Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2.

BACKGROUND AND AIMS: Secretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel-Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation. Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. METHODS: Human coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. RESULTS: Exposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. CONCLUSIONS: Our studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

The effect of intakes of fish and Camelina sativa oil on atherogenic and anti-atherogenic functions of LDL and HDL particles: A randomized controlled trial.

BACKGROUND AND AIMS: Omega-3 fatty acids are known to have several cardioprotective effects. Our aim was to investigate the effects of intakes of fish and Camelina sativa oil (CSO), rich in alpha-linolenic acid, on the atherogenic and anti-atherogenic functions of LDL and HDL particles. METHODS: Altogether, 88 volunteers with impaired glucose metabolism were randomly assigned to CSO (10 g of alpha-linolenic acid/day), fatty fish (4 fish meals/week), lean fish (4 fish meals/week) or control group for 12 weeks. 79 subjects completed the study. The binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determined in vitro. RESULTS: Intake of CSO decreased the binding of lipoproteins to aortic proteoglycans in a non-normalized model (p = 0.006). After normalizing with serum concentrations of non-HDL cholesterol, apolipoprotein B (apoB) or LDL cholesterol, which decreased in the CSO group, the change was no longer statistically significant. In the fish groups, there were no changes in the binding of lipoproteins to proteoglycans. Regarding other lipoprotein functions, there were no changes in any of the groups. CONCLUSIONS: Intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum LDL cholesterol concentration, which suggests that the level of apoB-containing lipoproteins in the circulation is the main driver of lipoprotein retention within the arterial wall. Intake of fish or CSO has no effects on other lipoprotein functions.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding.

BACKGROUND AND AIMS: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. METHODS: Mature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. RESULTS: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. CONCLUSIONS: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.

Chymase released from hypoxia-activated cardiac mast cells cleaves human apoA-I at Tyr192 and compromises its cardioprotective activity.

ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr192 and Phe229, but hypoxic activation at Tyr192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr192-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.

Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties.

OBJECTIVE: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. APPROACH AND RESULTS: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. CONCLUSIONS: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation.

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.

Acidification of the intimal fluid: the perfect storm for atherogenesis.

Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifies the proatherogenic and proinflammatory processes involved in atherogenesis.

Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells.

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH.

During atherogenesis, the extracellular pH of atherosclerotic lesions decreases. Here, we examined the effect of low, but physiologically plausible pH on aggregation of modified LDL, one of the key processes in atherogenesis. LDL was treated with SMase, and aggregation of the SMase-treated LDL was followed at pH 5.5-7.5. The lower the pH, the more extensive was the aggregation of identically prelipolyzed LDL particles. At pH 5.5-6.0, the aggregates were much larger (size >1 µm) than those formed at neutral pH (100-200 nm). SMase treatment was found to lead to a dramatic decrease in α-helix and concomitant increase in ß-sheet structures of apoB-100. Particle aggregation was caused by interactions between newly exposed segments of apoB-100. LDL-derived lipid microemulsions lacking apoB-100 failed to form large aggregates. SMase-induced LDL aggregation could be blocked by lowering the incubation temperature to 15°C, which also inhibited the changes in the conformation of apoB-100, by proteolytic degradation of apoB-100 after SMase-treatment, and by HDL particles. Taken together, sphingomyelin hydrolysis induces exposure of protease-sensitive sites of apoB-100, whose interactions govern subsequent particle aggregation. The supersized LDL aggregates may contribute to the retention of LDL lipids in acidic areas of atherosclerosis-susceptible sites in the arterial intima.

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1).

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu(2+) system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe(2+), Cu(2+) (0.05-1.0 microM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02-0.1 mM). Moreover, Cu(2+) alone inhibited the lactonase activity at concentrations as low as 1 microM. The ascorbate/Cu(2+)-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu(2+)-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu(2+)-catalyzed oxidation. Separately, ascorbate/Cu(2+)-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu(2+)-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu(2+) among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.

Degradation of very long chain dicarboxylic polyunsaturated fatty acids in mouse hepatocytes, a peroxisomal process.

