Long hydrocarbon chain diols and diacids with central ether or ketone moieties that favorably alter lipid disorders.

Long hydrocarbon chain derivatives with bis-terminal hydroxyl or carboxyl groups and various central moieties (ketone, ether, ester, amide, carbamate, etc.) have been synthesized and evaluated for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes as well as for their effects on lipid, glycemic and body weight variables in female obese Zucker fatty rats following one and two weeks of oral administration. The most active compounds were found to be symmetrical with four to five methylene groups separating the ether or ketone central functionality from the gem dimethyl, cycloalkyl or methyl/aryl substituents. Cycloalkyl substitution alpha to the carboxyl group in keto-acids lowered the in vitro activity to micromolar values. Furthermore, in vivo biological activity was found to be greatest for cyclopropyl-substituted ketone derivatives, particularly the ketodiacid with five methylene groups on each side of the central ketone functionality, which was identified as an HDL elevator and was also found to reduce insulin and glucose.

High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization.

BACKGROUND: Repeated doses of recombinant apolipoprotein A-I(Milano) phospholipid complex (apoA-I(m)) reduce atherosclerosis and favorably change plaque composition in rabbits and mice. In this study, we tested whether a single high dose of recombinant apoA-I(m) could rapidly mobilize tissue cholesterol and reduce plaque lipid and macrophage content in apoE-deficient mice. METHODS AND RESULTS: High cholesterol-fed, 26-week-old apoE-deficient mice received a single intravenous injection of saline (n=16), 1080 mg/kg dipalmitoylphosphatidylcholine (DPPC; n=14), or 400 mg/kg of recombinant apoA-I(m) complexed with DPPC (1:2.7 weight ratio; n=18). Blood was sampled before and 1 and 48 hours after injection, and aortic root plaques were evaluated for lipid content and macrophage content after oil-red O and immunostaining, respectively. One hour after injection, the plasma cholesterol efflux-promoting capacity was nearly 2-fold higher in recombinant apoA-I(m)-treated mice compared with saline and DPPC-treated mice (P<0.01). Compared with baseline values, serum free cholesterol, an index of tissue cholesterol mobilization, increased 1.6-fold by 1 hour after recombinant apoA-I(m) injection, and it remained significantly elevated at 48 hours (P<0.01). Mice receiving recombinant apoA-I(m) had 40% to 50% lower lipid content (P<0.01) and 29% to 36% lower macrophage content (P<0.05) in their plaques compared with the saline- and DPPC-treated mice, respectively. CONCLUSIONS: A single high dose of recombinant apoA-I(m) rapidly mobilizes tissue cholesterol and reduces plaque lipid and macrophage content in apoE-deficient mice. These findings suggest that this strategy could rapidly change plaque composition toward a more stable phenotype.

Expression of the mRNA encoding truncated PPAR alpha does not correlate with hepatic insensitivity to peroxisome proliferators.

Two alternatively spliced forms of human PPAR alpha mRNA, PPAR alpha1 and PPAR alpha2, have been identified. PPAR alpha1 mRNA gives rise to an active PPAR alpha protein while PPAR alpha2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR alpha2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR alpha1 and PPAR alpha2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR alpha2 mRNA abundance is approximately half that of PPAR alpha1 mRNA; a correlation analysis of PPAR alpha1 and PPAR alpha2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR alpha2/PPAR alpha1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR alpha2/PPAR alpha1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPAR alpha activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR alpha2/PPAR alpha1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR alpha2/PPAR alpha1 mRNA. These data suggest that selective PPAR alpha2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.

Rabbits possess a serum paraoxonase polymorphism similar to the human Q192R.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect low-density lipoprotein (LDL) against oxidation. These properties are influenced by a well-characterized polymorphism (Q192R) in human PON1. We now report the identification and characterization of a phenotypically similar, but genetically distinct polymorphism in rabbit PON1. This polymorphism in rabbits was detected by phenotyping sera obtained from 16 inbred rabbit strains and 20 outbred New Zealand White rabbits by paraoxonase/arylesterase activity. The genetic basis of the rabbit polymorphism was determined by DNA sequencing and found to reside in a region distinct from the human Q192R and M55L polymorphisms. Three variant nucleotides within exon 4 (corresponding to P82S, K93E and S1O1G) were found to segregate with the observed rabbit PON1 phenotypes (rPON1A and rPON1B). The rPON1A and rPON1B proteins were purified and compared to the two human isoforms (192Q and 192R). The human and rabbit PON1s displayed similar characteristics with respect to physical properties and substrate specificity. However, rPON1A and rPON1B hydrolysed a variety of substrates at different rates. The rPON1A was also at least three-fold more efficient at protecting LDL from oxidation than rPON1B. Our characterization of a rabbit PON1 polymorphism provides useful insights into important functional residues in PON1. In addition, due to the observed similarities between the rabbit and human polymorphisms, the rabbit may serve as a good model to examine the effect of human PON1 polymorphisms in disease development.

