RESUMO
BACKGROUND: The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. METHODS AND RESULTS: Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. CONCLUSIONS: Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.
Assuntos
Receptores Frizzled/antagonistas & inibidores , Insuficiência Cardíaca/prevenção & controle , Infarto do Miocárdio/complicações , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Wnt3A/farmacologia , Animais , Linhagem Celular Transformada , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Fibroblastos/efeitos dos fármacos , Receptores Frizzled/metabolismo , Insuficiência Cardíaca/etiologia , Humanos , Masculino , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Ratos , Relação Estrutura-Atividade , Remodelação Ventricular/efeitos dos fármacos , Proteína Wnt3A/antagonistas & inibidoresRESUMO
The biosynthesis of the mineralocorticoid hormone aldosterone involves a multistep hydroxylation of 11-deoxycorticosterone at the 11- and 18-positions, resulting in the formation of corticosterone and 18-hydroxycorticosterone, the final precursor of aldosterone. Two members of the cytochrome P450 11B family, CYP11B1 and CYP11B2, are known to catalyze these 11- and 18-hydroxylations, however, only CYP11B2 can oxidize 18-hydroxycorticosterone to aldosterone. It is unknown what sequence of hydroxylations leads to the formation of 18-hydroxycorticosterone. In this study we have investigated which of the possible conversion paths towards formation of 18-hydroxycorticosterone and aldosterone are most likely from the ligand perspective. Therefore, we combined quantum mechanical investigations on the steroid conformations of 11-deoxycorticosterone and its ensuing reaction intermediates with Fukui indices calculations to predict the reactivity of their carbon atoms for an attack by the iron-oxygen species. Both F(-) and F(0) were calculated to account for different mechanisms of substrate conversion. We show which particular initial conformations of 11-deoxycorticosterone and which conversion paths are likely to result in the successful synthesis of aldosterone, and thereby may be representative for the mechanism of aldosterone biosynthesis by CYP11B2. Moreover, we found that the most likely path for aldosterone synthesis coincides with the substrate conformation proposed in an earlier publication. To summarize, we show that on a theoretical and strictly ligand-directed basis only a limited number of reaction paths in the conversion of 11-deoxycorticosterone to aldosterone is possible. Despite its theoretical nature, this knowledge may help to understand the catalytic function of CYP11B1 and CYP11B2.
Assuntos
Aldosterona/biossíntese , Aldosterona/química , Citocromo P-450 CYP11B2/química , Ligantes , Teoria Quântica , Ferro/química , Estrutura Molecular , Oxigênio/químicaRESUMO
Wnt/beta-catenin signaling is an important regulator of cell polarity, proliferation, and stem cell maintenance during development and adulthood. Wnt proteins induce the nuclear accumulation of beta-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/beta-catenin signaling has been implicated in a plethora of pathologies and, most notably, underlies initiation and expansion of several cancers. Here, we apply enzyme fragment complementation to measure the nuclear accumulation of beta-catenin. beta-Catenin was tagged with a peptide fragment of beta-galactosidase and transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus. Stimulation of the cells with recombinant Wnt-3a restored beta-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses beta-catenin-driven reporter gene activity downstream of nuclear entry of beta-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce beta-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was identified as an activator of Wnt/beta-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous beta-catenin protein and can induce TCF/LEF-dependent gene transcription. Subsequent biochemical profiling of >200 kinases revealed both isoforms of glycogen synthase kinase 3, as previously unappreciated targets of sotrastaurin. We show that the beta-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/beta-catenin pathway and can be used to find new therapeutics targeting Wnt/beta-catenin signaling.-Verkaar, F., Blankesteijn, W. M., Smits, J. F. M., Zaman, G. J. R. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Teste de Complementação Genética/métodos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/farmacologia , beta Catenina/metabolismo , beta-Galactosidase/biossíntese , Animais , Polaridade Celular , Proliferação de Células , Regulação para Baixo/fisiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Células Jurkat , Camundongos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais/fisiologia , Fatores de Transcrição TCF , Transcrição Gênica , Transgenes/fisiologia , Proteína Wnt-5a , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genética , beta-Galactosidase/genéticaRESUMO
