Diabetes status modifies the long-term effect of lipoprotein-associated phospholipase A2 on major coronary events.

AIMS/HYPOTHESIS: Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity has an independent prognostic association with major coronary events (MCE). However, no study has investigated whether type 2 diabetes status modifies the effect of Lp-PLA2 activity or inhibition on the risk of MCE. We investigate the interaction between diabetes status and Lp-PLA2 activity with risk of MCE. Subsequently, we test the resulting hypothesis that diabetes status will play a role in modifying the efficacy of an Lp-PLA2 inhibitor. METHODS: A retrospective cohort study design was utilised in two study populations. Discovery analyses were performed in the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) cohort based in Scotland, UK. Participants were categorised by type 2 diabetes control status: poorly controlled (HbA1c ≥ 48 mmol/mol or ≥6.5%) and well-controlled (HbA1c < 48 mmol/mol or <6.5%) diabetes (n = 7420). In a secondary analysis of the Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy (STABILITY) trial of Lp-PLA2 inhibitor (darapladib) efficacy, 15,828 participants were stratified post hoc by type 2 diabetes diagnosis status (diabetes or no diabetes) at time of recruitment. Lp-PLA2 activity was then divided into population-specific quartiles. MCE were determined from linked medical records in GoDARTS and trial records in STABILITY. First, the interaction between diabetes control status and Lp-PLA2 activity on the outcome of MCE was explored in GoDARTS. The effect was replicated in the placebo arm of STABILITY. The effect of Lp-PLA2 on MCE was then examined in models stratified by diabetes status. This helped determine participants at higher risk. Finally, the effect of Lp-PLA2 inhibition was assessed in STABILITY in the higher risk group. Cox proportional hazards models adjusted for confounders were used to assess associations. RESULTS: In GoDARTS, a significant interaction between increased Lp-PLA2 activity (continuous and quartile divided) and diabetes control status was observed in the prediction of MCE (p < 0.0001). These effects were replicated in the placebo arm of STABILITY (p < 0.0001). In GoDARTS, stratified analyses showed that, among individuals with poorly controlled diabetes, the hazards of MCE for those with high (Q4) Lp-PLA2 activity was 1.19 compared with individuals with lower (Q1-3) Lp-PLA2 activity (95% CI 1.11, 1.38; p < 0.0001) and 1.35 (95% CI 1.16, 1.57; p < 0.0001) when compared with those with the lowest activity (Q1). Those in the higher risk group were identified as individuals with the highest Lp-PLA2 activity (Q4) and poorly controlled diabetes or diabetes. Based on these observations in untreated populations, we hypothesised that the Lp-PLA2 inhibitor would have more benefit in this higher risk group. In this risk group, Lp-PLA2 inhibitor use was associated with a 33% reduction in MCE compared with placebo (HR 0.67 [95% CI 0.50, 0.90]; p = 0.008). In contrast, Lp-PLA2 inhibitor showed no efficacy in individuals with low activity, regardless of diabetes status, or among those with no baseline diabetes and high Lp-PLA2 activity. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that diabetes status modifies the association between Lp-PLA2 activity and MCE. These results suggest that cardiovascular morbidity and mortality associated with Lp-PLA2 activity is especially important in patients with type 2 diabetes, particularly those with worse glycaemic control. Further investigation of the effects of Lp-PLA2 inhibition in diabetes appears warranted. DATA AVAILABILITY: STABILITY trial data are available from clinicaltrials.gov repository through the GlaxoSmithKline clinical study register https://clinicaltrials.gov/ct2/show/NCT00799903 . GoDARTS datasets generated during and/or analysed during the current study are available following request to the GoDARTS Access Managements Group https://godarts.org/scientific-community/ .

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.

Community-based statins and advanced carotid plaque: Role of CD163 positive macrophages in lipoprotein-associated phospholipase A2 activity in atherosclerotic plaque.

