Conjugated dienes: a critical trait of lipoprotein oxidizability in renal fibrosis.

BACKGROUND: We assessed whether a differential oxidizability of apolipoprotein B (apo B)-containing lipoproteins (LDL and VLDL) may explain the oxidative stress that we had observed at the onset of renal fibrosis in Zucker obese (ZO) rats (Nephrol Dial Transplant 2000, 15: 467--476). METHODS: Ex vivo copper-induced oxidation of lipoproteins was performed in 1-, 3-, and 9-month-old ZO and age-matched lean (ZL) rats. LDL/VLDL oxidizability was determined by spectrophotometry at 234 nm by monitoring the formation of conjugated diene hydroperoxides. RESULTS: A significant increase in lag time (reflecting the resistance to oxidation) was observed in ZO rats at 3 months while the maximal diene production (reflecting the amount of hydroperoxides formed during oxidation) was higher in ZO than in ZL rats as early as 1 month. Lipoproteins were larger in ZO than in ZL rats, as shown by their core to surface component ratio. Furthermore, ZO lipoproteins had increased vitamin E and polyunsaturated fatty acid (PUFA) content, with no change in vitamin E/PUFA ratio. CONCLUSIONS: Rather than oxidizability of apo B-containing lipoproteins, the ability of these molecules to produce high levels of conjugated dienes, which can act as toxic tissue messengers, appears to be a critical trait in the development of renal fibrosis in this rat model of obesity and renal fibrosis.

Inflammation is probably not a prerequisite for renal interstitial fibrosis in normoglycemic obese rats.

We examined the role of inflammation in the development of renal interstitial fibrosis in Zucker obese rats, which rapidly present kidney lesions in the absence of hypertension and hyperglycemia. Type I and III collagens were quantified using a polarized light and computer-assisted image analyzer. The expression of mRNA encoding matrix components, adhesion molecules, chemokines, and growth factors was followed by RT-PCR. The presence of synthesized proteins as well as lymphocytes and macrophages was determined by immunohistochemistry. Interstitial fibrosis developed in two phases. The first phase occurred as early as 3 mo and resulted from a neosynthesis of type III collagen and fibronectin and a reduction of extracellular matrix catabolism, in parallel with an overexpression of transforming growth factor-beta(1) and in the absence of any lymphocyte or macrophage infiltration. After 6 mo, interstitial fibrosis worsened with a large accumulation of type I collagen, concomitantly with a large macrophage infiltration. Thus inflammation cannot explain the onset of interstitial fibrosis that developed in young, insulinoresistant, normoglycemic, obese Zucker rats but aggravated this process afterward.

Oxidative stress occurs in absence of hyperglycaemia and inflammation in the onset of kidney lesions in normotensive obese rats.

BACKGROUND: Several factors favour the development of kidney lesions. We examined the role of oxidative stress in the onset of renal alterations that occur in Zucker obese (ZO) fa/fa rats. METHODS: Kidney structure, biological data, glycation parameters, advanced glycation end products (AGE), thiobarbituric acid-reactive substances (TBARS), circulating antibodies anti-malondialdehyde (MDA)-modified low-density lipoprotein (LDL), antioxidant defenses (Cu/Zn and Mn superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) activities, glutathione level), were determined in plasma and/or kidney of young and old ZO rats and lean (ZL) Fa/fa littermates. RESULTS: Renal lesions and functional decline appeared at 3 months in hyperlipidaemic, hyperinsulinaemic, normotensive ZO rats, independently of any macrophage-ED(1)(+)-cell infiltration. At 6 months and thereafter, kidney lesions and functional impairment worsened while numerous ED(1)(+)-cells invaded the interstitium. At 3 and 9 months, TBARS level in the LDL/very low-density lipoprotein fraction and in the kidney was higher in ZO than in ZL rats. Anti-MDA-LDL antibodies were increased in ZO rats. At 3 months, renal activity of Cu/Zn SOD was higher, and activities of catalase and GPx lower in ZO than in ZL rats, leading to an accumulation of hydrogen peroxide (H(2)O(2)). At 9 months, a decrease in Cu/Zn SOD activity and an increase in glutathione level were observed. Blood glucose and glycated proteins, as well as AGE in kidney, remained similar in both ZL and ZO rats, whatever their age. CONCLUSION: These data suggest that oxidative stress triggers, at an early age, the onset of kidney lesions and functional impairment in ZO rats, in absence of hyperglycaemia, hypertension and inflammation.