Baseline blood pressure (BP) is the strongest known determinant of progression to hypertension, but for an individualized prediction of the incidence of hypertension, the identification of additional biomarkers is crucial. In animal models of hypertension, renal nitric oxide (NO) handling modifies the systemic BP responses prior to the development of hypertension. This study aimed to evaluate whether urinary NO metabolites (NOx) predict the progression of hypertension in normotensive subjects. Among 62 participants enrolled in the Flemish Study on Environment, Genes and Health Outcomes, we assessed progression to hypertension over 4.6 years. In a case-control design, 49 normotensive subjects including 10 subjects with high-normal blood pressure were enrolled of whom 25 remained normotensive (controls), whereas 24 'progressed' to hypertension (progressors). Thirteen hypertensive patients served as negative controls. Urinary NOx concentration, renal function and the urinary excretion of electrolytes were assessed at baseline and follow-up. At baseline, progressors showed higher BP values than controls and urinary NOx concentration was significantly lower in progressors as compared to the normotensive controls (p < 0.01). In all initially normotensive subjects baseline urinary NOx concentration was associated with follow-up BP (r = -0.55, p < 0.001) and the relative increase of BP over time (r = -0.47, p < 0.001). In progressors baseline urinary NOx was associated with follow-up BP (r = -0.52, p < 0.009) and the relative increase of BP over time (r = -0.44, p = 0.033). Baseline urinary NOx and BP were independent predictors for the relative BP increase. A urinary NOx threshold of <130.5 mg/L predicted 75% of all progressors. In context with high-normal baseline BP, 87.5% of all progressors were identified. These findings indicate that urinary NO metabolites are associated with BP development in normotensive subjects. Moreover, urinary NOx predicts the progression to hypertension independent of baseline BP suggesting urinary NOx as a biomarker for individual new-onset hypertension.
Assuntos
Pressão Sanguínea , Hipertensão/fisiopatologia , Hipertensão/urina , Óxido Nítrico/urina , Adolescente , Adulto , Idoso , Bélgica , Biomarcadores/urina , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Progressão da Doença , Regulação para Baixo , Humanos , Rim/fisiopatologia , Modelos Lineares , Pessoa de Meia-Idade , Projetos Piloto , Modelos de Riscos Proporcionais , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Adulto JovemRESUMO
An excessive hypertrophic response of the heart to an increased workload is a leading cause of heart failure. At present, cardiac hypertrophy is treated with inhibitors of the renin-angiotensin system or with beta-adrenoceptor antagonists. These current therapeutic strategies inhibit prohypertrophic signaling pathways, but this therapy is inadequate in a substantial number of patients. However, the hypertrophic response of the heart is the net result of activation of prohypertrophic and antihypertrophic pathways. Glycogen synthase kinase-3 beta (GSK-3 beta) has a powerful antihypertrophic effect, but is inhibited by growth factors and hypertrophic stimuli through phosphorylation at the Ser9 residue of GSK-3 beta. Activation of the Wnt/frizzled pathway also results in inactivation of GSK-3 beta through sequestration of the kinase rather than phosphorylation at Ser9. In this Opinion article we will review the current evidence for the involvement of Wnt/frizzled signaling and the activation of GSK-3 beta in the regulation of cardiac hypertrophy, and subsequently discuss the potential of this pathway to serve as a novel therapeutic approach for cardiac hypertrophy.
