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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 anti-fibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting Protein Complex 1 (PC1). In vitro, IM156 inhibited TGFβ-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 (ECAR). IM156 significantly increased cellular 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, TCA 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 anti-fibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for IPF and other fibrotic diseases. Significance Statement Fibrosing Interstitial Lung Diseases (FILD) have a poor prognosis and current anti-fibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of OXPHOS with IM156, prevents the myofibroblast phenotype/collagen deposition and is a potentially effective and translational anti-fibrotic strategy.

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.

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 m2; blood pressure 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., Graphical 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.

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.

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.

Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels. Here, we demonstrate that a biomimetic topography, consisting of microhemispheres arrayed over the cell culture substrate, promotes podocyte differentiation in vitro. We define new methods for fabricating microscale curvature on various substrates, including a thin porous membrane. By growing podocytes on our topographic substrates, we found that these biophysical cues augmented nephrin gene expression, supported full-size nephrin protein expression, encouraged structural arrangement of F-actin and nephrin within the cell, and promoted process formation and even interdigitation compared to the flat substrates. Furthermore, the topography facilitated nephrin localization on curved structures while nuclei lay in the valleys between them. The improved differentiation was also evidenced by tracking barrier function to albumin over time using our custom topomembranes. Overall, our work presents accessible methods for incorporating microcurvature on various common substrates, and demonstrates the importance of biophysical stimulation in supporting higher-fidelity podocyte cultivation in vitro.

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.

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.

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.

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.

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 2 hours before stimulated with 100 nM angiotensin II (AngII), 10 μM indoxyl sulphate (IS) or 10 ng/ml transforming growth factor β1 (TGFβ1) for 48 hours. Neonatal rat cardiac myocytes (NCM) were pre-treated with G226 and G235 for 2 hours before being stimulated with 100 nM AngII for 60 hours, 10 μM IS, 10 ng/ml interleukin 1β (IL-1β) or tumour necrosis factor α (TNFα) for 48 hours. 3H-proline and 3H-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.

to the editor: We write in response to the recent article by White, Cibelli, Urban, Nilius, McGeown, and Nagy ([4][1]). We commend the authors for compiling such a wide literary review on this interesting ion channel, TRPV4. However, we have read with concern the description of the properties of the

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/ eIF 2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

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.

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.

Chronic neurohormonal and mechanical stresses are central fea-tures of heart disease. Increasing evidence supports a role forthe transient receptor potential canonical channels TRPC3 andTRPC6 in this pathophysiology. Channel expression for both is nor-mally very low but is increased by cardiac disease, and geneticgain- or loss-of-function studies support contributions to hypertro-phy and dysfunction. Selective small-molecule inhibitors remainscarce, and none target both channels, which may be useful giventhe high homology among them and evidence of redundant sig-naling. Here we tested selective TRPC3/6 antagonists (GSK2332255Band GSK2833503A; IC50,3–21 nM against TRPC3 and TRPC6) andfound dose-dependent blockade of cell hypertrophy signaling trig-gered by angiotensin II or endothelin-1 in HEK293T cells as well as inneonatal and adult cardiac myocytes. In vivo efficacy in mice andrats was greatly limited by rapid metabolism and high protein bind-ing, although antifibrotic effects with pressure overload were ob-served. Intriguingly, although gene deletion of TRPC3 or TRPC6alone did not protect against hypertrophy or dysfunction frompressure overload, combined deletion was protective, support-ing the value of dual inhibition. Further development of thispharmaceutical class may yield a useful therapeutic agent forheart disease management. Fil: Seo, Kinya. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos Fil: Rainer, Peter P.. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos. Medical University of Graz. Department of Medicine; Austria Fil: Shalkey Hahn, Virginia. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos Fil: Lee, Dong-ik. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos Fil: Jo, Su-Hyun. Kangwon National University School of Medicine; Corea del Sur. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos Fil: Andersen, Asger. Aarhus University Hospital. Department of Cardiology; Dinamarca Fil: Liu, Ting. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos Fil: Xu, Xiaoping. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados Unidos Fil: Willette, Robert N.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados Unidos Fil: Lepore, John J.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados Unidos Fil: Marino, Joseph P.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados Unidos Fil: Birnbaumer, Lutz. ational Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Schnackenberg, Christine G.. GlaxoSmithKline Heart Failure Discovery Performance Unit; Estados Unidos Fil: Kass, David A.. Johns Hopkins Medical Institutions. Department of Medicine; Estados Unidos

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.

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.

