Phospholipase A2 inhibitors in the treatment of atherosclerosis: a new approach moves forward in the clinic.

Phase II results of the trials of two phospholipase A2 inhibitors which may be of value in the treatment of atherosclerosis and cardiovascular disease have been reported in the past year. Darapladib (GlaxoSmithKline) is an inhibitor of lipoprotein-associated phospholipase A2 and varespladib (Anthera) inhibits several forms of the secreted phospholipase A2s. Despite the apparent similarity of mechanism, which is also built into the compounds' names, the role of the two types of phospholipase in atherogenesis is very different. Evidence for this comes from a range of preclinical studies and from epidemiological data which are summarised here. These data provide a basis for the Phase II studies and support decisions to move into Phase III, a decision which in the case of darapladib has been made and studies commenced (STABILITY trial). For varespladib the FRANCIS-ACS trial in acute coronary syndrome patients is in progress.

Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes.

The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO(4) (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80-90%), LDL receptor (-30-40%), CD36 (-40%) and phagocytosis (-35-40%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.

The return of two old targets?

Once the physiological function of an enzyme is understood, a rationale for therapeutic intervention often becomes apparent. It is much harder to find synthetic inhibitors with the required specificity and safety. Preclinical biological data packages are not always predictive of the response in humans. Rational targets, therefore, go in and out of fashion according to the status of candidate drugs. Acyl-CoA:cholesterol acyltransferase and squalene synthase inhibitors have been studied for many years, but safety and early clinical data did not support progression. In recent months, data have become available on new compounds for each target which suggest a way forward. This editorial reviews the difficulties and potential for each target in the light of recent reports.

Novel candidate genes in unstable areas of human atherosclerotic plaques.

OBJECTIVE: Comparison of gene expression in stable versus unstable atherosclerotic plaque may be confounded by interpatient variability. The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. METHODS AND RESULTS: Human carotid endarterectomy specimens were segmented and macroscopically classified using a morphological classification system. Two analytical methods, an intraplaque and an interplaque analysis, revealed 170 and 1916 differentially expressed genes, respectively using Affymetrix gene chip analysis. A total of 115 genes were identified from both analyses. The differential expression of 27 genes was also confirmed using quantitative-polymerase chain reaction on a larger panel of samples. Eighteen of these genes have not been associated previously with plaque instability, including the metalloproteinase, ADAMDEC1 (approximately 37-fold), retinoic acid receptor responder-1 (approximately 5-fold), and cysteine protease legumain (approximately 3-fold). Matrix metalloproteinase-9 (MMP-9), cathepsin B, and a novel gene, legumain, a potential activator of MMPs and cathepsins, were also confirmed at the protein level. CONCLUSIONS: The differential expression of 18 genes not previously associated with plaque rupture has been confirmed in stable and unstable regions of the same atherosclerotic plaque. These genes may represent novel targets for the treatment of unstable plaque or useful diagnostic markers of plaque instability.

Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants.

The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL3 or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [3H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [3H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins.

The PPARdelta agonist GW0742X reduces atherosclerosis in LDLR(-/-) mice.

Several lines of evidence suggest a biological role for peroxisome proliferator-activated receptor (PPARdelta) in the pathogenesis of atherosclerosis. Administration of synthetic PPARdelta agonists to obese rhesus monkeys elevates serum high-density lipoprotein (HDL) cholesterol as a result of increased reverse cholesterol transport whilst in vitro studies have suggested a role for PPARdelta in lipid uptake into macrophages. Recent studies have found that PPARdelta depletion from macrophages in LDL receptor (LDLR(-/-)) mice decreases lesion area via modulation of the inflammatory status of the macrophage, an effect also seen on pharmacological activation of PPARdelta in vitro. We demonstrate here that the PPARdelta agonist, GW0742X has potent anti-atherogenic activity in the LDLR(-/-) mouse, decreasing lesion area by up to 50%. Administration of GW0742X had no effect on total cholesterol, HDL or LDL cholesterol and modest effects on very low-density lipoprotein (VLDL). Treatment with GW0742X resulted in decreased expression of monocyte chemoattractant protein 1 (MCP-1) and intracellular adhesion moleculae 1 (ICAM-1) in the aortae of treated mice. In addition, GW0742X decreased tumour necrosis factor-alpha (TNFalpha) expression in peritoneal macrophages, aortae and adipose tissue in comparison with control animals. Changes in gene expression were reflected in decreased plasma levels of MCP-1. These observations support an atheroprotective effect of PPARdelta agonists in vivo.

