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1.
Circulation ; 124(15): 1626-35, 2011 Oct 11.
Article in English | MEDLINE | ID: mdl-21931076

ABSTRACT

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.


Subject(s)
Frizzled Receptors/antagonists & inhibitors , Heart Failure/prevention & control , Myocardial Infarction/complications , Peptide Fragments/pharmacology , Signal Transduction/drug effects , Wnt3A Protein/pharmacology , Animals , Cell Line, Transformed , Cells, Cultured , Chlorocebus aethiops , Cricetinae , Cricetulus , Fibroblasts/drug effects , Frizzled Receptors/metabolism , Heart Failure/etiology , Humans , Male , Mice , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/cytology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Rats , Structure-Activity Relationship , Ventricular Remodeling/drug effects , Wnt3A Protein/antagonists & inhibitors
2.
J Comput Chem ; 32(11): 2441-8, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21598279

ABSTRACT

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.


Subject(s)
Aldosterone/biosynthesis , Aldosterone/chemistry , Cytochrome P-450 CYP11B2/chemistry , Ligands , Quantum Theory , Iron/chemistry , Molecular Structure , Oxygen/chemistry
3.
Eur J Cardiovasc Prev Rehabil ; 18(4): 656-63, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21450631

ABSTRACT

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.


Subject(s)
Blood Pressure , Hypertension/physiopathology , Hypertension/urine , Nitric Oxide/urine , Adolescent , Adult , Aged , Belgium , Biomarkers/urine , Case-Control Studies , Chi-Square Distribution , Disease Progression , Down-Regulation , Humans , Kidney/physiopathology , Linear Models , Middle Aged , Pilot Projects , Proportional Hazards Models , Prospective Studies , Risk Assessment , Risk Factors , Time Factors , Young Adult
4.
Chem Biol ; 18(4): 485-94, 2011 Apr 22.
Article in English | MEDLINE | ID: mdl-21513885

ABSTRACT

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 ɛ, 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.


Subject(s)
Casein Kinase I/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Wnt Proteins/metabolism , beta Catenin/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Casein Kinase 1 epsilon/metabolism , Casein Kinase Idelta/metabolism , Cell Line , Drug Evaluation, Preclinical , Humans , Substrate Specificity
5.
Cardiovasc Res ; 87(3): 514-23, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20189955

ABSTRACT

AIMS: The Wnt/frizzled (Fzd) signal transduction cascade has been implicated in the proliferation, differentiation, and migration of many cell types, but the role of this pathway in cardiac fibroblast differentiation is not known. Our lab previously showed an up-regulation of Fzd-1 and -2 expression in myofibroblasts after myocardial infarction (MI), indicating a potential role for the Fzd receptor in fibroblast-myofibroblast differentiation. The present study was performed to further define the role of specific Wnt and Fzd proteins in the proliferation, migration, and differentiation of cardiac fibroblasts. METHODS AND RESULTS: Because primary fibroblasts become senescent after a few passages and are difficult to transfect, we immortalized rat cardiac fibroblasts with telomerase [cardiac fibroblasts immortalized with telomerase (CFIT)]. Proliferation of CFIT was not significantly influenced by Wnt/Fzd signalling. The migration, however, was attenuated by all Wnt/Fzd combinations tested. Also, specific Wnt/Fzd combinations modulated the expression of the following myofibroblast markers: collagen Ialpha1, collagen III, fibronectin and its splice variants, and alpha-smooth muscle actin. CONCLUSION: The results indicate that myofibroblast migration and differentiation, but not proliferation, can be modulated by interventions in Wnt/Fzd signalling. Therefore, Wnt/Fzd signalling may serve as a novel therapeutic target to ameliorate wound healing after MI.


Subject(s)
Cell Differentiation , Cell Movement , Fibroblasts/metabolism , Frizzled Receptors/metabolism , Myocardium/metabolism , Signal Transduction , Wnt Proteins/metabolism , Actins/metabolism , Animals , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Cell Line , Cell Proliferation , Collagen Type I/metabolism , Collagen Type III/metabolism , Fibronectins/metabolism , Frizzled Receptors/genetics , Humans , Male , Myocardium/cytology , RNA Interference , Rats , Rats, Inbred Lew , Receptors, G-Protein-Coupled/metabolism , Telomerase/genetics , Telomerase/metabolism , Time Factors , Transfection , Wnt Proteins/genetics , Wnt3 Protein
6.
J Med Chem ; 53(4): 1712-25, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20121113

ABSTRACT

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, this is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, we used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-yl)methyl)benzonitrile) displaying an IC(50) for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC(50) for CYP11B2 6.0 nM, selectivity 19.8). Molecular docking of the inhibitors in the models enabled us to generate posthoc hypotheses on their binding modes, providing a valuable basis for future studies and further design of CYP11B2 inhibitors.