Charge heterogeneity of LDL in asymptomatic hypercholesterolemic men is related to lipid parameters and variations in the ApoB and CIII genes.

This study was carried out to examine the relationship between the charge on low density lipoproteins (LDLs) and lipid and clinical parameters in 104 asymptomatic dyslipidemic men and to identify biochemical and genetic factors that could contribute to the charge variability of LDL. LDL charge heterogeneity was evaluated by relative electrophoretic mobility (REM) on preformed 0.5% agarose gels and by chromatographic quantification of a minor electronegative LDL subfraction designated LDL(-). The mean REM value for LDL was 0.147+/-0.016 and the mean LDL(-) subfraction percentage was 5.6+/-2.8%. Both were positively correlated with common atherosclerotic risk factors, especially total cholesterol [for REM, r=0.27, P<0.005; for LDL(-), r=0.28, P=0.008] and LDL cholesterol [for REM, r=0.27, P=0.007; for LDL(-), r=0.26, P=0.01)] levels, and REM was positively correlated with triglycerides (r=0.27, P<0.005) and negatively with apoAI levels (r=-0.30, P<0.002). The variations in LDL charge were not due to oxidation, as measured by the lag phase and binding to the LDL receptor. The results of the 2 methods used to measure LDL charge were significantly correlated and had some identical characteristics (eg, association with LDL apoCIII content and plasma triglyceride levels in borderline and IIb dyslipidemic subjects); these methods reflect different specific features of LDL charge. The percentage of LDL(-) was correlated positively with the LDL sialic acid content (P<0.0001), whereas the REM was related to at least 2 distinct chromosomal loci. Multiple logistic analysis showed that individuals carrying minor alleles of BsrDI (P<0.05), apoCIII/SacI (P<0.01), as well as the frequent allele of XbaI (P<0.05) at the apoB and CIII gene loci had high REMs. This result suggests that LDL charge heterogeneity, which is positively correlated with the atherogenic lipid profile, is influenced by both genetic and biochemical factors.

The effect of high density lipoprotein subfractions on endothelial eicosanoid secretion.

Epidemiological, clinical, and experimental studies have demonstrated that high density lipoproteins (HDL) are protective against atherosclerosis. However, the respective influence of two main HDL subfractions (HDL2 and HDL3) on atherosclerosis process is not yet clear. The present study was designed to determine, which HDL subfraction was antiatherogenic in terms of eicosanoid release by human umbilical vein endothelial cells (HUVEC). Endothelial cells were incubated for 4 hours with HDL2 or HDL3 and prostaglandins 6-keto-PGF1alpha, thromboxane B2 and prostaglandin E2 were measured by RIA in culture supernatant. HDL2 has a dose dependent stimulatory effect on 6-keto-PGF1alpha release without stimulatory effect on thromboxane B2 secretion. The 6-keto-PGF1alpha/thromboxane B2 ratio increased progressively from 1.65 to 4.65 for 0.39 to 6.25 mg HDL protein/ml. The pattern of prostanoid secretion under influence of HDL3 showed a predominant response in 6-keto-PGF1alpha and TxB2 release. As regards PGE2, both HDL subfractions stimulated considerably secretion of this prostanoid in a dose dependent manner. In terms of PGI2/TxA2 balance the better antiatherogenic effect was observed with HDL2 subfraction.

Sialic acid content of LDL in coronary artery disease: no evidence of desialylation in subjects with coronary stenosis and increased levels in subjects with extensive atherosclerosis and acute myocardial infarction: relation between desialylation and in vitro peroxidation.

