Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to oxidize alpha-amino acids to a family of reactive aldehydes. Mechanistic studies identifying labile intermediates along the reaction pathway

We have recently demonstrated that neutrophils oxidize nearly all of the amino acids commonly found in plasma to a corresponding family of aldehydes in high yield. The reaction is mediated by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H2O2-Cl- system of phagocytes. We now present evidence for the underlying mechanism of this reaction, including the structural requirements and reaction intermediates formed. Utilizing mass spectrometry and isotopically labeled amino acids, we rule out hydrogen atom abstraction from the alpha-carbon as the initial event in aldehyde formation during amino acid oxidation, a pathway known to occur with ionizing radiation. Aldehyde generation from amino acids required the presence of an alpha-amino moiety; beta- and epsilon-amino acids did not form aldehydes upon oxidation by either the myeloperoxidase system or HOCl, generating stable monochloramines instead. UV difference spectroscopy, high pressure liquid chromatography, and multinuclear (1H,15N) NMR spectroscopy established that the conversion of alpha-amino acids into aldehydes begins with generation of an unstable alpha-monochloramine, which subsequently decomposes to yield an aldehyde. Precursor product relationships between alpha-amino acid and alpha-monochloramine, and alpha-monochloramine and aldehyde were confirmed by high pressure liquid chromatography purification of the reaction intermediate and subsequent 1H and 15N NMR spectroscopy. Collectively, these results detail the chemical mechanism and reaction intermediates generated during conversion of amino acids into aldehydes by myeloperoxidase-generated HOCl.     

Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase

Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on L-tyrosine in vitro, it forms o, o'-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free tyrosyl radical (a2,6H = 6.3 G, a3,5H = 1.6 G, and abetaH = 15. 0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN = 15.6 G, a13C (2) = 8.0 G, a13C (1) = 7.1 G, a13C (1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring of L-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and L-tyrosine residues to the myeloperoxidase-H2O2-L-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert L-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of L-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.     

Botulinum toxin injections for voiding dysfunction following SCI

We used immunohistochemistry to localize canine Apolipoprotein B-100 (CApoB-100) and glutathione peroxidase (GSH-PO) in canine atherosclerotic lesions. CApoB-100 was deposited in the tunica intima and cytoplasms of infiltrating macrophages in early atherosclerotic lesions. In advanced atherosclerotic lesions, the cystic space of the lesions contained a large amount of CApoB-100 immunoreactive material. Expression of GSH-PO was recognized in the foamy cytoplasm of macrophages and smooth muscle cells in the early and advanced atherosclerotic lesions. These results indicate that expression of GSH-PO is closely associated with the deposition of CApoB-100. In addition, they suggest that, as in human atheromas, low-density lipoprotein (LDL) is peroxidized and changed into modified LDL. Deposition of modified LDL (oxidized or acetylated) may be a critical step in the formation of canine atherosclerotic lesions.     

Carotid intima-media thickening and in vivo LDL oxidation in patients with essential hypertension

Low density lipoproteins (LDL) from hypertensive patients are more prone to in vitro oxidation and undergo a more pronounced oxidation in vivo. Due to the pro-atherogenic activity of oxidatively modified LDL, the correlation between the carotid intima-media thickening (IMT) and the markers of in vivo LDL oxidation was investigated in hypertensive patients. A cross-sectional study on 101 normocholesterolemic patients with newly diagnosed and untreated essential hypertension was performed. The occurrence of in vivo LDL oxidation was evaluated by measuring the titers of autoantibodies against Cu(2+)-oxidised LDL (oxLDL) and malondialdehyde-derivatised LDL (MDA-LDL). The extent and degree of atherosclerosis and the IMT were measured by means of carotid and femoral ultrasonography with a duplex scanner equipped with a high resolution probe. We did not find significant correlations between in vivo LDL oxidation parameters and the extent of atherosclerotic lesion in the entire group of hypertensive patients. However, a significant direct correlation was detected between the carotid IMT and the titer of autoantibodies against both oxLDL and MDA-LDL in hypertensive patients without advanced atherosclerotic plaques. The results obtained support the hypothesis that enhanced LDL oxidation may be one of the pathophysiological events related to the formation and progression of early atherosclerotic lesions (IMT) in carotid arteries of hypertensive patients.     

