147 results on '"Willette RN"'
Search Results
2. GSK1562590, a slowly dissociating urotensin-II receptor antagonist, exhibits prolonged pharmacodynamic activity ex vivo
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Behm, DJ, primary, Aiyar, NV, additional, Olzinski, AR, additional, McAtee, JJ, additional, Hilfiker, MA, additional, Dodson, JW, additional, Dowdell, SE, additional, Wang, GZ, additional, Goodman, KB, additional, Sehon, CA, additional, Harpel, MR, additional, Willette, RN, additional, Neeb, MJ, additional, Leach, CA, additional, and Douglas, SA, additional
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- 2010
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3. Inhibition of p38 mitogen-activated protein kinase improves nitric oxide-mediated vasodilatation and reduces inflammation in hypercholesterolemia.
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Cheriyan J, Webb AJ, Sarov-Blat L, Elkhawad M, Wallace SM, Mäki-Petäjä KM, Collier DJ, Morgan J, Fang Z, Willette RN, Lepore JJ, Cockcroft JR, Sprecher DL, Wilkinson IB, Cheriyan, Joseph, Webb, Andrew J, Sarov-Blat, Lea, Elkhawad, Maysoon, Wallace, Sharon M L, and Mäki-Petäjä, Kaisa M
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- 2011
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4. Blood Pressure and Splanchnic Nerve Activity Are Reduced by a Vagally Mediated Opioid Action
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A J Krieger, Willette Rn, and Hreday N. Sapru
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Male ,Bradycardia ,medicine.medical_specialty ,Enkephalin ,Enkephalin, Methionine ,Biguanides ,Blood Pressure ,(+)-Naloxone ,Vagotomy ,Splanchnic nerves ,Internal medicine ,Animals ,Medicine ,Lung ,Pharmacology ,Vasomotor ,Naloxone ,business.industry ,Splanchnic Nerves ,Vagus Nerve ,Rats ,medicine.anatomical_structure ,Endocrinology ,Blood pressure ,Opioid ,Receptors, Opioid ,Reflex ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,medicine.drug - Abstract
An enkephalin analogue, D-Ala2-Met5-enkephalinamide (DAME), caused a fall in blood pressure (BP) following right atrial administration (RA) in urethane-anesthetized rats that were also atropinized, paralyzed, and artificially ventilated. This reduction in BP was not related to opioid-induced bradycardia. At a dose of 250 micrograms/kg RA the maximum percentage change was -36.0 +/- 3.9% occurring within 10-15 s subsequent to administration. The fall in BP was blocked by both pretreatment with naloxone HCl (75-100 micrograms/kg i.v.) and bilateral cervical vagotomy. Similar hypotensive responses (-37.3 +/- 2.3%) were obtained with phenyldiguanide (PDG) (40 micrograms/kg RA), a known stimulant of pulmonary J receptors (pulmonary C fibers). However, unlike DAME, the PDG response could not be blocked by naloxone. The fall in BP. obtained with DAME and PDG, paralleled a reduction in the spike frequency of the greater splanchnic nerve activity. This reflex fall in activity did not involve supramesencephalic structures because results were similar in both urethane-anesthetized and unanesthetized midcollicular decerebrate preparations. It was concluded that opioids can act peripherally, via pulmonary opiate receptors, to inhibit central vasomotor activity and reduce BP by eliciting a pulmonary chemoreflex.
- Published
- 1982
5. Modulation of Oxidative Phosphorylation with IM156 Attenuates Mitochondrial Metabolic Reprogramming and Inhibits Pulmonary Fibrosis.
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Willette RN, Mangrolia P, Pondell SM, Lee CYW, Yoo S, Rudoltz MS, Cowen BR, and Welsch DJ
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- Animals, Antifibrotic Agents chemistry, Antifibrotic Agents therapeutic use, Cell Line, Cellular Reprogramming physiology, Dose-Response Relationship, Drug, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Male, Metabolomics methods, Mice, Mice, Inbred C57BL, Pulmonary Fibrosis prevention & control, Antifibrotic Agents pharmacology, Cellular Reprogramming drug effects, Mitochondria drug effects, Mitochondria metabolism, Oxidative Phosphorylation drug effects, Pulmonary Fibrosis metabolism
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Metabolic reprogramming of the myofibroblast plays a fundamental role in the pathogenesis of fibrosing interstitial lung diseases. Here, we characterized the in vitro and in vivo metabolic and antifibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting protein complex 1. In vitro, IM156 inhibited transforming growth factor β (TGF β )-dependent increases in mitochondrial oxygen consumption rate and expression of myofibroblast markers in human pulmonary fibroblasts without altering cell viability or adding to TGF β -induced increases in the extracellular acidification rate. IM156 significantly increased cellular AMP-activated protein kinase (AMPK) phosphorylation and was 60-fold more potent than metformin. In vivo, chronic oral administration of IM156 was highly distributed to major peripheral organs (i.e., lung, liver, kidney, heart) and had significant dose-related effects on the plasma metabolome consistent with OXPHOS modulation and AMPK activation. IM156 increased glycolysis, lipolysis, β -oxidation, and amino acids and decreased free fatty acids, tricarboxylic acid cycle activity, and protein synthesis. In the murine bleomycin model of pulmonary fibrosis, daily oral administration of IM156, administered 7 days after lung injury, attenuated body/lung weight changes and reduced lung fibrosis and inflammatory cell infiltration. The plasma exposures of IM156 were comparable to well tolerated doses in human studies. In conclusion, the metabolic and antifibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for idiopathic pulmonary fibrosis and other fibrotic diseases. SIGNIFICANCE STATEMENT: Fibrosing interstitial lung diseases have a poor prognosis, and current antifibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of oxidative phosphorylation with IM156, prevents myofibroblast phenotype/collagen deposition and is a potentially effective and translational antifibrotic strategy., (Copyright © 2021 The Author(s).)
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- 2021
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6. Apoptosis signal-regulating kinase 1 inhibition reverses deleterious indoxyl sulfate-mediated endothelial effects.
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Savira F, Kompa AR, Magaye R, Xiong X, Huang L, Jucker BM, Willette RN, Kelly DJ, and Wang BH
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- Animals, Apoptosis drug effects, Cells, Cultured, Endothelial Cells metabolism, Endothelium drug effects, Endothelium, Vascular drug effects, Endothelium, Vascular metabolism, Indican adverse effects, Indican pharmacology, MAP Kinase Kinase Kinase 5 physiology, Male, NADPH Oxidases metabolism, Nitric Oxide Synthase Type III metabolism, Oxidative Stress drug effects, Rats, Rats, Sprague-Dawley, Reactive Oxygen Species metabolism, Signal Transduction drug effects, Vasodilation drug effects, Endothelium metabolism, MAP Kinase Kinase Kinase 5 antagonists & inhibitors, MAP Kinase Kinase Kinase 5 metabolism
- Abstract
Aims: Indoxyl sulfate (IS), a protein-bound uremic toxin, is implicated in endothelial dysfunction, which contributes to adverse cardiovascular events in chronic kidney disease. Apoptosis signal regulating kinase 1 (ASK1) is a reactive oxygen species-driven kinase involved in IS-mediated adverse effects. This study assessed the therapeutic potential of ASK1 inhibition in alleviating endothelial effects induced by IS., Main Methods: IS, in the presence and absence of a selective ASK1 inhibitor (GSK2261818A), was assessed for its effect on vascular reactivity in rat aortic rings, and cultured human aortic endothelial cells where we evaluated phenotypic and mechanistic changes., Key Findings: IS directly impairs endothelium-dependent vasorelaxation and endothelial cell migration. Mechanistic studies revealed increased production of reactive oxygen species-related markers, reduction of endothelial nitric oxide synthase and increased protein expression of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). IS also increases angiopoietin-2 and tumour necrosis factor α gene expression and promotes transforming growth factor β receptor abundance. Inhibition of ASK1 ameliorated the increase in oxidative stress markers, promoted autocrine interleukin 8 pro-angiogenic signalling and decreased anti-angiogenic responses at least in part via reducing TIMP1 protein expression., Significance: ASK1 inhibition attenuated vasorelaxation and endothelial cell migration impaired by IS. Therefore, ASK1 is a viable intracellular target to alleviate uremic toxin-induced impairment in the vasculature., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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7. High FGF23 Levels Failed to Predict Cardiac Hypertrophy in Animal Models of Hyperphosphatemia and Chronic Renal Failure.
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Moench I, Aravindhan K, Kuziw J, Schnackenberg CG, Willette RN, Toomey JR, and Gatto GJ Jr
- Abstract
Increased fibroblast growth factor 23 (FGF23) levels are an independent predictor for adverse cardiac events suggesting a role as a link that drives cardiomyopathic changes in cardiorenal syndrome. The search for the underlying mechanism driving this interaction has led to the hypothesis that FGF23 causes pathogenic changes in the heart. Increased serum FGF23 has been independently shown to cause increased cardiac morbidity, mortality, and hypertrophy by signalling through FGF receptor 4. This mechanistic concept was based on preclinical studies demonstrating inhibition of FGF23 signaling through FGF4, which led to suppression of left ventricular hypertrophy and fibrosis in a 2-week rat 5/6 nephrectomy study and a 12-week (2%) high-phosphate diet mouse model in which FGF23 levels were markedly elevated. In this report, renal dysfunction was observed in the 5/6 nephrectomy model, and FGF23 levels were significantly elevated, whereas no changes in left ventricular hypertrophy were observed at 2 or 4 weeks postnephrectomy. Mice placed on a high-phosphate diet that did not cause significant renal dysfunction resulted in significantly elevated FGF23 but no changes in left ventricular hypertrophy. The in vivo studies reported here, which were performed to recapitulate the observations of FGF23 as a driver of cardiac hypertrophy, did not lend support to the FGF23-driven cardiac remodelling hypothesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)
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- 2021
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8. RE: Blockade of apoptosis signal-regulating kinase 1 ameliorates cardiac dysfunction in cardiorenal syndrome via enhancing angiogenesis.
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Savira F, Kompa AR, Edgley AJ, Jucker BM, Willette RN, Kelly DJ, and Wang BH
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- Fibrosis, Humans, MAP Kinase Kinase Kinase 5, Cardio-Renal Syndrome diagnosis, Cardio-Renal Syndrome drug therapy, Heart Diseases
- Abstract
Competing Interests: Declaration of Competing Interest The authors declare that there is no duality of interest associated with this manuscript.
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- 2021
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9. RE: Inhibition of apoptosis signal-regulating kinase 1 might be a novel therapeutic target in the treatment of cardiorenal syndrome.