Subject(s)
Benzyl Compounds/chemical synthesis , Cytochrome P-450 CYP11B2/antagonists & inhibitors , Imidazoles/chemical synthesis , Models, Molecular , 18-Hydroxycorticosterone/chemistry , Animals , Benzyl Compounds/chemistry , Benzyl Compounds/pharmacology , Catalytic Domain , Cell Line , Cricetinae , Cricetulus , Cytochrome P-450 CYP11B2/chemistry , Fadrozole/chemistry , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Molecular Dynamics Simulation , Protein Binding , Stereoisomerism , Structure-Activity Relationship
7.
FASEB J ; 24(4): 1205-17, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19940259

ABSTRACT

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.


Subject(s)
Down-Regulation/drug effects , Genetic Complementation Test/methods , Signal Transduction/drug effects , Wnt Proteins/pharmacology , beta Catenin/metabolism , beta-Galactosidase/biosynthesis , Animals , Cell Polarity , Cell Proliferation , Down-Regulation/physiology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Humans , Jurkat Cells , Mice , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Quinazolines/pharmacology , Signal Transduction/physiology , TCF Transcription Factors , Transcription, Genetic , Transgenes/physiology , Wnt-5a Protein , Wnt3 Protein , Wnt3A Protein , beta Catenin/genetics , beta-Galactosidase/genetics
8.
Cardiovasc Res ; 84(2): 273-82, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19542177

ABSTRACT

AIMS: Our objective was to study the effect of the genetic background on the wound healing process after myocardial infarction (MI) in mice. METHODS AND RESULTS: MI was induced in five different mouse strains (BalbC, C57Bl6, FVB, 129S6, and Swiss). At 3, 14, and 28 days after MI, cardiac dimensions were monitored by echocardiography and histology, whereas cardiac function was determined by direct intraventricular pressure measurements (dP/dt). Furthermore, matrix metalloproteinases were measured by zymography, and mRNA expression by quantitative PCR. Infarct rupture, which typically occurred at 3-6 days post-MI, was most frequent in 129S6 mice (62%), followed by C57Bl6 (36%), FVB (29%), Swiss (23%), and BalbC (5%). The high incidence of infarct rupture in 129S6 mice was associated with high systolic blood pressure and increased influx of inflammatory cells. Cardiac dilatation was most marked in Swiss mice and least prominent in 129S6 mice. The degree of dilatation was associated with a reduced ejection fraction and decreased dP/dt values at 14 and 28 days post-MI. At day 14 and 28 post-MI, secondary thinning of the infarct area was marked in BalbC, FVB, and Swiss, but absent in C57Bl6 and 129S6 mice. In the latter two groups, this was paralleled by the highest number of myofibroblasts at day 14 post-MI. CONCLUSION: The outcome of infarct healing in mice strongly depends on genetic background. On the basis of our results, we suggest that for studies on infarct rupture, the 129S6 mouse is the background of choice, whereas BalbC and Swiss mice are the preferred models to study infarct thinning post-MI.


Subject(s)
Heart Rupture, Post-Infarction/genetics , Myocardial Infarction/genetics , Myocardium/pathology , Wound Healing/genetics , Animals , Antihypertensive Agents/pharmacology , Atrial Natriuretic Factor/genetics , Blood Pressure/genetics , Disease Models, Animal , Echocardiography , Gene Expression Regulation, Enzymologic , Heart Rupture, Post-Infarction/pathology , Heart Rupture, Post-Infarction/physiopathology , Heart Rupture, Post-Infarction/prevention & control , Inflammation/genetics , Inflammation/pathology , Inflammation/physiopathology , Male , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/enzymology , Natriuretic Peptide, Brain/genetics , Polymerase Chain Reaction , RNA, Messenger/metabolism , Species Specificity , Stroke Volume/genetics , Time Factors , Ventricular Function, Left/genetics , Ventricular Pressure/genetics
9.
JACC Cardiovasc Imaging ; 2(2): 187-98, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19356555

