Myeloperoxidase-mediated LDL oxidation and endothelial cell toxicity of oxidized LDL: attenuation by (−)-epicatechin

Recent data suggest an inverse epidemiological association between intake of flavanol-rich cocoa products and cardiac mortality. Potential beneficial effect of cocoa may be attributed to flavanol-mediated improvement of endothelial function, as well as to enhancement of bioavailability and bioactivity of nitric oxide in vivo. ( ? )-Epicatechin is one bioactive flavanol found in cocoa. This review deals with protective actions of ( ? )-epicatechin on two key processes in atherogenesis, oxidation of LDL and damage to endothelial cell by oxidized LDL (oxLDL), with emphasis on data from this laboratory. ( ? )-Epicatechin not only abrogates or attenuates LDL oxidation but also counteracts deleterious actions of oxLDL on vascular endothelial cells. These protective actions are only partially shared by other vasoprotective agents such as vitamins C and E or aspirin. Thus, ( ? )-epicatechin appears to be a pleiotropic protectant for both LDL and endothelial cells.     

Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase

Intake of flavanol-rich food or beverages was previously shown to ameliorate endothelial function and to enhance bioactivity of nitric oxide with individuals at risk for cardiovascular disease. Here, we examined whether the major dietary flavanol, (-)-epicatechin, counteracts the action of oxidized LDL on endothelial cells, an action considered pivotal for endothelial dysfunction in the pathogenesis of atherosclerosis. Oxidation by myeloperoxidase plus nitrite rendered human LDL cytotoxic towards endothelial cells, more so than oxidation by Cu2+. Oxidized LDL also caused a marked loss of endothelial NO synthase protein which did not occur in the presence of a proteasome inhibitor, lactacystin. Both actions of oxidized LDL, which were not evoked by native LDL, were effectively counteracted by (-)-epicatechin. We conclude that dietary flavanols contribute to protection of the integrity of endothelial cells not only by scavenging free radicals but also by maintaining endothelial NO synthase.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation

Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis.     

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.     

Human urine: epicatechin metabolites and antioxidant activity after cocoa beverage intake

Associations between cocoa consumption in humans, excreted metabolites and total antioxidant capacity (TAC) have been scarcely investigated. The aims of the study were to investigate the epicatechin (( - )-Ec) metabolites excreted in urine samples after an intake of 40 g of cocoa powder along with the TAC of these urine samples and the relation between both the analyses. Each of the 21 volunteers received two interventions, one with a polyphenol-rich food (PRF) and one with a polyphenol-free food (PFF) in a randomized cross-over study. Urine samples were taken before and during 24 h at 0-6, 6-12 and 12-24 h periods after test intake. The excreted ( - )-Ec metabolites and the TAC were determined in urine samples by LC-MS/MS and TEAC assay, respectively. The maximum excretion of ( - )-Ec metabolites and the maximum TAC value were observed in urine samples excreted between 6 and 12 h after PRF consumption. Significance of TAC increase was found in urine samples excreted during 0-6 and 6-12 h (66.6 and 72.67%, respectively, with respect to the 0 h).     

Autoantibodies against oxidized LDL are associated with severe chest pain attacks in patients with coronary heart disease

Autoantibodies against oxidized low-density lipoprotein (oxLDL) predict the progression of atherosclerosis. Several studies have shown that oxLDL is present in atherosclerotic lesions and that several factors present in active atherosclerotic plaques can oxidatively modify LDL. Oxidation of LDL induces production of autoantibodies against oxLDL (oxLDLab) that can be measured using an EIA test. Our aim was to see whether oxLDLab are associated with severe chest pain attacks in coronary heart disease (CHD) patients. Patients having two- or three-vessel CHD, as assessed by coronary angiography, and their siblings were recruited into the study (n = 568, mean age 55.8 years, range 29.3–83.2 years). Nondiabetic patients having a history of severe chest pain attacks had significantly higher oxLDLab levels (0.611 ± 0.56) than those who did not have a history of severe chest pain attacks (0.487 ± 0.40) (p = 0.027), even though age, cholesterol level, body mass index, and blood pressure were similar in both groups. However, no difference was found in oxLDLab levels in diabetic patients with or without a history of severe chest pain attacks. Increased levels of oxLDL autoantibodies are associated with severe chest pain attacks in nondiabetic patients with CHD.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Myeloperoxidase-induced lipid peroxidation of LDL in the presence of nitrite. Protection by cocoa flavanols

Lipid peroxidation (LPO) of low-density lipoprotein (LDL) is believed to be a pivotal process rendering this plasma lipoprotein atherogenic. Several endogenous factors have been proposed to mediate LPO of LDL, among them myeloperoxidase (MPO), which is active in atherosclerotic lesions, and the plasma level of which has been proposed to be a prognostic parameter for cardiac events. Nitrite, a major oxidation product of nitric oxide, is substrate of MPO and a cofactor of MPO-mediated LPO under physiological conditions. Dietary flavonoids including (-)-epicatechin, a major flavan-3-ol in cocoa products, grapes and wine, are substrates of MPO as well as potent inhibitors of LPO in LDL at micromolar concentrations. Moreover, they strongly suppress protein tyrosine nitration of LDL by MPO/nitrite or peroxynitrite. By blunting undesirable MPO-mediated actions of nitrite, presumably via scavenging of the strong prooxidant and nitrating *NO2 radical, dietary flavonoids modulate NO metabolism in a favorable direction and thus counteract endothelial dysfunction. This article gives a survey on recent progress in this field with special reference to own recently published work.     

