Lea Sarov-Blat

Effects of p38 Mitogen-Activated Protein Kinase Inhibition on Vascular and Systemic Inflammation in Patients With Atherosclerosis

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).

Losmapimod, a novel p38 mitogen-activated protein kinase inhibitor, in non-ST-segment elevation myocardial infarction: a randomised phase 2 trial

BACKGROUND p38 MAPK inhibition has potential myocardial protective effects. We assessed losmapimod, a potent oral p38 MAPK inhibitor, in patients with non-ST-segment elevation myocardial infarction (NSTEMI) in a double-blind, randomised, placebo-controlled trial. METHODS From October, 2009, to November, 2011, NSTEMI patients were assigned oral losmapimod (7·5 mg or 15·0 mg loading dose followed by 7·5 mg twice daily) or matching placebo in a 3:3:2 ratio. Safety outcomes were serious adverse events and alanine aminotransferase (ALT) concentrations over 12 weeks, and cardiac events (death, myocardial infarction, recurrent ischaemia, stroke, and heart failure) at 90 days. Efficacy outcomes were high-sensitivity C-reactive protein (hsCRP) and B-type natriuretic peptide (BNP) concentrations at 72 h and 12 weeks, and troponin I area under the curve (AUC) over 72 h. The losmapimod groups were pooled for analysis. This trial is registered with ClinicalTrials.gov, number NCT00910962. FINDINGS Of 535 patients enrolled, 526 (98%) received at least one dose of study treatment (losmapimod n=388 and placebo n=138). Safety outcomes did not differ between groups. HsCRP concentrations at 72 h were lower in the losmapimod group than in the placebo group (geometric mean 64·1 nmol/L, 95% CI 53·0-77·6 vs 110·8 nmol/L, 83·1-147·7; p=0·0009) but were similar at 12 weeks. Early geometric mean BNP concentrations were similar at 72 h but significantly lower in the losmapimod group at 12 weeks (37·2 ng/L, 95% CI 32·3-42·9 vs 49·4 ng/L, 38·7-63·0; p=0·04). Mean troponin I AUC values did not differ. INTERPRETATION p38 MAPK inhibition with oral losmapimod was well tolerated in NSTEMI patients and might improve outcomes after acute coronary syndromes. FUNDING GlaxoSmithKline.

Cathepsin D, a lysosomal protease, regulates ABCA1-mediated lipid efflux.

To identify genes involved in the regulation of plasma high density lipoprotein (HDL) cholesterol (HDL-C) levels, patients with low HDL-C and age- and sex-matched controls (normal HDL-C) were extensively characterized. Comparative transcriptome analysis was carried out in cholesterol-loaded monocyte-derived macrophages from low HDL subjects segregated into groups with or without cholesterol efflux defects or ABCA1 mutations. Clusters of differentially regulated genes were evident in the low HDL groups as compared with controls. Of particular note, expression of cathepsin D (CTSD), a lysosomal proteinase, was reduced by ∼50% in monocyte-derived macrophages of low HDL-C subjects, most significantly those with cholesterol efflux defects but without mutations in ABCA1 (p < 0.01). These results were verified by reverse transcription-PCR and replicated in a second cohort. We show here that blocking the activity or expression of CTSD, by pepstatin or CTSD small interfering RNA, respectively, reduced ABCA1 expression and protein abundance in both macrophages and CHO cells and apolipoprotein A-I-mediated lipid efflux by more than 70%. Conversely, expression of CTSD increased both ABCA1 mRNA expression and cellular ABCA1 protein. Consistent with its role in the proteolytic processing of prosaposin, inactivation of CTSD function resulted in the accumulation of glycosphingo-lipid and free cholesterol in late endosomes/lysosomes, a phenotype similar to NPC1 deficiency. Inhibition of CTSD also caused retention of ABCA1 in lysosomal compartments, reducing its trafficking to the plasma membrane. These studies demonstrate a novel and potentially important role for CTSD in intracellular cholesterol trafficking and ABCA1-mediated efflux. Therefore, decreased CTSD expression may contribute to low plasma HDL-C levels.

Inhibition of p38 Mitogen-Activated Protein Kinase Improves Nitric Oxide–Mediated Vasodilatation and Reduces Inflammation in Hypercholesterolemia

