Colin H. Macphee

Resistin is expressed in human macrophages and directly regulated by PPARγ activators

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

Lipoprotein-Associated Phospholipase A2 as an Independent Predictor of Coronary Heart Disease

BACKGROUND Chronic inflammation is believed to increase the risk of coronary events by making atherosclerotic plaques in coronary vessels prone to rupture. We examined blood constituents potentially affected by inflammation as predictors of risk in men with hypercholesterolemia who were enrolled in the West of Scotland Coronary Prevention Study, a trial that evaluated the value of pravastatin in the prevention of coronary events. METHODS A total of 580 men who had had a coronary event (nonfatal myocardial infarction, death from coronary heart disease, or a revascularization procedure) were each matched for age and smoking status with 2 control subjects (total, 1160) from the same cohort who had not had a coronary event. Lipoprotein-associated phospholipase A2, C-reactive protein, and fibrinogen levels, and the white-cell count were measured at base line, along with other traditional risk factors. The association of these variables with the risk of coronary events was tested in regression models and by dividing the range of values according to quintiles. RESULTS Levels of C-reactive protein, the white-cell count, and fibrinogen levels were strong predictors of the risk of coronary events; the risk in the highest quintile of the study cohort for each variable was approximately twice that in the lowest quintile. However, the association of these variables with risk was markedly attenuated when age, systolic blood pressure, and lipoprotein levels were included in multivariate models. Levels of lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase), the expression of which is regulated by mediators of inflammation, had a strong, positive association with risk that was not confounded by other factors. It was associated with almost a doubling of the risk in the highest quintile as compared with the lowest quintile. CONCLUSIONS Inflammatory markers are predictors of the risk of coronary events, but their predictive ability is attenuated by associations with other coronary risk factors. Elevated levels of lipoprotein-associated phospholipase A2 appear to be a strong risk factor for coronary heart disease, a finding that has implications for atherogenesis and the assessment of risk.

Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group.

BACKGROUND Chronic inflammation is believed to increase the risk of coronary events by making atherosclerotic plaques in coronary vessels prone to rupture. We examined blood constituents potentially affected by inflammation as predictors of risk in men with hypercholesterolemia who were enrolled in the West of Scotland Coronary Prevention Study, a trial that evaluated the value of pravastatin in the prevention of coronary events. METHODS A total of 580 men who had had a coronary event (nonfatal myocardial infarction, death from coronary heart disease, or a revascularization procedure) were each matched for age and smoking status with 2 control subjects (total, 1160) from the same cohort who had not had a coronary event. Lipoprotein-associated phospholipase A2, C-reactive protein, and fibrinogen levels, and the white-cell count were measured at base line, along with other traditional risk factors. The association of these variables with the risk of coronary events was tested in regression models and by dividing the range of values according to quintiles. RESULTS Levels of C-reactive protein, the white-cell count, and fibrinogen levels were strong predictors of the risk of coronary events; the risk in the highest quintile of the study cohort for each variable was approximately twice that in the lowest quintile. However, the association of these variables with risk was markedly attenuated when age, systolic blood pressure, and lipoprotein levels were included in multivariate models. Levels of lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase), the expression of which is regulated by mediators of inflammation, had a strong, positive association with risk that was not confounded by other factors. It was associated with almost a doubling of the risk in the highest quintile as compared with the lowest quintile. CONCLUSIONS Inflammatory markers are predictors of the risk of coronary events, but their predictive ability is attenuated by associations with other coronary risk factors. Elevated levels of lipoprotein-associated phospholipase A2 appear to be a strong risk factor for coronary heart disease, a finding that has implications for atherogenesis and the assessment of risk.

Role of Lipoprotein-Associated Phospholipase A2 in Atherosclerosis Biology, Epidemiology, and Possible Therapeutic Target

The development of atherosclerotic vascular disease is invariably linked to the formation of bioactive lipid mediators and accompanying vascular inflammation. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme that is produced by inflammatory cells, co-travels with circulating low-density lipoprotein (LDL), and hydrolyzes oxidized phospholipids in LDL. Its biological role has been controversial with initial reports purporting atheroprotective effects of Lp-PLA2 thought to be a consequence of degrading platelet-activating factor and removing polar phospholipids in modified LDL. Recent studies, however, focused on pro-inflammatory role of Lp-PLA2 mediated by products of the Lp-PLA2 reaction (lysophosphatidylcholine and oxidized nonesterified fatty acids). These bioactive lipid mediators, which are generated in lesion-prone vasculature and to a lesser extent in the circulation (eg, in electronegative LDL), are known to elicit several inflammatory responses. The proinflammatory action of Lp-PLA2 is also supported by a number of epidemiology studies suggesting that the circulating level of the enzyme is an independent predictor of cardiovascular events, despite some attenuation of the effect by inclusion of LDL, the primary carrier of Lp-PLA2, in the analysis. These observations provide a rationale to explore whether inhibiting Lp-PLA2 activity and consequent interference with the formation of bioactive lipid mediators will abrogate inflammation associated with atherosclerosis, produce favorable changes in intermediate cardiovascular end points (eg, biomarkers, imaging, and endothelial function), and ultimately reduce cardiovascular events in 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.

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

Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 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-PLA2 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.

Tumor suppressor and anti-inflammatory actions of PPARγ agonists are mediated via upregulation of PTEN

The PTEN tumor suppressor gene modulates several cellular functions, including cell migration, survival, and proliferation [1] by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling cascades. Mechanisms by which the expression of PTEN is regulated are, however, unclear. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) [2] has been shown to regulate differentiation and/or cell growth in a number of cell types [3, 4, 5], which has led to the suggestion that PPARgamma, like PTEN [1, 6], could act as a tumor suppressor. PPARgamma has also been implicated in anti-inflammatory responses [7, 8], although downstream mediators of these effects are not well defined. Here, we show that the activation of PPARgamma by its selective ligand, rosiglitazone, upregulates PTEN expression in human macrophages, Caco2 colorectal cancer cells, and MCF7 breast cancer cells. This upregulation correlated with decreased PI 3-kinase activity as measured by reduced phosphorylation of protein kinase B. One consequence of this was that rosiglitazone treatment reduced the proliferation rate of Caco2 and MCF7 cells. Antisense-mediated disruption of PPARgamma expression prevented the upregulation of PTEN that normally accompanies monocyte differentiation and reduced the proportion of macrophages undergoing apoptosis, while electrophoretic mobility shift assays showed that PPARgamma is able to bind two response elements in the genomic sequence upstream of PTEN. Our results demonstrate a role for PPARgamma in regulating PI 3-kinase signaling by modulating PTEN expression in inflammatory and tumor-derived cells.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease

A specific and robust immunoassay for the lipoprotein-associated phospholipase A2 (Lp-PLA2), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA2 levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA2 participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA2 was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA2 levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA2 appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA2 mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.

Lipoprotein-Associated Phospholipase A2, Platelet-Activating Factor Acetylhydrolase, Is Expressed by Macrophages in Human and Rabbit Atherosclerotic Lesions

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Purification, Properties, Sequencing, and Cloning of a Lipoprotein-Associated, Serine-Dependent Phospholipase Involved in the Oxidative Modification of Low-Density Lipoproteins

A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.