We recently showed that sialic acid content of LDL was not a marker of early cardiovascular disease (Arterioscler Thromb Vasc Biol. 1995;15:334-339). Here, we investigated this parameter in patients with advanced coronary artery disease (CAD). We first examined 100 patients having undergone coronary angiography. The distribution of LDL sialic acid values was very similar in subjects with no coronary stenosis (31.3+/-3.7 nmol/mg LDL protein, mean+/-SD) and those with > or = 75% stenosis in at least one main coronary artery or > or = 50% stenosis in at least two main coronary arteries (32.1+/-5.5 nmol/mg LDL protein). In contrast, LDL sialic acid content was significantly increased in patients with both coronary stenosis and peripheral arterial atherosclerotic lesions compared with those with either no lesion or only one or the other type of lesion. We then examined LDL sialic acid content in 20 patients with acute myocardial infarction. LDL sialic acid content was significantly higher (35.9+/-3.2 nmol/mg LDL protein) than that in the CAD(-) control group. These data suggest that LDL sialic acid content increases with the extension of atherosclerosis and its progression to acute complications. To explain the discordance with Orekhov and coworkers (Atherosclerosis. 1991;86:153-161), who showed that LDL sialic acid content in patients with advanced CAD was lower than that in healthy subjects, we studied the time courses of sialic acid, TBARS, and vitamin E levels in LDL dialyzed in different experimental conditions. A continuous decrease in both sialic acid and vitamin E levels and an increase in TBARS levels were observed in LDL samples containing less than 1 mmol/L EDTA, the intensity and rapidity of which varied with the EDTA concentration in the buffer. Our data support the idea that desialylation may result from in vitro peroxidation of LDL.

[Atherogenic and anti-atherogenic plasma lipoproteins modulate secretion of prostanoids by endothelial cells in vitro]. / Aterogénne a antiaterogénne lipoproteíny plazmy modulujú sekréciu prostanoidov endotelovou bunkou in vitro.

The authors present the evidence of atherogenic properties of VLDL and LDL potentiation on the model of endothelial cells-human umbilical vein endothelial cells, by preferable stimulation of the endothelial cell to thromboxane A1 production at in vitro conditions by atherogenic lipoproteins. The vasoconstrictive, thrombogenic and atherogenic effects of TXA2 are exerted on the vessel in this way. The ratio prostacycline/thromboxane, decisive for the maintenance of vascular homeostasis, is less than 1, this means the beneficial effect of prostacycline can not be applied. Protective, antiatherogenic effect of HDL and its subfractions HDL2 and HDL3/predominantly through their function in the reverse cholesterol transport from the periphery to the liver, antioxidative influence on LDL, as far as antiaggregation and fibrinolytic effects of HDL/is multiplied by the fact that HDL preferably stimulates the secretion of prostacycline by the endothelial cell. The ratio prostacycline/thromboxane A2 is higher than 1, that means beneficial vasodilative, antiaggregation and antiatherogenic effect of prostacycline on the vessel wall predominate. Quantitative evaluation of antiatherogenic effects of HDL subfractions (HDL2 and HDL3) revealed more significant antiatherogenic effect in HDL2 subfraction-in the sense of prostacycline secretion stimulation and exertion of its beneficial effects on the vessel. (Fig. 5, Ref. 33.)

Oxidative modification of low-density lipoprotein by the human hepatoma cell line HepG2.

The human hepatoblastoma cell line HepG2 is a liver model commonly used for lipid metabolism studies. Numerous cell types have been found to oxidize low-density lipoprotein (LDL) but, to our knowledge, the effects of HepG2 cells on LDL have not been investigated. We found that LDL is modified by HepG2 cells through a peroxidative mechanism, as judged by an increase in TBARS content (which was prevented in the presence of the antioxidants vitamin E, 2,6-di-tertbutyl-cresol and probucol), increased degradation by J774 macrophages, decreased internalization by MRC5 fibroblasts, and aggregation of apo B. Aspirin and allopurinol, which inhibit cyclooxygenase and xanthine-oxidase activities, respectively, had no effect on HepG2-induced LDL modification, and neither did catalase, which dismutates hydrogen peroxide; or mannitol, which scavenges hydroxyl radicals. In contrast, superoxide dismutase, a superoxide anion scavenger, and glutamate and threonine, which alter cellular cystine uptake, prevented LDL modifications, as did the removal of cysteine/cystine from the culture medium. Oxidation of LDL by HepG2 cells might thus involve superoxide anion production and/or thiol metabolism.