Elevated hepatic apolipoprotein A-I transcription is associated with diet-induced hyperalphalipoproteinemia in rabbits.

Past studies have shown that a high saturated fatty acid diet containing coconut oil elevates plasma HDL cholesterol and apolipoprotein A-I (apoA-1) in rabbits through a mechanism involving increased synthesis. We have extended those studies by investigating expression of the hepatic apolipoprotein A-I gene and other lipid related genes in that model. Rabbits fed a diet containing 14% coconut oil for 4 weeks showed HDL-C elevations of 170% to 250% over chow-fed controls with peak differences occurring at 1 week. Plasma apoA-I levels were also increased over this time frame (160% to 180%) reflecting the HDL-C changes. After 4 weeks, there were no differences in plasma VLDL-C or LDL-C levels in chow versus coconut oil-fed rabbits. Hepatic levels of apoA-I mRNA in coconut oil-fed animals were elevated 150% after 4 weeks compared to chow-fed controls; hepatic mRNA levels for ten other genes either decreased slightly (apoB, LCAT, hepatic lipase, albumin, ACAT, and HMG CoA reductase) or were unchanged (CETP, apoE, LDL-receptor, and acyl CoA oxidase). Nuclear run-on transcription assays revealed that coconut oil feeding for 4 weeks caused a 220% increase in hepatic apoA-I transcription rate compared to controls; no change was observed for CETP and apoE. Treatment of cultured rabbit liver cells with various saturated fatty acids and sera from chow-fed and coconut oil-fed rabbits did not alter apoA-I mRNA levels as observed in vivo. These data demonstrate that coconut oil elevates plasma HDL-C and apoA-I by increasing hepatic apoA-I transcription while expression of other genes involved in lipid metabolism are reduced or unchanged in response to coconut oil feeding.

9-cis retinoic acid induces monocyte chemoattractant protein-1 secretion in human monocytic THP-1 cells.

Monocyte migration and activation are regulated by monocyte chemoattractant protein-1 (MCP-1). Prior studies have shown MCP-1 expression is modulated by a variety of ligands that act through extracellular receptors. In the current study, we show 9-cis retinoic acid (RA), a ligand for the nuclear hormone receptor retinoid X receptor (RXR) and retinoic acid receptor (RAR), markedly induces the expression of MCP-1. In human THP-1 monocytic leukemia cells cultured with RA (0.05 to 500 nmol/L), MCP-1 expression was induced rapidly, significantly, and dose-dependently by as much as 165-fold. MCP-1 RNA level was also increased in RA-treated cells. Expression of PPARgamma, a heterodimer partner of RXR, is also markedly induced by RA in THP-1 cells. However, BRL49653, a PPARgamma ligand, failed to induce MCP-1 secretion either alone or to modify the expression level induced by RA. In contrast, BRL49653 significantly increased MCP-1 (biotinylated MCP-1) binding to THP-1 cells, whereas RA had no effect. Other peroxisome proliferator activated receptor (PPAR) ligands, 15d-PGJ(2) and troglitazone (PPARgamma), Wy14,643 (PPARalpha), and PD195599 (PPARbeta) inhibited the induction of MCP-1 by RA. RA's effect on MCP-1 expression in human elutriated monocytes were similar to that observed in the THP-1 cells. These studies identify RA as a nuclear signal for MCP-1 induction in undifferentiated human monocytic cells. These studies also suggest monocyte MCP-1 expression induced through RA may modulate cell migration.

Human serum Paraoxonase/Arylesterase's retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids : apolipoprotein A-I stabilizes activity.

In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.

Expression, purification, and biochemical characterization of SAG, a ring finger redox-sensitive protein.

We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis. Here we report the expression, purification, and biochemical characterization of SAG. Bacterially expressed SAG is brown in color and dithiothreitol (DTT)-sensitive. SAG forms large oligomers without DTT that can be reduced into a monomer in the presence of DTT. These features help us to purify SAG using the chromatography with or without DTT. Likewise, purified SAG is redox sensitive. Upon H2O2 exposure, SAG forms oligomers as well as monomer doublets due to the formation of the inter- or intramolecular disulfide bonds, respectively. This process can be reversed by DTT or prevented by pretreatment with the alkylating reagent, N-ethylmaleimide (NEM). Although SAG contains two putative heme-binding sites and a RING finger domain, the protein appears not to bind with heme and to lack transcription factor activity as determined in a Gal4-fusion/transactivation assay. Wildtype, but not RING finger domain-disrupted SAG mutants, prevents copper-induced lipid peroxidation. These results, along with our previous observations, suggest that SAG is an intracellular antioxidant molecule that may act as a redox sensor to buffer oxidative-stress induced damage.

Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase.

Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears to protect low density lipoproteins (LDL) from oxidation. Mature PON1 retains its N-terminal hydrophobic signal sequence, which may be needed for binding to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding to lipoproteins. As a model system, we studied binding of mutant and wild type PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoproteins whereas the A19A20 mutant did not. These results show that the N-terminus is required for binding to either apolipoproteins or phospholipids. Furthermore, we showed that wild type enzyme can bind to phospholipids directly without apolipoproteins. To determine if lipid binding is a requirement for PON1's protection against LDL oxidation, we used a copper ion-induced oxidation system and found that the wild type enzyme and A19A20 mutant showed similar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association with serum lipoproteins.

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants.

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.

Increased atherosclerosis in ApoE and LDL receptor gene knock-out mice as a result of human cholesteryl ester transfer protein transgene expression.

The plasma cholesteryl ester transfer protein (CETP) plays a major role in the catabolism of HDL cholesteryl ester (CE). CETP transgenic mice have decreased HDL cholesterol levels and have been reported to have either increased or decreased early atherosclerotic lesions. To evaluate the impact of CETP expression on more advanced forms of atherosclerosis, we have cross-bred the human CETP transgene into the apoE knock-out (apoE0) background with and without concomitant expression of the human apo A-I transgene. In this model the CETP transgene is induced to produce plasma CETP levels 5 to 10 times normal human levels. CETP expression resulted in moderately reduced HDL cholesterol (34%) in apoE0 mice and markedly reduced HDL cholesterol (76%) in apoE0/apoA1 transgenic mice. After injection of radiolabeled HDL CE, the CETP transgene significantly delayed the clearance of CE radioactivity from plasma in apoE0 mice, but accelerated the clearance in apoE0/apoA1 transgenic mice. ApoE0/CETP mice displayed an increase in mean atherosclerotic lesion area on the chow diet (approximately 2-fold after 2 to 4 months, and 1.4- to 1.6-fold after 7 months) compared with apoE0 mice (P<0.02). At 7 months apoA1 transgene expression resulted in a 3-fold reduction in mean lesion area in apoE0 mice (P<0.001). In the apoE0/apoA1 background, CETP produced an insignificant 1.3- to 1.7-fold increase in lesion area. In further studies the CETP transgene was bred onto the LDL receptor knock-out background (LDLR0). After 3 months on the Western diet, the mean lesion area was increased 1.8-fold (P<0.01) in LDLR0/CETP mice, compared with LDLR0 mice. These studies indicate that CETP expression leads to a moderate increase in atherosclerosis in apoE0 and LDLR0 mice, and suggest a proatherogenic effect of CETP activity in metabolic settings in which clearance of remnants or LDL is severely impaired. However, apoA1 overexpression has more dramatic protective effects on atherosclerosis in apoE0 mice, which are not significantly reversed by concomitant expression of CETP.

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents.

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.

Lipoprotein lipase greatly enhances the retention of lipoprotein(a) to endothelial cell-matrix.

The trapping of apolipoprotein (apo)B containing lipoproteins within the arterial subendothelial matrix (ECM) is an early event in atherosclerosis. When lipoprotein lipase, a constituent of the ECM, is prebound to ECM both LDL and oxidized LDL binding is greatly enhanced. In this study we compared the binding of lipoprotein(a) (Lp(a)), a lipoprotein correlated with atherosclerosis and restenosis, to ECM in the presence of varying concentrations of LPL. Without LPL, Lp(a) binding was low and non-saturable. In the presence of LPL, Lp(a) retention increased from 2.7 x 10(-7) to 1.13 x 10(-4) nmoles. Scatchard analysis demonstrated that the affinities of both Lp(a) and LDL to lipase were similar. In competition experiments, LDL, apoE, polymers of lysine and arginine were all capable of preventing the lipase specific [125I]Lp(a) retention. However, neither collagen nor fibronectin were capable of blocking or displacing [125I]Lp(a) from the lipase bound to ECM. In a separate set of experiments, when ECM was not saturated with lipase, both fibronectin and collagen (at 10-fold protein excess) prevented approximately 40% of total [125I]Lp(a) retention to ECM. These data suggest, in the absence of lipase, apo(a) may regulate the binding of Lp(a) to ECM. Whereas, lipase enhanced the binding of Lp(a) to ECM, most probably through the apoB moiety of the Lp(a) particle.