BACKGROUND AND AIMS: Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzymatic inflammatory biomarker primarily bound to low-density lipoprotein cholesterol, is associated with an approximate twofold increased risk of cardiovascular disease and stroke. Despite indications that circulating Lp-PLA2 is sensitive to statins, it remains largely unknown whether statin usage exerts local effects on Lp-PLA2 expression at the site of atheromatous plaque. METHODS: Carotid plaques (n = 38) were prospectively collected from symptomatic (n = 18) and asymptomatic (n = 20) patients with (n = 20) or without (n = 18) documented statin history. In all cases, endarterectomy was performed where the primary stenosis was removed in an undisturbed manner. Serial cryosections of the presenting lesion were assessed histologically for macrophages, Lp-PLA2, and cell death (apoptotic index). RESULTS: Symptomatic lesions exhibited less calcification, with greater inflammation characterized by increased expression of CD68+ and CD163+ macrophage subsets, and Lp-PLA2. Symptomatic plaques also exhibited greater necrotic core area and increased apoptosis, as compared with asymptomatic lesions. In contrast, statin treatment did not appear to influence any of these parameters, except for the extent of apoptosis, which was less in statin treated as compared with statin naïve lesions. Overall, Lp-PLA2 expression correlated positively with necrotic core area, CD68+ and CD163+ macrophage area, and cell death. Finally, in vitro assays and dual immunofluorescence staining confirmed CD163-expressing monocytes/macrophages are also a major source of Lp-PLA2. CONCLUSIONS: Statin treatment has no effect on local atherosclerotic lesion Lp-PLA2 activity, therefore, the addition of anti-inflammatory treatments to further decrease macrophage Lp-PLA2 expression in atherosclerotic lesions may reduce lesional inflammation and cell death, and prevent necrotic core expansion and lesion progression.

Retinal pathology is associated with increased blood-retina barrier permeability in a diabetic and hypercholesterolaemic pig model: Beneficial effects of the LpPLA2 inhibitor Darapladib.

Using a porcine model of diabetes mellitus and hypercholesterolaemia, we previously showed that diabetes mellitus and hypercholesterolaemia is associated with a chronic increase in blood-brain barrier permeability in the cerebral cortex, leading to selective binding of immunoglobulin G and deposition of amyloid-beta1-42 peptide in pyramidal neurons. Treatment with Darapladib (GlaxoSmithKline, SB480848), an inhibitor of lipoprotein-associated phospholipase-A2, alleviated these effects. Here, investigation of the effects of chronic diabetes mellitus and hypercholesterolaemia on the pig retina revealed a corresponding increased permeability of the blood-retina barrier coupled with a leak of plasma components into the retina, alterations in retinal architecture, selective IgG binding to neurons in the ganglion cell layer, thinning of retinal layers due to cell loss and increased glial fibrillary acidic protein expression in Müller cells, all of which were curtailed by treatment with Darapladib. These findings suggest that chronic diabetes mellitus and hypercholesterolaemia induces increased blood-retina barrier permeability that may be linked to altered expression of blood-retina barrier-associated tight junction proteins, claudin and occludin, leading to structural changes in the retina consistent with diabetic retinopathy. Additionally, results suggest that drugs with vascular anti-inflammatory properties, such as Darapladib, may have beneficial effects on eye diseases strongly linked to vascular abnormalities such as diabetic retinopathy and age-related macular degeneration.

Atherosclerotic plaque inflammation varies between vascular sites and correlates with response to inhibition of lipoprotein-associated phospholipase A2.

BACKGROUND: Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site-specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs). METHODS AND RESULTS: Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site-specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp-PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site-specific differences in plaque severity. In the AAs, normalized plaque area was 4.4-fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5-fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib-induced attenuation of normalized plaque area was site-specific, as CORs responded 2.9-fold more than AAs (P=0.045). CONCLUSIONS: While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp-PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti-inflammatory therapeutics.

Effect of darapladib treatment on endarterectomy carotid plaque lipoprotein-associated phospholipase A2 activity: a randomized, controlled trial.