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Savira F, Edgley AJ, Jucker BM, Willette RN, Krum H, Kelly DJ, Kompa AR, and Wang BH
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- Fibrosis, Humans, MAP Kinase Kinase Kinase 5, Cardio-Renal Syndrome drug therapy
- Abstract
Competing Interests: Declaration of Competing Interest The authors declare that there is no duality of interest associated with this manuscript.
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- 2021
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10. RE: ASK1, a new target in treating cardiorenal syndrome (CRS).
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Savira F, Kompa AR, Edgley AJ, Jucker BM, Willette RN, Kelly DJ, and Wang BH
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- Animals, Fibrosis, Hypertrophy, MAP Kinase Kinase Kinase 5, Rats, Cardio-Renal Syndrome diagnosis, Cardio-Renal Syndrome therapy, Ventricular Dysfunction, Left
- Abstract
Competing Interests: Declaration of Competing Interest The authors declare that there is no duality of interest associated with this manuscript.
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- 2020
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11. Inhibition of apoptosis signal-regulating kinase 1 ameliorates left ventricular dysfunction by reducing hypertrophy and fibrosis in a rat model of cardiorenal syndrome.
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Savira F, Wang BH, Edgley AJ, Jucker BM, Willette RN, Krum H, Kelly DJ, and Kompa AR
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- Animals, Fibrosis, Hypertrophy, Left Ventricular, MAP Kinase Kinase Kinase 5, Male, Rats, Rats, Sprague-Dawley, Cardio-Renal Syndrome drug therapy, Ventricular Dysfunction, Left
- Abstract
Background: Cardiorenal syndrome (CRS) is a major health burden worldwide in need of novel therapies, as current treatments remain suboptimal. The present study assessed the therapeutic potential of apoptosis signal-regulating kinase 1 (ASK1) inhibition in a rat model of CRS., Methods: Adult male Sprague-Dawley rats underwent surgery for myocardial infarction (MI) (week 0) followed by 5/6 subtotal nephrectomy (STNx) at week 4 to induce to induce a combined model of heart and kidney dysfunction. At week 6, MI + STNx animals were randomized to receive either 0.5% carboxymethyl cellulose (Vehicle, n = 15, Sham = 10) or G226 (15 mg/kg daily, n = 11). Cardiac and renal function was assessed by echocardiography and glomerular filtration rate (GFR) respectively, prior to treatment at week 6 and endpoint (week 14). Haemodynamic measurements were determined at endpoint prior to tissue analysis., Results: G226 treatment attenuated the absolute change in left ventricular (LV) fractional shortening and posterior wall thickness compared to Vehicle. G226 also attenuated the reduction in preload recruitable stroke work. Increased myocyte cross sectional area, cardiac interstitial fibrosis, immunoreactivity of cardiac collagen-I and III and cardiac TIMP-2 activation, were significantly reduced following G226 treatment. Although we did not observe improvement in GFR, G226 significantly reduced renal interstitial fibrosis, diminished renal collagen-I and -IV, kidney injury molecule-1 immunoreactivity as well as macrophage infiltration and SMAD2 phosphorylation., Conclusion: Inhibition of ASK1 ameliorated LV dysfunction and diminished cardiac hypertrophy and cardiorenal fibrosis in a rat model of CRS. This suggests that ASK1 is a critical pathway with therapeutic potential in the CRS setting., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2020
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12. Assessing the Impact of Losmapimod on Proteinuria in Idiopathic Focal Segmental Glomerulosclerosis.
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Gipson DS, Hladunewich MA, Lafayette R, Sedor JR, Rovin BH, Barbour SJ, McMahon A, Jennette JC, Nachman PH, Willette RN, Paglione M, Gao F, Ross Terres JA, Vallow S, Holland MC, Thorneloe KS, and Sprecher DL
- Abstract
Introduction: Idiopathic focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease. In preclinical models and biopsies of human FSGS kidneys, p38 mitogen-activated protein kinase (MAPK) has demonstrated enhanced activity; and p38 MAPK inhibition has improved disease markers. This proof-of-concept trial aimed to assess efficacy, safety, tolerability, and pharmacokinetics of losmapimod, an oral p38 MAPK inhibitor, in humans with FSGS., Methods: A single-arm, multicenter, open-label, Phase II trial (NCT02000440) was conducted in adults with FSGS; proteinuria ≥2.0 g/d; estimated glomerular filtration rate (eGFR) ≥45 ml/min per 1.73 m
2 ; blood pressure <140/90 mm Hg. Collapsing and genetic forms of FSGS were excluded. The primary endpoint was number of patients with ≥50% proteinuria reduction and eGFR ≥70% of baseline after receiving losmapimod twice-daily for 16 to 24 weeks., Results: Seventeen patients received ≥1 losmapimod dose. No patients achieved the primary endpoint; therefore, the study was terminated following a prespecified interim analysis. At week 24, proteinuria reductions between 20% and <50% were observed in 4 patients and proteinuria increases >20% in 3 patients. One patient achieved a proteinuria response (≥50% reduction) at week 2 but subsequently relapsed. Losmapimod pharmacokinetics were consistent with prior studies. No serious adverse events (AEs) were reported., Conclusion: p38 MAPK inhibition with losmapimod did not result in ≥50% reduction of proteinuria in patients with FSGS. However, study population heterogeneity may have contributed to our negative findings and therefore this does not eliminate the potential to demonstrate benefit in a population more sensitive to p38 MAPK inhibition if identifiable in the future by precision-medicine methods., (© 2020 Published by Elsevier, Inc., on behalf of the International Society of Nephrology.)- Published
- 2020
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13. Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.
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Guo Y, Sui JY, Kim K, Zhang Z, Qu XA, Nam YJ, Willette RN, Barnett JV, Knollmann BC, Force T, and Lal H
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- Animals, Biomarkers metabolism, Heart Failure genetics, Heart Failure pathology, MAP Kinase Kinase 1 genetics, MAP Kinase Kinase 1 metabolism, Mice, Mice, Knockout, Myocardium pathology, Protein Serine-Threonine Kinases genetics, Algorithms, Gene Expression Profiling, Heart Failure enzymology, MAP Kinase Signaling System, Myocardium enzymology, Oligonucleotide Array Sequence Analysis, Protein Serine-Threonine Kinases metabolism
- Abstract
Background: Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown., Methods: We used the Expression2Kinase algorithm for the screening of kinase targets. To determine the role of HIPK2 in the heart, we generated cardiomyocyte (CM)-specific HIPK2 knockout and heterozygous mice. Heart function was examined by echocardiography, and related cellular and molecular mechanisms were examined. Adeno-associated virus serotype 9 carrying cardiac-specific constitutively active MEK1 (TnT-MEK1-CA) was administrated to rescue cardiac dysfunction in CM-HIPK2 knockout mice., Results: To our knowledge, this is the first study to define the role of HIPK2 in cardiac biology. Using multiple HIPK2 loss-of-function mouse models, we demonstrated that reduction of HIPK2 in CMs leads to cardiac dysfunction, suggesting a causal role in heart failure. It is important to note that cardiac dysfunction in HIPK2 knockout mice developed with advancing age, but not during development. In addition, CM-HIPK2 knockout mice and CM-HIPK2 heterozygous mice exhibited a gene dose-response relationship of CM-HIPK2 on heart function. HIPK2 expression in the heart was significantly reduced in human end-stage ischemic cardiomyopathy in comparison to nonfailing myocardium, suggesting a clinical relevance of HIPK2 in cardiac biology. In vitro studies with neonatal rat ventricular CMscorroborated the in vivo findings. Specifically, adenovirus-mediated overexpression of HIPK2 suppressed the expression of heart failure markers, NPPA and NPPB , at basal condition and abolished phenylephrine-induced pathological gene expression. An array of mechanistic studies revealed impaired extracellular signal-regulated kinase 1/2 signaling in HIPK2-deficient hearts. An in vivo rescue experiment with adeno-associated virus serotype 9 TnT-MEK1-CA nearly abolished the detrimental phenotype of knockout mice, suggesting that impaired extracellular signal-regulated kinase signaling mediated apoptosis as the key factor driving the detrimental phenotype in CM-HIPK2 knockout mice hearts., Conclusions: Taken together, these findings suggest that CM-HIPK2 is required to maintain normal cardiac function via extracellular signal-regulated kinase signaling.
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- 2019
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14. Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery.
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Feric NT, Pallotta I, Singh R, Bogdanowicz DR, Gustilo MM, Chaudhary KW, Willette RN, Chendrimada TP, Xu X, Graziano MP, and Aschar-Sobbi R
- Abstract
Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology.)
- Published
- 2019
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15. CXA-10, a Nitrated Fatty Acid, Is Renoprotective in Deoxycorticosterone Acetate-Salt Nephropathy.
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Arbeeny CM, Ling H, Smith MM, O'Brien S, Wawersik S, Ledbetter SR, McAlexander A, Schopfer FJ, Willette RN, and Jorkasky DK
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- Animals, Desoxycorticosterone Acetate pharmacokinetics, Kidney metabolism, Kidney Diseases metabolism, Male, Mice, Nitro Compounds pharmacokinetics, Oleic Acids pharmacokinetics, Oxidative Stress drug effects, Tissue Distribution, Cytoprotection drug effects, Desoxycorticosterone Acetate pharmacology, Kidney drug effects, Kidney pathology, Kidney Diseases chemically induced, Kidney Diseases pathology, Nitro Compounds pharmacology, Oleic Acids pharmacology, Salts adverse effects
- Abstract
Underlying pathogenic mechanisms in chronic kidney disease (CKD) include chronic inflammation, oxidant stress, and matrix remodeling associated with dysregulated nuclear factor- κ B, nuclear factor- κ B, and SMAD signaling pathways, respectively. Important cytoprotective mechanisms activated by oxidative inflammatory conditions are mediated by nitrated fatty acids that covalently modify proteins to limit inflammation and oxidant stress. In the present study, we evaluated the effects of chronic treatment with CXA-10 (10-nitro-9(E)-octadec-9-enoic acid) in the uninephrectomized deoxycorticosterone acetate-high-salt mouse model of CKD. After 4 weeks of treatment, CXA-10 [2.5 millligrams per kilogram (mpk), p.o.] significantly attenuated increases in plasma cholesterol, heart weight, and kidney weight observed in the model without impacting systemic arterial blood pressure. CXA-10 also reduced albuminuria, nephrinuria, glomerular hypertrophy, and glomerulosclerosis in the model. Inflammatory MCP-1 and fibrosis (collagen, fibronectin, plasminogen activator inhibitor-1, and osteopontin) renal biomarkers were significantly reduced in the CXA-10 (2.5 mpk) group. The anti-inflammatory and antifibrotic effects, as well as glomerular protection, were not observed in the enalapril-treated group. Also, CXA-10 appears to exhibit hormesis as all protective effects observed in the low-dose group were absent in the high-dose group (12.5 mpk). Taken together, these findings demonstrate that, at the appropriate dose, the nitrated fatty acid CXA-10 exhibits anti-inflammatory and antifibrotic effects in the kidney and limits renal injury in a model of CKD., (Copyright © 2019 The Author(s).)
