RESUMO
Recent evidence indicates that the GTPase activated Rho/Rho-kinase pathway contributes angiotensin II-induced cardiac hypertrophy and vascular remodeling. We tested this hypothesis in vivo by determining the effects of fasudil, a Rho-kinase inhibitor, on angiotensin II-induced cardiac hypertrophy, coronary vascular remodeling, and ventricular dysfunction. Six-month-old apolipoprotein E deficient (apoE-KO) mice were subcutaneously infused with angiotensin II (1.44 mg/kg/day) using an osmotic mini-pump. Mice were randomly assigned to either vehicle or fasudil (136 or 213 mg/kg/day in drinking water) group. Infusion of angiotensin II for 4 weeks resulted in cardiac enlargement, myocyte hypertrophy, and myocardial interstitial and coronary artery perivascular fibrosis. These changes were accompanied by reduced aortic flow velocity and acceleration rate. Cardiac gene expression levels of atrial natriuretic peptide (ANP) and collagen type III detected by real-time reverse transcriptase polymerase chain reaction were significantly increased in angiotensin II-infused mice. Treatment with fasudil dose-dependently attenuated angiotensin II-induced cardiac hypertrophy, prevented perivascular fibrosis, blunted the increase in ANP and collagen type III expression, and improved cardiac function, without changing blood pressure. These data are consistent with a role for Rho-kinase activation in angiotensin II-induced cardiac remodeling and vascular wall fibrosis.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Angiotensina II/farmacologia , Apolipoproteínas E/genética , Cardiomegalia/prevenção & controle , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apolipoproteínas E/metabolismo , Fator Natriurético Atrial/genética , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Colágeno Tipo III/genética , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Relação Dose-Resposta a Droga , Fibrose/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Quinases Associadas a rhoRESUMO
BACKGROUND: Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein-coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II-induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were infused with Ang II (1.44 mg x kg(-1) x d(-1)) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg x kg(-1) x d(-1) in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. CONCLUSIONS: These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II-induced AAA through inhibition of both apoptosis and proteolysis pathways.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/tratamento farmacológico , Apolipoproteínas E/deficiência , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Apoptose , Endotélio Vascular/citologia , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Inibidores de Proteases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinases Associadas a rhoRESUMO
Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Humanos , Concentração de Íons de Hidrogênio , Micelas , Microquímica/métodosRESUMO
The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.
Assuntos
Aorta/citologia , Apolipoproteínas E/genética , Macrófagos/citologia , Receptores de Superfície Celular/metabolismo , Vasculite/fisiopatologia , Animais , Aorta/imunologia , Arteriosclerose/imunologia , Arteriosclerose/fisiopatologia , Antígeno CD11b/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Humanos , Rim/citologia , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Macrófagos/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais/imunologia , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vasculite/imunologiaRESUMO
Sequence profile and fold recognition methods identified mammalian purple acid phosphatase (PAP), a member of a dimetal-containing phosphoesterase (DMP) family, as a remote homolog of human acid sphingomyelinase (ASM). A model of the phosphoesterase domain of ASM was built based on its predicted secondary structure and the metal-coordinating residues of PAP. Due to the low sequence identity between ASM and PAP (approximately 15%), the highest degree of confidence in the model resides in the metal-binding motifs. The ASM model predicts residues Asp 206, Asp 278, Asn 318, His 425, and His 457 to be dimetal coordinating. A putative orientation for the phosphorylcholine head group of the ASM substrate, sphingomyelin (SM), was made based on the predicted catalysis of the phosphorus-oxygen bond in the active site of ASM and on a structural comparison of the PAP-phosphate complex to the C-reactive protein-phosphorylcholine complex. These complexes revealed similar spatial interactions between the metal-coordinating residues, the metals, and the phosphate groups, suggesting a putative orientation for the head group in ASM consistent with the mechanism considerations. A conserved sequence motif in ASM, NX3CX3N, was identified (Asn 381 to Asn 389) and is predicted to interact with the choline amine moiety in SM. The resulting ASM model suggests that the enzyme uses an SN2-type catalytic mechanism to hydrolyze SM, similar to other DMPs. His 319 in ASM is predicted to protonate the ceramide-leaving group in the catalysis of SM. The putative functional roles of several ASM Niemann-Pick missense mutations, located in the predicted phosphoesterase domain, are discussed in context to the model.
Assuntos
Modelos Moleculares , Esfingomielina Fosfodiesterase/química , Fosfatase Ácida/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Glicoproteínas/química , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Estrogens have been shown to exert significant benefits on the cardiovascular system both in animals and in postmenopausal women. However, the exact mechanism of these effects are, for the most part, still unknown. The goal of this paper is to evaluate the role of estrogen receptors (ER) in mediating some of the cardiovascular beneficial actions of 17 beta-estradiol (E2). This analysis was possible because of the availability of ER alpha (ER alpha KO) and ER beta-deficient (ER beta KO) mice, and access to a patient with ER alpha-deficiency. Experimental results obtained in our laboratory demonstrated that the ER alpha subtype mediates E2-induced increase in endothelial nitric oxide production and facilitation of fibroblast growth factor-elicited angiogenesis in vivo. Others have confirmed these findings. Experiments using a novel ER-antagonist and ApoExER alpha double-knockout mice proved that ER alpha mediates some of the antiatherosclerotic effects of E2 as well. In contrast, both the ER alpha and ER beta subtypes appear to mediate the beneficial effects of E2 on vascular smooth muscle proliferation after vessel injury. The young male patient with ER alpha-deficiency exhibited reduced endothelial nitric oxide production and premature coronary arteriosclerosis. These studies in mice and a male human subject suggest that absence of functional ER may represent a novel risk factor for cardiovascular diseases.
Assuntos
Sistema Cardiovascular/metabolismo , Receptores de Estrogênio/fisiologia , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Estradiol/farmacologia , Estradiol/uso terapêutico , Humanos , Receptores de Estrogênio/genéticaRESUMO
Animal studies evaluating gender difference, the effects of gonadectomy and estrogen replacement and clinical studies in post-menopausal women with and without estrogen replacement therapy (ERT) proved that estrogen exerts significant benefits on the cardiovascular system. Since effects on the plasma lipoprotein profile is responsible for only approximately 25-40% of the cardiovascular protection exerted by estrogens, it is postulated that direct effects of estrogen on the vascular wall must play an important role. Indeed, experimental and clinical evidence accumulated over the past decade, and reviewed briefly here, indicate that at least a part of cardiovascular benefits of 17 beta-estradiol can be attributed to the direct effect of the ovarian sex steroid hormone on vascular endothelial cells. Maintenance and upregulation of endothelial nitric oxide production and suppression of EDCF generation by 17 beta-estradiol may play an important role in preventing or reversing endothelial dysfunction, associated with atherosclerosis, hypertension and other cardiovascular diseases. Stimulation of angiogenesis (especially collateral vessel formation in ischemic tissues) by the ovarian steroid hormone could be beneficial in coronary artery disease, peripheral vascular disease, cerebral ischemia (stroke) and congestive heart failure. Despite these indisputable beneficial effects, several key questions remain to be answered in the future, including the better understanding of the apparently opposite effects of estrogen on prevention of cardiovascular disease vs. treatment of existing disease.