Assuntos
Cardiomegalia/tratamento farmacológico , Receptores Frizzled/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Animais , Cardiomegalia/etiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Receptor Cross-Talk , beta Catenina/fisiologiaRESUMO
INTRODUCTION: Intrapericardial (IPC) delivery of antiarrhythmic agents is an appealing idea to increase the therapeutic width and reduce side effects of drugs, particularly in the thin atria. The aim of this study was to determine the effects of IPC versus intravenous (IV) d,l-sotalol and flecainide infusion on transmural atrial electrophysiology and sustained atrial fibrillation (AF) in the goat. METHODS AND RESULTS: Effects of IPC and IV sotalol and flecainide infusion on epi- and endocardial atrial electrophysiology, ECG, and tissue drug concentrations were studied in goats without and with persistent AF (>24 hours). Epicardial atrial refractory period (AERP, bcl 400 ms) increased after 120 minutes of 1 mg/kg/hour IPC sotalol with 61 +/- 8 ms (P = 0.02), whereas the endocardial AERP was not affected. One mg/kg/hour IPC flecainide increased the epicardial pacing threshold and the epicardial AERP with 4 +/- 0.5 mA (P = 0.003) and 33 +/- 11 ms (P = 0.05), respectively. Endocardial values were unchanged. Marked ST-elevations in the precordial ECG leads were observed after IPC flecainide. In the AF group, IPC drugs did not prolong AF cycle length to a greater extent than IV delivery. The number of cardioversions was not different between the two delivery routes. A steep transmural drug concentration gradient after IPC sotalol and flecainide was observed in all heart chambers. CONCLUSION: IPC sotalol and flecainide infusion in goats markedly affects epicardial atrial electrophysiology. IPC delivery, however, does not prolong AFCL or terminate AF to a greater extent than IV infusion. This suggests that the perpetuation of AF is not dominated by the epicardial and sub epicardial atrial layers.
Assuntos
Antiarrítmicos/farmacologia , Fibrilação Atrial/tratamento farmacológico , Cabras/fisiologia , Pericárdio , Sotalol/farmacologia , Animais , Antiarrítmicos/administração & dosagem , Antiarrítmicos/farmacocinética , Fibrilação Atrial/fisiopatologia , Estimulação Elétrica , Eletrocardiografia , Eletrofisiologia , Flecainida/administração & dosagem , Flecainida/farmacocinética , Flecainida/farmacologia , Átrios do Coração , Injeções , Injeções Intravenosas , Pericárdio/fisiopatologia , Sotalol/administração & dosagem , Sotalol/farmacocinéticaRESUMO
Reversal of cardiac fibrosis is a major determinant of the salutary effects of mineralocorticoid receptor antagonists in heart failure. Recently, R-fadrozole was coined as an aldosterone biosynthesis inhibitor, offering an appealing alternative to mineralocorticoid receptor antagonists to block aldosterone action. The present study aimed to evaluate the effects of R- and S-fadrozole on plasma aldosterone and urinary aldosterone excretion rate and to compare their effectiveness vs. the mineralocorticoid receptor antagonist potassium canrenoate to reverse established cardiac fibrosis. Male lean spontaneously hypertensive heart failure (SHHF) rats (40 wk) were treated for 8 wk by sc infusions of low (0.24 mg/kg.d) or high (1.2 mg/kg.d) doses of R- or S-fadrozole or by potassium canrenoate via drinking water (7.5 mg/kg.d). At the high dose, plasma aldosterone levels were decreased similarly by R- and S-fadrozole, whereas urinary aldosterone excretion rate was reduced only by S-fadrozole. In contrast, whereas at the high dose, R-fadrozole effectively reversed preexistent left ventricular interstitial fibrosis by 50% (vs. 42% for canrenoate), S-fadrozole was devoid of an antifibrotic effect. The low doses of the fadrozole enantiomers did not change cardiac fibrosis or plasma aldosterone but similarly reduced urinary aldosterone excretion rate. In conclusion, R-fadrozole may possess considerable therapeutic merit because of its potent antifibrotic actions in the heart. However, the observed discordance between the aldosterone-lowering and antifibrotic effects of the fadrozole enantiomers raises some doubt about the mechanism by which R-fadrozole diminishes cardiac collagen and about the generality of the concept of lowering aldosterone levels to treat the diseased heart.
