Clinical applications of circulating oxidized low-density lipoprotein biomarkers in cardiovascular disease

PURPOSE OF REVIEW: The aim of this article is to review, analyze and interpret the growing body of evidence on circulating oxidized low-density lipoprotein and its relationship to diagnosis and prognosis of cardiovascular disease. RECENT FINDINGS: Previous studies focused on indirect measures of oxidative stress such as susceptibility of low-density lipoprotein to oxidation and measurement of autoantibodies to oxidized low-density lipoprotein. The generation of monoclonal antibodies recognizing distinct oxidation-specific epitopes has allowed the development of sensitive and specific assays to measure circulating oxidized low-density lipoprotein. Recent work in human populations has demonstrated that circulating oxidized low-density lipoprotein is associated with preclinical atherosclerosis, coronary and peripheral arterial atherosclerosis, acute coronary syndromes and vulnerable plaques. Several studies have also suggested that elevated levels of oxidized low-density lipoprotein are a prognostic indicator of cardiovascular outcomes. In addition, it has been shown that lipoprotein(a) is the primary carrier of oxidized phospholipids in the circulation of humans, suggesting additional mechanisms through which lipoprotein(a) may be pro-atherogenic. SUMMARY: Research on circulating oxidized low-density lipoprotein biomarkers is rapidly accelerating and providing novel insights into the pathophysiology of cardiovascular disease. Future studies will further assess the clinical utility of oxidized low-density lipoprotein biomarkers by determining their prognostic value in the diagnosis and prognosis of cardiovascular disease and will also evaluate the relative merit of specific assays by performing comparative studies.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Oxidized low-density lipoprotein-dependent platelet-derived microvesicles trigger procoagulant effects and amplify oxidative stress

The fundamental mechanisms that underlie platelet activation in atherothrombosis are still obscure. Oxidative stress is involved in central features of atherosclerosis. Platelet-derived microvesicles (PMVs) could be important mediators between oxidative stress and platelet activation. CD36 could be a receptor of PMVs, thus generating a PMV–CD36 complex. We aimed to investigate the detailed pathway by which oxidative damage contributes to platelet activation by the PMV–CD36 complex. We found that oxidized low-density lipoprotein stimulated the generation of PMVs. PMVs enhanced normal platelet activation, as assessed by the expression of integrin α_IIbβ₃, secretion of soluble P-selectin and platelet aggregation, but CD36-deficient platelets were not activated by PMVs. The function of the PMV–CD36 complex was mediated by the MKK4/JNK2 signaling axis. Meanwhile, PMVs increased the level of 8-iso-prostaglandin-F2α, a marker of oxidative stress, in a CD36- and phosphatidylserine-dependent manner. We concluded that PMVs are important mediators between oxidative stress and platelet activation. PMVs and CD36 may be effective targets for preventing platelet activation in cardiovascular diseases.     

Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator?

Sphingosine 1-phosphate (S1P) concentration in plasma and serum has been estimated to be within 200-900 nM. Among plasma and serum components, S1P is concentrated in lipoprotein fractions with a rank order of high-density lipoprotein (HDL)>low-density lipoprotein (LDL)>very low-density lipoprotein (VLDL)>lipoprotein-deficient plasma (LPDP) when expressed as the per unit amount of protein. It is well known that LDL, especially oxidized LDL, is closely correlated and HDL is inversely correlated, with the risk of cardiovascular disease, such as atherosclerosis. Evidence was presented that a part of HDL-induced actions previously reported are mediated by the lipoprotein-associated S1P. Furthermore, S1P content in LDL was markedly decreased during its oxidation. This paper will discuss whether S1P is an atherogenic mediator or an anti-atherogenic mediator.     

Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis

The interaction of C-reactive protein with low-density lipoprotein is considered to be one of the key properties that link C-reactive protein with atherosclerosis. However the data obtained to date are controversial, and hence make it difficult to conclude actual physiological or pathological impact of such interaction. The incompatible findings could be ascribed to the different structural state of C-reactive protein and/or low-density lipoprotein. We investigated in detail the interaction of various C-reactive protein isoforms with native and modified low-density lipoprotein. Our data showed "C-reactive protein" could indeed interact with each of native low-density lipoprotein, oxidized or enzymatically modified low-density lipoprotein, but that interaction occurs primarily when C-reactive protein is conformed in a modified form and not pentameric structure. Low level of modified C-reactive protein "contaminant" could confer C-reactive protein obvious low-density lipoprotein binding capacity. Interaction of modified C-reactive protein and low-density lipoprotein was mediated synergistically by both electrostatic association with ApoB and hydrophobic insertion into lipid layer. When complexed with modified C-reactive protein, macrophage binding/uptake of native and oxidized low-density lipoprotein was either increased 150% or decreased 35%, respectively. Thus the interaction of modified C-reactive protein with low-density lipoprotein may contribute to the regulation of low-density lipoprotein metabolism and foam cell formation in arterial wall. These results highlight an active role of modified C-reactive protein in atherosclerotic process.     

Death-signaling pathways in human myeloid cells by oxLDL and its cytotoxic components 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide

Oxidized low-density lipoprotein contains many potentially proatherogenic molecules, including oxysterols, which have been shown to induce apoptosis in various cell lines. The aim of this study was to investigate the pathway of apoptosis induced by oxidized low-density lipoprotein and the oxysterols, 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide, in two human monocytic cell lines. The HL-60 cells appeared to be more sensitive to oxidized low-density lipoprotein than U937 cells, whereas the isolated oxysterols were more potent inducers of apoptosis in the U937 cells. Caspase-2 inhibition decreased the number of viable cells in oxidized low-density lipoprotein-treated samples; however, it protected against cholesterol-5beta,6beta-epoxide-induced cell death. Western blot analysis was utilized to examine the effect of caspase-2 inhibition on the expression of the antiapoptotic protein Bcl-2. Pretreatment with the inhibitor protected against the decrease in Bcl-2 expression in oxidized low-density lipoprotein- and 7beta-hydroxycholesterol-treated U937 cells. In HL-60 cells, Bcl-2 was overexpressed in oxidized low-density lipoprotein-treated cells, but in the presence of the inhibitor Bcl-2 expression was returned to control levels. Depleted ATP concentrations in the cells suggest that both apoptosis and necrosis may have occurred simultaneously. Our results highlight differences in the signaling pathways induced by oxidized low-density lipoprotein, 7beta-hydroxycholesterol, and cholesterol-5beta,6beta-epoxide in U937 and HL-60 cells.     

Effects of flavonol-rich green tea cultivar (Camellia sinensis L.) on plasma oxidized LDL levels in hypercholesterolemic mice

To examine the possible benefits of tea flavonols, we compared anti-atherogenic effects between common and flavonol-rich tea cultivars. The tea infusion made from a flavonol-rich cultivar, but not a common cultivar, significantly decreased the plasma oxidized low-density lipoprotein level in mice fed a high-cholesterol diet. The result suggests that tea flavonols have the potential to protect against cardiovascular diseases.     

Formation of methionine sulfoxide-containing specific forms of oxidized high-density lipoproteins

Atherosclerosis is characterized by the accumulation of both lipoprotein-derived lipids and inflammatory cells in the affected vascular wall that results in a state of heightened oxidative stress and that is reflected by the accumulation of oxidized lipoproteins. Circulating oxidized low-density lipoprotein (oxLDL) is used as a surrogate marker for coronary artery disease, although the 'escape' of oxLDL from the vessel wall is hindered by the large size of this lipoprotein and its specific retention by the extracellular matrix. Also, the oxidation of lipoproteins in human atherosclerotic lesions is not limited to LDL. In fact, the lipids of all classes of lipoproteins are oxidized to a comparable extent. Examining the fate of lipid hydroperoxides, the primary lipid peroxidation products, in high-density lipoproteins (HDL) undergoing oxidation, revealed that they become reduced to the corresponding alcohols by specific Met residues of apolipoprotein A-I (apoA-I) and apoA-II. As a consequence, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. This review describes the characterization of specifically oxidized HDL with an emphasis on MetO formation, the structural and functional consequences of such oxidation, and the potential utility of specifically oxidized HDL as a surrogate marker of atherosclerosis.     