Characterisation of the lipoprotein structure in the St. Thomas' Mixed Hyperlipidaemic (SMHL) rabbit.

Familial combined hyperlipidaemia (FCHL) is a complex genetic disorder of unknown aetiology. Study of this human condition over many decades has been hampered by likely genetic heterogeneity. In order to find better phenotypic markers, we have characterised the structures of VLDL, IDL and LDL in the St. Thomas' Mixed Hyperlipidaemic (SMHL) rabbit--an animal model of FCHL in which the hyperlipidaemia is caused primarily by an increased production rate of apolipoprotein B (apoB)--containing lipoproteins-and compared them with those in the Watanabe Heritable Hyperlipidaemic (WHHL) rabbit, in which hyperlipidaemia is caused mainly by a defect in lipoprotein clearance, and those in the normolipidaemic New Zealand White (NZW) animal. All three rabbit strains were fed a cholesterol-enriched (0.08%, w/w) diet for at least 3 months prior to blood sampling. Both SMHL and WHHL rabbits showed combined hyperlipidaemia as evidenced by significantly increased levels of plasma cholesterol and triglycerides. Raised plasma lipids in the SMHL rabbit were attributable mainly to an overabundance of lipoprotein particles with the same lipid composition as those in NZW rabbits. VLDL and IDL in the SMHL rabbit showed a significantly increased sphingomyelin to phosphatidyl choline ratio. In the WHHL rabbit there was a high concentration of particles that were significantly enriched in cholesteryl esters and depleted in triglycerides. Phospholipids in all lipoprotein fractions from WHHL rabbits contained significantly more sphingomyelin and less phosphatidyl choline resulting in a significantly increased sphingomyelin to phosphatidyl choline ratio. We found that the VLDL of SMHL rabbits could be distinguished from that of NZW rabbits on the basis of the cholesterol:apoB and the sphingomyelin:phosphatidylcholine ratios, and from that of WHHL rabbits by the sphingomyelin:triglyceride ratio. Extrapolating these findings to the human condition, an assessment of particle core composition, together with the proportion of sphingomyelin in phospholipids especially in VLDL might help in the differentiation of the combined hyperlipidaemia of FCHL into disorders of lipoprotein overproduction versus decreased clearance.

Short term arterial remodelling in the aortae of cholesterol fed New Zealand white rabbits shown in vivo by high-resolution magnetic resonance imaging - implications for human pathology.

High-resolution, non-invasive imaging methods are required to monitor progression and regression of atherosclerotic plaques. We investigated the use of MRI to measure changes in plaque volume and vessel remodelling during progression and regression of atherosclerosis in New Zealand White rabbits. Atherosclerotic lesions were induced in the abdominal aorta by balloon injury and cholesterol feeding. MR images (2D) of the abdominal aorta were acquired with cardiac and respiratory gating using a fast spin echo sequence with and without fat-suppression. In an initial study on rabbits treated for 30 weeks we imaged the aortae with a spatial resolution of 250x250 micrometers with a slice thickness of 2 mm and achieved a close correlation between MRI-derived measurements and those made on perfusion pressure-fixed histological sections (r(1) = 0.83, slope p(1) < 0.01). We subsequently imaged 18 rabbits before and periodically during 12 weeks of cholesterol feeding (progression) followed by 12 weeks on normal diet (regression). Aortic wall (atherosclerotic lesion) volume increased significantly during progression and decreased during regression. In contrast, lumen volume increased during progression and did not change during regression. In conclusion, this study confirms that non-invasive, high-resolution MRI can be used to monitor progression and regression of atherosclerosis, each within 3 months and shows, for the first time in a short-term model, that positive remodelling occurs early during progression and persists through regression of atherosclerotic lesions.

Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages.

The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.

Protection of chylomicron remnants from oxidation by incorporation of probucol into the particles enhances their uptake by human macrophages and increases lipid accumulation in the cells.

The effects of protection of chylomicron remnants from oxidation on their uptake and induction of lipid accumulation in macrophages were investigated using chylomicron remnant-like particles (CRLPs) containing the lipophilic antioxidant drug, probucol, and macrophages derived from the human monocyte cell line, THP-1. The total lipid content of THP-1 macrophages was markedly higher (x2.2) after 48 h of incubation of THP-1 macrophages with CRLPs containing probucol (pCRLPs) when compared to CRLPs without probucol, and this was because of increases in triacylglycerol (x2.3) and cholesterol (x1.8) levels, while cholesteryl ester concentrations were not significantly changed. Determination of the uptake of CRLPs and pCRLPs by the cells using particles labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethylindo-carbocyanine perchlorate showed that pCRLPs are taken up at a faster rate than CRLPs. The synthesis of triacylglycerol, as measured by the incorporation of [(3)H]oleate and [(3)H]glycerol, was also increased in macrophages incubated with pCRLPs as compared to CRLPs without probucol, but phospholipid and cholesteryl ester formation from [(3)H]oleate was unaffected. In addition, no differences between the effects of CRLPs and pCRLPs on the expression of mRNA for a range of genes believed to be involved in lipoprotein uptake, intracellular lipid metabolism and the efflux of cholesterol from macrophages was detected. These results suggest that antioxidants carried in chylomicron remnants enhance lipid accumulation in macrophages by increasing the rate of uptake of the particles and raising the intracellular synthesis of triacylglycerol, but not cholesteryl ester, and that these effects are brought about by changes at the post-transcriptional level. Antioxidants carried in chylomicron remnants therefore may promote the development of atherosclerosis, and this is likely to be particularly important in conditions where clearance of remnants from the circulation is delayed.

Chylomicron remnants and oxidised low density lipoprotein have differential effects on the expression of mRNA for genes involved in human macrophage foam cell formation.

The effects of chylomicron remnants (non-oxidised or oxidised) and oxidised low density lipoprotein (oxLDL) on the expression of mRNA for a wide range of genes believed to play a role in macrophage foam cell formation were compared using macrophages derived from the human monocyte cell line THP-1. Chylomicron remnant-like particles (CMR-LPs), oxidised CMR-LPs (oxCMR-LPs) and oxLDL were incubated with THP-1 macrophages, and the relative abundance of mRNA transcripts for genes involved in lipoprotein uptake, intracellular lipid metabolism, transport and storage and cholesterol efflux from macrophages was determined. The results show that CMR-LPs and oxLDL differ markedly in their effects on the expression of mRNA for a number of the genes tested. OxLDL increased mRNA levels for the scavenger receptors CD36 (x3.2) and lectin-like oxLDL receptor 1 (x2.1), and peroxisome proliferator-activated receptor gamma while CMR-LPs did not. In contrast, the expression of mRNA for the LDL receptor-like protein was raised by CMR-LPs (x1.8) but not oxLDL. Furthermore, down-regulation of mRNA levels for the ATP-binding cassette transporter (ABC) A1 was observed with CMR-LPs (x0.6), compared to the up-regulation found with oxLDL (x4.4). In addition, a number of significant differences were found between the effects of CMR-LPs and oxCMR-LPs, with the oxidised particles causing a striking rise in mRNA expression for the multi-drug resistance 1 gene (x13.7), but otherwise showing pattern more similar to that seen with oxLDL. These findings provide evidence to indicate that chylomicron remnants cause lipid accumulation in macrophages by influencing the expression of genes which regulate lipid metabolism at the transcriptional level, and that the mechanisms involved differ in important respects from those triggered by oxLDL.

