A new, highly selective murine peroxisome proliferator-activated receptor δ agonist increases responsiveness to thermogenic stimuli and glucose uptake in skeletal muscle in obese mice.

AIM: We investigated how GW800644, the first pharmacologically selective murine peroxisome proliferator-activated receptor δ (PPARδ) agonist, affects energy balance, glucose homeostasis and fuel utilization by muscle in obese mice. METHODS: Potencies were determined in transactivation assays. Oral glucose tolerance was determined after 14 and 22 days' administration (10 mg/kg body weight, twice daily) to Lep(ob)/Lep(ob) mice. Food intake and energy expenditure were measured during a 26-day experiment, and plasma metabolites and 2-deoxyglucose uptake in vivo at termination. Palmitate oxidation and 2-deoxyglucose uptake by isolated soleus muscles were measured after 14 (in lean and obese mice) and 26 days. RESULTS: GW800644 activated murine PPARδ (EC(50) 2 nM), but caused little to no activation of PPARα or PPARγ up to 10 µM. It did not increase liver weight. GW800644 reduced food intake and body weight in obese mice after 8 days. It did not affect resting energy expenditure, but, compared to pair-fed mice, it increased the response to a ß(3)-adrenoceptor agonist. It improved glucose tolerance. GW800644, but not pair-feeding, reduced plasma glucose, insulin and triglyceride concentrations. It increased 2-deoxyglucose uptake in vivo in adipose tissue, soleus muscle, heart, brain and liver, and doubled 2-deoxyglucose uptake and palmitate oxidation in isolated soleus muscle from obese but not lean mice. CONCLUSIONS: PPARδ agonism reduced food intake and independently elicited metabolic effects that included increased responsiveness to ß(3)-adrenoceptor stimulation, increased glucose utilization and fat oxidation in soleus muscle of Lep(ob)/Lep(ob) but not lean mice and increased glucose utilization in vivo in Lep(ob)/Lep(ob) mice.

Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E.

Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signalling phenomena. ApoE-deficient (apoE(-/-)) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE(-/-) mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trial for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (epsilon3) and a defective receptor-binding mutant (epsilon2) human apoE transgene in apoE(-/-) mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (ie <10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.

PPAR agonists as direct modulators of the vessel wall in cardiovascular disease.

Successful management of cardiovascular (CV) disease and associated metabolic syndromes, such as diabetes, is a major challenge to the clinician. Reducing CV risk factors, such as abnormal lipid profiles, insulin resistance or hypertension is the foundation of such therapy. A relatively new class of therapeutic agent, activators of peroxisome proliferator-activated receptors (PPAR), is poised to make a major impact with regard to several areas of risk factor management. However, there is growing evidence that PPAR agonists may also influence the CV system directly by modulating vessel wall function. These observations suggest that additional benefit, in the treatment of CV disease, may derive not only from the ability of agents to modify risk factors but also to influence directly the cellular mechanisms of disease within the vessel wall. A precedent for this dual action comes from examination of the effects of inhibitors of HMG CoA reductase (statins), where risk factor modulation is accompanied by direct actions on the vessel wall. In this review, we summarize the evidence suggesting that PPAR agonists may directly modulate vessel wall function, and that these may parallel those effects reported recently for the statins.

Apolipoprotein E and regulation of cytokine-induced cell adhesion molecule expression in endothelial cells.

Atherosclerotic plaques develop in the arterial wall from complex multicellular processes following the early recruitment of circulating monocytes. Infiltration of monocytes is mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly induced in endothelial cells in response to cytokines. Apolipoprotein E (apo E), a 34-kDa polypeptide, helps protect against atherosclerosis, in part, because apo E phospholipid particles secreted by macrophages may have local protective effects within lesions. Here we have investigated whether purified plasma apo E, complexed with dimyristoyl phosphatidylcholine (DMPC) vesicles, can inhibit cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs). Expression of VCAM-1 in endothelial cells after exposure to tumour necrosis factor-alpha (TNF-alpha) or interleukin 1beta (IL-1beta) was quantified by ELISA and shown to be partially inhibited by 17beta-estradiol (40-60% inhibition) or by S-nitroso-L-glutathione, a nitric oxide donor (20-25%). However, preincubations with physiological concentrations (10-100 microg protein/ml) of apo E DMPC did not downregulate VCAM-1 expression, even with extended preincubation times. These findings were confirmed using a fluorescence-activated cell sorter (FACS) for analysis which indicated additionally that apo E-DMPC had no effect on sub-populations within the HUVEC cultures. Finally, apo E-DMPC vesicles were also unable to suppress TNF-alpha-induced upregulation of E-selectin or intercellular adhesion molecule-1 (ICAM-1). We conclude that plasma apo E is unlikely to be important in limiting endothelial activation.