Toxicity of enzymically-oxidized low-density lipoprotein

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.     

Air-pollutant chemicals and oxidized lipids exhibit genome-wide synergistic effects on endothelial cells

Background  

Ambient air pollution is associated with increased cardiovascular morbidity and mortality. We have found that exposure to ambient ultrafine particulate matter, highly enriched in redox cycling organic chemicals, promotes atherosclerosis in mice. We hypothesize that these pro-oxidative chemicals could synergize with oxidized lipid components generated in low-density lipoprotein particles to enhance vascular inflammation and atherosclerosis.     

Lectin-like oxidized LDL receptor-1 as extracellular chaperone receptor: its versatile functions and human diseases

Well-known coronary risk factors such as hyperlipidemia, hypertension, smoking, and diabetes are reported to induce the oxidative stress. Under the oxidative stress, low-density lipoprotein (LDL) is oxidatively modified in the vasculature, and formed oxidized LDL induces endothelial dysfunction, expression of adhesion molecules and apoptosis of vascular smooth muscle cells. It has become evident that these cellular responses induced by oxidized LDL are mediated by lectin-like oxidized LDL receptor-1 (LOX-1). LOX-1 was originally identified from cultured aortic endothelial cells as a receptor for oxidized LDL; however, recent investigations revealed that LOX-1 has diverse roles in the host-defense system and inflammatory responses, and it is involved in the pathogenesis of various diseases such as atherosclerosis-based cardiovascular diseases and septic shock. Beside oxidized LDL, LOX-1 recognizes multiple ligands including apoptotic cells, platelets, advanced glycation end products, bacteria, and heat shock proteins (HSPs). The HSPs function as a chaperone to affect protein folding of newly synthesized or denatured proteins. There are accumulating evidences that the HSPs released into the extracellular space have potent biological activities and it may work as a kind of cytokines. It is demonstrated that LOX-1 works as a receptor for HSP70, since it has high affinity for HSP70. The interaction of LOX-1 with HSP70 is involved in the cross-presentation of antigen. Given the potent and wide variety of biological activities, more understanding their interaction provides potential therapeutic strategy for various human diseases.     

Adsorption kinetics of low-density lipoproteins with Langmuir monolayer

The present work utilizes the Langmuir monolayer technique to detect the adsorption kinetics of native low-density lipoproteins and their oxidized form with the lipid monolayer. We found that low-density lipoproteins and oxidized low-density lipoproteins are able to penetrate the LM up to pressure π?=?9.9 and 11.6 mN/m. Also, the adsorption constants of both particles were found to depend strongly on the monolayer initial pressure. It is found that less compressed lipid monolayers could accommodate more native low-density lipoproteins than the oxidized ones due their higher binding affinity toward monolayers. The probable α-helical regions along the apoproteinB-100 secondary structure and average hydrophobicity could explain partially their adsorption kinetics into lipid monolayers. This simplified ‘in vitro’ study of low-density lipoprotein–monolayer interaction may serve as a step further to understand the mechanism and bioactivity of the atherosclerotic process. Also, it may shed light on the oxidized low-density lipoprotein’s role in plaque formation in the innermost arterial wall in blood vessels.     

Effect of oxidized low density lipoproteins on the structure of platelet membrane. Use of electron paramagnetic resonance]

The effect of low-density lipoproteins on the structure of platelet plasma membrane was studied by electron paramagnetic resonance spectroscopy. Low-density lipoproteins were incubated with platelet rich plasma at a volume ratio 1:1. Plasma incubated with buffer served as a control. After incubation, the fluidity of platelet plasma membrane was determined by electron spin resonance probes 5-doxylstearate and 16-doxylstearate, which were immobilized in membranes of cells subjected to triple precipitation. Significant differences in the order parameter S, which characterizes the spectrum of the 5-doxylstearate probe, for samples incubated with the buffer and oxidized low-density lipoproteins were found. The dependence of the parameter on incubation time and the extend of oxidation of low-density lipoproteins were obtained. No significant differences in rotational correlation time of 16-doxylstearate between platelets incubated with and without oxidized low-density lipoproteins was observed within the limits of experimental error; however, the changes in the half-width of the low-field component may be considered reliable. The interaction of oxidized low-density lipoproteins with platelets leads to an increase in plasma membrane fluidity, thereby mediating the activating action on platelets.     