Polyunsaturated fatty acids can be omega-oxidized to dicarboxylic polyunsaturated fatty acids (DC-PUFA), bioactive compounds which cause vasodilatation and activation of PPARalpha and gamma. DC-PUFA can be shortened by beta-oxidation, and to determine whether mitochondria and/or peroxisomes are responsible for this degradation 20-carboxy-[1-(14)C]-eicosatetraenoic acid (20-COOH-AA) was synthesized and given to hepatocytes from mouse models with peroxisomal dysfunctions. In contrast to wild type cells, hepatocytes from mice with liver-selective elimination of peroxisomes, due to Pex5p deficiency, failed to produce (14)CO(2) and labeled acid-soluble oxidation products, indicating that peroxisomes are involved in the degradation of 20-COOH-AA. Subsequently, the oxidation of 20-COOH-AA was analyzed in hepatocytes lacking multifunctional protein 1 (MFP1) or MFP2, key enzymes of the peroxisomal beta-oxidation. Degradation of 20-COOH-AA was partially impaired in MFP1, but not in MFP2 knockout hepatocytes. Taken together, peroxisomes and not mitochondria are the site of beta-oxidation of DC-PUFA, and MFP1 is involved in this process.

Rice albumin N-terminal (Asp-His-His-Gln) prevents against copper ion-catalyzed oxidations.

Chromatographic separation of soluble proteins from rice (Oryza sativa L.) yielded a major albumin protein (16 kDa), with the DHHQVYSPGEQ sequence in the N terminus, showing antioxidant action. The rice albumin was more potent than other rice proteins in preventing Cu2+-induced low-density lipoprotein (LDL) oxidation. Additionally, it also exhibited a remarkable suppression of HOCl oxidation. In a further study, albumin inhibited Cu2+-induced oxidation of LDL in a stoichiometric manner with an EC50 value of 4.3 microM, close to that of serum albumins. Moreover, after digestion with trypsin or chymotrypsin, it maintained its antioxidant action. In an experiment to see the involvement of the N terminus in antioxidant action, a synthetic tetrapeptide, equivalent to the N terminus DHHQ, was found to inhibit Cu2+-induced LDL oxidation or degradation of apolipoprotein B, similar to that of rice albumin. In mechanistic analyses, the action of rice albumin or tetrapeptide is primarily due to the removal of Cu2+, as suggested from its inhibitory effect on Cu2+/diphenylcarbohydrazide (DPCH) complex formation. However, despite its similar inhibitory effect on Cu2+-induced oxidation of LDL, rice albumin was less effective than serum albumin in inhibiting Cu2+/DPCH complex formation, suggesting that the number of Cu2+-binding sites in rice albumin may be less than that in serum albumins. Taken together, rice albumin exerts a potent preventive action against Cu2+-induced oxidations, which is due to the Cu2+ binding by DHHQ in the N-terminal sequence. Such a role as a Cu2+ chelator would add up to the application of rice albumin protein.

Differential effect of lysophospholipids on activities of human plasma paraoxonase1, either soluble or lipid-bound.

Interaction of paraoxonase1 (PON1) with lysophospholipids was examined with respect to activity regulation and binding property. Paraoxonase activity of purified PON1 was partially inhibited by palmitoyl-lysophosphatidyl-glycerol (palmitoyl-lysoPG) and lysophosphatidylinositol (lysoPI), which had a stimulatory effect on arylesterase and diazoxonase activities. The selective inhibition of paraoxonase activity by palmitoyl-lysoPG, characterized by noncompetitiveness and charge interaction, was also observed with HDL- or dimyristoylphosphatidylcholine (DMPC)-bound PON1. Meanwhile, lysophosphatidylcholine (lysoPC) stimulated all three activities of purified PON1, although it stimulated DMPC-bound or HDL-bound PON1 to a lesser extent. The stimulatory action of lysophospholipids was observed around their CMC, suggesting that micelle formation of lysophospholpids might be involved in the stimulation of PON1 activity. Presumably in support of this, the tryptophan fluorescence intensity of PON1 was increased by lysophospholipids at concentrations required for the stimulation of PON1 activity. Separately, lysoPC stimulation was less remarkable for DMPC-bound PON1 than for either dimyristoylphosphatidylserine (DMPS)- or dimyristoylphosphatidylglycerol-bound PON1, suggesting a tight association between PON1 and DMPC. In support of this, the stimulatory role of apolipoprotein A-I was less prominent for DMPC-bound PON1 than for DMPS-bound PON1. Taken together, these data suggest that the inhibition of paraoxonase activity by lysoPG or lysoPI may be due to binding to a site distinct from the active center, whereas the stimulation by lysophospholipid may be ascribed to the micelle formation around the lipid-associable region of PON1.