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- 2019
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16. Inhibition of Apoptosis Signal-Regulating Kinase 1 Attenuates Myocyte Hypertrophy and Fibroblast Collagen Synthesis.
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Yang W, Wang BH, Wang I, Huang L, Savira F, Kompa A, Jucker BM, Willette RN, Kelly D, Krum H, Li Z, and Fu Q
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- Animals, Animals, Newborn, Cardiomegaly metabolism, Cardiomegaly pathology, Cells, Cultured, Disease Models, Animal, Fibroblasts metabolism, MAP Kinase Kinase Kinase 5 antagonists & inhibitors, MAP Kinase Kinase Kinase 5 biosynthesis, Myocytes, Cardiac metabolism, Polymerase Chain Reaction, Rats, Rats, Sprague-Dawley, Signal Transduction, Cardiomegaly genetics, Collagen biosynthesis, Gene Expression Regulation, Developmental, MAP Kinase Kinase Kinase 5 genetics, Myocytes, Cardiac pathology, RNA genetics
- Abstract
Background: Cardiac remodelling is a dynamic process whereby structural and functional changes occur within the heart in response to injury or inflammation. Recent studies have demonstrated reactive oxygen species sensitive MAPK, apoptosis signal-regulating kinase 1 (ASK1) plays a critical role in cardiac remodelling. This study aims to determine the effectiveness of small molecule ASK1 inhibitors on these processes and their therapeutic potential., Methods: Neonatal rat cardiac fibroblasts (NCF) were pre-treated with ASK1 inhibitors, G2261818A (G226) and G2358939A (G235), for 2hours before stimulated with 100nM angiotensin II (AngII), 10μM indoxyl sulphate (IS) or 10ng/ml transforming growth factor β
1 (TGFβ1 ) for 48hours. Neonatal rat cardiac myocytes (NCM) were pre-treated with G226 and G235 for 2hours before being stimulated with 100nM AngII for 60hours, 10μM IS, 10ng/ml interleukin 1β (IL-1β) or tumour necrosis factor α (TNFα) for 48hours.3 H-proline and3 H-leucine incorporation was used to assess collagen turnover and hypertrophy, respectively. Pro-fibrotic, pro-hypertrophic and THP-1 inflammatory cytokine gene expressions were determined by RT-PCR., Results: Both G226 and G235 dose-dependently attenuated AngII-, IS-, IL-1β- and TNFα-stimulated NCM hypertrophy and hypertrophic gene expression, IS-, AngII- and TGFβ1 -stimulated NCF collagen synthesis and AngII- and TGFβ1 -stimulated pro-fibrotic gene expression. Inhibition of ASK1 by G226 and G235 inhibited lipopolysaccharides-stimulated inflammatory cytokine gene expression in THP-1 cells., Conclusions: Selective ASK1 inhibition confers anti-hypertrophic and anti-fibrotic effects in cardiac cells, and anti-inflammation in monocytic cells. ASK1 inhibitors may represent novel therapeutic agents to alleviate cardiac remodelling post cardiac injury where hypertrophy, fibrosis and inflammation play critical roles., (Copyright © 2017 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)- Published
- 2019
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17. Apoptosis signal-regulating kinase 1 inhibition attenuates cardiac hypertrophy and cardiorenal fibrosis induced by uremic toxins: Implications for cardiorenal syndrome.
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Savira F, Cao L, Wang I, Yang W, Huang K, Hua Y, Jucker BM, Willette RN, Huang L, Krum H, Li Z, Fu Q, and Wang BH
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- Animals, Cells, Cultured, Cresols pharmacology, Fibrosis, Indican pharmacology, Mitogen-Activated Protein Kinases metabolism, NF-kappa B metabolism, Protein Kinase Inhibitors pharmacology, Rats, Sulfuric Acid Esters pharmacology, Cardio-Renal Syndrome pathology, Cardiomegaly prevention & control, MAP Kinase Kinase Kinase 5 antagonists & inhibitors, Toxins, Biological toxicity
- Abstract
Intracellular accumulation of protein-bound uremic toxins in the setting of cardiorenal syndrome leads to adverse effects on cardiorenal cellular functions, where cardiac hypertrophy and cardiorenal fibrosis are the hallmarks. In this study, we sought to determine if Apoptosis Signal-Regulated Kinase 1 (ASK1), an upstream regulator of cellular stress response, mediates cardiac hypertrophy and cardiorenal fibrosis induced by indoxyl sulfate (IS) and p-cresol sulfate (PCS) in vitro, and whether ASK1 inhibition is beneficial to ameliorate these cellular effects. PCS augmented cardiac myocyte hypertrophy and fibroblast collagen synthesis (as determined by 3H-leucine and 3H-proline incorporation, respectively), similar to our previous finding with IS. IS and PCS also increased collagen synthesis of proximal tubular cells and renal mesangial cells. Pro-hypertrophic (α-skeletal muscle actin and β-MHC) and pro-fibrotic genes (TGF-β1 and ctgf) were induced by both IS and PCS. Western blot analyses revealed the activation of ASK1 and downstream mitogen activated protein kinases (MAPKs) (p38MAPK and ERK1/2) as well as nuclear factor-kappa B (NF-κB) by IS and PCS. ASK1, OAT1/3, ERK1/2 and p38MAPK inhibitors suppressed all these effects. In summary, IS and PCS exhibit pro-hypertrophic and pro-fibrotic properties, at least in part, via the activation of ASK1 and its downstream pathways. ASK1 inhibitor is an effective therapeutic agent to alleviate protein-bound uremic toxin-induced cardiac hypertrophy and cardiorenal fibrosis in vitro, and may be translated further for cardiorenal syndrome therapy.
- Published
- 2017
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18. Toward improved myocardial maturity in an organ-on-chip platform with immature cardiac myocytes.
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Sheehy SP, Grosberg A, Qin P, Behm DJ, Ferrier JP, Eagleson MA, Nesmith AP, Krull D, Falls JG, Campbell PH, McCain ML, Willette RN, Hu E, and Parker KK
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- Animals, Cell Differentiation, Cells, Cultured, Gene Expression Profiling, Lab-On-A-Chip Devices, Myocardial Contraction physiology, Rats, Rats, Sprague-Dawley, Heart Ventricles cytology, Microchip Analytical Procedures methods, Myocytes, Cardiac cytology, Myocytes, Cardiac physiology, Tissue Engineering methods, Ventricular Function physiology
- Abstract
In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the β-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.
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- 2017
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19. PROPERTIES OF THE TRPV4 AGONIST GSK1016790A AND the TRPV4 ANTAGONIST GSK2193874.
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Thorneloe KS, Cheung M, Holt DA, and Willette RN
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- Leucine analogs & derivatives, Sulfonamides, TRPV Cation Channels
- Published
- 2017
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20. The prolyl 4-hydroxylase inhibitor GSK360A decreases post-stroke brain injury and sensory, motor, and cognitive behavioral deficits.
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Zhou J, Li J, Rosenbaum DM, Zhuang J, Poon C, Qin P, Rivera K, Lepore J, Willette RN, Hu E, and Barone FC
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- Administration, Oral, Animals, Brain drug effects, Brain metabolism, Brain pathology, Brain Injuries blood, Brain Injuries physiopathology, Cognition Disorders etiology, Erythropoietin blood, Erythropoietin genetics, Glycine administration & dosage, Glycine pharmacokinetics, Glycine pharmacology, Glycine therapeutic use, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Infarction, Middle Cerebral Artery blood, Infarction, Middle Cerebral Artery complications, Infarction, Middle Cerebral Artery pathology, Infarction, Middle Cerebral Artery physiopathology, Male, Organ Specificity drug effects, Prolyl Hydroxylases metabolism, Prolyl-Hydroxylase Inhibitors administration & dosage, Prolyl-Hydroxylase Inhibitors pharmacology, Quinolones administration & dosage, Quinolones pharmacokinetics, Quinolones pharmacology, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Sprague-Dawley, Sensation drug effects, Stroke blood, Stroke physiopathology, Vascular Endothelial Growth Factor A blood, Vascular Endothelial Growth Factor A genetics, Behavior, Animal drug effects, Brain Injuries drug therapy, Brain Injuries etiology, Cognition Disorders drug therapy, Glycine analogs & derivatives, Motor Activity drug effects, Prolyl-Hydroxylase Inhibitors therapeutic use, Quinolones therapeutic use, Stroke complications
- Abstract
There is interest in pharmacologic preconditioning for end-organ protection by targeting the HIF system. This can be accomplished by inhibition of prolyl 4-hydroxylase (PHD). GSK360A is an orally active PHD inhibitor that has been previously shown to protect the failing heart. We hypothesized that PHD inhibition can also protect the brain from injuries and resulting behavioral deficits that can occur as a result of surgery. Thus, our goal was to investigate the effect of pre-stroke surgery brain protection using a verified GSK360A PHD inhibition paradigm on post-stroke surgery outcomes. Vehicle or an established protective dose (30 mg/kg, p.o.) of GSK360A was administered to male Sprague-Dawley rats. Initially, GSK360A pharmacokinetics and organ distribution were determined, and then PHD-HIF pharmacodynamic markers were measured (i.e., to validate the pharmacological effects of the GSK360A administration regimen). Results obtained using this validated PHD dose-regimen indicated significant improvement by GSK360A (30mg/kg); administered at 18 and 5 hours prior to transient middle cerebral artery occlusion (stroke). GSK360A exposure and plasma, kidney and brain HIF-PHD pharmacodynamics endpoints (e.g., erythropoietin; EPO and Vascular Endothelial Growth Factor; VEGF) were measured. GSK360A provided rapid exposure in plasma (7734 ng/ml), kidney (45-52% of plasma level) and brain (1-4% of plasma level), and increased kidney EPO mRNA (80-fold) and brain VEGF mRNA (2-fold). We also observed that GSK360A increased plasma EPO (300-fold) and VEGF (2-fold). Further assessments indicated that GSK360A reduced post-stroke surgery neurological deficits (47-64%), cognitive dysfunction (60-75%) and brain infarction (30%) 4 weeks later. Thus, PHD inhibition using GSK360A pretreatment produced long-term post-stroke brain protection and improved behavioral functioning. These data support PHD inhibition, specifically by GSK360A, as a potential strategy for pre-surgical use to reduce brain injury and functional decline due to surgery-related cerebral injury.