Small dense low-density lipoprotein and its role as an independent predictor of cardiovascular disease

PURPOSE OF REVIEW: This review discusses whether the relationship of small dense low-density lipoprotein to cardiovascular risk is direct, due to the atherogenic properties of the particle, or a reflection of concomitant abnormalities in high-density lipoprotein and plasma triglyceride. RECENT FINDINGS: Recent studies have examined whether low-density lipoprotein size distribution or concentration of small low-density lipoprotein is related more strongly to risk. It appears that the latter is a better predictor in major surveys, although in smaller cohort studies particle size shows a strong association with atherosclerosis burden. While the main causes of the formation of small dense low-density lipoprotein are relatively well understood, novel metabolic factors may also play a role, and pharmacologic interventions such as glitazones may have a direct regulatory impact. SUMMARY: Evidence links abnormalities in low-density lipoprotein structure to cardiovascular risk. The plasma concentration of small dense low-density lipoprotein is likely to be more informative than relative low-density lipoprotein particle size, and although methods are available for quantitation of this subfraction, there is considerable room for improvement. It is not yet clear how knowledge of the small dense low-density lipoprotein concentration may add to risk prediction.     

The role of oxidized phospholipids in mediating lipoprotein(a) atherogenicity

PURPOSE OF REVIEW: To review emerging data on the relationship between lipoprotein(a) and oxidized phospholipids. RECENT FINDINGS: We have recently proposed that a unique physiological role of lipoprotein(a) may be to bind and transport proinflammatory oxidized phospholipids and that this interaction may mediate a common biological influence on cardiovascular disease. In a large series of clinical studies performed to date, a very strong correlation was found between plasma levels of lipoprotein(a) and the content of oxidized phospholipids on apolipoprotein B-100 particles (OxPL/apoB), measured by monoclonal antibody E06, which binds the phosphocholine head group of oxidized phospholipids but not native phospholipids. The correlation of OxPL/apoB to lipoprotein(a) is very strong in individuals with small apolipoprotein(a) isoforms (r = approximately 0.95) and modest in individuals with large isoforms (r = approximately 0.60). In-vitro studies have demonstrated that the vast majority of oxidized phospholipids detected by E06 are bound to lipoprotein(a) in human plasma. A similarly strong association with oxidized phospholipids was also documented in transgenic mice overexpressing lipoprotein(a), even in mice not fed atherogenic diets or with overt atherosclerosis. SUMMARY: A better understanding of the ability of human lipoprotein(a) to bind oxidized phospholipids may allow clinically important insights into the role of oxidized phospholipids and lipoprotein(a) in human atherogenesis and cardiovascular disease and may provide novel diagnostic tools and therapeutic interventions aimed at measuring and treating elevated levels of OxPL/apoB and lipoprotein(a).     

Platelet agonist F11 receptor is a member of the immunoglobulin superfamily and identical with junctional adhesion molecule (JAM): regulation of expression in human endothelial cells and macrophages

The stimulatory mAb F11 binds two platelet membrane proteins of 32 and 35 kDa and causes activation of platelets when cross-linked with the FcgammaRII receptor. We used bioinformatics to identify expressed sequence tags from libraries of cytokine-stimulated human endothelial cell (EC) cDNAs. The protein sequence deduced from full-length F11 cDNA was identical to partial sequences of peptides derived from affinity-purified platelet F11 antigen. F11 mRNA is expressed in human EC, macrophages, and a variety of non-hematopoietic vascular tissues. Expression of F11 mRNA is modulated by cytokines in EC and is up-regulated by oxidized low-density lipoprotein in human macrophages. The F11 receptor contains two immunoglobulin-like domains in its 236-amino-acid-long extracellular region, and has identity to the recently described junctional adhesion molecule. The data indicate that the F11 antigen is a novel receptor or cell adhesion molecule belonging to the immunoglobulin superfamily.     