BACKGROUND: The aim of this study was to assess the effects of darapladib, a selective oral investigational lipoprotein-associated phospholipase A2 inhibitor, on both plasma and plaque lipoprotein-associated phospholipase A2 activity. METHODS: Patients undergoing elective carotid endarterectomy were randomized to darapladib 40 mg (n = 34), 80 mg (n = 34), or placebo (n = 34) for 14 days, followed by carotid endarterectomy 24 hours after the last dose of study medication. RESULTS: Darapladib 40 mg and 80 mg reduced plasma lipoprotein-associated phospholipase A2 activity by 52% and 81%, respectively, versus placebo (both P<0.001). Significant reductions in plaque lipoprotein-associated phospholipase A2 activity were also observed compared with placebo (P<0.0001), which equated to a 52% and 80% decrease compared with placebo. No significant differences were observed between groups in plaque lysophosphatidylcholine content or other biomarkers, although a dose-dependent decrease in plaque matrix metalloproteinase-9 mRNA expression was observed with darapladib 80 mg (P = 0.053 vs placebo). In a post-hoc analysis, plaque caspase-3 (P<0.001) and caspase-8 (P<0.05) activity were found to be significantly lower in the darapladib 80-mg group versus placebo. No major safety concerns were identified in the study. CONCLUSIONS: Short-term treatment (14 ± 4 days) with darapladib produced a robust, dose-dependent reduction in plasma lipoprotein-associated phospholipase A2 activity. More importantly, darapladib demonstrated placebo-corrected reductions in carotid plaque lipoprotein-associated phospholipase A2 activity of similar magnitude. Darapladib was generally well tolerated and no safety concerns were identified. Additional studies of longer duration are needed to explore whether these pharmacodynamic effects are associated with improved clinical outcomes, as might be hypothesized.

Platelet aggregation unchanged by lipoprotein-associated phospholipase A2 inhibition: results from an in vitro study and two randomized phase I trials.

BACKGROUND: We explored the theorized upregulation of platelet-activating factor (PAF)- mediated biologic responses following lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition using human platelet aggregation studies in an in vitro experiment and in 2 clinical trials. METHODS AND RESULTS: Full platelet aggregation concentration response curves were generated in vitro to several platelet agonists in human plasma samples pretreated with rilapladib (selective Lp-PLA2 inhibitor) or vehicle. This was followed by a randomized, double-blind crossover study in healthy adult men (n = 26) employing a single-agonist dose assay of platelet aggregation, after treatment of subjects with 250 mg oral rilapladib or placebo once daily for 14 days. This study was followed by a second randomized, double-blind parallel-group trial in healthy adult men (n = 58) also treated with 250 mg oral rilapladib or placebo once daily for 14 days using a full range of 10 collagen concentrations (0-10 µg/ml) for characterizing EC50 values for platelet aggregation for each subject. Both clinical studies were conducted at the GlaxoSmithKline Medicines Research Unit in the Prince of Wales Hospital, Sydney, Australia. EC50 values derived from multiple agonist concentrations were compared and no pro-aggregant signals were observed during exposure to rilapladib in any of these platelet studies, despite Lp-PLA2 inhibition exceeding 90%. An increase in collagen-mediated aggregation was observed 3 weeks post drug termination in the crossover study (15.4% vs baseline; 95% confidence interval [CI], 3.9-27.0), which was not observed during the treatment phase and was not observed in the parallel-group study employing a more robust EC50 examination. CONCLUSIONS: Lp-PLA2 inhibition does not enhance platelet aggregation. TRIAL REGISTRATION: 1) Study 1: ClinicalTrials.gov NCT01745458 2) Study 2: ClinicalTrials.gov NCT00387257.

Diabetes and hypercholesterolemia increase blood-brain barrier permeability and brain amyloid deposition: beneficial effects of the LpPLA2 inhibitor darapladib.