Evidence for a paraoxonase-independent inhibition of low-density lipoprotein oxidation by high-density lipoprotein.

One mechanism by which plasma high-density lipoprotein (HDL) may protect against atherogenesis is by inhibiting the oxidation of low-density lipoprotein (LDL). Recent evidence suggests that paraoxonase, an HDL-associated, calcium-dependent enzyme, may be responsible for the antioxidant action of HDL (Mackness et al., Atherosclerosis 1993;104:129; Mackness et al., FEBS Lett 1991;286:152; Watson et al., J Clin Invest 1995;96:2882; Navab et al., Arterio Thromb Vasc Biol 1996;16:831); in particular, paraoxonase activity inhibits the formation of 'minimally oxidized' LDL by hydrolyzing biologically active oxidized phospholipids (Watson et al., J Clin Invest 1995;96:2882; Navab et al., Arterio Thromb Vasc Biol 1996;16:831). However, antioxidant effects of HDL have also been demonstrated under calcium-free conditions, arguing that this enzyme may not be the only mechanism by which HDL inhibits LDL oxidation (Tribble et al., J Lipid Res 1995;36:2580). Here we have evaluated the role of paraoxonase in prevention of LDL oxidation by using HDL subfractions, isolated from human serum or EDTA-plasma, which display markedly different levels of paraoxonase activity; the abilities of modified forms of HDL to prevent LDL oxidation by cultured human (THP-1) macrophages were also assessed. Paraoxonase activity was substantially lower in HDL prepared from plasma compared to serum HDL; moreover, virtually all of the lipoprotein-associated paraoxonase activity was located in the HDL3 fraction, with HDL2 retaining only 1-5% of the total activity. Despite possessing 5-fold differences in paraoxonase activity, HDL3 isolated from plasma or serum was equally effective in inhibiting LDL oxidation by THP-1 macrophages; furthermore, although plasma HDL3 was more protective than plasma HDL2, the latter did significantly inhibit LDL oxidation. Non-paraoxonase antioxidant constituents of plasma HDL3 were investigated further. ApoHDL3, the totally delipidated form of HDL3, was much less effective than native HDL3; when examined individually, purified apolipoprotein A-II gave greater protection than apo A-I, although this effect was not evident in apo A-II-enriched HDL3. Partial delipidation of HDL3, which removes both neutral lipids and alpha-tocopherol, did not significantly diminish its ability to inhibit LDL oxidation by THP-1 macrophages; phospholipid vesicles prepared from partially delipidated HDL3 also inhibited LDL oxidation effectively. We conclude that, in this model of cellular LDL oxidation, the phospholipid fraction of HDL exerts inhibitory effects which are independent of HDL paraoxonase activity.

Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages.

The formation of macrophage-derived foam cells is central to the development of fatty streaks within the arterial wall, and to the progression of atherosclerosis. The unregulated deposition of cholesteryl esters, as lipid droplets within the cytoplasm of these cells, is responsible for the formation of foam cells; this process is thought to be regulated by the balance between cholesterol esterification, by acyl CoA:cholesterol acyltransferase (ACAT), and hydrolysis, by neutral cholesteryl ester hydrolase (nCEH). This study examines the importance of the balance between these two enzymes in determining the efflux of cholesterol from human (THP-1) macrophages. The presence of modified lipoprotein, or of 25-hydroxycholesterol, markedly increased cholesterol esterification in these cells and these effects were potently inhibited by the presence of the ACAT inhibitor, 447C88. In the absence of HDL, an acceptor particle, there was little or no hydrolysis of the cholesteryl ester pool and no efflux of cholesterol to the extracellular milieu; addition of HDL led to a partial (36%) reduction in cholesteryl esters, an effect which was not enhanced by the inhibition of ACAT. This suggested that the stored cholesteryl esters in human (THP-1) macrophages, unlike those in mouse peritoneal macrophages, were relatively resistant to removal by efflux to HDL. Efflux of newly synthesised free cholesterol from these macrophages was increased by HDL in a saturable manner, suggesting that the lack of reduction of stored cholesteryl esters was due to impaired mobilisation of cholesteryl esters to free cholesterol via nCEH. Indeed, nCEH activity in these macrophages was much lower than in mouse peritoneal macrophages, and appeared to be down-regulated in the presence of 25-hydroxycholesterol or modified lipoproteins; this loss of nCEH activity was prevented by the ACAT inhibitor 447C88. The efflux of stored cholesteryl esters from THP-1 macrophages therefore appears to be limited by the activity of nCEH.

Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages.

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381-388, 1992) using THP-1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP-1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reduction in cholesterol biosynthesis.

Three probe flow cytometry of a human foam-cell forming macrophage.

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.

5-[6-1 -(Cyclohexyl-1 H-tetrazol-5-YL)hexyl]-1,8-naphthyridin-2-(1H)-one, SC-44368, a Potent Anti-aggregatory Agent which Selectively Inhibits Platelet Cyclic AMP Phosphodiesterase.

SC-44368 (5-[6-(1-cyclohexyl-1H-tetrazol-5-y)hexyl]-1,8-naphthyridin-2(1H)-one) is a potent and selective competitive inhibitor of platelet cyclic AMP-dependent phosphodiesterase (cAMP-PDE) (Ki: 1.65 µM). For the phosphodiesterase isoenzyms from human platelets SC-44368 shows a 26-fold selectivity (IC50 ratio) for the inhibition of the cAMP-PDE over the cyclic GMP-dependent phosphodiesterase (cGMP-PDE). By comparison, 3-isobutyl-1-methyl-xanthine (IBMX) inhibited the cAMP-PDE and cGMP-PDE from human platelets with approximately equal efficacy. Broad inhibitory activity was evident against human platelet aggregatory responses in vitro. IC50 values of 18.1 ± 5.3 µM (25 nM platelet activating factor, PAF), 17.3 ± 3.0 µM (1.0 µg/ml collagen) and 24.2 ± 10.3 µM (1µM ADP) were obtained against maximum increases in platelet-rich plasma (PRP) light transmission achieved by each agonist. SC-44368 potentiated the prostacyclin-induced increase of intra-platelet cAMP levels but did not potentiate the sodium nitroprusside-induced increase of intraplatelet cGMP levels. In an ex vivo model of platelet aggregation SC-44368 (3 mg/kg, i.v.) produced a potent inhibition of collagen-induced platelet aggregation. SC-44368 produced only weak hypotensive activity in the rat. Thus, SC-44368 is a novel cAMP-PDE inhibitor which possesses potent, broad spectrum anti-aggregatory properties.

CD11/CD18 cell surface adhesion glycoproteins: discordance of monocyte function and expression in response to stimulation.

The adherence of blood leukocytes to vascular endothelium precedes their diapedesis into the extravascular space. These processes require the expression of adherence glycoproteins on the cell surface of the leukocyte. The relative importance of these adherence molecules is so far poorly understood. However, there is evidence to suggest that a disparity exists between the surface receptor expression of these glycoproteins and leukocyte adherence to vascular endothelial cells in culture. We have investigated the importance of each of the adhesion glycoproteins CD11a, CD11b, and CD11c in mediating the adherence of human monocytes to endothelial cells in culture. We have also investigated the chronological relationship between changes in monocyte adherence to endothelial cells and the surface expression of CD11a, CD11b, and CD11c following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The increase in adherence occurred within 1 minute, but declined if monocytes were preincubated with fMLP for up to 30 minutes. The surface expression of adherence molecules demonstrated a significant increase in CD11a and CD11b in the presence of fMLP after 10 min and was maintained while monocyte adherence to endothelium declined. These changes in surface receptor expression were quantitated using an immunolabeling technique. It is suggested that fMLP stimulation of monocyte adherence is unlikely to be solely dependent on increased surface receptor expression of adhesion molecules.

Nitric oxide and prostacyclin. Divergence of inhibitory effects on monocyte chemotaxis and adhesion to endothelium in vitro.