ABSTRACT

OBJECTIVES: Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination. BACKGROUND: The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m-labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI. METHODS: CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition. RESULTS: Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 +/- 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 +/- 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 +/- 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 +/- 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake. CONCLUSIONS: Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Cardiovascular Agents/pharmacology , Mineralocorticoid Receptor Antagonists/pharmacology , Myocardial Infarction/drug therapy , Myocardium/pathology , Tomography, Emission-Computed, Single-Photon , Ventricular Remodeling/drug effects , Animals , Captopril/pharmacology , Carbocyanines , Disease Models, Animal , Drug Therapy, Combination , Echocardiography , Fibrillar Collagens/metabolism , Fibroblasts/diagnostic imaging , Fibroblasts/drug effects , Fibrosis , Losartan/pharmacology , Mice , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/physiopathology , Myocardium/metabolism , Oligopeptides , Predictive Value of Tests , Spironolactone/pharmacology , Technetium , Ventricular Function, Left/drug effects
10.
J Cardiovasc Electrophysiol ; 20(2): 207-15, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19175851

ABSTRACT

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.


Subject(s)
Anti-Arrhythmia Agents/pharmacology , Atrial Fibrillation/drug therapy , Goats/physiology , Pericardium , Sotalol/pharmacology , Animals , Anti-Arrhythmia Agents/administration & dosage , Anti-Arrhythmia Agents/pharmacokinetics , Atrial Fibrillation/physiopathology , Electric Stimulation , Electrocardiography , Electrophysiology , Flecainide/administration & dosage , Flecainide/pharmacokinetics , Flecainide/pharmacology , Heart Atria , Injections , Injections, Intravenous , Pericardium/physiopathology , Sotalol/administration & dosage , Sotalol/pharmacokinetics
11.
Cell Signal ; 21(1): 22-33, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18929644

ABSTRACT

Signal transduction via the Frizzled family of seven-transmembrane receptors controls important developmental processes. Aberrant signaling caused by altered Frizzled receptor activity or by mutations in downstream signaling components has been implicated in several adult pathologies. A diverse array of intracellular signaling pathways has been suggested to transduce the signals exerted in cells when secreted ligands of the Wnt family bind to Frizzled receptors. Studies with a chimeric receptor composed of Frizzled-2 and the beta2-adrenergic receptor have suggested that the binding of Wnt-5a to Frizzled-2 results in the activation of G proteins of the Galpha(i) family, the mobilization of calcium from intracellular stores and the induction of gene transcription through nuclear factor of activated T cells. In this report, we demonstrate by using beta-lactamase reporter gene technology that full-length, wild-type human Frizzled-2 does not couple to calcium-mediated signaling in HEK293 cells following stimulation with purified recombinant mouse Wnt-5a. In contrast, when stimulated with recombinant mouse Wnt-3a, Frizzled-2 activates the canonical Wnt/Frizzled signaling pathway, involving the transcriptional modulator beta-catenin. Our report underlines the importance of using cell lines stably overexpressing wild-type Frizzled receptors and the use of purified ligands when studying receptor pharmacology. This approach has allowed us to measure the half-maximal concentration for activation of human Frizzled-2 (1.5+/-0.4 nM; avg.+/-SD) and human Frizzled-1 (1.3+/-0.5 nM) following stimulation by Wnt-3a. Our results suggest that there is receptor redundancy with regard to Wnt-3a reception. In addition, we introduce beta-lactamase reporter gene technology as an alternative to luciferase-based reporters to measure Frizzled receptor modulation for the discovery of Frizzled receptor-interacting drugs.


Subject(s)
Calcium/metabolism , Frizzled Receptors/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , beta Catenin/metabolism , Animals , Cell Line , Genes, Reporter , Humans , Luminescent Measurements , Mice , Penicillinase/metabolism , Rats , Receptors, Adrenergic, beta-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Wnt Proteins/metabolism
12.
Cardiovasc Pathol ; 18(1): 37-43, 2009.
Article in English | MEDLINE | ID: mdl-18402833

ABSTRACT

BACKGROUND: Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. METHODS: Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. RESULTS: The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. CONCLUSIONS: These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans.