Diabetes mellitus (DM) and hypercholesterolemia (HC) have emerged as major risk factors for Alzheimer's disease, highlighting the importance of vascular health to normal brain functioning. Our previous study showed that DM and HC favor the development of advanced coronary atherosclerosis in a porcine model, and that treatment with darapladib, an inhibitor of lipoprotein-associated phospholipase A2, blocks atherosclerosis progression and improves animal alertness and activity levels. In the present study, we examined the effects of DM and HC on the permeability of the blood-brain barrier (BBB) using immunoglobulin G (IgG) as a biomarker. DMHC increased BBB permeability and the leak of microvascular IgG into the brain interstitium, which was bound preferentially to pyramidal neurons in the cerebral cortex. We also examined the effects of DMHC on the brain deposition of amyloid peptide (Aß42), a well-known pathological feature of Alzheimer's disease. Nearly all detectable Aß42 was contained within cortical pyramidal neurons and DMHC increased the density of Aß42-loaded neurons. Treatment of DMHC animals with darapladib reduced the amount of IgG-immunopositive material that leaked into the brain as well as the density of Aß42-containing neurons. Overall, these results suggest that a prolonged state of DMHC may have chronic deleterious effects on the functional integrity of the BBB and that, in this DMHC pig model, darapladib reduces BBB permeability. Also, the preferential binding of IgG and coincident accumulation of Aß42 in the same neurons suggests a mechanistic link between the leak of IgG through the BBB and intraneuronal deposition of Aß42 in the brain.

Specificity of lipoprotein-associated phospholipase A(2) toward oxidized phosphatidylserines: liquid chromatography-electrospray ionization mass spectrometry characterization of products and computer modeling of interactions.

Ca(2+)-independent lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a member of the phospholipase A(2) superfamily with a distinguishing characteristic of high specificity for oxidatively modified sn-2 fatty acid residues in phospholipids that has been especially well characterized for peroxidized species of phosphatidylcholines (PC). The ability of Lp-PLA(2) to hydrolyze peroxidized species of phosphatidylserine (PS), acting as a recognition signal for clearance of apoptotic cells by professional phagocytes, as well as the products of the reaction has not been investigated. We performed liquid chromatography-electrospray ionization mass spectrometry-based structural characterization of oxygenated, hydrolyzed molecular species of PS-containing linoleic acid in either the sn-2 position (C(18:0)/C(18:2)) or in both sn-1 and sn-2 positions (C(18:2)/C(18:2)), formed in the cytochrome c- and H(2)O(2)-driven enzymatic oxidation reaction. Cytochrome c has been chosen as a catalyst of peroxidation reactions because of its likely involvement in PS oxidation in apoptotic cells. We found that Lp-PLA(2) catalyzed the hydrolysis of both nontruncated and truncated (oxidatively fragmented) species of oxidized PS species, albeit with different efficiencies, and performed detailed characterization of the major reaction products: oxygenated derivatives of linoleic acid as well as nonoxygenated and oxygenated species of lyso-PS. Among linoleic acid products, derivatives oxygenated at the C(9) position, including 9-hydroxyoctadecadienoic acid (9-HODE), a potent ligand of G protein-coupled receptor G2A, were the most abundant. Computer modeling of interactions of Lp-PLA(2) with different PS-oxidized species indicated that they are able to bind in the proximity (<5 Å) of Ser273 and His351 of the catalytic triad. For 9-hydroxy and 9-hydroperoxy derivatives of oxidized PS, the sn-2 ester bond was positioned very close (<3 Å) to the Ser273 residue, a nucleophile directly attacking the sn-2 bond, thus favoring the hydrolysis reaction. We suggest that oxidatively modified free fatty acids and lyso-PS species generated by Lp-PLA(2) may represent important signals facilitating and regulating the execution of apoptotic and phagocytosis programs essential for the control of inflammation.

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.

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.

Lipoprotein-associated phospholipase A(2) and atherosclerosis.