Assuntos
Aldosterona/sangue , Fadrozol/química , Fadrozol/uso terapêutico , Insuficiência Cardíaca/prevenção & controle , Coração/efeitos dos fármacos , Miocárdio/patologia , Aldosterona/urina , Animais , Ácido Canrenoico/farmacologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Avaliação Pré-Clínica de Medicamentos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/urina , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Miocárdio/metabolismo , Ratos , Ratos Endogâmicos SHR , Estereoisomerismo , Relação Estrutura-Atividade , Resultado do TratamentoRESUMO
BACKGROUND: Aldosterone plays a detrimental role in the pathology of chronic heart failure. An important mechanism resides in its ability to evoke extensive fibrosis in the heart. However, the locations of the aldosterone interaction sites responsible for triggering cardiac fibrosis are puzzling. Extra-cardiac aldosterone actions are known to contribute to cardiac fibrosis but whether local cardiac aldosterone actions are involved is unclear. AIMS: This study aimed to investigate whether local aldosterone actions contribute to cardiac fibrosis in vivo. METHODS: Saline treated male Wistar rats were intravenously (systemically) or intrapericardially (locally) infused for 8 weeks with 75 or 750 ng/h aldosterone to monitor end point left ventricular epicardial collagen levels (histology). RESULTS: Perivascular fibrosis was observed only at high dose aldosterone infusions and was not different for the administration routes. Regarding interstitial fibrosis however, clear differences between the administration routes were seen. Intrapericardial aldosterone infusions increased interstitial collagen levels 1.72x (P<0.05) at low dose, but -surprisingly- had no significant effect at high dose. In contrast, intravenous aldosterone had no significant effect at low dose but increased interstitial collagen 1.72x (P<0.05) at high dose. CONCLUSION: Our data suggest that local cardiac aldosterone actions contribute to the development of left ventricular interstitial fibrosis.
Assuntos
Aldosterona/efeitos adversos , Fibrose/induzido quimicamente , Ventrículos do Coração/efeitos dos fármacos , Pericárdio/efeitos dos fármacos , Disfunção Ventricular Esquerda/induzido quimicamente , Aldosterona/administração & dosagem , Animais , Modelos Animais de Doenças , Fibrose/fisiopatologia , Ventrículos do Coração/patologia , Infusões Intravenosas , Infusões Parenterais , Masculino , Ratos , Ratos Wistar , Fatores de Risco , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Conventional cell-based assays for seven-transmembrane receptors, also known as G protein-coupled receptors, rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins. New assay methods have become available that are not based on G protein activation, but that apply the molecular mechanism underlying the attenuation of G protein signaling mediated by beta-arrestin. beta-arrestin is a cytoplasmic protein that targets receptors to clathrin-coated endocytotic vesicles for degradation or recycling. This process has been visualized and quantified in high-content imaging assays using receptor- or beta-arrestin-chimeras with green fluorescent protein. Other assay methods use bioluminescence resonance energy transfer, enzyme fragment complementation, or a protease-activated transcriptional reporter gene, to measure receptor-beta-arrestin proximity. beta-arrestin recruitment assays have been applied successfully for receptors coupling to Galpha(q), Galpha(s) and Galpha(i) proteins, thus providing a generic assay platform for drug discovery on G protein-coupled receptors. The best understood signal transduction pathway elicited by the seven-transmembrane Frizzled receptors does not involve G proteins. The activation of Frizzleds by their cognate ligands of the Wnt family recruits the phosphoprotein dishevelled. Dishevelled regulates a protein complex involved in the destruction of beta-catenin. Activation of Frizzled blocks degradation of beta-catenin, which translocates to the nucleus to activate transcription of Wnt-responsive genes. The cytoplasm-to-nuclear translocation of beta-catenin forms the basis of several high-content assays to measure Wnt/Frizzled signal transduction. Interestingly, Frizzled receptors have recently been shown to internalize and to recruit beta-arrestin. This suggests that beta-arrestin recruitment assays may be applied for drug discovery on seven-transmembrane receptors beyond G protein-coupled receptors.