Background— Oxidized low-density lipoprotein reduces endothelial nitric oxide production (an important mediator of vasoregulation) and activates p38 mitogen-activated protein kinase (MAPK), a mediator of vascular inflammation. Animal models of vascular stress have previously predicted improvements in vascular function after p38 MAPK inhibition. We hypothesized that a selective p38&agr;/&bgr; MAPK inhibitor (losmapimod; GW856553) would improve compromised nitric oxide–mediated vasoregulation in patients with hypercholesterolemia. Methods and Results— Untreated hypercholesterolemic patients (low-density lipoprotein cholesterol >4.1 mmol/L) were randomized to receive losmapimod 7.5 mg (n=27) or placebo (n=29) twice daily for 28 days. Patients with known vascular disorders (eg, diabetes mellitus, coronary heart disease) were excluded. Forearm blood flow was measured by venous occlusion plethysmography in response to serial intra-arterial infusion of acetylcholine, sodium nitroprusside, and NG-monomethyl-l-arginine (L-NMMA). Acetylcholine and L-NMMA responses were significantly impaired (P=0.01 and P=0.03) compared with responses in control subjects (n=12). In hypercholesterolemic patients treated with losmapimod, responses to acetylcholine were improved by 25% (95% confidence interval, 5 to 48; P=0.01), to sodium nitroprusside by 20% (95% confidence interval, 3 to 40; P=0.02), and to L-NMMA by 10% (95% confidence interval, −1 to 23; P=0.07) compared with placebo. C-reactive protein was reduced by 57% (95% confidence interval, −81 to −6%; P<0.05) in patients treated with losmapimod compared with placebo. Conclusions— Losmapimod improves nitric oxide–mediated vasodilatation in hypercholesterolemic patients, which is consistent with findings in previous translational animal models. These data support the hypothesis that attenuating the inflammatory milieu by inhibiting p38 MAPK activity improves NO activity. This suggests p38 MAPK as a novel target for patients with cardiovascular disease. Clinical Trial Registration— URL: http://clinicaltrials.gov. Unique identifier: NCT00474864.

In Vivo Activation of Peroxisome Proliferator-Activated Receptor-δ Protects the Heart from Ischemia/Reperfusion Injury in Zucker Fatty Rats

Peroxisome proliferator-activated receptor (PPAR)-δ is a transcription factor that belongs to the PPAR family. PPAR-δ is abundantly expressed in the heart, and its role in the heart is largely unknown. We tested whether pharmacological activation of PPAR-δ 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-δ 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-δ 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.

Effect of treatment for 12 weeks with rilapladib, a lipoprotein-associated phospholipase A2 inhibitor, on arterial inflammation as assessed with 18F-fluorodeoxyglucose-positron emission tomography imaging.

To the Editor: Previous reports have demonstrated that lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzymatic inflammatory biomarker, is associated with increased risk of cardiovascular events [(1)][1]. Lp-PLA2 mediates formation of bioactive mediators (lysophosphatidyl choline and

Inhibition of p38 Mitogen-Activated Protein Kinase Reduces Inflammation After Coronary Vascular Injury in Humans

Objective—To evaluate whether a p38&agr;/&bgr; 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. Conclusion—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.

Predominance of a Proinflammatory Phenotype in Monocyte-Derived Macrophages From Subjects With Low Plasma HDL-Cholesterol

Objective—Reduced plasma concentrations of high-density lipoprotein-cholesterol (HDL-C) are a significant risk factor for cardiovascular disease. Mechanisms that regulate HDL-C concentrations represent an important area of investigation. Methods and Results—Comparative transcriptome analyses of monocyte-derived macrophages (MDM) from a large population of low HDL-C subjects and age- and sex-matched controls revealed a cluster of inflammatory genes highly expressed in low HDL-C subjects. The expression levels of peroxisome proliferator activated receptor (PPAR) γ and several antioxidant metallothionein genes were decreased in MDM from all low HDL-C groups compared with controls, as was the expression of other genes regulated by PPARγ, including CD36, adipocyte fatty acid binding protein (FABP4), and adipophilin (ADFP). In contrast, PPARδ expression was increased in MDM from low HDL-C groups. Quantitative RT-PCR corroborated all major findings from the microarray analysis in two separate patient cohorts. Expression of several inflammatory cytokine genes including interleukin 1β, interleukin 8, and tumor necrosis factor α were highly increased in low HDL-C subjects. Conclusions—The activated proinflammatory state of monocytes and MDM in low HDL-C subjects constitutes a novel parameter of risk associated with HDL deficiency, related to altered expression of metallothionein genes and the reciprocal regulation of PPARγ and PPARδ.

PPAR?? Activation Normalizes Cardiac Substrate Metabolism and Reduces Right Ventricular Hypertrophy in Congestive Heart Failure

Previously, it was shown that selective deletion of peroxisome proliferator activated receptor δ (PPARδ) 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 PPARδ in a model of congestive heart failure. PPARδ-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 1-13C 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 PPARδ agonist normalizes cardiac substrate metabolism and reduces RV hypertrophy and pulmonary congestion consistent with improvement in congestive heart failure.

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively. These proteins are required for SOS mutagenesis, although their mechanism of action is unknown. By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E. coli. To examine the interaction at the protein level, the MucA, MucA′, and MucB proteins were overproduced, purified in denatured state, and refolded. Purified MucA and MucA′ each formed homodimers, whereas MucB was a monomer under native conditions. RecA promoted the cleavage of MucA to MucA′, and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins. Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins. Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide. Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB. These results suggest that MucB induces a major conformational change in the SSB·ssDNA complex but does not promote massive release of SSB from the DNA. The interaction with SSB might be related to the role of MucB in SOS-regulated mutagenesis.