Enhanced modifications of low-density lipoproteins (LDL) by endothelial cells from smokers: a possible mechanism of smoking-related atherosclerosis.

OBJECTIVE: The aim of the study was to investigate LDL modifications by cultured human umbilical vein endothelial cells (HUVEC) from women smokers and non-smokers. METHODS: Modifications of LDL by HUVEC were studied by determining the values of thiobarbituric acid-reactive substances (TBARS) and the percentage of the most electronegative oxidized LDL fraction (fraction C) by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC). We also studied the cellular production of superoxide anion, the effect of various inhibitors and cysteine, and determined total intracellular glutathione content and cell growth. RESULTS: LDL exposed to HUVEC from smokers for 48 h showed significantly greater modifications than LDL exposed to HUVEC from non-smokers, as assessed by TBARS determination (19.4 +/- 1.2, mean +/- s.e.m., n = 20 versus 15.4 +/- 0.7 nmol/mg LDL, n = 19; P < 0.01) and by FPLC (percentage of fraction C: 39 +/- 7, n = 29 versus 14 +/- 3, n = 34; P < 0.001). Moreover, HUVEC from smokers produced significantly more superoxide anion than those from non-smokers (0.46 +/- 0.13 nmol/10(5) cell/min, n = 9 versus 0.22 +/- 0.05, n = 10; P < 0.05). Superoxide production, like cell-induced modification of LDL, was strongly dependent on the presence of cysteine in the medium. Furthermore, HUVEC from smokers had a significantly (P < 0.05) higher total intracellular glutathione content than those from non-smokers (39.9 +/- 3.1 nmol/mg, n = 9 versus 31.8 +/- 2.2, n = 7). Finally, HUVEC from smokers and non-smokers showed similar growth at 48 h. CONCLUSION: HUVEC from smokers converted significantly more LDL into an atherogenic form than HUVEC from non-smokers, a phenomenon that was not due to altered cell growth. HUVEC-mediated LDL modifications were strongly thiol-dependent, as both LDL modifications and superoxide anion production were inhibited in cysteine-free medium. Stimulation of cystine uptake by HUVEC, reflected by the enhanced total glutathione content, could account for the enhanced superoxide anion production. All these observations may be relevant to the pathophysiology of smoking-related cardiovascular disease.

A cytotoxic electronegative LDL subfraction is present in human plasma.

By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926.

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64 +/- 0.18 (m +/- SE, n = 11) and 3.12 +/- 0.24 (n = 11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14 +/- 3% (n = 34) and 22 +/- 13% (n =10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12 +/- 0.04 (n = 11) and 0.07 +/- 0.01 nmol/min/mg cell protein (n = 11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.

[Charge heterogeneity of low density lipoproteins]. / Hétérogénéité de charge des lipoprotéines de basse densité (LDL).

Traditionally, low-density lipoprotein (LDL) are separated with respect to their size, density and apolipoprotein composition. Fractionation of LDL according to their electrical charge is also interesting as modified LDL have been implicated in the onset of atherosclerosis. This review discusses possible mechanisms underlying charge heterogeneity of human plasma LDL, such as oxidation, glycation, conjugation with aldehydes, carbamylation and changes in sialic acid content and protein composition.

Characteristics of ten charge-differing subfractions isolated from human native low-density lipoproteins (LDL). No evidence of peroxidative modifications.