Elevated low-density lipoprotein in Alzheimer's disease correlates with brain abeta 1-42 levels.

Sera obtained in the immediate postmortem from 100 individuals, 64 neuropathologically diagnosed Alzheimer's disease (AD) cases and 36 nondemented controls, were analyzed for cholesterol, lipoproteins, apolipoproteins (Apo), and triglycerides. All individuals were ApoE genotyped, and the amounts of Abeta (N-40 and N-42) in cerebral cortex of AD and control subjects were determined. When compared to controls, AD individuals had significantly higher LDL cholesterol (P = 0.006), ApoB (P = 0.018), Abeta N-40 (P = 0.024) and Abeta N-42 (P < 0.001), and significantly lower HDL cholesterol (P = 0.040). There were positive correlations between the levels of serum total cholesterol (r = 0.359, P = 0.004), LDL cholesterol (r = 0.328, P = 0.008), and ApoB (r = 0.395, P = 0.001) to the amount of Abeta N-42 in AD brains, but not to Abeta N-40. These correlations were independent of ApoE genotype and were not seen in the control group. The present results suggest for the first time that elevated serum cholesterol, especially in the form of LDL, influences the expression of AD-related pathology.

Induction of PPARgamma1 expression in human THP-1 monocytic leukemia cells by 9-cis-retinoic acid is associated with cellular growth suppression.

9-cis-Retinoic acid (RA) and peroxisome proliferator activated receptor gamma (PPARgamma) regulates cellular growth and differentiation. In THP-1 cells, a human monocytic leukemia cell line, RA markedly induced PPARgamma1 RNA, nuclear PPARgamma1 protein and suppressed cell growth. The PPARgamma ligand, BRL49653 enhanced RA's growth suppression ability. With BRL49653 alone, THP-1 cell growth was only marginally suppressed. Cell cycle analysis revealed the G1 phase cell population was significantly increased when cells were treated with both ligands. RA induced growth suppression did not differentiate the THP-1 cells to macrophages. Phorbol ester (PMA) induced differentiation of cells to macrophage also induced PPARgamma1 expression, however when RA is given either simultaneously or sequentially to these cells, no further increase in expression of the nuclear receptor was observed. Overall, these data suggest RA induction of PPARgamma1 may block cell growth and may have application for the treatment of proliferative diseases.

Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation.

Increased atherosclerosis risk in hyperlipidemic patients may be a result of the enhanced oxidizability of their plasma lipoproteins. We have previously shown that hypocholesterolemic drug therapy, including the 3-hydroxy-3-methyl-glutaryl CoenzymeA (HMG-CoA) reductase inhibitors, and the hypotriglyceridemic drug bezafibrate, significantly reduced the enhanced susceptibility to oxidation of low density lipoprotein (LDL) isolated from hyperlipidemic patients. Although this antioxidative effect could not be obtained in vitro with all of these drugs, the active drug metabolites, which are formed in vivo, could affect lipoprotein oxidizability. We thus sought to analyze the effect of atorvastatin and gemfibrozil, as well as specific hydroxylated metabolites, on the susceptibility of LDL, very low density lipoprotein (VLDL), and high density lipoprotein (HDL) to oxidation. LDL oxidation induced by either copper ions (10 microM CuSO4), by the free radical generator system 2'-2'-azobis 2-amidino propane hydrochloride (5 mM AAPH), or by the J-774A.1 macrophage-like cell line, was not inhibited by the parent forms of atorvastatin or gemfibrozil, but was substantially inhibited (57-97%), in a concentration-dependent manner, by pharmacological concentrations of the o-hydroxy and the p-hydroxy metabolites of atorvastatin, as well as by the p-hydroxy metabolite (metabolite I) of gemfibrozil. On using the atorvastatin o-hydroxy metabolite and gemfibrozil metabolite I in combination an additive inhibitory effect on LDL oxidizability was found. Similar inhibitory effects (37-96%) of the above metabolites were obtained for the susceptibility of VLDL and HDL to oxidation in the oxidation systems outlined above. The inhibitory effects of these metabolites on LDL, VLDL, and HDL oxidation could be related to their free radical scavenging activity, as well as (mainly for the gemfibrozil metabolite I) to their metal ion chelation capacities. In addition, inhibition of HDL oxidation was associated with the preservation of HDL-associated paraoxonase activity. We conclude that atorvastatin hydroxy metabolites, and gemfibrozil metabolite I possess potent antioxidative potential, and as a result protect LDL, VLDL, and HDL from oxidation. We hypothesize that in addition to their beneficial lipid regulating activity, specific metabolites of both drugs may also reduce the atherogenic potential of lipoproteins through their antioxidant properties.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

A novel compound that elevates high density lipoprotein and activates the peroxisome proliferator activated receptor.