Subject(s)
Heart Rupture, Post-Infarction/enzymology , Matrix Metalloproteinase 8/metabolism , Matrix Metalloproteinase 9/metabolism , Myocardial Infarction/enzymology , Aged , Aged, 80 and over , Cell Count , Female , Heart Rupture, Post-Infarction/etiology , Heart Rupture, Post-Infarction/pathology , Humans , Immunohistochemistry , Male , Matrix Metalloproteinase 2/metabolism , Middle Aged , Myocardial Infarction/complications , Myocardial Infarction/pathology , Myocarditis/pathology
13.
J Am Coll Cardiol ; 52(24): 2017-28, 2008 Dec 09.
Article in English | MEDLINE | ID: mdl-19055994

ABSTRACT

OBJECTIVES: The purpose of this study was to evaluate interstitial alterations in myocardial remodeling using a radiolabeled Cy5.5-RGD imaging peptide (CRIP) that targets myofibroblasts. BACKGROUND: Collagen deposition and interstitial fibrosis contribute to cardiac remodeling and heart failure after myocardial infarction (MI). Evaluation of myofibroblastic proliferation should provide indirect evidence of the extent of fibrosis. METHODS: Of 46 Swiss-Webster mice, MI was induced in 41 by coronary artery occlusion, and 5 were unmanipulated. Of the 41 mice, 6, 6, and 5 received intravenous technetium-99m labeled CRIP for micro-single-photon emission computed tomography imaging 2, 4, and 12 weeks after MI, respectively; 8 received captopril or captopril with losartan up to 4 weeks after MI. Scrambled CRIP was used 4 weeks after MI in 6 mice; the remaining 10 of 46 mice received unradiolabeled CRIP for histologic characterization. RESULTS: Maximum CRIP uptake was observed in the infarct area; quantitative uptake (percent injected dose/g) was highest at 2 weeks (2.75 +/- 0.46%), followed by 4 (2.26 +/- 0.09%) and 12 (1.74 +/- 0.24%) weeks compared with that in unmanipulated mice (0.59 +/- 0.19%). Uptake was higher at 12 weeks in the remote areas. CRIP uptake was histologically traced to myofibroblasts. Captopril alone (1.78 +/- 0.31%) and with losartan (1.13 +/- 0.28%) significantly reduced tracer uptake; scrambled CRIP uptake in infarct area (0.74 +/- 0.17%) was similar to CRIP uptake in normal myocardium. CONCLUSIONS: Radiolabeled CRIP allows for noninvasive visualization of interstitial alterations during cardiac remodeling, and is responsive to antiangiotensin treatment. If proven clinically feasible, such a strategy would help identify post-MI patients likely to develop heart failure.


Subject(s)
Carbocyanines , Endomyocardial Fibrosis/complications , Fibroblasts , Heart Failure/etiology , Myocardial Infarction/diagnostic imaging , Myocardium/pathology , Ventricular Remodeling , Animals , Collagen/metabolism , Coronary Artery Disease , Endomyocardial Fibrosis/diagnostic imaging , Feasibility Studies , Heart Failure/diagnostic imaging , Integrins , Male , Mice , Models, Animal , Myocardial Infarction/complications , Myocardial Infarction/pathology , Radiopharmaceuticals , Time Factors , Tomography, Emission-Computed, Single-Photon , Tomography, X-Ray Computed , Ultrasonography
14.
Eur J Heart Fail ; 10(12): 1166-71, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19000945

ABSTRACT

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.


Subject(s)
Aldosterone/adverse effects , Fibrosis/chemically induced , Heart Ventricles/drug effects , Pericardium/drug effects , Ventricular Dysfunction, Left/chemically induced , Aldosterone/administration & dosage , Animals , Disease Models, Animal , Fibrosis/physiopathology , Heart Ventricles/pathology , Infusions, Intravenous , Infusions, Parenteral , Male , Rats , Rats, Wistar , Risk Factors , Ventricular Dysfunction, Left/physiopathology
15.
Biotechnol Annu Rev ; 14: 253-74, 2008.
Article in English | MEDLINE | ID: mdl-18606367

ABSTRACT

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.