Assuntos
Arrestinas/metabolismo , Bioensaio/métodos , Desenho de Fármacos , Proteínas de Ligação ao GTP/metabolismo , Preparações Farmacêuticas/administração & dosagem , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Técnicas Biossensoriais/métodos , beta-ArrestinasRESUMO
BACKGROUND: Hypertensive target organ damage shows characteristics of accelerated cell turnover and aging. This might have developed during the evolution of hypertension. In the kidney, high cell turnover is mainly restricted to tubular cells. It was the aim of this study to investigate whether a transient intervention in spontaneously hypertensive rats (SHRs) leads to reduced tubular cell turnover and attenuates the renal aging process and tubulo-interstitial damage in the long-term. METHODS: SHRs (i) were prehypertensively (weeks 4-8) treated with losartan (ii) or hydralazine (iii) (20 and 4 mg/kg/day, respectively) and compared to Wistar-Kyoto (WKY) rats (iv). Groups were investigated at weeks 8 and 72 (except iii). At both time points tubular cell proliferation (proliferative cell nuclear antigen) and systolic blood pressure (SBP) were evaluated. At week 72, aging parameters such as telomere length were assessed. Renal damage was semiquantitatively assessed (scale: 0-4) by measuring the parenchyma (atrophy) and vasculature (media thickness). RESULTS: Treatments equipotently reduced SBP in young SHRs (P < 0.01) but only losartan reduced renal proliferation (proliferative cell nuclear antigen: (i) 2.8 +/- 0.8, (ii) 1.3 +/- 0.3, (iii) 3.0 +/- 0.6, (iv) 0.1 +/- 0.1 cells/mm(2)). In SHRs treated with losartan(SHR-Los) tubular proliferation remained reduced and renal telomere length was significantly greater than in untreated SHRs (fold: (i) 1.0 +/- 0.1, (ii) 2.8 +/- 0.3, P < 0.01). Untreated SHRs (median 2.0, range 1-3; P < 0.007), but not SHR-Los (median 1.0, range 0-2; P = 0.06) demonstrated more tubular atrophy than WKY rats (median 0.5, range 0-1). CONCLUSIONS: Transient losartan treatment reduces cell-turnover not only acutely but also for a prolonged period after drug withdrawal. This results in the long-term in reduced aging and attenuated tubulo-interstitial damage, suggesting there exists a modulating effect of angiotensin II (ANGII)-antagonism on long-term cell turnover.
Assuntos
Envelhecimento/fisiologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Hipertensão Renal/tratamento farmacológico , Hipertensão Renal/fisiopatologia , Túbulos Renais/efeitos dos fármacos , Losartan/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Hiperplasia , Hipertensão Renal/patologia , Túbulos Renais/patologia , Túbulos Renais/fisiologia , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Proteins from the Wnt family have been implicated in cell-cell communication in a wide variety of developmental and physiological processes. Wnt signaling is required for different aspects of cardiac and vascular development, including myocardial specification, cardiac morphogenesis and cardiac valve formation as well as endothelial and vascular smooth muscle cell proliferation. Defective Wnt signaling can result in different cardiac and vascular abnormalities. In the adult heart and blood vessels, Wnt signaling activity is quite low under normal conditions. However, this pathway is reactivated during the pathological cardiac remodeling induced by pressure overload, in injured arteries and after myocardial infarction. Genetically modified animal models have shown that inhibition of Wnt signaling results in increased angiogenesis, better infarct healing and an attenuated hypertrophic response of the heart. This suggests that pharmacological inhibition of Wnt signaling could provide a novel therapeutic strategy to prevent excessive cardiac and vascular remodeling.