Objectives This study sought to determine the effects of a p38 mitogen-activated protein kinase inhibitor, losmapimod, on vascular inflammation, by 18F-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)

Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.

BACKGROUND AND PURPOSE Recently identified antagonists of the urotensin–II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. EXPERIMENTAL APPROACH The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. KEY RESULTS GSK1440115 (pKi= 7.34–8.64 across species) and GSK1562590 (pKi= 9.14–9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA2= 5.59–7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pKb= 8.93–10.12 across species), but a competitive antagonist in monkey arteries (pKb= 8.87–8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. CONCLUSIONS AND IMPLICATIONS Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

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).GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1PHD2 = 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).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.

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 P P Conclusion— An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

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.

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.

Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca2+-activated K+ channels (BKCa) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BKCaalphabeta1 (CHO BKCaalphabeta1) and human BKCaalpha (CHO BKCaalpha). Acetic acid activated BKCa currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BKCa current at +30 mV by 2764+/-918% (n=8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V1/2 of conductance-voltage curve by 64+/-14 (n=5), 128+/-14 (n=5), and 126+/-12 mV (n=4) in CHO BKCaalpha, CHO BKCaalphabeta1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BKCaalphabeta1 by acetic acid. In conclusion, acetic acid directly activates BKCa channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.

Transient receptor potential channel vanilloid 4 (TRPV4) is a non-selective Ca2+ permeable cation channel, recently implicated by computational methods as a key signalling component during myocardial infarction. We previously showed that pharmacological TRPV4 inhibition can prevent and resolve pulmonary edema in pre-clinical heart failure animal models. Here, we examined the impact of genetic deletion of TRPV4 (TRPV4-/-) on cardiac function, both basally and following myocardial infarction. Cardiac function was similar in wild type and TRPV4-/- mice under normal conditions. By contrast, following myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, left ventricular systolic and diastolic volumes were reduced and ejection fraction was significantly improved in TRPV4-/- when compared to wild type mice. Consistent with the differences in chamber volumes between TRPV4-/- and wild type mice, myocardial infarction induced a significant increase in heart weight and left ventricular mass index in wild type, but not in TRPV4-/- mice. In a separate cohort of mice, we also investigated the effect of genetic deletion of TRPV4 on infarct size after a 30 min myocardial ischemia and 24 hours reperfusion. There were no differences in myocardial infarct size or area at risk in TRPV4-/- and wild type mice after ischemia/reperfusion injury. These results suggest that TRPV4 does not mediate acute myocardial ischemic injury, but does play an important role in ventricular remodelling known to correlate with poor outcomes following acute myocardial infarction.

Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.

Inflammation and leukocyte activation/infiltration play a major role in the initiation and progression of cardiovascular diseases including atherosclerosis and heart failure. Acute p38 mitogen-activated protein kinase (MAPK) pathway inhibition attenuates tissue damage and leukocyte accumulation in myocardial ischemia/reperfusion injury, although its effect on the acute phase of leukocyte recruitment has not been elucidated. The purpose of this study was to test the hypothesis that acute treatment of rats with a selective p38 inhibitor, SB-239063, inhibits ischemia/reperfusion-induced leukocyte-endothelial adhesion in vivo. Male Sprague–Dawley rats were treated with either SB-239063 (10 mg kg−1), dexamethasone (3 mg kg−1) or vehicle 1 h prior to ischemia. Postcapillary venules were observed microscopically in exteriorized, superfused cremaster tissue. Leukocytes were fluorescently labeled in vivo using intravenous rhodamine 6G. Leukocyte adhesion, rolling, and rolling velocities were quantitated prior to 30 min ischemia, and at several time points during a 90 min reperfusion period. Ischemia caused a 3-fold increase in adherent leukocytes 5 min following reperfusion, a response that was maintained throughout the monitoring period (90 min) in vehicle-treated animals. SB-239063, at a dose known to inhibit p38 MAPK activity in vivo (10 mg kg−1), had no effect on ischemia/reperfusion-induced leukocyte adhesion, the number of rolling leukocytes, rolling velocities during the reperfusion period or adhesion molecule expression (P-, E-selectin, VCAM-1, ICAM-1). In contrast, dexamethasone completely blocked leukocyte adhesion in response to ischemia/reperfusion, and reduced expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). We conclude that p38 MAPK may not play a role in initial leukocyte recruitment in response to ischemia/reperfusion injury, but could affect leukocyte emigration, thereby resulting in increased leukocyte accumulation in ischemic-reperfused tissue.