Apolipoprotein A-I-mimetic peptides with antioxidant actions.

To augment antioxidant action of apolipoprotein A-I (Apo A-I)-mimetic peptide, the peptide F3,6,14,18 18A (DWFKAFYDKVAEKFKEAF) was modified by incorporating antioxidant amino acid residues. Introduction of His residue at position 2 or 3 at N-terminal of the peptide remarkably enhanced antioxidant action against Cu2+ oxidation of LDL and the capability of sequestering Cu2+. Likewise, the substitution of Ala for Cys residue at position 12 increased antioxidant action against Cu2+ oxidation of LDL. Additionally, the Cys substitution contributed to enhanced capabilities in the removal of hypochlorous acid (HOCl) and 13-hydroperoxyoctadecadienoic acid. Furthermore, the combined incorporation of His and Cys residues enhanced antioxidant actions in preventing Cu2+ oxidation and reducing HOCl and hydroperoxide levels. Separately, in solubilizing phosphatidylcholine, either peptides with His residue at N-terminal position 2 or 3, or those containing Cys residue at position 11 or 12 were equipotent to peptide F3,6,14,18 18A. Further, the lipid-solubilizing ability of those containing both His and Cys residues was comparable to that of peptide F3,6,14,18 18A. In support of this, a similar structural importance was observed with Trp fluorescence study illustrating the penetration of peptides in phosphatidylcholine liposome. Besides, the modified peptides were also comparable to peptide F3,6,14,18 18A in restoring phosphatidylserine-induced loss of PON1 activity. These results indicate that the insertion of His or Cys residue into peptide F3,6,14,18 18A at appropriate positions could lead to enhanced antioxidant action with no significant change of lipid-solubilizing action.

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position.

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (> or =0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.

Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1.

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu(2+)-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu(2+), peroxyl radicals or HOCl. When Cu(2+)-binding property of PON1 was examined spectrophotometrically, the maximal Cu(2+) binding was achieved at 1:1 molar ratio of PON1: Cu(2+). Additionally, Cu(2+)-catalyzed oxidative inactivation of PON1 was prevented by Ca(2+)-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu(2+)-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu(2+)-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu(2+)-induced LDL oxidation (24 h), indicating a tight binding of Cu(2+) by peptides. In support of this, the peptide/Cu(2+) complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu(2+), 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation.

Paraoxonasel (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu(2+)-mediated inactivation of PON1 was examined. Cu(2+)-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu(2+)-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu2+, but not Fe2+. The DOPA/Cu(2+)-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu(2+)-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu2+. Separately, it was found that DOPA at low concentrations (1-6 microM) acted as a pro-oxidant by enhancing Cu(2+)-induced oxidation of HDL, while it exhibited an antioxidant action at > or = 10 microM. In addition, Cu(2+)-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu(2+)-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu2+ in the presence of DOPA (60 or 120 microM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 microM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu(2+)-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.

Preferential inhibition of paraoxonase activity of human paraoxonase 1 by negatively charged lipids.

To determine the causes responsible for a preferential decrease of paraoxonase activity, which has been observed in the serum of patients with cardiovascular diseases, the inactivation or inhibition of paraoxonase 1 (PON1) by various endogenous factors was examined using paraoxon or phenyl acetate as a substrate. When purified PON1 was incubated with various endogenous oxidants or aldehydes, they failed to cause a preferential reduction of paraoxonase activity, suggesting no participation of the inactivation mechanism in the preferential loss of paraoxonase activity. Next, when we examined the inhibition of PON1 activity by endogenous lipids, monoenoic acids such as palmitoleic acid or oleic acid inhibited paraoxonase activity preferentially, in contrast to a parallel inhibition of both activities by polyunsaturated or saturated acids. Noteworthy, oleoylglycine inhibited paraoxonase activity, but not arylesterase activity, complying with the selective inhibition of paraoxonase activity. Moreover, such a selective inhibition of paraoxonase activity was also expressed by lysophosphatidylglycerol or lysophosphatidylinositol, but not by lysophosphatidylserine or lysophosphatidylcholine, indicating the importance of the type of head group. Furthermore, such a preferential or selective inhibition of paraoxonase activity was also observed with PON1 associated with HDL or plasma. These data suggest that some negatively charged lipids may correspond to factors causing the preferential inhibition of paraoxonase activity of PON1.