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- 2017
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21. Activation of the Amino Acid Response Pathway Blunts the Effects of Cardiac Stress.
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Qin P, Arabacilar P, Bernard RE, Bao W, Olzinski AR, Guo Y, Lal H, Eisennagel SH, Platchek MC, Xie W, Del Rosario J, Nayal M, Lu Q, Roethke T, Schnackenberg CG, Wright F, Quaile MP, Halsey WS, Hughes AM, Sathe GM, Livi GP, Kirkpatrick RB, Qu XA, Rajpal DK, Faelth Savitski M, Bantscheff M, Joberty G, Bergamini G, Force TL, Gatto GJ Jr, Hu E, and Willette RN
- Subjects
- Amino Acids deficiency, Amino Acyl-tRNA Synthetases antagonists & inhibitors, Amino Acyl-tRNA Synthetases metabolism, Animals, Autophagy drug effects, Cells, Cultured, Disease Models, Animal, Dose-Response Relationship, Drug, Fibroblasts metabolism, Fibroblasts pathology, Fibrosis, Heart Failure metabolism, Heart Failure pathology, Heart Failure physiopathology, Humans, Hypertrophy, Left Ventricular metabolism, Hypertrophy, Left Ventricular pathology, Hypertrophy, Left Ventricular physiopathology, Hypertrophy, Left Ventricular prevention & control, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells metabolism, Male, Mice, Inbred C57BL, Myocytes, Cardiac metabolism, Myocytes, Cardiac pathology, Protein Serine-Threonine Kinases metabolism, Time Factors, Ventricular Function, Left drug effects, Ventricular Remodeling drug effects, Amino Acids metabolism, Enzyme Inhibitors pharmacology, Fibroblasts drug effects, Heart Failure prevention & control, Myocytes, Cardiac drug effects, Piperidines pharmacology, Protein Synthesis Inhibitors pharmacology, Quinazolinones pharmacology, Stress, Physiological
- Abstract
Background: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure., Methods and Results: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner., Conclusions: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress., (© 2017 The Authors and GlaxoSmithKline. Published on behalf of the American Heart Association, Inc., by Wiley.)
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- 2017
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22. Discovery of GSK2193874: An Orally Active, Potent, and Selective Blocker of Transient Receptor Potential Vanilloid 4.
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Cheung M, Bao W, Behm DJ, Brooks CA, Bury MJ, Dowdell SE, Eidam HS, Fox RM, Goodman KB, Holt DA, Lee D, Roethke TJ, Willette RN, Xu X, Ye G, and Thorneloe KS
- Abstract
Transient Receptor Potential Vanilloid 4 (TRPV4) is a member of the Transient Receptor Potential (TRP) superfamily of cation channels. TRPV4 is expressed in the vascular endothelium in the lung and regulates the integrity of the alveolar septal barrier. Increased pulmonary vascular pressure evokes TRPV4-dependent pulmonary edema, and therefore, inhibition of TRPV4 represents a novel approach for the treatment of pulmonary edema associated with conditions such as congestive heart failure. Herein we report the discovery of an orally active, potent, and selective TRPV4 blocker, 3-(1,4'-bipiperidin-1'-ylmethyl)-7-bromo- N -(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide (GSK2193874, 28 ) after addressing an unexpected off-target cardiovascular liability observed from in vivo studies. GSK2193874 is a selective tool for elucidating TRPV4 biology both in vitro and in vivo .
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- 2017
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23. Cardioprotection Resulting from Glucagon-Like Peptide-1 Administration Involves Shifting Metabolic Substrate Utilization to Increase Energy Efficiency in the Rat Heart.
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Aravindhan K, Bao W, Harpel MR, Willette RN, Lepore JJ, and Jucker BM
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- Animals, Carbon Isotopes metabolism, Cardiotonic Agents administration & dosage, Cyclic AMP metabolism, Energy Metabolism physiology, Fatty Acids metabolism, Glucagon-Like Peptide 1 administration & dosage, Glucose metabolism, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Myocytes, Cardiac metabolism, Oxygen Consumption physiology, Rats, Cardiotonic Agents pharmacology, Energy Metabolism drug effects, Glucagon-Like Peptide 1 pharmacology, Myocardium metabolism, Reperfusion Injury metabolism
- Abstract
Previous studies have shown that glucagon-like peptide-1 (GLP-1) provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. Here we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes (CM). Relative carbohydrate and fat oxidation levels were measured in both normal and ischemic hearts using a 1-13C glucose clamp coupled with NMR-based isotopomer analysis, as well as in adult rat CMs by monitoring pH and O2 consumption in the presence of glucose or palmitate. In normal heart, GLP-1 increased glucose uptake (↑64%, p<0.05) without affecting glycogen levels. In ischemic hearts, GLP-1 induced metabolic substrate switching by increasing the ratio of carbohydrate versus fat oxidation (↑14%, p<0.01) in the LV area not at risk, without affecting cAMP levels. Interestingly, no substrate switching occurred in the LV area at risk, despite an increase in cAMP (↑106%, p<0.05) and lactate (↑121%, p<0.01) levels. Furthermore, in isolated CMs GLP-1 treatment increased glucose utilization (↑14%, p<0.05) and decreased fatty acid oxidation (↓15%, p<0.05) consistent with in vivo finding. Our results show that this benefit may derive from distinct and complementary roles of GLP-1 treatment on metabolism in myocardial sub-regions in response to this injury. In particular, a switch to anaerobic glycolysis in the ischemic area provides a compensatory substrate switch to overcome the energetic deficit in this region in the face of reduced tissue oxygenation, whereas a switch to more energetically favorable carbohydrate oxidation in more highly oxygenated remote regions supports maintaining cardiac contractility in a complementary manner.
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- 2015
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24. TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury.
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Balakrishna S, Song W, Achanta S, Doran SF, Liu B, Kaelberer MM, Yu Z, Sui A, Cheung M, Leishman E, Eidam HS, Ye G, Willette RN, Thorneloe KS, Bradshaw HB, Matalon S, and Jordt SE
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- Acute Lung Injury drug therapy, Animals, Anti-Inflammatory Agents pharmacology, Bronchoalveolar Lavage Fluid chemistry, Chlorine toxicity, HEK293 Cells, Humans, Hydrochloric Acid toxicity, Male, Mice, Pneumonia drug therapy, Rats, TRPV Cation Channels agonists, TRPV Cation Channels deficiency, Acute Lung Injury chemically induced, TRPV Cation Channels antagonists & inhibitors
- Abstract
The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function., (Copyright © 2014 the American Physiological Society.)
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- 2014
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25. Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy.
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Seo K, Rainer PP, Shalkey Hahn V, Lee DI, Jo SH, Andersen A, Liu T, Xu X, Willette RN, Lepore JJ, Marino JP Jr, Birnbaumer L, Schnackenberg CG, and Kass DA
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- Animals, HEK293 Cells, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorylation, Rats, TRPC Cation Channels genetics, TRPC6 Cation Channel, Cardiomegaly genetics, TRPC Cation Channels antagonists & inhibitors
- Abstract
Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3-21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management., Competing Interests: X.X, R.N.W., J.J.L., J.P.M., and C.G.S. are employees of Glaxo Smith Kline and contributed substantial resources in developing the new TRPC3/6 channel blockers.
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- 2014
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26. Cardioprotection by systemic dosing of thymosin beta four following ischemic myocardial injury.
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Bao W, Ballard VL, Needle S, Hoang B, Lenhard SC, Tunstead JR, Jucker BM, Willette RN, and Pipes GT
- Abstract
Thymosin beta 4 (Tβ4) was previously shown to reduce infarct size and improve contractile performance in chronic myocardial ischemic injury via two phases of action: an acute phase, just after injury, when Tβ4 preserves ischemic myocardium via antiapoptotic or anti-inflammatory mechanisms; and a chronic phase, when Tβ4 activates the growth of vascular or cardiac progenitor cells. In order to differentiate between the effects of Tβ4 during the acute and during the chronic phases, and also in order to obtain detailed hemodynamic and biomarker data on the effects of Tβ4 treatment suitable for use in clinical studies, we tested Tβ4 in a rat model of chronic myocardial ischemia using two dosing regimens: short term dosing (Tβ4 administered only during the first 3 days following injury), and long term dosing (Tβ4 administered during the first 3 days following injury and also every third day until the end of the study). Tβ4 administered throughout the study reduced infarct size and resulted in significant improvements in hemodynamic performance; however, chamber volumes and ejection fractions were not significantly improved. Tβ4 administered only during the first 3 days following injury tended to reduce infarct size, chamber volumes and improve hemodynamic performance. Plasma biomarkers of myocyte injury were significantly reduced by Tβ4 treatment during the acute injury period, and plasma ANP levels were significantly reduced in both dosing groups. Surprisingly, neither acute nor chronic Tβ4 treatment significantly increased blood vessel density in peri-infarct regions. These results suggest the following: repeated dosing may be required to achieve clinically measureable improvements in cardiac function post-myocardial infarction (MI); improvement in cardiac function may be observed in the absence of a high degree of angiogenesis; and that plasma biomarkers of cardiac function and myocardial injury are sensitive pharmacodynamic biomarkers of the effects of Tβ4.
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- 2013
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27. Novel fusion of GLP-1 with a domain antibody to serum albumin prolongs protection against myocardial ischemia/reperfusion injury in the rat.