Assuntos
Sistema Cardiovascular/crescimento & desenvolvimento , Receptores Frizzled/fisiologia , Proteínas Wnt/fisiologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Cardiomegalia/fisiopatologia , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Receptores Frizzled/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Humanos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Wnt/efeitos dos fármacosRESUMO
OBJECTIVE: We previously demonstrated that when the renin-angiotensin system (RAS) is transiently blocked by an angiotensin receptor blocker (ARB) in young spontaneously hypertensive rats (SHR), this results in a prolonged blood pressure decrease and protection against target organ damage. Aldosterone is an essential hormone in the RAS, and contributes to pathologic remodeling. Thus, part of the protective effects of the ARB may be due to inhibition of aldosterone-mediated effects. To test this hypothesis, in young SHR, we compared the effectiveness of transient treatment with the mineralocorticoid receptor blocker spironolactone with those obtained by the ARB losartan. METHODS: SHR were transiently (i.e. between 4-8 weeks of age) treated with spironolactone (SHR-Spiro: 1 mg/kg per day), losartan (SHR-Los: 20 mg/kg per day) or saline. Rats were followed up until week 72 of age and cardiovascular parameters were repeatedly assessed by echocardiography, radiotelemetry of blood pressure and 24-h urine collection. End-point measurements included direct left ventricular contractility and relaxation, as well as cardiac and renal histomorphology. RESULTS: Transient spironolactone treatment reduced blood pressure up to 36 weeks of age and cardiac and renal collagen deposition and tubular atrophy up to 72 weeks of age compared to untreated SHR. Pulse pressure was higher in SHR-Spiro compared to SHR-Los. Cardiac hypertrophy, albuminuria and glomerulosclerosis were not attenuated in SHR-Spiro compared to untreated SHR up to 72 weeks of age, whereas the effects in SHR-Los were ameliorated. CONCLUSIONS: Although transient spironolactone treatment leads to prolonged blood pressure reduction and reduced collagen deposition, long-term organ protection only partially exists. Thus, transient spironolactone treatment is less effective than transient losartan treatment.
Assuntos
Hipertensão/tratamento farmacológico , Losartan/uso terapêutico , Espironolactona/uso terapêutico , Fatores Etários , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Sequência de Bases , Pressão Sanguínea/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Primers do DNA/genética , Coração/efeitos dos fármacos , Hipertensão/patologia , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Endogâmicos SHRRESUMO
OBJECTIVE: In young spontaneously hypertensive rats (SHR), transient angiotensin II type 1 receptor (AT1R) blockade decreases blood pressure for a prolonged period. We tested the hypothesis that transient AT1R blockade in SHR leads to cardiac protection until advanced age. METHOD: Wistar-Kyoto rats, SHR and transiently losartan-treated SHR (SHR-Los) (20 mg/kg per day; weeks 4-8 of age) were followed up until week 72 (n=9 each group), including repeated echocardiography, radiotelemetric investigations and 24-h urine collection. End-point measurements comprised left ventricular function parameters, left ventricular histomorphology and molecular biology (types I and III collagen, brain natriuretic peptide, AT1R mRNA) as well as renal morphology. RESULTS: Prehypertensive treatment with losartan, but not with the general vasodilator hydralazine, reduced blood pressure until age 48 weeks. In untreated SHR, the end-diastolic volume increased from week 36 and the left ventricular ejection fraction fell from week 48. In contrast, age-related changes in end-diastolic volume and ejection fraction were comparable in SHR-Los and Wistar-Kyoto rats up to age 60 weeks. At age 72 weeks, the ejection fraction was reduced in SHR-Los but higher than that in untreated SHR (ejection fraction: Wistar-Kyoto rats, 58 +/- 3%; SHR, 39 +/- 3%; SHR-Los, 46 +/- 3%; P < 0.01 and P < 0.05, respectively). The heart weight/body weight ratio (SHR-Los, 4.7 +/- 0.1 g/kg; SHR, 5.2 +/- 0.2 g/kg) and cardiac brain natriuretic peptide mRNA levels were improved by treatment. Left ventricular histomorphology and 24-h albuminuria were significantly improved in SHR-Los (41 +/- 5 mg/day; SHR, 80 +/- 22 mg/day; P < 0.05). CONCLUSION: In young SHR, transient AT1R blockade, not blood pressure lowering, attenuates the development of hypertension and exerts cardioprotective effects up to age 72 weeks.