In the current studies we describe the effects of PD 72953 and related compounds on lipoprotein levels in chow-fed male rats. After 2 weeks, 10 mg/kg of PD 72953 daily was as effective as 100 mg/kg gemfibrozil for elevating HDL-cholesterol. At 100 mg/kg, PD 72953 further elevated HDL-cholesterol to 232% of control levels, and was associated with increased HDL size and plasma apoE (169% of control), despite no change in hepatic apoE mRNA. ApoA-I rose transiently (at 1 week), but by 2 weeks only apoE remained elevated. PD 72953 dose-dependently reduced plasma apoB, VLDL-cholesterol, LDL-cholesterol, and triglyceride. Hepatic apoC-III mRNA reduction parallelled triglyceride lowering. After 1 week, 30 and 100 mg/kg per day PD 72953 reduced plasma apo-CIII levels by 30 and 34%, and triglycerides by 60 and 83%, respectively. PD 72953 treatment had no effect on triglyceride production rates; however, 125I-labeled VLDL apoB disappearance was enhanced. We compared PD 72953 to a structurally similar diacid, PD 69405, that also reduced VLDL and LDL, but had no effect on HDL elevation. Compared to PD 72953, PD 69405 further accelerated 125I-labeled VLDL apoB disappearance, decreased triglyceride production, and elevated the ratio of post-heparin hepatic to lipoprotein lipase activity. Whole animal studies, transient transfection studies in HepG2 cells, and chimeric receptor studies in kidney 293 cells suggest that PD 72953 is a ligand for the peroxisomal proliferation activated receptor alpha (PPARalpha), and PPARgamma. Overall, PD 72953 may act through a peroxisomal proliferation activated receptor and result in plasma triglycerides and apoB-containing lipoprotein reduction, while also raising HDL cholesterol. Reduced apoC-III may allow triglyceride-rich remnants to more efficiently bind and present substrate to peripheral tissue lipoprotein lipase, and therefore allow enhanced shedding of remnant phospholipid surface for HDL production.

Discovering HDL-elevating drugs: are there simplistic approaches to a polygenic disorder?

High density lipoproteins (HDL) are thought to serve a protective role in the development of atherosclerosis. For this reason, there have been intensive efforts over the years to discover and develop compounds that would successfully elevate HDL levels in humans. However, unlike the HMG-CoA reductase inhibitors, which lower low density lipoproteins (LDL), there has been a dearth of compounds specifically indicated for HDL elevation which have progressed significantly through drug development. Nonetheless, recent research into the mechanism of action of HDL elevators in preclinical animal models has provided some valuable insights for targeted strategies. In this review, these strategies are discussed: firstly data are described on the known effects of these compounds in animal models and at the cellular and molecular level; secondly, a working hypothesis for identifying and testing putative HDL-elevating drugs is provided; and finally, the future roles of genomics, transcriptomics and proteomics in HDL drug discovery research will be discussed.

High density lipoprotein: are elevated levels desirable and achievable?

In this review we focus on addressing two questions concerning high density lipoproteins (HDL). First, are elevated levels of HDL a desirable clinical plasma endpoint and secondly, if so, can strategies be devised that would allow the identification of agents to elevate HDL. To address the first question we briefly review the human epidemiologic and prospective data that identifies HDL as a risk factor for coronary heart disease (CHD). To introduce HDL elevating strategies, we next provide a brief review of the structural and enzymatic features of HDL followed by a discussion on the current thinking of the metabolic origin of the lipoprotein. We then turn to discussions on the key plasma and cell associated proteins involved in the synthesis, catabolism, and remodeling of HDL by analyzing data derived from human mutations, genetically engineered animal models with altered HDL metabolism and in vitro experimental systems. Lastly, we propose approaches to raise HDL that are either based on identification of small organic molecules or more unconventional approaches such as gene therapy or delivery of biologicals into plasma. This last section is based on an evaluation of the putative mechanism of actions of both old and new HDL elevating compounds. Our review concludes with an optimistic view that agents can be identified which may have promise in the treatment of human hypoalphalipoproteinemia and CHD.