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Bao W, Holt LJ, Prince RD, Jones GX, Aravindhan K, Szapacs M, Barbour AM, Jolivette LJ, Lepore JJ, Willette RN, DeAngelis E, and Jucker BM
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- Animals, Cardiotonic Agents administration & dosage, Cardiotonic Agents blood, Cardiotonic Agents pharmacokinetics, Disease Models, Animal, Exenatide, Glucagon-Like Peptide 1 administration & dosage, Glucagon-Like Peptide 1 blood, Glucagon-Like Peptide 1 pharmacokinetics, Glucagon-Like Peptide-1 Receptor, Immunoconjugates administration & dosage, Immunoconjugates blood, Immunoconjugates pharmacokinetics, Injections, Subcutaneous, Male, Myocardial Contraction drug effects, Myocardial Infarction metabolism, Myocardial Infarction pathology, Myocardial Infarction physiopathology, Myocardial Reperfusion Injury metabolism, Myocardial Reperfusion Injury pathology, Myocardial Reperfusion Injury physiopathology, Myocardium metabolism, Myocardium pathology, Peptide Fragments administration & dosage, Peptide Fragments blood, Peptide Fragments pharmacokinetics, Peptides pharmacology, Rats, Rats, Sprague-Dawley, Receptors, Glucagon agonists, Receptors, Glucagon genetics, Single-Domain Antibodies administration & dosage, Single-Domain Antibodies blood, Venoms pharmacology, Ventricular Function, Left drug effects, Cardiotonic Agents pharmacology, Glucagon-Like Peptide 1 pharmacology, Immunoconjugates pharmacology, Myocardial Infarction prevention & control, Myocardial Reperfusion Injury prevention & control, Peptide Fragments pharmacology, Serum Albumin immunology, Single-Domain Antibodies pharmacology
- Abstract
Background: Glucagon-like peptide-1 (GLP-1) and its mimetics reduce infarct size in the setting of acute myocardial ischemia/reperfusion (I/R) injury. However, the short serum half-life of GLP-1 and its mimetics may limit their therapeutic use in acute myocardial ischemia. Domain antibodies to serum albumin (AlbudAbs) have been developed to extend the serum half-life of short lived therapeutic proteins, peptides and small molecules. In this study, we compared the effect of a long acting GLP-1 agonist, DPP-IV resistant GLP-1 (7-36, A8G) fused to an AlbudAb (GAlbudAb), with the effect of the GLP-1 mimetic, exendin-4 (short half-life GLP-1 agonist) on infarct size following acute myocardial I/R injury., Methods: Male Sprague-Dawley rats (8-week-old) were treated with vehicle, GAlbudAb or exendin-4. Myocardial ischemia was induced 2 h following the final dose for GAlbudAb and 30 min post the final dose for exendin-4. In a subgroup of animals, the final dose of exendin-4 was administered (1 μg/kg, SC, bid for 2 days) 6 h prior to myocardial ischemia when plasma exendin-4 was at its minimum concentration (C(min)). Myocardial infarct size, area at risk and cardiac function were determined 24 h after myocardial I/R injury., Results: GAlbudAb and exendin-4 significantly reduced myocardial infarct size by 28% and 23% respectively, compared to vehicle (both p < 0.01 vs. vehicle) after I/R injury. Moreover, both GAlbudAb and exendin-4 markedly improved post-ischemic cardiac contractile function. Body weight loss and reduced food intake consistent with the activation of GLP-1 receptors was observed in all treatment groups. However, exendin-4 failed to reduce infarct size when administered 6 h prior to myocardial ischemia, suggesting continuous activation of the GLP-1 receptors is needed for cardioprotection., Conclusions: Cardioprotection provided by GAlbudAb, a long acting GLP-1 mimetic, following myocardial I/R injury was comparable in magnitude, but more sustained in duration than that produced by short-acting exendin-4. Very low plasma concentrations of exendin-4 failed to protect the heart from myocardial I/R injury, suggesting that sustained GLP-1 receptor activation plays an important role in providing cardioprotection in the setting of acute myocardial I/R injury. Long-acting GLP-1 agonists such as GAlbudAb may warrant additional evaluation as novel therapeutic agents to reduce myocardial I/R injury during acute coronary syndrome.
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- 2013
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28. Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype.
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Ivashchenko CY, Pipes GC, Lozinskaya IM, Lin Z, Xiaoping X, Needle S, Grygielko ET, Hu E, Toomey JR, Lepore JJ, and Willette RN
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- Cell Differentiation physiology, Cell Line, Humans, Ion Channels physiology, Myocytes, Cardiac classification, Phenotype, Pluripotent Stem Cells classification, Action Potentials physiology, Calcium Signaling physiology, Gene Expression Regulation, Developmental physiology, Myocytes, Cardiac cytology, Myocytes, Cardiac physiology, Pluripotent Stem Cells cytology, Pluripotent Stem Cells physiology
- Abstract
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.
- Published
- 2013
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29. The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore.
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Washburn DG, Holt DA, Dodson J, McAtee JJ, Terrell LR, Barton L, Manns S, Waszkiewicz A, Pritchard C, Gillie DJ, Morrow DM, Davenport EA, Lozinskaya IM, Guss J, Basilla JB, Negron LK, Klein M, Willette RN, Fries RE, Jensen TC, Xu X, Schnackenberg CG, and Marino JP Jr
- Subjects
- Diglycerides metabolism, Drug Discovery, HEK293 Cells, Humans, TRPC Cation Channels metabolism, TRPC6 Cation Channel, Aniline Compounds chemistry, Aniline Compounds pharmacology, TRPC Cation Channels antagonists & inhibitors, Thiazoles chemistry, Thiazoles pharmacology
- Abstract
Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease., (Published by Elsevier Ltd.)
- Published
- 2013
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30. Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction after aortic constriction in rats and mice.
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Morgan LA, Olzinski AR, Upson JJ, Zhao S, Wang T, Eisennagel SH, Hoang B, Tunstead JR, Marino JP Jr, Willette RN, Jucker BM, and Behm DJ
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- Animals, Aorta pathology, Cardiomegaly drug therapy, Cardiomegaly pathology, Constriction, Pathologic, Disease Models, Animal, Fibrosis, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Species Specificity, Ventricular Remodeling drug effects, Aorta drug effects, Cyclohexylamines pharmacology, Enzyme Inhibitors pharmacology, Epoxide Hydrolases antagonists & inhibitors, Piperidines pharmacology, Triazines pharmacology
- Abstract
Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.
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- 2013
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31. Unrestrained p38 MAPK activation in Dusp1/4 double-null mice induces cardiomyopathy.
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Auger-Messier M, Accornero F, Goonasekera SA, Bueno OF, Lorenz JN, van Berlo JH, Willette RN, and Molkentin JD
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- Animals, Calcium metabolism, Calcium-Binding Proteins deficiency, Calcium-Binding Proteins genetics, Cardiomyopathies diagnosis, Cardiomyopathies genetics, Cardiomyopathies physiopathology, Cardiomyopathies prevention & control, Cells, Cultured, Disease Models, Animal, Dual Specificity Phosphatase 1 genetics, Enzyme Activation, Fibroblasts enzymology, Gene Expression Regulation, Hemodynamics, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Myocardial Contraction, Myocytes, Cardiac drug effects, Myocytes, Cardiac pathology, Phosphorylation, Protein Kinase Inhibitors pharmacology, Protein Tyrosine Phosphatases, Time Factors, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, Cardiomyopathies enzymology, Dual Specificity Phosphatase 1 deficiency, Myocytes, Cardiac enzymology, Signal Transduction, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
Rationale: Mitogen-activated protein kinases (MAPKs) are activated in the heart by disease-inducing and stress-inducing stimuli, where they participate in hypertrophy, remodeling, contractility, and heart failure. A family of dual-specificity phosphatases (DUSPs) directly inactivates each of the MAPK terminal effectors, potentially serving a cardioprotective role., Objective: To determine the role of DUSP1 and DUSP4 in regulating p38 MAPK function in the heart and the effect on disease., Methods and Results: Here, we generated mice and mouse embryonic fibroblasts lacking both Dusp1 and Dusp4 genes. Although single nulls showed no molecular effects, combined disruption of Dusp1/4 promoted unrestrained p38 MAPK activity in both mouse embryonic fibroblasts and the heart, with no change in the phosphorylation of c-Jun N-terminal kinases or extracellular signal-regulated kinases at baseline or with stress stimulation. Single disruption of either Dusp1 or Dusp4 did not result in cardiac pathology, although Dusp1/4 double-null mice exhibited cardiomyopathy and increased mortality with aging. Pharmacological inhibition of p38 MAPK with SB731445 ameliorated cardiomyopathy in Dusp1/4 double-null mice, indicating that DUSP1/4 function primarily through p38 MAPK in affecting disease. At the cellular level, unrestrained p38 MAPK activity diminished cardiac contractility and Ca2+ handling, which was acutely reversed with a p38 inhibitory compound. Poor function in Dusp1/4 double-null mice also was partially rescued by phospholamban deletion., Conclusions: Our data demonstrate that Dusp1 and Dusp4 are cardioprotective genes that play a critical role in the heart by dampening p38 MAPK signaling that would otherwise reduce contractility and induce cardiomyopathy.
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- 2013
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32. Chronic inhibition of 11 β -hydroxysteroid dehydrogenase type 1 activity decreases hypertension, insulin resistance, and hypertriglyceridemia in metabolic syndrome.
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Schnackenberg CG, Costell MH, Krosky DJ, Cui J, Wu CW, Hong VS, Harpel MR, Willette RN, and Yue TL
- Subjects
- 11-beta-Hydroxysteroid Dehydrogenase Type 1 antagonists & inhibitors, Animals, Humans, Hypertension metabolism, Hypertension pathology, Hypertriglyceridemia pathology, Insulin Resistance genetics, Liver enzymology, Liver metabolism, Liver physiopathology, Metabolic Syndrome pathology, Obesity blood, Obesity enzymology, Obesity physiopathology, Rats, Receptors, Leptin genetics, Receptors, Leptin metabolism, Weight Gain, 11-beta-Hydroxysteroid Dehydrogenase Type 1 biosynthesis, Glucocorticoids metabolism, Hypertension enzymology, Hypertriglyceridemia enzymology, Metabolic Syndrome enzymology
- Abstract
Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11 β -hydroxysteroid dehydrogenase type 1 (11 β -HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11 β -HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11 β -HSD1. Compound 11 significantly decreased 11 β -HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11 β -HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.
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- 2013
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33. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.
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Thorneloe KS, Cheung M, Bao W, Alsaid H, Lenhard S, Jian MY, Costell M, Maniscalco-Hauk K, Krawiec JA, Olzinski A, Gordon E, Lozinskaya I, Elefante L, Qin P, Matasic DS, James C, Tunstead J, Donovan B, Kallal L, Waszkiewicz A, Vaidya K, Davenport EA, Larkin J, Burgert M, Casillas LN, Marquis RW, Ye G, Eidam HS, Goodman KB, Toomey JR, Roethke TJ, Jucker BM, Schnackenberg CG, Townsley MI, Lepore JJ, and Willette RN
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- Administration, Oral, Animals, Blood Pressure drug effects, Calcium metabolism, Disease Models, Animal, Diuretics pharmacology, Endothelium drug effects, Endothelium metabolism, Endothelium pathology, Heart Failure pathology, Heart Failure physiopathology, Heart Rate drug effects, Humans, In Vitro Techniques, Ion Channel Gating drug effects, Lung drug effects, Lung metabolism, Lung pathology, Membrane Transport Modulators chemistry, Membrane Transport Modulators pharmacology, Mice, Mice, Knockout, Permeability drug effects, Protein Transport drug effects, Pulmonary Edema etiology, Pulmonary Edema pathology, Rats, TRPV Cation Channels metabolism, Water-Electrolyte Balance drug effects, Heart Failure complications, Membrane Transport Modulators administration & dosage, Membrane Transport Modulators therapeutic use, Pulmonary Edema drug therapy, Pulmonary Edema prevention & control, TRPV Cation Channels antagonists & inhibitors
- Abstract
Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.