PURPOSE OF REVIEW: There is substantial data from over 50 000 patients that increased lipoprotein-associated phospholipase A2 (Lp-PLA2) mass or activity is associated with an increased risk of cardiac death, myocardial infarction, acute coronary syndromes and ischemic stroke. However, only recently have data emerged demonstrating a role of Lp-PLA2 in development of advanced coronary artery disease. Indeed, Lp-PLA2 may be an important link between lipid homeostasis and the vascular inflammatory response. RECENT FINDINGS: Lp-PLA2, also known as platelet-activating factor acetylhydrolase, rapidly cleaves oxidized phosphatidylcholine molecules produced during the oxidation of LDL and atherogenic lipoprotein Lp(a), generating the soluble proinflammatory and proapoptotic lipid mediators, lyso-phosphatidylcholine and oxidized nonesterified fatty acids. These proinflammatory lipids play an important role in the development of atherosclerotic necrotic cores, the substrate for acute unstable coronary disease by recruiting and activating leukocytes/macrophages, inducing apoptosis and impairing the subsequent removal of dead cells. Selective inhibition of Lp-PLA2 reduces development of necrotic cores and may result in stabilization of atherosclerotic plaques. SUMMARY: Recent data have shown that immune pathways play a major role in the development and progression of high-risk atherosclerosis, which leads to ischemic sudden death, myocardial infarction, acute coronary syndromes and ischemic strokes. Persistent and sustained macrophage apoptosis appears to play a major role in the resulting local inflammatory response in part by effects elicited by Lp-PLA2. Selective inhibition of Lp-PLA2 has been postulated to reduce necrotic core progression and the clinical sequelae of advanced, unstable atherosclerosis.

Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development.

Increased lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA(2) is a causative agent. Here we show that selective inhibition of Lp-PLA(2) with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA(2) activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA(2) inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein.

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA(2) levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA(2) inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA(2) inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA(2). A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA(2) substrates. The major PC products of Lp-PLA(2), saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA(2) inhibitor therapy.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells.

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.

Role of lipoprotein-associated phospholipase A2 in atherosclerosis and its potential as a therapeutic target.

Despite substantial progress in preventing adverse cardiovascular events with current therapeutic strategies, there remains an extensive residual risk of clinical events, particularly in high-risk patients. Because of the evidence implicating inflammation in the pathogenesis of atherosclerosis, identifying and targeting inflammatory pathways could help further reduce cardiovascular risk. There has been controversy regarding the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in atherosclerosis, partly because of the lack of simple animal models with a human-like pattern of Lp-PLA2 lipoprotein distribution. However, accumulating evidence from pathology, biology and epidemiology studies favors a pro-atherogenic rather than an atheroprotective role for the enzyme. In particular, Lp-PLA2 might play an important role in plaque vulnerability. As a result, additional studies are warranted to determine whether Lp-PLA2 inhibition improves plaque stability and ultimately clinical outcomes for high-risk patients.

Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo.

BACKGROUND: Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metabolism and inflammation. Synthetic LXR agonists have been shown to inhibit the progression of atherosclerosis in mice, but the mechanism is uncertain. LXR agonism upregulates the genes encoding ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in macrophages, thus promoting efflux of cholesterol; it also upregulates liver and intestinal ABCG5 and ABCG8, helping to promote biliary and fecal excretion of cholesterol. Thus, LXR agonism may inhibit atherosclerosis through promotion of reverse cholesterol transport (RCT) in vivo, but this has not been proven. We previously described an in vivo method to trace the movement of cholesterol from 3H-cholesterol-labeled J774 macrophages into plasma, into liver, and ultimately into the bile and feces as free cholesterol or bile acids. In the present study we used this approach to test the hypothesis that administration of the synthetic LXR agonist GW3965 would increase the rate of macrophage RCT in vivo. METHODS AND RESULTS: Three different mouse models-wild-type C57BL/6 mice, LDLR/apobec-1 double knockout mice, and human apolipoprotein (apo)B/cholesteryl ester transfer protein (CETP) double transgenic mice-were treated with either vehicle or GW3965. Mice were injected intraperitoneally with 3H-cholesterol-labeled and cholesterol-loaded macrophages and monitored for the appearance of 3H-tracer in plasma, liver, and feces. Administration of GW3965 significantly increased the levels of 3H-tracer in plasma and feces in all 3 mouse models. CONCLUSIONS: These results demonstrate that administration of the LXR agonist GW3965 increases the rate of RCT from macrophages to feces in vivo.

