. CONCLUSIONS: Circulating IL-6 is elevated within the aorta in patients with aneurysms and corresponds to aneurysm position. Furthermore, aneurysm surface area and mean plasma IL-6 are correlated. In the absence of any evidence of systemic inflammation in the form of elevated hs-CRP, these data support the hypothesis that aneurysms secrete IL-6 into the circulation. This may contribute to the high cardiovascular mortality observed in patients with aneurysms.