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- 2012
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34. Effects of p38 mitogen-activated protein kinase inhibition on vascular and systemic inflammation in patients with atherosclerosis.
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Elkhawad M, Rudd JH, Sarov-Blat L, Cai G, Wells R, Davies LC, Collier DJ, Marber MS, Choudhury RP, Fayad ZA, Tawakol A, Gleeson FV, Lepore JJ, Davis B, Willette RN, Wilkinson IB, Sprecher DL, and Cheriyan J
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- Aged, Aortitis blood, Aortitis diagnostic imaging, Aortitis enzymology, Atherosclerosis blood, Atherosclerosis diagnostic imaging, Atherosclerosis enzymology, Carotid Artery Diseases blood, Carotid Artery Diseases diagnostic imaging, Carotid Artery Diseases enzymology, Double-Blind Method, Female, Fluorodeoxyglucose F18, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors therapeutic use, Inflammation Mediators blood, Intra-Abdominal Fat diagnostic imaging, Intra-Abdominal Fat drug effects, Male, Middle Aged, Multimodal Imaging, Odds Ratio, Positron-Emission Tomography, Predictive Value of Tests, Radiopharmaceuticals, Subcutaneous Fat diagnostic imaging, Subcutaneous Fat drug effects, Time Factors, Tomography, X-Ray Computed, Treatment Outcome, United Kingdom, p38 Mitogen-Activated Protein Kinases metabolism, Anti-Inflammatory Agents therapeutic use, Aortitis drug therapy, Atherosclerosis drug therapy, Carotid Artery Diseases drug therapy, Cyclopropanes therapeutic use, Protein Kinase Inhibitors therapeutic use, Pyridines therapeutic use, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
Objectives: This study sought to determine the effects of a p38 mitogen-activated protein kinase inhibitor, losmapimod, on vascular inflammation, by (18)F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography imaging., Background: The p38 mitogen-activated protein kinase cascade plays an important role in the initiation and progression of inflammatory diseases, including atherosclerosis., Methods: Patients with atherosclerosis on stable statin therapy (n = 99) were randomized to receive losmapimod 7.5 mg once daily (lower dose [LD]), twice daily (higher dose [HD]) or placebo for 84 days. Vascular inflammation was assessed by FDG positron emission tomography/computed tomography imaging of the carotid arteries and aorta; analyses focused on the index vessel (the artery with the highest average maximum tissue-to-background ratio [TBR] at baseline). Serum inflammatory biomarkers and FDG uptake in visceral and subcutaneous fat were also measured., Results: The primary endpoint, change from baseline in average TBR across all segments in the index vessel, was not significantly different between HD and placebo (ΔTBR: -0.04 [95% confidence interval [CI]: -0.14 to +0.06], p = 0.452) or LD and placebo (ΔTBR: -0.02 [95% CI: -0.11 to +0.06], p = 0.579). However, there was a statistically significant reduction in average TBR in active segments (TBR ≥1.6) (HD vs. placebo: ΔTBR: -0.10 [95% CI: -0.19 to -0.02], p = 0.0125; LD vs. placebo: ΔTBR: -0.10 [95% CI: -0.18 to -0.02], p = 0.0194). The probability of a segment being active was also significantly reduced for HD when compared with placebo (OR: 0.57 [95% CI: 0.41 to 0.81], p = 0.002). Within the HD group, reductions were observed in placebo-corrected inflammatory biomarkers including high-sensitivity C-reactive protein (% reduction: -28% [95% CI: -46 to -5], p = 0.023) as well as FDG uptake in visceral fat (ΔSUV: -0.05 [95% CI: -0.09 to -0.01], p = 0.018), but not subcutaneous fat., Conclusions: Despite nonsignificant changes for the primary endpoint of average vessel TBR, HD losmapimod reduced vascular inflammation in the most inflamed regions, concurrent with a reduction in inflammatory biomarkers and FDG uptake in visceral fat. These results suggest a systemic anti-inflammatory effect. (A Study to Evaluate the Effects of 3 Months Dosing With GW856553, as Assessed FDG-PET/CT Imaging; NCT00633022)., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
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- 2012
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35. Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress.
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Costell MH, Ancellin N, Bernard RE, Zhao S, Upson JJ, Morgan LA, Maniscalco K, Olzinski AR, Ballard VL, Herry K, Grondin P, Dodic N, Mirguet O, Bouillot A, Gellibert F, Coatney RW, Lepore JJ, Jucker BM, Jolivette LJ, Willette RN, Schnackenberg CG, and Behm DJ
- Abstract
Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.
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- 2012
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36. Serial MRI characterization of the functional and morphological changes in mouse lung in response to cardiac remodeling following myocardial infarction.
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Alsaid H, Bao W, Rambo MV, Logan GA, Figueroa DJ, Lenhard SC, Kotzer CJ, Burgert ME, Willette RN, Ferrari VA, and Jucker BM
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- Animals, Lung pathology, Lung physiopathology, Male, Mice, Mice, Inbred C57BL, Pulmonary Edema complications, Ventricular Dysfunction, Left complications, Magnetic Resonance Imaging methods, Pulmonary Edema pathology, Pulmonary Edema physiopathology, Ventricular Dysfunction, Left pathology, Ventricular Dysfunction, Left physiopathology, Ventricular Remodeling physiology
- Abstract
The temporal evolution of heart failure and associated pulmonary congestion in rodent heart failure models has not yet been characterized simultaneously and noninvasively. In this study, MRI was used to assess the serial progression of left-ventricular dysfunction and lung congestion in mice following myocardial infarction (MI). Cardiac and lung (1) H MRI was performed at baseline and every 3 days up to 13 days postsurgery in sham and MI mice. Respiratory parameters and terminal lung mechanics were assessed followed by histological analysis. MRI revealed that the MI induced significant pulmonary congestion/edema as detected by increased MRI signal intensity and was associated with increased lung volume and reduced cardiac contractility. Pulmonary function was also depressed in MI-mice, reflected by a reduced tidal volume and a low minute ventilation rate. Additionally, MI significantly increased lung resistance, markedly reduced lung compliance and total lung capacity and significantly increased lung weights by 57%. Significant correlations were observed between the MRI measured lung congestion, lung volume, ejection fraction, and lung wet-weight parameters. This study demonstrates that MRI may be of significant value in evaluating therapies aimed at primary intervention for lung congestion and secondary prevention of unfavorable cardiac remodeling., (Copyright © 2011 Wiley-Liss, Inc.)
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- 2012
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37. Albiglutide, a long lasting glucagon-like peptide-1 analog, protects the rat heart against ischemia/reperfusion injury: evidence for improving cardiac metabolic efficiency.
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Bao W, Aravindhan K, Alsaid H, Chendrimada T, Szapacs M, Citerone DR, Harpel MR, Willette RN, Lepore JJ, and Jucker BM
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- Animals, Blood Glucose metabolism, Body Weight drug effects, Cyclic AMP metabolism, Energy Metabolism drug effects, Feeding Behavior drug effects, Glucagon-Like Peptide 1 administration & dosage, Glucagon-Like Peptide 1 blood, Glucagon-Like Peptide 1 pharmacology, Heart, Heart Function Tests, Hemodynamics drug effects, In Vitro Techniques, Insulin blood, Lactic Acid blood, Male, Metabolic Networks and Pathways drug effects, Metabolic Networks and Pathways genetics, Myocardial Infarction complications, Myocardial Infarction pathology, Myocardial Infarction physiopathology, Myocardial Reperfusion Injury complications, Myocardial Reperfusion Injury physiopathology, Principal Component Analysis, Rats, Rats, Sprague-Dawley, Transcription, Genetic drug effects, Cardiotonic Agents pharmacology, Glucagon-Like Peptide 1 analogs & derivatives, Myocardial Reperfusion Injury prevention & control, Myocardium metabolism
- Abstract
Background: The cardioprotective effects of glucagon-like peptide-1 (GLP-1) and analogs have been previously reported. We tested the hypothesis that albiglutide, a novel long half-life analog of GLP-1, may protect the heart against I/R injury by increasing carbohydrate utilization and improving cardiac energetic efficiency., Methods/principal Findings: Sprague-Dawley rats were treated with albiglutide and subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Left ventricle infarct size, hemodynamics, function and energetics were determined. In addition, cardiac glucose disposal, carbohydrate metabolism and metabolic gene expression were assessed. Albiglutide significantly reduced infarct size and concomitantly improved post-ischemic hemodynamics, cardiac function and energetic parameters. Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake while reducing lactate efflux. Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation which in part was due to an increase in both glucose and lactate oxidation. Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic myocardium by albiglutide., Conclusion/significance: Albiglutide reduced myocardial infarct size and improved cardiac function and energetics following myocardial I/R injury. The observed benefits were associated with enhanced myocardial glucose uptake and a shift toward a more energetically favorable substrate metabolism by increasing both glucose and lactate oxidation. These findings suggest that albiglutide may have direct therapeutic potential for improving cardiac energetics and function.
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- 2011
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38. Inhibition of p38 mitogen-activated protein kinase reduces inflammation after coronary vascular injury in humans.
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Sarov-Blat L, Morgan JM, Fernandez P, James R, Fang Z, Hurle MR, Baidoo C, Willette RN, Lepore JJ, Jensen SE, and Sprecher DL
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- Aged, C-Reactive Protein analysis, Coronary Artery Disease therapy, Double-Blind Method, Female, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors therapeutic use, Inflammation blood, Male, Middle Aged, Prosthesis Implantation adverse effects, Vascular System Injuries blood, Vascular System Injuries etiology, Angioplasty, Balloon, Coronary adverse effects, Anti-Inflammatory Agents therapeutic use, Coronary Vessels injuries, Stents adverse effects, Vascular System Injuries prevention & control, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
Objective: To evaluate whether a p38α/β mitogen-activated protein kinase inhibitor, SB-681323, would limit the elevation of an inflammatory marker, high-sensitivity C-reactive protein (hsCRP), after a percutaneous coronary intervention (PCI)., Methods and Results: Coronary artery stents provide benefit by maintaining lumen patency but may incur vascular trauma and inflammation, leading to myocardial damage. A key mediator for such stress signaling is p38 mitogen-activated protein kinase. Patients with angiographically documented coronary artery disease receiving stable statin therapy and about to undergo PCI were randomly selected to receive SB-681323, 7.5 mg (n=46), or placebo (n=46) daily for 28 days, starting 3 days before PCI. On day 3, before PCI, hsCRP was decreased in the SB-681323 group relative to the placebo group (29% lower; P=0.02). After PCI, there was a statistically significant attenuation in the increase in hsCRP in the SB-681323 group relative to the placebo group (37% lower on day 5 [P=0.04]; and 40% lower on day 28 [P=0.003]). There were no adverse safety signals after 28 days of treatment with SB-681323., Conclusions: In the setting of statin therapy, SB-681323 significantly attenuated the post-PCI inflammatory response, as measured by hsCRP. This inflammatory dampening implicates p38 mitogen-activated protein kinase in the poststent response, potentially defining an avenue to limit poststent restenosis.