Incorporation of lycopene into chylomicron remnant-like particles enhances their induction of lipid accumulation in macrophages.

Lipid accumulation in macrophages exposed to chylomicron remnant-like particles containing the dietary antioxidant lycopene was investigated. After incubation with THP-1 macrophages (48h), chylomicron remnant-like particles containing lycopene (lycCRLPs) as compared to those without (CRLPs) caused significantly more lipid accumulation in the cells, and this was due to increases in both the triacylglycerol (+100%) and cholesterol (+62%) content. In addition, expression of mRNA for diacylglycerol acyltransferase (DGAT), a key enzyme in triacylglycerol synthesis, was significantly decreased by lycCRLPs, but not CRLPs. These findings suggest that lycopene from the diet may promote, rather than retard, lipid accumulation in macrophages during its transport in the blood in chylomicron remnants.

Repeated three-dimensional magnetic resonance imaging of atherosclerosis development in innominate arteries of low-density lipoprotein receptor-knockout mice.

BACKGROUND: In vivo methods to evaluate the size and composition of atherosclerotic lesions in animal models of atherosclerosis would assist in the testing of antiatherosclerotic drugs. We have developed an MRI method of detecting atherosclerotic plaque in the major vessels at the base of the heart in low-density lipoprotein (LDL) receptor-knockout (LDLR(-/-)) mice on a high-fat diet. METHODS AND RESULTS: Three-dimensional fast spin-echo magnetic resonance images were acquired at 7 T by use of cardiac and respiratory triggering, with approximately 140- micro m isotropic resolution, over 30 minutes. Comparison of normal and fat-suppressed images from female LDLR(-/-) mice 1 week before and 8 and 12 weeks after the transfer to a high-fat diet allowed visualization and quantification of plaque development in the innominate artery in vivo. Plaque mean cross-sectional area was significantly greater at week 12 in the LDLR(-/-) mice (0.14+/-0.086 mm2 [mean+/-SD]) than in wild-type control mice on a normal diet (0.017+/-0.031 mm2, P<0.01). In the LDLR(-/-) mice, but not control mice, increase in plaque burden at week 12 relative to week 1 was also highly significant (P=0.001). Lumen cross section was not significantly different between time points or groups. MRI and histological assessments of plaque size were closely correlated (R=0.8). The lumen of proximal coronary arteries could also be visualized. CONCLUSIONS: This is the first report of in vivo detection of aortic arch atherosclerosis in any animal model. The method could significantly assist rapid evaluation of experimental antiatherosclerotic therapies.

Lipoprotein-associated phospholipase A2: a target directed at the atherosclerotic plaque.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is so named because it is found in human plasma largely associated with low-density lipoprotein (LDL). It is secreted by macrophages and able to hydrolyse oxidised fatty acids from oxidised phospholipids in LDL thereby releasing pro-atherogenic lysophosphatidylcholine and fatty acids. Inhibition of this enzyme activity was proposed to be antiatherogenic and this hypothesis has been confirmed both in vitro and in animal studies using specific inhibitors. In addition, the enzyme has been shown to be present in human atherosclerotic plaques and to be a potential risk factor for coronary heart disease in epidemiological studies. However, Lp-PLA(2) is identical to platelet-activating factor acetylhydrolase (PAF-AH), whose activity is regarded as antiatherogenic. The role of this enzyme in humans, represented as Lp-PLA(2) or PAF-AH, remains to be clarified. Specific and potent inhibitors of Lp-PLA(2) have been described and help address this question. This is a novel approach directed specifically towards processes in atherogenesis which take place in the artery wall. Innovative strategies for clinical development are required to progress novel molecular strategies such as this.