Detrimental vascular effects of lysophosphatidylcholine is limited by other phospholipid components of low-density lipoprotein

Current consensus suggests that lysophosphatidylcholine is the major detrimental factor in oxidized low-density lipoprotein that may contribute to the alterations of vasomotor responses associated with atherosclerosis. This study investigated the influences of lysophosphatidylcholine and major lipid components in oxidized low-density lipoprotein on vascular relaxation. We also determine if there was any interaction between these phospholipid components on relaxation. Porcine coronary artery rings were incubated with lysophosphatidylcholine, phosphatidylcholine or sphingomyelin. After contraction by the thromboxane A₂ mimetic U46619, rings were relaxed with bradykinin and calcium ionophore A23187. Lysophosphatidylcholine with a higher proportion of stearoyl-lysophosphatidylcholine to palmitoyl-lysophosphatidylcholine ratio caused greater reduction of relaxational responses. While phosphatidylcholine and sphingomyelin had no effect on vascular relaxation, they reduced the ability of lysophosphatidylcholine to impair vascular relaxation. Our results thus suggested that the effectiveness of oxidized low-density lipoprotein at inhibiting vasodilatory responses may be determined by the relative proportion of different types of lysophosphatidylcholine as well as the amount of other phospholipid components: phosphatidylcholine and sphingomyelin.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

C-reactive protein inhibits in vitro oxidation of low-density lipoprotein

C-reactive protein (CRP) is elevated in cardiovascular disease and binds to oxidized phosphatidylcholine (oxPtC) in the low-density lipoprotein (LDL) surface. In the present study, we tested if CRP influences the susceptibility of LDL to oxidation. At physiological concentrations of 1-7mug/ml, CRP strongly inhibited copper-mediated oxidation of LDL and phospholipid liposomes in a concentration-dependent manner. Similar concentrations of different monoclonal antibodies or albumin did not influence LDL oxidation. Antioxidant activity of CRP was inhibited by phosphocholine (PC), indicating that the observed activity involves binding of CRP to oxPtC. These results suggest that CRP may limit atherogenic oxidation of LDL in vivo.     

Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells

Elevated levels of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)] have been considered strong risk factors for atherosclerotic cardiovascular disease. Increased production of plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of thrombosis and atherosclerosis. Previous studies by our group and others demonstrated that oxidation enhances LDL- and Lp(a)-induced production of PAI-1 in human umbilical vein endothelial cells (HUVEC). The present study examined the involvement of protein kinase C (PKC) and its isoform in vascular endothelial cells (EC) induced by native or oxidized LDL and Lp(a). Treatment with Lp(a) or LDL transiently increased PKC activity at 15 min and 5.5 h after the start of lipoprotein treatment in EC. Copper-oxidized LDL and Lp(a) induced greater PKC activation in EC compared with comparable forms of those lipoproteins. Additions of 1 microM calphostin C, a PKC-specific inhibitor, at the beginning or > or =5 h, but not > or = 9 h, after the initiation of lipoprotein treatment, blocked native and oxidized LDL- or Lp(a)-induced increases in PKC activity and PAI-1 production. Treatment of LDL, Lp(a), or their oxidized forms was induced in translocation of PKC-beta1 from cytosol to membrane in HUVEC. Treatments with 60 nM 379196, a PKC-beta-specific inhibitor, effectively prevented PAI-1 production induced by LDL, Lp(a), or their oxidized forms in HUVEC and human coronary artery EC. The results suggest that activation of PKC-beta may mediate the production of PAI-1 in cultured arterial and venous EC induced by LDL, Lp(a), or their oxidized forms.