Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

Cu2(+)-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein.

We previously reported that the expression of an epitope of apolipoprotein B (apoB), mapped to the C-terminus and defined by antibody Bsol7, increased during Cu2(+)-mediated oxidation of isolated low density lipoprotein (LDL). We describe now the properties of Bsol7 as a marker of LDL oxidation in whole plasma in relation to other effects of oxidative treatment of plasma, such as the distribution of apoA-I and cholesteryl ester transfer protein (CETP). In dialyzed plasma, no LDL oxidation was detected at Cu2+ concentrations (5 microM) sufficient for extensive oxidation of isolated LDL. At a higher Cu2+ concentration (50 microM), an increased expression of the Bsol7 epitope was observed; at 250 microM Cu2+, other evidence of LDL oxidation was found. The pattern of LDL response to Cu2+ observed in dialyzed plasma could be reproduced by adding 3% bovine serum albumin to isolated LDL. We demonstrate that the effect of albumin most likely results from its ability to bind copper ions. Incubation of plasma with increasing concentrations of Cu2+ resulted first in the disappearance of alpha 2-migrating HDL, the usual carrier of CEPT; free CETP and high molecular weight apoA-I-containing particles were also generated during oxidation. Addition of oxidized, but not native, LDL to plasma resulted in a transfer to LDL of some of the CETP initially associated with apoA-I. In conclusion, the increased immunoreactivity of the Bsol7 epitope was the most sensitive parameter of LDL oxidation, but other parameters, such as the presence of alpha 2-HDL and CETP-lipoprotein associations were even more sensitive evidence of lipoprotein oxidation.     

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.     

Lipid peroxide and transition metals are required for the toxicity of oxidized low density lipoprotein to cultured endothelial cells

The toxicity of oxidized low density lipoprotein (Ox-LDL) to cultured vascular endothelial cells was investigated. The modification of low density lipoprotein (LDL) by copper led to the production of thiobarbituric acid-reacting substance (TBARS) and lipid hydroperoxide (LPO). TBARS was distributed not only in lipoprotein, but also in the aqueous phase, whereas LPO was observed only in the lipoprotein particle. During the incubation of LDL with copper, the copper bound to lipoprotein and formed a complex. The toxicity of products resulting from the oxidation of LDL to endothelial cells was recognized in Ox-LDL particles, not in the aqueous phase. Following dialysis of Ox-LDL against EDTA, copper which had bound to the Ox-LDL particle was released and the toxicity of Ox-LDL disappeared. The addition of copper to the dialyzed Ox-LDL restored the cytotoxicity. To a lesser extent this effect was also observed with the addition of iron. A study of the time-course of LDL oxidation showed that the toxicity of Ox-LDL depends upon the level of LPO, not upon the content of TBARS, the extent of negative charge or the protein adduct of aldehydes. These results demonstrate that transition metal is required for Ox-LDL toxicity and that the toxic moiety of the products resulting from LDL oxidation is LPO associated with the Ox-LDL particle.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

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.     

Comparison of the effects of O2*-/HO* free radical- and copper ions-oxidized LDL or lipoprotein(a) on the endothelial cell releases of tissue plasminogen activator and plasminogen activator inhibitor-1.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O2*-/HO* free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O2*-/HO* free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O2*-/HO* free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7beta-hydroxycholesterol, 5,6beta-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O2*-/HO* oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.     

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.     

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.     

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

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

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

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.     

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu²⁺ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.     

Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein.

Low-density lipoprotein (LDL) is oxidized by cellular and noncellular mechanisms, both leading to an increased binding to collagen. We have investigated the effect of serum on lipid peroxidation, apoprotein oxidation and the binding of oxidized apoprotein to collagen. During noncellular oxidation, lipoprotein-deficient serum strongly inhibited all three processes. The serum fraction of M(r) > 100,000 was equally inhibitory; this effect was not due to alpha 1 or gamma globulins, alpha 2 macroglobulins, haptoglobins or ceruloplasmin. The serum fraction of M(r) 30,000-100,000 stimulated the binding of oxidized apoprotein but the albumin in this fraction inhibited lipid peroxidation and apoprotein oxidation. Serum ultrafiltrate (M(r) < 1000) inhibited lipid and protein oxidation, and binding; the inhibitory effect was abolished by deionization which removed histidine. The effects of lipoprotein-deficient serum and its fractions on cellular oxidation were similar but weaker than those on noncellular oxidation, HDL inhibited noncellular oxidation as well as binding of oxidized apoprotein. VLDL also inhibited oxidation; this could not be accounted for by its content of apo B. If present in vivo, these inhibitory effects would completely suppress both cellular and noncellular oxidation of LDL and its subsequent binding to collagen.     

氧化修饰高密度脂蛋白的研究进展

血浆高密度脂蛋白(HDL)与低密度脂蛋白(LDL)一样可以在体内外发生氧化修饰,引起其理化性质发生一系列的改变,如多不饱和脂肪酸过氧化,卵磷脂水解,蛋白质发生聚合或分解等.活体内HDL可能在动脉壁巨噬细胞、内皮细胞及中性粒细胞、单核细胞的作用下发生氧化修饰.氧化修饰HDL可能通过清道夫受体途径代谢.氧化修饰HDL产生多种致动脉粥样硬化作用.维生素E、C的摄入可能有助于防止脂蛋白的氧化.     

Modulation of nonselective cation current by oxidized LDL and lysophosphatidylcholine and its inhibitory contribution to endothelial damage

AimsThis study examined the effects of oxidized low-density lipoprotein (LDL) and its major lipid constituent lysophosphatidylcholine (LPC) on nonselective cation (NSC) current and its inhibitory contribution to LPC-induced cytotoxicity in cultured human umbilical endothelial cells (HUVECs).Main methodsPatch-clamp technique and the resazurin-based cell viability assay were used.Key findingsIn voltage-clamped cells, oxidized LDL or LPC slowly activated NSC current. NSC current was also activated by loading cells with Ca²⁺ solution buffered at various concentrations using a patch pipette or by applying the sarcoplasmic reticulum Ca²⁺ pump blocker 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), the metabolic inhibitor CN^? or the hydroperoxide donor tert-butyl hydroperoxide (TBHP). On the contrary, when intracellular Ca²⁺ was strongly buffered with 12 mM BAPTA or cells were loaded with superoxide dismutase using a patch pipette, LPC or BHQ did not activate NSC current. Furthermore, NSC current activated by LPC, TBHP or CN^? was inhibited by the antioxidant tempol or extracellular Ca²⁺ depletion and NSC current activated by intracellular Ca²⁺ was further augmented by oxidized LDL or LPC. LPC or oxidized LDL released Ca²⁺ from intracellular stores and further enhanced store-operated Ca²⁺ entry. LPC-induced cytotoxicity was augmented by inhibiting Ca²⁺ influx and NO synthesis.SignificanceOxidized LDL or its main component LPC activated Ca²⁺-permeable NSC current via releasing Ca²⁺ from intracellular stores and producing ROS and thereby increased Ca²⁺ influx. Ca²⁺ influx through NSC channel might protect endothelial cells by producing NO.     

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did.We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.     

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.