Assuntos
Envelhecimento/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Doenças Cardiovasculares/prevenção & controle , Hipertensão/tratamento farmacológico , Losartan/farmacologia , Fatores Etários , Albuminúria/etiologia , Albuminúria/prevenção & controle , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/etiologia , Cardiomegalia/prevenção & controle , Doenças Cardiovasculares/etiologia , Modelos Animais de Doenças , Progressão da Doença , Esquema de Medicação , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Hidralazina/farmacologia , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/patologia , Losartan/administração & dosagem , Losartan/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Vasodilatadores/farmacologia , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Arterial function after long-term hypertension is characterized by remodeling, endothelial dysfunction and reduction of previously enhanced contractile responses. We investigated whether transient prehypertensive renin-angiotensin-aldosterone system (RAAS) blockade modifies long-term arterial function. Wistar Kyoto rats (WKY) (i) and spontaneously hypertensive rats (SHR) (ii) were prehypertensively (week 4-8) treated with losartan (iii) or spironolactone (iv) (20 and 0.5 mg/kg/day, respectively) and investigated at 8 and 72 weeks of age. Systolic blood pressure (SBP) was measured intra-arterially. In isolated mesenteric arteries, active wall stress (AWS), relaxation in response to acetylcholine and wall-to-lumen ratio (W/L) were assessed. Western blotting and immunofluorescent staining of whole-mount arterial preparations and two photon laser scanning microscopy (TPLSM) were performed to quantify endothelial nitric oxide synthase (eNOS) and analyze its intracellular distribution. In 8-week-old SHR treatments were found to have reduced SBP. Relaxation, contractile responses and vascular morphology remained unaffected irrespective of treatment. At 72 weeks, SBP was similar in all SHR groups ((i) 129+/-6, (ii) 222 +/- 10, (iii) 210 +/- 16, (iv) 214 +/- 9 mmHg). Relaxation and maximum AWS were enhanced after treatments. W/L demonstrated hypertrophy ((i) 0.10 +/- 0.01, (ii) 0.16 +/- 0.02, (iii) 0.15 +/- 0.01, (iv) 0.17 +/- 0.01). Untreated SHR (p<0.01), SHR treated with losartan and SHR treated with spironolactone (p<0.05) showed less eNOS as compared to WKY. In treated SHR eNOS was concentrated in a perinuclear endothelial cell compartment. In conclusion, these findings demonstrate that transient prehypertensive blockade results in a long-lasting and blood pressure independent improvement of arterial contractility and endothelium-dependent vasodilatation that persists in aging SHR. This might be associated with an intracellular redistribution of eNOS in the endothelial cell layer.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Hipertensão/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Sistema Renina-Angiotensina/fisiologia , Animais , Aorta/metabolismo , Pressão Sanguínea/fisiologia , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Músculo Liso Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatação/fisiologiaRESUMO
BACKGROUND: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. RESULTS: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. CONCLUSION: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.
Assuntos
Artefatos , Perfilação da Expressão Gênica/métodos , Ventrículos do Coração/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , Fatores de Transcrição/genética , Animais , Simulação por Computador , Genes Reporter , Modelos Genéticos , Modelos Estatísticos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19+/-0.01) versus normal ejection fraction (0.11+/-0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.