Objective : Evidence suggests important relationships among chronic inflammatory processes, endothelial dysfunction, hypertension and target organ damage. The present study examined the effects of chronic treatment with an anti-inflammatory p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-239063AN) in the N ω-nitro-l-arginine methyl ester-treated spontaneously hypertensive rat (SHR+l-NAME) model of severe hypertension and accelerated target organ damage. Methods: SHRs were divided into control ( n =16), l-NAME ( n =26) and l-NAME+SB-239063AN ( n =24) groups. l-NAME was delivered by the drinking water ad lib (50 mg/L) and SB-239063AN was administered by the diet (1200 ppm) for 4 weeks. Arterial blood pressure (telemetry) and target organ damage (kidney, heart, and vasculature) were examined. Results: The introduction of l-NAME to the drinking water elicited a severe/sustained increase in blood pressure and significant morbidity and mortality. Chronic treatment with SB-239063AN had no effect on the initial blood pressure response (7 days) to l-NAME but attenuated subsequent increases in diastolic blood pressure and significantly reduced morbidity/mortality (42% vs. 5%, p

Purpose To compare atherosclerotic plaque uptake of a first (ferumoxtran-10) and second generation (ferumoxytol) ultrasmall superparamagnetic iron oxide (USPIO) contrast agent with different pharmacokinetic/pharmacodynamic properties. Materials and Methods New Zealand White rabbits maintained on a high cholesterol/fat diet were subjected to balloon injury to the abdominal aorta. Ferumoxtran-10 or ferumoxytol (500 μmol/kg) was administered at 2, 4, and 8 weeks following injury. In vivo magnetic resonance imaging (MRI) was performed immediately prior to, immediately after, and 6 days post-contrast administration. Ex vivo MRI, histologic, and inductively coupled plasma-mass spectrometry (ICP-MS) iron analyses were performed on the excised vessels. Results The blood pool clearance of ferumoxytol (t½ ≤ 6 hours) was more rapid than that of ferumoxtran-10 (t½ ≤ 48 hours). Decreased in vivo MRI signal intensity in the abdominal aorta was observed at 2, 4, and 8 weeks following injury with ferumoxtran-10, but not with ferumoxytol. Consistent with these observations, ex vivo MRI signal intensity was decreased in the ferumoxtran-10 vessels, and to a lesser degree in the ferumoxytol vs. control vessels (− contrast agent). In contrast, in vitro macrophage phagocytosis of USPIO was four to six fold greater with ferumoxytol than with ferumoxtran-10. Additionally, the absolute iron content correlated with ex vivo MRI signal intensity in all vessels (r = −0.86, P < 0.0001). Conclusions These data suggest that the exposure period of atherosclerotic plaque to USPIO rather than the kinetics of the USPIO uptake by plaque alone is a critical criterion for experimental design of in vivo studies. J. Magn. Reson. Imaging 2005;21:432–442. © 2005 Wiley-Liss, Inc.

Objectives: This study examined the effects of diet-induced hypercholesterolemia on the extent of postischemic myocyte apoptosis and elucidated the potential mechanisms involved. Furthermore, the effects of rosiglitazone (RSG) on postischemic myocardial apoptosis in HC rabbits were investigated. Methods: Male New Zealand rabbits were fed normal or high cholesterol diets for 8 weeks. Three weeks after being fed a high cholesterol diet, HC rabbits were randomized to receive vehicle or RSG during the remaining 5 weeks. Rabbits were then subjected to 60 min of coronary occlusion followed by 4 h of reperfusion. Results: Compared with rabbits fed with a normal diet, HC rabbits had increased caspase-3 activity, apoptotic cell death and retarded contractile function recovery after reperfusion. HC increased iNOS expression, total NOx and nitrotyrosine contents, indicating that an increased nitrative stress occurred in HC myocardial tissue. Activity of a hypertrophic/anti-apoptotic mitogen-activated protein kinase (MAPK), ERK1/2, was significantly decreased while activation of a pro-apoptotic MAPK, p38, was increased. Treatment with RSG in HC rabbits attenuated postischemic myocardial nitrative stress, restored a beneficial balance between pro- and anti-apoptotic MAPK signaling, reduced postischemic myocardial apoptosis, and improved cardiac functional recovery. Conclusions: Our results demonstrated that HC increased postischemic myocardial apoptosis likely by increasing the production of pro-apoptotic molecules, activating pro-apoptotic signaling pathways and inhibiting anti-apoptotic signaling. In addition, our results suggest that peroxisome proliferator-activated receptor (PPAR) γ agonists may not only attenuate the formation of atherosclerosis associated with hypercholesterolemia as previously reported, but may also protect the heart from subsequent ischemic/reperfusion-induced myocardial apoptosis.