Native plasma low-density lipoproteins (LDL) were fractionated into ten subfractions with increasingly negative charges (LDL-1, the least electronegative, to LDL-10) using an anion-exchange column coupled to a fast protein-liquid chromatography system. Prior to fractionation, contaminating Lp(a) and apo A-I-containing lipoproteins were removed from LDL preparations by immunoaffinity chromatography. No significant difference in thiobarbituric acid-reactive substances, vitamin E or free aminogroup was found among subfractions, and no peptide with a higher molecular weight than apo B was observed on SDS-PAGE. We observed a gradual increase in cholesterol esters and a concomitant decrease in triglycerides from LDL-1 to LDL-7, and a reverse tendency from LDL-8 to LDL-10 (P < 0.01). Free cholesterol increased linearly from LDL-1 to LDL-10 (P < 0.01). LDL-1 to -3 had a homogeneous density profile, while other more electronegative subfractions showed a bimodal distribution with a second, minor peak of slightly higher density. A gradual increase in apolipoprotein C-III content related to LDL electronegativity was observed (P < 0.001). Apolipoprotein E content was also increased in the last two subfractions (P < 0.01). LDL subfractions displayed a similar binding fate on human fibroblasts, with the exception of the most electronegative subfractions [LDL-(9 + 10)], which bound more actively to apo B/E receptors (P < 0.05). This study shows that charge heterogeneity of native LDL is not related to lipid peroxidation or derivatization of free aminogroups of apolipoprotein B. In contrast, the enrichment of LDL in apolipoproteins other than apo B may explain, in part, the difference in their particle charge.

Evaluation of the sialic acid content of LDL as a marker of coronary calcification and extracoronary atherosclerosis in asymptomatic hypercholesterolemic subjects. PCVMETRA Group.

Recent studies have shown that the sialic acid content of LDL isolated from patients with angiographically demonstrated advanced coronary atherosclerosis is lower than that of LDL isolated from healthy subjects. These observations raise the question as to whether LDL sialic acid content could be used as an early marker of atherosclerosis. We screened for carotid, aortic, and femoral plaques by ultrasonography and for coronary calcifications by ultrafast computed tomography in 160 hypercholesterolemic subjects free of cardiovascular disease to investigate the relation between LDL sialic acid content and the prevalence of these early atherosclerotic lesions. LDL sialic acid values varied from 19.6 to 46.6 nmol/mg LDL protein (33.9 +/- 4.4, mean +/- SD) in the whole population, but the distribution was very similar: (1) in subjects with no plaque (34.1 +/- 4.9) relative to those with one or several plaques at one (34.2 +/- 4.4), two (33.0 +/- 3.6), or three (34.8 +/- 3.4) different arterial sites; (2) in subjects with (33.9 +/- 3.7) and without (34.1 +/- 4.8) coronary calcification; and (3) in subjects with both extracoronary and coronary lesions (33.8 +/- 3.9) relative to those with no arterial lesions (34.2 +/- 4.5). LDL sialic acid content was not related to sex, age, body mass index, smoking, blood pressure, or serum total cholesterol and lipoprotein(a) levels but correlated negatively with serum triglyceride levels (P < .001). These results suggest that LDL sialic acid content is not a discriminant marker of early atherosclerosis in asymptomatic hypercholesterolemic subjects.

[Biology of the endothelial cell and atherogenesis]. / Biologie de la cellule endothéliale et athérogenèse.

Through their specific biological properties, vascular endothelial cells play a major role in maintaining the homeostasis of the cardiovascular system. The vascular endothelium participates actively in coagulation and fibrinolysis, contributes to regulating vascular tone, is involved in inflammation and immunological responses, produces several different stromal components, plays a crucial role in angiogenesis and wound-healing, and interacts with plasma lipoproteins. These physiological functions of endothelial cells are triggered by different endothelium derived mediators and are regulated by numerous environmental factors that can markedly modulate the functional state of these cells by affecting their biosynthetic capabilities, giving them different phenotypes in native, activated and injured states. Excessive endothelial activation leads to changes in endothelial cell gene expression, leading to what is referred to as a dysfunctional state. In this non-adaptative functional state, endothelial cells lose the ability to adjust, within the physiological range, some of their constitutive functions and express newly induced molecules, some of which act as proatherosclerotic factors. Activated endothelial cells thus participate actively in the pathogenesis of atherosclerosis.