Cellular thiol production and oxidation of low-density lipoprotein

Compelling evidence suggests that low-density lipoprotein (LDL) is oxidized by cells within the arterial intima and that, once oxidized, it is profoundly atherogenic. The precise mechanism(s) by which cells promote the oxidation of LDL in vivo are not known; in vitro, however, oxidation of LDL can be enhanced by a number of differing mechanisms, including reaction with free and protein-bound metal ions, thiols, reactive oxygen species, lipoxygenase, myeloperoxidase and peroxynitrite. This review is concerned with the mechanisms by which cells enhance the oxidation of LDL in the presence of transition metals; in particular, the regulation, pro- and anti-oxidant consequences, and mechanism of action of cellular thiol production are examined, and contrasted with thiol-independent oxidation of LDL in the presence of transition metals.     

In vivo transfer of lipoprotein(a) into human atherosclerotic carotid arterial intima

The aim of this study was to compare the atherogenic potential of lipoprotein(a) [Lp(a)] and LDL by measuring the intimal clearance of these two plasma lipoproteins in the atherosclerotic intima of the human carotid artery in vivo. Autologous 131I-Lp(a) and 125I-LDL were mixed and reinjected intravenously 3 hours before elective surgical removal of the arterial intima in four patients. The intimal clearance of Lp(a) and LDL was 229+/-48 and 405+/-127 nL/cm2 per hour, respectively (paired t test; P=.12). The mass accumulation of Lp(a) (114+/-32 ng/cm2 per hour) was on average one 15th that of LDL (paired t test; P=.06), mainly reflecting a low plasma concentration of Lp(a) compared with LDL in the human subjects studied. In accordance with our previous observation in rabbits, there was a positive association between the intimal clearance of LDL and that of Lp(a) (r=.97, P=.03). Accordingly, high plasma levels of Lp(a) may share with LDL the potential for causing lipid accumulation in the arterial intima in humans.     

Local application of LDL promotes intimal thickening in the collared carotid artery of the rabbit

Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 micrograms h-1, n = 16 arteries), oxLDL (Cu2+ oxidized, 7 micrograms h-1, n = 16), or phosphate-buffered saline (5 microL h-1, n = 16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of alpha-SMC actin-immunoreactive cells in the intima, thereby increasing the intimal thickness from 5 +/- 1 to 26 +/- 5 microns. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5 +/- 2% in the discrete collar-induced intima to approximately 20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.     

Antiatherogenic effects of the antioxidant BO-653 in three different animal models

Antioxidants have been proposed to have antiatherogenic potential by their inhibition of low density lipoprotein (LDL) oxidation. Here, we report an antioxidant, BO-653 (2,3-dihydro-5-hydroxy-2, 2-dipentyl-4,6-di-tert-butylbenzofuran), designed to exhibit antioxidative potency comparable to that of alpha-tocopherol, but yet possess a high degree of lipophilicity comparable to that of probucol. BO-653 exhibits a high affinity for LDL and is well distributed in aortic vessels in vivo. In atherosclerosis models of rabbits and mice, BO-653 has been shown to be able to suppress the formation of atherosclerotic lesions without untoward side effects. Specifically, there was no reduction of high density lipoprotein levels. This antioxidant provides additional evidence in support of the oxidized-LDL hypothesis, and itself is a promising candidate antioxidant for clinical use.     

Metabolism of native and naturally occurring multiple modified low density lipoprotein in smooth muscle cells of human aortic intima

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.     

C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries

There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation.     

Effect of antioxidant in endothelial cells exposed to oxidized low-density lipoproteins

Antioxidants such as probucol and alpha-tocopherol have been shown to attenuate the oxidation of low-density lipoproteins (LDL) and atherosclerotic lesions in animal models of atherosclerosis. The purpose of this study is to determine the protection effect of antioxidants on endothelial cells when exposed to oxidized and native LDL. In a cell-free system, we found that probucol, alpha-tocopherol, and ascorbic acid inhibited copper-induced LDL oxidation by a dose-dependent fashion (from 1 microM to 10 mM). In porcine aortic endothelial cells, antioxidants alone did not change basal endothelin-1 (ET-1) secretion. When porcine aortic endothelial cells were exposed to LDL and oxidized-LDL, both of them stimulated ET-1 secretion dose-dependently, whereas oxidized-LDL elicited higher ET-1 secretion. However, probucol, alpha-tocopherol, and ascorbic acid did not prevent LDL or oxidized-LDL induced ET-1 secretion. Furthermore, nimodipine inhibited both of native and oxidized LDL induced ET-1 secretion. Since Ca2+ channel blocker reduced the elevation of induced ET-1 secretion, the [Ca2+]i is possibly involved for the regulation of ET-1 secretion. Our results suggest that antioxidants can only prevent the oxidation of LDL rather than oxidized and native LDL-induced ET-1 secretion in vascular endothelial cells. The increase in the [Ca2+]i of endothelial cells through the opening of voltage-dependent Ca2+ channels may be involved in the LDL-induced ET-1 release.     