The low-density lipoprotein (LDL)-associated PAF-acetylhydrolase activity and the extent of LDL oxidation are important determinants of the autoantibody titers against oxidized LDL in patients with coronary artery disease

The oxidation of low-density lipoprotein (LDL) induces immunogenic epitopes, many of which are due to oxidatively modified phospholipids (oxPL). Lysophosphatidylcholine (lyso-PC) which is generated during LDL oxidation through the hydrolysis of oxPL by LDL-associated PAF-acetylhydrolase (PAF-AH) is also immunogenic. We investigated whether the LDL-associated PAF-AH and the extent of LDL oxidation influence the autoantibody titers against oxidized LDL (oxLDL) in patients with stable angina as well as in apparently healthy volunteers. Three types of copper-oxidized LDL, were prepared at the end of the lag, propagation or decomposition phase (oxLDL(L), oxLDL(P) and oxLDL(D), respectively). Similar types of oxidized LDL were prepared after previous inactivation of endogenous PAF-AH [oxLDL(-)]. All these types of oxLDL as well as malondialdehyde-modified LDL (MDA-LDL) were used as antigens. Antibody titers against the above antigens were measured with an ELISA method in the serum of 65 patients with stable angina and 47 apparently healthy volunteers. Both groups exhibited higher autoantibody titers against each type of oxLDL(-) compared to the respective type of oxLDL (P<0.00001). In both groups autoantibody titers were higher when the oxLDL(P) and oxLDL(D) or oxLDL(-)(P) and oxLDL(-)(D) were used as antigens compared to oxLDL(L) (P<0.04) or to oxLDL(-)(L), respectively (P<0.0001 for all comparisons). Patients had significantly higher titers against all types of oxLDL (enriched in lyso-PC) and oxLDL(-) (enriched in intact oxPL) compared to controls. Autoantibody titers against MDA-LDL did not differ between patients and controls. Multivariate logistic regression analysis showed that among the autoantibody titers measured only those towards oxLDL(P) are associated with a significantly higher risk for coronary artery disease. LDL-associated PAF-AH activity may play an important role in decreasing the overall immunogenicity of oxLDL, whereas the extent of LDL oxidation seems to modulate the epitopes formed on oxLDL. Lyso-PC, a major component of oxLDL(P), could be mainly responsible for the elevated autoantibody titers against oxLDL in patients with stable angina.     

Flavanols: digestion,absorption and bioactivity

Flavanols, or flavan-3-ols, are a family of bioactive compounds present in cocoa, red wine, green tea, red grapes, berries and apples. With a basic monomer unit of (−)-epicatechin or (+)-catechin, flavanols can be present in foods and beverages as monomers or oligomers (procyanidins). Most, but not all, procyanidins are degraded into monomer or dimer units prior to absorption. The bioavailability of flavanols can be influenced by multiple factors, including food processing, cooking, digestion, and biotransformation. Flavanols are potent antioxidants, scavenging free radicals in vitro and in vivo. While some of the actions of flavanols can be linked to antioxidant activities, other modes of action may also occur, including modulation of intracellular signaling, effects on membrane fluidity and regulation of cytokine release or action. Physiologically, flavanol-rich foods and beverages can affect platelet aggregation, vascular inflammation, endothelial nitric oxide metabolism, and may confer protective effects against neurodegeneration. Epidemiological data suggests that intake of cocoa, a rich source of flavanols, is inversely associated with 15-year cardiovascular and all-cause mortality in older males. (−)-Epicatechin and its metabolite, epicatechin-7-O-glucuronide, have been identified as independent predictors of some of the vascular effects associated with the consumption of a flavanol-rich beverage. Targeted dietary components and nutrition supplements that can influence the vascular system will be of great value in the prevention and treatment of chronic disease.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Proteomic approach to study the effects of various oxidatively modified low-density lipoprotein on regulation of protein expression in human umbilical vein endothelial cell

Circulating low-density lipoprotein (LDL) isolated by our laboratory, a new form of modified LDL and designated as L5, has been reported to be cytotoxic by inducing apoptosis of vascular endothelial cells in vitro. The objective of this study was to compare the biological functions of three different forms of oxidatively modified LDL on human umbilical vein endothelial cells (HUVEC) by proteomic approaches. HUVEC were incubated with serum-free medium, native LDL (N-LDL), L5 isolated from familial hypercholesterolemic subjects (FH-L5), copper-oxidized LDL (Cu-ox-LDL), and atheroma-derived LDL (a-LDL) at 37 °C for 24 h. We found that HUVEC incubated with FH-L5 expressed approximately 3 fold higher concentration of MCP-1 than did cells subject to other treatments. All modified LDL significantly suppressed ATP synthase, Grp58, Grp78, and Prdx3. However, the expression of hnRNP C1/C2 was significantly enhanced by FH-L5 and a-LDL; glutathione transferase was significantly enhanced only by FH-L5. A concordant pattern of protein expression was observed between immunoblotting and 2D electrophoresis. Different forms of oxidatively modified LDL regulated HUVEC protein expression in different patterns, suggesting different roles for different oxLDL forms in inducing atherogenesis.     

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.03 to 0.37 +/- 0.05 and from 0.15 +/- 0.01 to 0.31 +/- 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 13.8 +/- 3 and from 10.7 +/- 2.1 to 16.3 +/- 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin

The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

LDL protein nitration: implication for LDL protein unfolding

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL⁻) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL⁻ assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC).The plasma LDL⁻ fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL⁻ revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the α-helical structures and β₂ sheet as well as cysteine oxidation to cysteic acid in β₁ sheet. Circular dichroism analyses showed that the α-helical content of LDL⁻ was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL⁻ showed greater content of β-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO⁻) or SIN-1: similar amino acid modifications as well as conformational changes (loss of α-helical structure and gain in β-sheet structure) were observed. Both LDL⁻ and ONOO⁻-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO⁻-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R.It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.