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- 2010
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39. Chronic inhibition of hypoxia-inducible factor prolyl 4-hydroxylase improves ventricular performance, remodeling, and vascularity after myocardial infarction in the rat.
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Bao W, Qin P, Needle S, Erickson-Miller CL, Duffy KJ, Ariazi JL, Zhao S, Olzinski AR, Behm DJ, Pipes GC, Jucker BM, Hu E, Lepore JJ, and Willette RN
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- Animals, Cell Line, Coronary Vessels metabolism, Coronary Vessels physiopathology, Glycine pharmacology, Hemodynamics drug effects, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Male, Myocardial Infarction metabolism, Myocardial Infarction physiopathology, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Coronary Vessels drug effects, Glycine analogs & derivatives, Hypoxia-Inducible Factor 1 metabolism, Myocardial Infarction drug therapy, Procollagen-Proline Dioxygenase antagonists & inhibitors, Quinolones pharmacology, Ventricular Remodeling drug effects
- Abstract
Background: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI)., Methods and Results: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group)., Conclusions: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.
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- 2010
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40. Pharmacological inhibition of C-C chemokine receptor 2 decreases macrophage infiltration in the aortic root of the human C-C chemokine receptor 2/apolipoprotein E-/- mouse: magnetic resonance imaging assessment.
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Olzinski AR, Turner GH, Bernard RE, Karr H, Cornejo CA, Aravindhan K, Hoang B, Ringenberg MA, Qin P, Goodman KB, Willette RN, Macphee CH, Jucker BM, Sehon CA, and Gough PJ
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- Angiotensin II administration & dosage, Animals, Anti-Inflammatory Agents pharmacokinetics, Aortic Diseases immunology, Aortic Diseases pathology, Apolipoproteins E genetics, Atherosclerosis immunology, Atherosclerosis pathology, Contrast Media, Dextrans, Dietary Fats administration & dosage, Disease Models, Animal, Ferrosoferric Oxide, Humans, Immunohistochemistry, Infusion Pumps, Implantable, Macrophages immunology, Macrophages pathology, Magnetite Nanoparticles, Mice, Mice, Knockout, Mice, Transgenic, Naphthyridines pharmacokinetics, Peritonitis immunology, Peritonitis prevention & control, Receptors, CCR2 genetics, Receptors, CCR2 metabolism, Time Factors, Anti-Inflammatory Agents pharmacology, Aortic Diseases diet therapy, Apolipoproteins E deficiency, Atherosclerosis drug therapy, Macrophages drug effects, Magnetic Resonance Imaging, Naphthyridines pharmacology, Receptors, CCR2 antagonists & inhibitors
- Abstract
Unlabelled: Purpose- This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model., Methods and Results: Apolipoprotein E(-/-) mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05)., Conclusions: An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.
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- 2010
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41. Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease.
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Willette RN, Eybye ME, Olzinski AR, Behm DJ, Aiyar N, Maniscalco K, Bentley RG, Coatney RW, Zhao S, Westfall TD, and Doe CP
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- Aldosterone blood, Animals, Blood Pressure drug effects, Cardiovascular Diseases enzymology, Cyclooxygenase 1 blood, Cyclooxygenase 2 blood, Cytokines antagonists & inhibitors, Electrocardiography drug effects, Endothelium, Vascular drug effects, Interleukin-1beta blood, Kidney Function Tests, Lipid Metabolism drug effects, Male, Rats, Rats, Inbred SHR, Renin blood, Vasodilation drug effects, Ventricular Remodeling drug effects, Cardiovascular Diseases drug therapy, Cyclooxygenase 2 Inhibitors pharmacology, Cyclopropanes pharmacology, Enzyme Inhibitors pharmacology, Lactones pharmacology, Pyridines pharmacology, Sulfones pharmacology, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.
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- 2009
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42. Assessment of macrophage infiltration in a murine model of abdominal aortic aneurysm.
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Turner GH, Olzinski AR, Bernard RE, Aravindhan K, Boyle RJ, Newman MJ, Gardner SD, Willette RN, Gough PJ, and Jucker BM
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- Analysis of Variance, Angiotensin II administration & dosage, Angiotensin II pharmacology, Animals, Apolipoproteins E deficiency, Contrast Media pharmacokinetics, Disease Models, Animal, Image Processing, Computer-Assisted, Inflammation diagnosis, Inflammation metabolism, Infusion Pumps, Implantable, Magnetite Nanoparticles, Male, Mice, Aortic Aneurysm, Abdominal diagnosis, Aortic Aneurysm, Abdominal metabolism, Dextrans pharmacokinetics, Ferrosoferric Oxide pharmacokinetics, Macrophages metabolism, Magnetic Resonance Imaging methods
- Abstract
Purpose: To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model., Materials and Methods: Osmotic pumps were implanted subcutaneously in apoE(-/-) mice for continuous infusion of Angiotensin II (Ang-II). Weekly bright-blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 mumol/kg of the USPIO contrast agent ferumoxtran-10 (Combidex) followed by in vivo scanning 24 h post-USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis., Results: Reduced signal intensity was evident in the post-USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by means of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage-driven cytokine gene expression was up-regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model., Conclusion: These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms.
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- 2009
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43. In vivo serial assessment of aortic aneurysm formation in apolipoprotein E-deficient mice via MRI.
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Turner GH, Olzinski AR, Bernard RE, Aravindhan K, Karr HW, Mirabile RC, Willette RN, Gough PJ, and Jucker BM
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- Angiotensin II pharmacology, Animals, Aorta, Abdominal pathology, Aortic Aneurysm, Abdominal metabolism, Aortic Aneurysm, Abdominal pathology, Doxycycline pharmacology, Male, Matrix Metalloproteinase Inhibitors, Matrix Metalloproteinases metabolism, Mice, Mice, Knockout, Aortic Aneurysm, Abdominal diagnosis, Apolipoproteins E deficiency, Magnetic Resonance Imaging
- Abstract
Background: Hyperlipidimic mice administered angiotensin II have been used for the study of abdominal aortic aneurysms (AAAs). The purpose of this study was to examine the use of MRI for studying AAA development and for examining the effects of pharmacological intervention on AAA development in the apolipoprotein E-deficient mouse., Methods and Results: Suprarenal aortic aneurysms were generated in apolipoprotein E-deficient mice administered angiotensin II (1000 ng/kg per min) for up to 28 days. In vivo MRI was performed serially (once weekly) to assess AAA development and rupture. Comparison of AAA size as measured by in vivo and ex vivo MRI resulted in excellent agreement (r=0.96, P<0.0001). In addition, MRI correlated with histology-derived AAA area assessment (in vivo versus histology: r=0.84, P<0.0001; ex vivo versus histology: r=0.89, P<0.0001). In a separate study, angiotensin II-administered apolipoprotein E-deficient mice were treated with doxycycline (broad-based matrix metalloproteinase inhibitor; 30 mg/kg per day for 28 days). MRI was able to noninvasively assess a reduced rate of AAA development (46% versus 71%, P<0.05), a decreased AAA area (2.56 versus 4.02 mm(2), P<0.01), and decreased incidence of rupture (43% versus 100%) in treated versus control animals. Inhibition of aorta matrix metalloproteinase 2/9 activity was observed in the treated animals., Conclusions: These results demonstrate the use of MRI to noninvasively and temporally assess AAA development on pharmacological intervention in this preclinical cardiovascular disease model.
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- 2008
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44. 1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a potent and highly selective small molecule blocker of the large-conductance voltage-gated and calcium-dependent K+ channel.
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Zeng H, Gordon E, Lin Z, Lozinskaya IM, Willette RN, and Xu X
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- Action Potentials drug effects, Animals, CHO Cells, Calcium Channels drug effects, Cricetinae, Cricetulus, ERG1 Potassium Channel, Ether-A-Go-Go Potassium Channels drug effects, Guinea Pigs, Humans, Indoles pharmacology, KCNQ1 Potassium Channel drug effects, Large-Conductance Calcium-Activated Potassium Channels chemistry, Large-Conductance Calcium-Activated Potassium Channels physiology, Peptides pharmacology, Rabbits, Sodium Channels drug effects, Indazoles pharmacology, Large-Conductance Calcium-Activated Potassium Channels antagonists & inhibitors, Potassium Channel Blockers pharmacology
- Abstract
The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.
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- 2008
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45. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.
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Willette RN, Bao W, Nerurkar S, Yue TL, Doe CP, Stankus G, Turner GH, Ju H, Thomas H, Fishman CE, Sulpizio A, Behm DJ, Hoffman S, Lin Z, Lozinskaya I, Casillas LN, Lin M, Trout RE, Votta BJ, Thorneloe K, Lashinger ES, Figueroa DJ, Marquis R, and Xu X
- Subjects
- Animals, Aorta, Thoracic metabolism, Capillary Permeability drug effects, Cell Adhesion drug effects, Cell Line, Dogs, Dose-Response Relationship, Drug, Endothelium, Vascular metabolism, Female, Humans, Immunohistochemistry, Leucine adverse effects, Leucine pharmacokinetics, Male, Mice, Mice, Knockout, Molecular Structure, Patch-Clamp Techniques, Rats, Rats, Sprague-Dawley, Sulfonamides pharmacokinetics, TRPV Cation Channels genetics, Vasoconstriction drug effects, Aorta, Thoracic drug effects, Endothelium, Vascular drug effects, Hemodynamics drug effects, Leucine analogs & derivatives, Sulfonamides adverse effects, TRPV Cation Channels agonists, Ventricular Function, Left drug effects
- Abstract
The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.
- Published
- 2008
- Full Text
- View/download PDF
46. In vivo activation of peroxisome proliferator-activated receptor-delta protects the heart from ischemia/reperfusion injury in Zucker fatty rats.