A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells

Addition of leumedin, N-[9H-(2,7-dimethylfluorenyl-9-methoxy) carbon]-L-leucine at 30-60 microM together with LDL almost completely prevented the induction of monocyte chemotactic protein mRNA, reduced monocyte chemotactic protein 1 levels by 84%, and inhibited monocyte migration into the subendothelial space of cocultures of human aortic wall cells by < or = 98%. LDL incubated with leumedin formed a stable complex that remained intact even after refloating in an ultracentrifuge. Leumedin at 50 microM did not change conjugated diene formation during coculture modification of LDL or Cu++ catalyzed oxidation of LDL. Unlike LDL from control rabbits, LDL isolated from rabbits that were injected with 20 mg/kg leumedin was remarkably resistant to modification by the coculture and did not induce monocyte migration to a significant degree. Moreover, HDL isolated from rabbits injected with leumedin was far more effective in protecting against LDL modification by the artery wall cocultures than HDL from control rabbits. We conclude that leumedins can associate with lipoproteins in vivo, rendering LDL resistant to biological modification and markedly amplifying the protective capacity of HDL against in vitro LDL oxidation by artery wall cells.     

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a)

A systematic immunohistochemical study of different stages of atherosclerosis in human aortas was performed using several antibodies. Because oxidation of lipoproteins could be a key event in atherogenesis, an antibody against apolipoprotein B (apoB) from low-density lipoprotein (LDL) modified with the lipid peroxidation-specific aldehyde, 4-hydroxynonenal (4-HNE) (anti-4-HNE-apoB), was raised in rabbits. This antibody recognizing 4-HNE protein adducts was used in concert with an antibody to apo(a) from lipoprotein(a), considered also potentially atherogenic, as well as with an antibody and a monoclonal antibody (mAb) to apoB. Autopsy material from 12 corpses was investigated. The immunohistochemical investigation by the alkaline-phosphatase technique included control specimens regarding postmortem artifacts by autolysis and oxidation. The results from six specimens from five corpses are presented. A positive staining with the antibody to apoB but not with anti-4-HNE-apoB was seen in the normal intima. The thickened intima of early, transitional, and advanced atherosclerotic lesions and atheromata showed a predominantly extracellular staining with all antibodies and the applied mAb. To test the specificity of the staining, antibodies preadsorbed by the appropriate antigens and nonimmune sera were used, giving negative results. These findings indicated a colocalization of epitopes derived from lipid peroxidation of polyunsaturated fatty acids and epitopes specific for apoB and apo(a) during atherogenesis in humans.     

Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.     

Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions

To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.     

Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells

Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O2.-) and oxidize LDL in vitro; however, the role of O2.- in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 microg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5+/-1.1 versus 6.3+/-0.2 [mean+/-SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 microg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2.- contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.     

Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance

The oxidation of low density lipoproteins (LDLs) is thought to take place in the arterial intima when the particles have become isolated from circulating water-soluble antioxidants. We hypothesized that isoflavonoid antioxidants derived from soy could be incorporated into lipoproteins and possibly could protect them against oxidation, which is regarded as atherogenic. Six healthy volunteers received 3 soy bars [containing the isoflavonoid antioxidants genistein (12 mg) and daidzein (7 mg)] daily for 2 weeks. LDLs were isolated from blood drawn at the the end of a 2-week dietary baseline period, after 2 weeks on soy, and after discontinuation of soy. Large increases in plasma isoflavonoid levels occurred during soy feeding, but only minute amounts were stably associated with lipoproteins (less than 1% of plasma isoflavonoids in the LDL fraction). The LDLs were subjected to copper-mediated oxidation in vitro. Compared with off soy values, lag phases of LDL oxidation curves were prolonged by a mean of 20 min (P < 0.02) during soy intake, indicating a reduced susceptibility to oxidation. The results suggest that intake of soy-derived antioxidants, such as genistein and daidzein, may provide protection against oxidative modification of LDL. As only very small amounts of these substances were detected in purified LDL, modified LDL particles may have been produced in vivo by circulating isoflavonoids promoting resistance to oxidation ex vivo.