Subject(s)
Arrestins/metabolism , Biological Assay/methods , Drug Design , GTP-Binding Proteins/metabolism , Pharmaceutical Preparations/administration & dosage , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Biosensing Techniques/methods , beta-Arrestins
16.
J Am Coll Cardiol ; 51(22): 2184-92, 2008 Jun 03.
Article in English | MEDLINE | ID: mdl-18510968

ABSTRACT

OBJECTIVES: Our goal was to evaluate intercellular adhesion complex proteins in myocardium in human infarct rupture. BACKGROUND: Infarct rupture, a fatal complication of myocardial infarction (MI), has been attributed to a defective cell adhesion complex in a transgenic mouse model. METHODS: Heart samples were collected from autopsies from infarct rupture and control (nonrupture) MI patients. Both infarcted and remote areas were included. Cell adhesion proteins including alphaE-catenin, beta-catenin, gamma-catenin, and N-cadherin were characterized by immunohistochemistry and immunoblotting. Genetic analysis was undertaken to evaluate mutations and polymorphisms in the alphaE-catenin gene. In addition, infarct rupture was studied in transgenic mice heterozygous for alphaE-catenin C-terminal deficiency, mimicking the situation in human infarct rupture patients. RESULTS: No alphaE-catenin was detected in 70% of remote samples of infarct rupture hearts compared with 20% in control MI by immunohistochemistry. The immunoblot analysis confirmed a significant reduction in remote areas, and complete absence of alphaE-catenin in infarct areas from infarct rupture patients. No mutation or polymorphism of the alphaE-catenin gene was discovered. Other cell adhesion proteins were not significantly affected in remote areas of infarct rupture hearts. Three-fourths of the heterozygous alphaE-catenin C-terminal truncated mice died of infarct rupture, compared with one-fourth of the wild-type littermates. CONCLUSIONS: The data show a reduced expression and defective localization of alphaE-catenin in the intercalated disc region in patients dying of infarct rupture. The mechanism of lower expression of alphaE-catenin remains to be elucidated.


Subject(s)
Heart Rupture, Post-Infarction/etiology , Intercellular Adhesion Molecule-1/metabolism , Myocardium/metabolism , Aged , Aged, 80 and over , Animals , Cadaver , Cadherins/metabolism , Case-Control Studies , Catenins/metabolism , Female , Humans , Male , Mice , Middle Aged , Models, Animal , Myocytes, Cardiac/metabolism , Risk Factors
17.
Eur J Pharmacol ; 585(2-3): 338-45, 2008 May 13.
Article in English | MEDLINE | ID: mdl-18417121

ABSTRACT

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.


Subject(s)
Cardiovascular System/growth & development , Frizzled Receptors/physiology , Wnt Proteins/physiology , Animals , Blood Vessels/drug effects , Cardiomegaly/drug therapy , Cardiomegaly/physiopathology , Cardiovascular Physiological Phenomena/drug effects , Frizzled Receptors/drug effects , Heart/growth & development , Heart Diseases/drug therapy , Heart Diseases/physiopathology , Humans , Myocardial Infarction/drug therapy , Myocardial Infarction/physiopathology , Signal Transduction/drug effects , Signal Transduction/physiology , Wnt Proteins/drug effects
18.
Trends Pharmacol Sci ; 29(4): 175-80, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18342376

ABSTRACT

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.


Subject(s)
Cardiomegaly/drug therapy , Frizzled Receptors/physiology , Glycogen Synthase Kinase 3/physiology , Signal Transduction/physiology , Wnt Proteins/physiology , Animals , Cardiomegaly/etiology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Humans , Receptor Cross-Talk , beta Catenin/physiology
19.
Am J Hypertens ; 21(2): 177-82, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18188163

ABSTRACT

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.


Subject(s)
Aging/physiology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Hypertension, Renal/drug therapy , Hypertension, Renal/physiopathology , Kidney Tubules/drug effects , Losartan/pharmacology , Animals , Cell Cycle/drug effects , Cell Division/drug effects , Hyperplasia , Hypertension, Renal/pathology , Kidney Tubules/pathology , Kidney Tubules/physiology , Male , Rats , Rats, Inbred SHR , Rats, Inbred WKY
20.
Endocrinology ; 149(1): 28-31, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17884944

ABSTRACT

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.


Subject(s)
Aldosterone/blood , Fadrozole/chemistry , Fadrozole/therapeutic use , Heart Failure/prevention & control , Heart/drug effects , Myocardium/pathology , Aldosterone/urine , Animals , Canrenoic Acid/pharmacology , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Collagen Type III/genetics , Collagen Type III/metabolism , Drug Evaluation, Preclinical , Fibrosis , Gene Expression Regulation/drug effects , Heart Failure/urine , Male , Mineralocorticoid Receptor Antagonists/pharmacology , Myocardium/metabolism , Rats , Rats, Inbred SHR , Stereoisomerism , Structure-Activity Relationship , Treatment Outcome
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