Assuntos
Baixo Débito Cardíaco/etiologia , Matriz Extracelular/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Miocárdio/metabolismo , Trombospondinas/biossíntese , Angiotensina II/antagonistas & inibidores , Angiotensina II/toxicidade , Animais , Animais Geneticamente Modificados , Baixo Débito Cardíaco/genética , Baixo Débito Cardíaco/metabolismo , Cardiomiopatias/induzido quimicamente , Colagenases/metabolismo , Progressão da Doença , Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Ruptura Cardíaca/induzido quimicamente , Ruptura Cardíaca/patologia , Humanos , Hipertensão/complicações , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/genética , Metaloproteinase 9 da Matriz , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Renina/genética , Volume Sistólico , Trombospondinas/genética , Trombospondinas/fisiologia , Regulação para CimaRESUMO
OBJECTIVE: To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE-/- and t-ACE+/+ mice. RESULTS- In CA of t-ACE-/- mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE-/- than t-ACE+/+ mice. In UA of t-ACE-/- mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains. CONCLUSIONS: t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.
Assuntos
Artérias Carótidas/enzimologia , Hemorreologia , Proteínas de Membrana/fisiologia , Peptidil Dipeptidase A/fisiologia , Útero/irrigação sanguínea , Alelos , Angiotensina II/fisiologia , Animais , Artérias/anatomia & histologia , Artérias/enzimologia , Artérias Carótidas/patologia , Feminino , Hiperplasia , Hipertrofia , Ligadura , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/deficiência , Peptidil Dipeptidase A/genética , Período Pós-Parto , Gravidez , Estrutura Terciária de Proteína , Deleção de Sequência , Estresse MecânicoRESUMO
OBJECTIVE: Angiotensin II, generated from angiotensin I by angiotensin converting enzyme (ACE), induces multiple effects including vasoconstriction, positive cardiac inotropy, hypertrophy of cardiomyocytes and proliferation of fibroblasts. ACE exists both in a tissue-bound (t-ACE) and a soluble form. The functional importance of locally produced angiotensin II is still unclear. In the present study, mice lacking tissue-bound angiotensin converting enzyme (t-ACE -/-) were used to investigate the importance of t-ACE during cardiac remodeling after myocardial infarction. METHODS: Mice were subjected to coronary artery occlusion or sham surgery. At 14 days after MI, stroke volume (SV) was determined with an electromagnetic flow probe around the ascending aorta. Mean arterial pressure (MAP) was measured through a cannula in the abdominal aorta. Both parameters were determined at rest and after a volume loading of 2.5 ml warm (37 degrees C) Ringer's solution in 60 s. Hearts were dissected and formalin-fixed to measure infarct size, cardiac dimensions and collagen concentration. Tissue levels of angiotensin I and II were determined in hearts and kidneys. RESULTS: At rest, under pentobarbital anaesthesia, t-ACE -/- mice (n=12) exhibited a significantly lower MAP (26+/-3 vs. 45+/-3 mmHg) than t-ACE +/+ (n=11). SV was similar in both strains. Maximal SV was significantly reduced after MI. Furthermore, infarcted t-ACE -/- (n=6) exhibited a significantly lower maximal SV compared to infarcted t-ACE +/+ mice (n=5; 20.4+/-1.5 vs. 29.6+/-2.3 microl). Structural cardiac parameters as well as cardiac and renal angiotensin II levels in t-ACE -/- and t-ACE +/+ were comparable. CONCLUSIONS: These results suggest that the structural adaptations of the heart that follow MI are independent of t-ACE. However, the presence of t-ACE is necessary for maintenance of cardiac function.
Assuntos
Infarto do Miocárdio/fisiopatologia , Peptidil Dipeptidase A/fisiologia , Remodelação Ventricular/fisiologia , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Colágeno/metabolismo , Hemodinâmica , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Sistema Renina-Angiotensina/fisiologiaRESUMO
Wnt/ß-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a ß-galactosidase fragment complementation assay measuring ß-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated ß-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/ß-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated ß-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and É, which are known activators of Wnt/ß-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of ß-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/ß-catenin signaling.