Monocyte-endothelial interactions are of fundamental importance in determining the movement of monocytes from the blood stream into the vessel wall. This study reports that two endothelium-derived factors, nitric oxide and prostacyclin, alter in vitro monocyte behavior. Nitric oxide (greater than 10(-5) M) inhibited monocyte adhesion to porcine aortic endothelial cell monolayers, whereas prostacyclin (10(-9) to 10(-5) M) had no effect. Both nitric oxide and prostacyclin inhibited monocyte chemotaxis stimulated by N-formyl-methionyl-leucyl-phenylalanine and induced dose-dependent increases in intracellular cyclic guanosine monophosphate and cyclic adenosine monophosphate concentrations, respectively. The cell surface expression of the CD11b/CD18 adhesion receptor, a glycoprotein complex known to mediate monocyte intracellular adhesion, was not altered by either nitric oxide or by prostacyclin. Thus, endothelium-derived nitric oxide and prostacyclin may have a physiological role in modulating monocyte-vascular wall interactions. Alterations in this system may contribute to the increased monocyte emigration from the blood stream into the vessel wall observed in atherogenesis.

MAC-1 mediates adherence of human monocytes to endothelium via a protein kinase C dependent mechanism.

Leukocyte adherence is mediated by a superfamily of glycoproteins denoted LFA-1 (the lymphocyte function-associated antigen-1), Mac-1 (macrophage antigen-1) and p150,95. The relative importance of these in mediating human monocyte adherence to endothelium, and the biochemical mechanisms which modulate these events, are not understood. In this report, the role of protein kinase C (pkC) in regulating human monocyte adherence to endothelial cells has been investigated. Addition of phorbol 12,13-dibutyrate (PDBu), which specifically stimulates pkC, caused a dose-dependent increase in their adherence to monolayers of bovine aortic endothelial cells. 4 alpha-phorbol didecanoate (4 alpha-PDD), a structural analogue of PDBu which does not stimulate pkC, failed to increase monocyte adhesion. PDBu also produced a dose-dependent increase in the expression of both Mac-1 and p150,95. The pkC-stimulated adherence of monocytes to endothelium was inhibited by the presence of a monoclonal antibody to Mac-1, while monoclonal antibodies to p150,95 and LFA-1 did not influence adherence. It is concluded that monocyte adherence to endothelial cells is regulated through a pkC-dependent mechanism; moreover, this process is mediated primarily via the Mac-1 adhesion glycoprotein.

Detection of a Protein in Human Platelet Membranes which Binds Low-density Lipoproteins.

Sensitisation of human blood platelets by plasma low-density lipoproteins (LDL) is thought to involve an initial interaction between LDL and the platelet surface membrane. When washed, fully responsive platelets were incubated with radio-iodinated LDL, two phases of binding were identified; one due to saturable sites, binding about 1965 ± 177 (mean ± SEM) LDL molecules per platelet, and the other of low affinity but high capacity. The saturable sites could be distinguished from the LDL receptors of nucleated cells by their presence in patients with homozygous familial hypercholesterolaemia, by their resistance to pronase digestion, by their reduced affinity for apolipoprotein E (apoE), and by the absence of a requirement for calcium ions. Binding of LDL by platelets was rapid and apparently mediated by recognition of positively charged arginine and lysine residues within the protein constituent (apoB) of LDL, findings previously reported as characteristics of LDL stimulation of platelet aggregation. Blotting of electrophoretically separated platelet membrane proteins with radio-iodinated LDL revealed a single component, presumably the saturable, high affinity binding site, whose apparent molecular weight of approx. 140 000 was lower than that of the classical LDL receptor. We conclude that occupation of (glyco)proteins on the platelet surface membrane by LDL molecules enhances platelet reactivity.

Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation.

A new rabbit model of atherosclerosis is described in which several of the features seen in early human atherosclerosis are generated within a period of 7 days. The positioning of a hollow silastic collar around the carotid artery of a cholesterol-fed rabbit results in macrophage and smooth muscle cell infiltration into the arterial subendothelium, foam cell formation and the deposition of extracellular lipid. A time-dependent accumulation of extracellular cholesteryl ester occurs within the arterial wall. Each of these changes occurs in the presence of a morphologically intact endothelium as assessed using light microscopy, scanning and transmission electron microscopy. A high cholesterol diet did not affect the extent of proliferation but exacerbated cholesteryl ester accumulation. It is proposed that the changes induced by the collar may be mediated by obstruction of the adventitial vasa vasorum with the creation of a localised ischaemic region.