- Author
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Yue TL, Nerurkar SS, Bao W, Jucker BM, Sarov-Blat L, Steplewski K, Ohlstein EH, and Willette RN
- Subjects
- Animals, Blood Pressure drug effects, Cytokines genetics, Disease Models, Animal, Fatty Acids blood, Fatty Acids metabolism, Heart physiopathology, Ketones metabolism, Male, Metabolic Syndrome blood, Metabolic Syndrome physiopathology, Myocardial Reperfusion Injury blood, Myocardial Reperfusion Injury physiopathology, PPAR delta physiology, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger metabolism, Rats, Rats, Zucker, Troponin I blood, Cardiotonic Agents therapeutic use, Myocardial Reperfusion Injury prevention & control, PPAR delta agonists, Thiazoles therapeutic use
- Abstract
Peroxisome proliferator-activated receptor (PPAR)-delta is a transcription factor that belongs to the PPAR family. PPAR-delta is abundantly expressed in the heart, and its role in the heart is largely unknown. We tested whether pharmacological activation of PPAR-delta protects the heart from ischemia/reperfusion (I/R) injury in male Zucker fatty rats, a rodent model of obesity and dyslipidemia. A highly selective PPAR-delta agonist, [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl] thio]-2-methylphenoxy]acetic acid (GW0742), was administered for 7 days at 10 mg/kg/day (p.o., once a day). Ischemic injury was produced by occlusion of the left anterior descending artery for 30 min followed by reperfusion for up to 24 h. Treatment with GW0742 reduced serum levels of cardiac troponin-I and infarct size by 63% (p < 0.01) and 32% (p < 0.01), respectively, and improved left ventricular function. Treatment with GW0742 up-regulated gene expression involved in cardiac fatty acid oxidation, increased fat use in the heart, and reduced serum levels of free fatty acids. The enhanced cardiac expression of interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 induced by I/R were significantly attenuated by GW0742. Treatment with GW0742 also reduced apoptotic cardiomyocytes by 34% and cardiac caspase-3 activity by 61% (both p < 0.01 versus vehicle). GW0742 differentially regulated Bcl family members, favoring cell survival, and attenuated I/R-induced cardiac mitochondrial damage. In addition, GW0742 treatment augmented the cardiac Akt signaling pathway, as reflected by enhanced phospho-3-phosphoinositide-dependent kinase-1 and p-Akt. The results indicate that activation of PPAR-delta protected the heart from I/R injury in Zucker fatty rats, and multiple mechanisms including amelioration of lipotoxicity, anti-inflammation, and up-regulation of prosurvival signaling contribute together to the cardioprotection.
- Published
- 2008
- Full Text
- View/download PDF
47. 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243) causes instantaneous current through human ether-a-go-go-related gene potassium channels.
- Author
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Gordon E, Lozinskaya IM, Lin Z, Semus SF, Blaney FE, Willette RN, and Xu X
- Subjects
- Action Potentials drug effects, Action Potentials physiology, Animals, Binding Sites, CHO Cells, Cricetinae, Cricetulus, Data Interpretation, Statistical, Dose-Response Relationship, Drug, Electric Conductivity, Ether-A-Go-Go Potassium Channels genetics, Heart Ventricles cytology, Humans, Hydrophobic and Hydrophilic Interactions, Isoindoles chemistry, Kinetics, Male, Mice, Microelectrodes, Models, Molecular, Molecular Structure, Myocytes, Cardiac drug effects, Myocytes, Cardiac physiology, Niacin chemistry, Nicotinic Acids chemistry, Patch-Clamp Techniques, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Rabbits, Transfection, Ether-A-Go-Go Potassium Channels drug effects, Ether-A-Go-Go Potassium Channels physiology, Isoindoles pharmacology, Niacin analogs & derivatives, Niacin pharmacology, Nicotinic Acids pharmacology
- Abstract
Long and short QT syndromes associated with loss and gain of human ether-a-go-go-related gene (hERG) channel activity, respectively, can cause life-threatening arrhythmias. As such, modulation of hERG channel activity is an important consideration in the development of all new therapeutic agents. In the present study, we investigated the mechanisms of action of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243), a known hERG channel activator, on hERG channels stably expressed in Chinese hamster ovary (CHO) cells using the patch-clamp technique. In the whole-cell recordings, the extracellular application of PD-307243 concentration-dependently increased the hERG current and markedly slowed hERG channel deactivation and inactivation. PD-307243 had no effect on the selectivity filter of hERG channels. The activity of PD-307243 was use-dependent. PD-307243 (3 and 10 muM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an I(to)-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K(+) current (I(Kr)) in rabbit ventricular myocytes were similar to those observed in hERG channel-transfected CHO cells. Inside-out patch experiments revealed that PD-307243 increased hERG tail currents by 2.1 +/- 0.6 (n = 7) and 3.4 +/- 0.3-fold (n = 4) at 3 and 10 muM, respectively, by slowing the channel deactivation but had no effect on channel activation. During a voltage-clamp protocol using a prerecorded cardiac action potential, 3 muM PD-307243 increased the total potassium ions passed through hERG channels by 8.8 +/- 1.0-fold (n = 5). Docking studies suggest that PD-307243 interacts with residues in the S5-P region of the channel.
- Published
- 2008
- Full Text
- View/download PDF
48. p38 MAPK inhibition reduces aortic ultrasmall superparamagnetic iron oxide uptake in a mouse model of atherosclerosis: MRI assessment.
- Author
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Morris JB, Olzinski AR, Bernard RE, Aravindhan K, Mirabile RC, Boyce R, Willette RN, and Jucker BM
- Subjects
- Angiotensin II administration & dosage, Animals, Apolipoproteins E genetics, Atherosclerosis diagnosis, Contrast Media pharmacokinetics, Enzyme Inhibitors pharmacology, Feasibility Studies, Inflammation diagnosis, Male, Mice, Mice, Knockout, Vasoconstrictor Agents administration & dosage, Aorta pathology, Ferrosoferric Oxide pharmacokinetics, Imidazoles pharmacology, Magnetic Resonance Angiography methods, Metal Nanoparticles, Pyrimidines pharmacology, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
Objective: Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE-/- mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake., Methods and Results: ApoE-/- mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (increased 103%, P<0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (increased 6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment., Conclusions: The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II-infused apoE-/- mice.
- Published
- 2008
- Full Text
- View/download PDF
49. PPARdelta activation normalizes cardiac substrate metabolism and reduces right ventricular hypertrophy in congestive heart failure.
- Author
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Jucker BM, Doe CP, Schnackenberg CG, Olzinski AR, Maniscalco K, Williams C, Hu TC, Lenhard SC, Costell M, Bernard R, Sarov-Blat L, Steplewski K, and Willette RN
- Subjects
- Animals, Biological Transport, Diuresis drug effects, Dose-Response Relationship, Drug, Energy Metabolism, Gene Expression drug effects, Heart Failure etiology, Heart Failure physiopathology, Hypertrophy, Right Ventricular etiology, Hypertrophy, Right Ventricular physiopathology, Lipids blood, Magnetic Resonance Spectroscopy, Male, Myocardial Infarction complications, Oxidation-Reduction, PPAR delta metabolism, Pulmonary Edema drug therapy, Pulmonary Edema etiology, Rats, Rats, Sprague-Dawley, Reverse Transcriptase Polymerase Chain Reaction, Ventricular Function, Left drug effects, Heart Failure drug therapy, Hypertrophy, Right Ventricular drug therapy, PPAR delta agonists
- Abstract
Previously, it was shown that selective deletion of peroxisome proliferator activated receptor delta (PPARdelta) in the heart resulted in a cardiac lipotoxicity, hypertrophy, and heart failure. The aim of the present study was to determine the effects of chronic and selective pharmacological activation of PPARdelta in a model of congestive heart failure. PPARdelta-specific agonist treatment (GW610742X at 30 and 100 mg/kg/day for 6-9 weeks) was initiated immediately postmyocardial infarction (MI) in Sprague-Dawley rats. Magnetic resonance imaging/spectroscopy was used to assess cardiac function and energetics. A 1-(13)C glucose clamp was performed to assess relative cardiac carbohydrate versus fat oxidation. Additionally, cardiac hemodynamics and reverse-transcription polymerase chain reaction gene expression analysis was performed. MI rats had significantly reduced left ventricle (LV) ejection fractions and whole heart phosphocreatine/adenosine triphosphate ratio compared with Sham animals (reduction of 43% and 14%, respectively). However, GW610742X treatment had no effect on either parameter. In contrast, the decrease in relative fat oxidation rate observed in both LV and right ventricle (RV) following MI (decrease of 58% and 54%, respectively) was normalized in a dose-dependent manner following treatment with GW610742X. These metabolic changes were associated with an increase in lipid transport/metabolism target gene expression (eg, CD36, CPT1, UCP3). Although there was no difference between groups in LV weight or infarct size measured upon necropsy, there was a dramatic reduction in RV hypertrophy and lung congestion (decrease of 22-48%, P<0.01) with treatment which was associated with a >7-fold decrease (P<0.05) in aterial natriuretic peptide gene expression in RV. Diuretic effects were not observed with GW610742X. In conclusion, chronic treatment with a selective PPARdelta agonist normalizes cardiac substrate metabolism and reduces RV hypertrophy and pulmonary congestion consistent with improvement in congestive heart failure.
- Published
- 2007
- Full Text
- View/download PDF
50. Effects of p38 MAPK Inhibitor on angiotensin II-dependent hypertension, organ damage, and superoxide anion production.
- Author
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Bao W, Behm DJ, Nerurkar SS, Ao Z, Bentley R, Mirabile RC, Johns DG, Woods TN, Doe CP, Coatney RW, Ohlstein JF, Douglas SA, Willette RN, and Yue TL
- Subjects
- Animals, Aorta, Abdominal drug effects, Aorta, Abdominal enzymology, Aorta, Abdominal metabolism, Blood Pressure drug effects, Carotid Arteries drug effects, Carotid Arteries enzymology, Carotid Arteries metabolism, Echocardiography, Endothelium, Vascular drug effects, Hypertension chemically induced, Hypertension enzymology, Hypertension metabolism, Intracellular Signaling Peptides and Proteins, Male, Membrane Glycoproteins metabolism, Mice, Mice, Knockout, Myocardium enzymology, Myocardium metabolism, NADPH Oxidase 2, NADPH Oxidases metabolism, Protein Kinases genetics, Protein Serine-Threonine Kinases, Rats, Rats, Sprague-Dawley, Vasodilation drug effects, p38 Mitogen-Activated Protein Kinases biosynthesis, Angiotensin II adverse effects, Enzyme Inhibitors administration & dosage, Enzyme Inhibitors pharmacology, Enzyme Inhibitors therapeutic use, Hypertension drug therapy, Imidazoles administration & dosage, Imidazoles pharmacology, Imidazoles therapeutic use, Pyrimidines administration & dosage, Pyrimidines pharmacology, Pyrimidines therapeutic use, Superoxides metabolism, Ventricular Remodeling drug effects, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91 in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91 mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.
- Published
- 2007
- Full Text
- View/download PDF
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