Urotensin-II, a potent mammalian vasoconstrictor, may play a role in the etiology of essential hypertension. However, a species suitable for assessing such a role, one where a "classical" systemic hypertensive response (increase in mean blood pressure and systemic vascular resistance) is observed following bolus i.v. urotensin-II administration, has yet to be identified. The present study demonstrates that the cat may represent such a species since urotensin-II potently (pEC(50)s 9.68+/-0.24-8.73+/-0.08) and efficaciously (E(max) 73+/-15%-205+/-21% KCl) constricts all feline isolated arteries studied (aortae, renal, femoral, carotid, and mesenteric conduit/resistance). Accordingly, exogenous urotensin-II (1 nmol/kg, i.v.) effectively doubles both mean blood pressure (from 99+/-14 to 183+/-15 mmHg) and systemic vascular resistance (from 0.36+/-0.12 to 0.86+/-0.20 mmHg/ml/min) in the anaesthetized cat (without altering heart rate or stroke volume). Thus, in view of these profound contractile effects, the cat could be suitable for determining the effects of urotensin-II receptor antagonism on cardiovascular homeostasis in both normal and diseased states.

Recent evidence suggests p38 mitogen-activated protein kinase (MAPK) signal transduction plays an important role in the pathogenesis of progressive renal disease. Using dynamic contrast enhanced magnetic resonance imaging (MRI), we evaluated chronic treatment with a p38 MAPK inhibitor, trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole (SB-239063), on renal function in a hypertension model of progressing renal dysfunction. Spontaneously hypertensive-stroke prone rats were placed on a high salt/fat diet (SFD) or maintained on normal chow diet (ND). SFD animals with albuminuria at 4 to 8 weeks (or =10 mg/day inclusion criteria), were randomized into p38 MAPK inhibitor treatment (SB-239063, 1200 ppm in diet) or vehicle groups. The progression of blood pressure and albuminuria during the treatment period (approximately 6 weeks) was decreased by 12 and 60%, respectively, in the SFD + SB-239063 versus SFD control group. Renal perfusion and filtration were assessed by in vivo MRI at the end of the study. Relative cortical perfusion was increased in the SFD + SB-239063 group compared with the SFD control group as reflected by a 29% decrease in time to peak of contrast agent in the cortex. Additionally, the regional renal glomerular filtration rate index (Kcl) was increased by 39% in the SFD + SB-239063 versus SFD control group and was normalized to the ND control group. Greater functional heterogeneity was observed in the SFD control versus SFD + SB-239063 or ND control group. All alterations of renal function were supported by histopathological findings. In conclusion, chronic treatment with a p38 MAPK inhibitor, SB-239063, attenuates functional and structural renal degeneration in a hypertensive model of established renal dysfunction.

Numerous mediators, believed to play a role in endothelial dysfunction (e.g., neurohormones, cytokines, hypoxia, and stretch), have been shown to activate p38 mitogen-activated protein kinase (MAPK) in a variety of cell types. The purpose of the present study was to examine the regulation of p38 MAPK in endothelium and its role in endothelial dysfunction and salt sensitivity. In cultured human umbilical vein endothelial cells (HUVECs), tumor necrosis factor-alpha and lipopolysaccharide increased phosphorylation of p38 MAPK (P-p38 MAPK) and increased ICAM-1 expression. Preincubation with highly selective p38 MAPK inhibitors, 1-(1,3-dihydroxyprop-2-yl)-4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl] imidazole (SB-239063AN) or SB-239063, dose dependently reduced intercellular adhesion molecule-1 expression in HUVECs. In spontaneously hypertensive-stroke prone rats (SHR-SP), P-p38 MAPK was localized by immunohistochemistry to the aortic endothelium and adventitia but was undetectable in aortae from normotensive rats. Introduction of a salt/fat diet (SFD) to the SHR-SP strain induced endothelial dysfunction (ex vivo vascular reactivity analysis), albuminuria, and an increase in blood pressure within 4 weeks. Chronic dietary dosing (approx. 100 mg/kg/day) with SB-239063AN inhibited the SFD diet-induced hypertension. In addition, delayed treatment also significantly improved survival and restored nitric oxide-mediated endothelium-dependent relaxation in SFD-SHR-SPs with established endothelial dysfunction. These results suggest an important role for p38 MAPK in endothelial inflammation and dysfunction as well as providing the first evidence for p38 MAPK-dependent hypertension.