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure.

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

Rosiglitazone protects against ischemia/reperfusion-induced leukocyte adhesion in the zucker diabetic fatty rat.

Increased susceptibility to atherosclerosis increases the risk of mortality in type 2 diabetic patients. Leukocyte adhesion to the endothelium is a critical step in atherogenesis. In addition to its insulin-sensitizing effects, rosiglitazone (RSG) possesses anti-inflammatory properties. However, the effects of RSG on the initial phase of leukocyte recruitment (rolling, adhesion) have not been studied in vivo. This study tested the hypothesis that RSG treatment of Zucker diabetic fatty (ZDF) rats inhibits ischemia/reperfusion-induced leukocyte adhesion to the endothelium. Male ZDF rats (16 weeks) were treated with RSG (3 mg/kg/day, p.o.) 7 days before experimentation. Leukocyte-endothelial interactions in cremaster venules were recorded using intravital microscopy prior to 30 min of ischemia and during a 90-min reperfusion period. Although blood pressure, plasma glucose, and insulin were not different between treatment groups, RSG treatment was associated with reduced leukocyte rolling and inhibition of leukocyte adhesion throughout the reperfusion period (P < 0.01). Cremaster mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) was reduced by 35% in RSG-treated animals (P < 0.01), whereas P- and E-selectin and intercellular adhesion molecule-1 (ICAM-1) were unchanged. Immunostaining for P-selectin, E-selectin, and VCAM-1 was reduced by 21, 61, and 50%, respectively (for all, P < 0.05), in RSG-treated animals. Inhibition of ischemia/reperfusion-induced leukocyte adhesion might contribute to the utility of RSG as a therapy for vascular disease.

Synthetic LXR agonists increase LDL in CETP species.

Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.

Lipoprotein-associated phospholipase A2 as a target of therapy.

PURPOSE OF REVIEW: Considerable discussion continues regarding the precise role that secreted lipoprotein-associated phospholipase A2 (Lp-PLA2), also called platelet-activating factor acetylhydrolase, plays in atherosclerosis. Since interest in this enzyme as a putative drug target has been based primarily upon its association with low-density lipoprotein (LDL) in human plasma, this review will focus on Lp-PLA2 and human coronary heart disease. RECENT FINDINGS: Recent reports have linked Lp-PLA2 enrichment not only to the most atherogenic of LDL particles but also to the most advanced, rupture-prone, plaques. Electronegative LDL has been shown to be highly enriched in Lp-PLA2; and in advanced atheroma, Lp-PLA2 levels are highly upregulated, colocalizing with macrophages in both the necrotic core and fibrous cap. Lp-PLA2 is well placed, whether on an oxidation susceptible LDL particle or in the highly oxidative environment of an advanced rupture-prone plaque, to hydrolyse oxidized phospholipid and generate significant quantities of the two pro-inflammatory mediators, lysophosphatidylcholine and oxidized nonesterified fatty acid. Several studies have confirmed that Lp-PLA2 is an independent risk factor for cardiovascular events (i.e. myocardial infarction and stroke). Although epidemiology studies consistently support a relationship between plasma Lp-PLA2 levels and susceptibility to coronary heart disease this is not the case for Lp-PLA2 polymorphisms. Two clinical studies have linked the Ala-379-->Val polymorphism with a reduced risk of myocardial infarction, but functional differences between the AA and VV polymorphs have yet to be demonstrated. SUMMARY: Lp-PLA2 is intimately associated with several aspects of human atherogenesis. Although various lipid-lowering therapies, such as statins, have been shown to reduce plasma levels of Lp-PLA2, none has been studied in terms of its ability to lower the large macrophage-mediated upregulation of Lp-PLA2 within advanced plaques.