Monocyte-lymphocyte discrimination in a new microtitre-based adhesion assay.

A new, rapid and reproducible method is described for the quantification of in vitro monocyte adherence in microtitre 96-well ELISA plates. The assay is based on the measurement of the myeloperoxidase activity in monocytes that have adhered to either plastic or bovine/porcine aortic endothelial cells. The assay shows a linear response to monocyte concentrations over the range 5 x 10(4)-5 x 10(5) cells/well. The method provides good discrimination between monocyte and and lymphocytes and thus allows a study of mononuclear cell populations to be made. The assay works equally well with polymorphonuclear cells. The coefficients of variation are: intra-assay, 7%; interassay, 13%.

Intracellular mechanisms in the activation of human platelets by low-density lipoproteins.

Low-density lipoproteins (LDL) have been shown to cause aggregation of human blood platelets at concentrations above 2 g of protein/l. The secretion of the contents of platelet dense granules was detected, but not that of the lysosomes. LDL gave rise to a mobilization of [3H]arachidonic acid from phospholipids and the appearance of products of the cyclo-oxygenase pathway after only 10 s. LDL-promoted aggregation was inhibited by both aspirin and indomethacin. There was an increase in 3H-labelled diacylglycerols and the phosphorylation of 47 kDa proteins. LDL therefore shares at least some of the mechanisms of stimulus/response coupling with those of other agonists.

The effect of lanosterol on platelet aggregation in human platelets.

It has been reported that lanosterol can sensitize isolated rat platelets to agonists such as ADP and thrombin (4). The purpose of this paper was to determine whether lanosterol had similar effects on human platelets and whether this was achieved by changes in membrane fluidity. Lanosterol did increase the sensitivity of human platelets, particularly to adrenaline and ADP at concentrations as low as 5 mg.L-1 when added from solutions in ethanol. At similar concentrations cholesterol, 4-cholesten-3-one or ethynyloestradiol had either no effect or were inhibitory. Measurement of membrane fluidity with diphenylhexatriene indicated that lanosterol did not affect membrane fluidity. Incubation of platelets with [4C]-mevalonic acid gave rise to a very small incorporation into lanosterol, squalene and farnesol. Sudden activation of the platelets did not accelerate lanosterol synthesis during or after platelet aggregation. It was concluded that lanosterol could only influence platelet behaviour if it came from the plasma. However the concentration of the steroid in both platelets and plasma is ten fold less than that required to sensitise the platelets.

The effect of low-density lipoproteins on the synthesis of cyclic nucleotides induced by prostacyclin in isolated platelets.

Isolated platelets are strongly sensitized by the presence of low-density lipoproteins (LDL) so that they aggregate with very low concentrations of other agonists or exhibit spontaneous aggregation. Prostacyclin (PGI2) is a potent inhibitor of aggregation, but its action was reversed by LDL. This effect of LDL was accompanied by a decrease in the synthesis of cyclic AMP induced by PGI2, but its efficacy depended on the relative concentrations of LDL and PGI2. PGI2 also enhanced the synthesis of cyclic GMP, but this was completely reversed by the presence of LDL. LDL did not remove inhibitory prostaglandins, e.g. E1, from their receptor sites. The lipoproteins also decreased cyclic AMP synthesis induced by forskolin, which has its effect on the GTP-sensitive protein or the catalytic unit of the adenylate cyclase enzyme complex. It is proposed that LDL may act on the enzyme catalytic unit via an inhibitory GTP-sensitive protein or by a separate mechanism which indirectly impedes the production of cyclic AMP.

The increased sensitivity of platelets to prostacyclin in marathon runners.

Platelet-rich plasma was obtained 24 hr after the race ended from athletes who ran in the London marathon. The platelets were only marginally less sensitive to adrenaline than were those of non-runners using conventional aggregation tests. However, the runners' platelets were much more sensitive to inhibition by prostacyclin, a prostaglandin synthesized by endothelial cells. It appeared that this effect was due to a greater activity in the platelets of the membrane-bound adenylate cyclase enzyme which generates intracellular cyclic AMP. Cyclic AMP production is known to be stimulated by prostacyclin and to cause the inhibition of platelet aggregation. The results indicate another possible protective effect of exercise against cardiovascular disease which is independent of the known changes in lipoprotein concentrations previously observed in athletes.