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Background and Aims: Secretory phospholipase A 2 (PLA 2 ) hydrolyzes LDL phospholipids generating modified LDL particles (PLA 2 -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 PLA 2 -LDL on exocytosis of WPBs., Methods: Human coronary artery endothelial cells (HCAECs) were stimulated with PLA 2 - LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA 2 -LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice., Results: Exposure of HCAECs to PLA 2 -LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA 2 -LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA 2 -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 PLA 2 -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 PLA 2 -LDL-dependent promotion of vascular inflammation and atherosclerosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 Tyr 192 and Phe 229 , but hypoxic activation at Tyr 192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr 192 -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., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

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.

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 β-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.

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.

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.

Paraoxon-hydrolyzing activity of HDL (HDL-PON1) is known to lose its activity under oxidative stress condition. Here, we attempted to elucidate the possible causes for the oxidative inactivation of HDL-PON1 in vivo system. Of various oxidative systems, ascorbate/Cu2+ system was the most potent in inactivating the paraoxon-hydrolyzing activity of purified PON1 (PON1). The inclusion of Cu2+ (0.5-2.0 microM) remarkably enhanced the ascorbate (0.5 mM)-induced inactivation of purified PON1. A similar inactivation was also obtained with HDL-PON1, although to a less extent. The inactivation of PON1, either purified or HDL-bound, by ascorbate/Cu2+ was prevented by catalase or thiols, but not general hydroxyl radical scavengers, suggesting the involvement of Cu(2+)-catalyzed oxidation in PON1 inactivation. In addition, some lipids such as oleic acid or dioleoylphophatidylglycerol expressed a partial protection. Noteworthy, HDL-PON1, but not purified PON1, was inactivated significantly in a concentration-dependent manner by Cu2+ alone, and the inactivation of HDL-PON1 by Cu2+ was prevented by catalase, consistent with the intermediacy of H2O2 in Cu(2+)-induced inactivation of HDL-PON1. Separately, PON1, either purified or HDL-bound, was found to be susceptible to hypochlorite oxidation. While the susceptibility to hypochlorite below 1 mM was similar between purified PON1 and HDL-PON1, the inactivation of HDL-PON1 by hypochlorite at > 1 mM seemed to be interfered by the membrane. Moreover, the sequential inclusion of hypochlorite and ascorbate/Cu2+ showed a cooperative action in inactivating HDL-PONI. Based on these results, it is proposed that copper ion-catalyzed oxidation and hypochlorite oxidation may be mainly responsible for the loss of HDL-associated paraoxonasel activity.