Role of the 12-lipoxygenase pathway in diabetes pathogenesis and complications.

12-lipoxygenase (12-LOX) is one of several enzyme isoforms responsible for the metabolism of arachidonic acid and other poly-unsaturated fatty acids to both pro- and anti-inflammatory lipid mediators. Mounting evidence has shown that 12-LOX plays a critical role in the modulation of inflammation at multiple checkpoints during diabetes development. Due to this, interventions to limit pro-inflammatory 12-LOX metabolites either by isoform-specific 12-LOX inhibition, or by providing specific fatty acid substrates via dietary intervention, has the potential to significantly and positively impact health outcomes of patients living with both type 1 and type 2 diabetes. To date, the development of truly specific and efficacious inhibitors has been hampered by homology of LOX family members; however, improvements in high throughput screening have improved the inhibitor landscape. Here, we describe the function and role of human 12-LOX, and mouse 12-LOX and 12/15-LOX, in the development of diabetes and diabetes-related complications, and describe promise in the development of strategies to limit pro-inflammatory metabolites, primarily via new small molecule 12-LOX inhibitors.

STAT4 contributes to adipose tissue inflammation and atherosclerosis.

Adipose tissue (AT) inflammation is an emerging factor contributing to cardiovascular disease. STAT4 is a transcription factor expressed in adipocytes and in immune cells and contributes to AT inflammation and insulin resistance in obesity. The objective of this study was to determine the effect of STAT4 deficiency on visceral and peri-aortic AT inflammation in a model of atherosclerosis without obesity. Stat4(-/-)Apoe(-/-) mice and Apoe(-/-) controls were kept either on chow or Western diet for 12 weeks. Visceral and peri-aortic AT were collected and analyzed for immune composition by flow cytometry and for cytokine/chemokine expression by real-time PCR. Stat4(-/-)Apoe(-/-) and Apoe(-/-) mice had similar body weight, plasma glucose, and lipids. Western diet significantly increased macrophage, CD4+, CD8+, and NK cells in peri-aortic and visceral fat in Apoe(-/-) mice. In contrast, in Stat4(-/-)Apoe(-/-) mice, a Western diet failed to increase the percentage of immune cells infiltrating the AT. Also, IL12p40, TNFa, CCL5, CXCL10, and CX3CL1 were significantly reduced in the peri-aortic fat in Stat4(-/-)Apoe(-/-) mice. Importantly, Stat4(-/-)Apoe(-/-) mice on a Western diet had significantly reduced plaque burden vs Apoe(-/-) controls. In conclusion, STAT4 deletion reduces inflammation in peri-vascular and visceral AT and this may contribute via direct or indirect effects to reduced atheroma formation.

Interaction between cytokines and inflammatory cells in islet dysfunction, insulin resistance and vascular disease.

Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in sustained inflammation in diabetes and atherosclerosis. 12- and 15-lipoxygenase (LO), such as 12/15-LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12- and 15-LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue-specific knockout of 12/15-LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12- and 15-LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12- and 15-LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)-12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1) in islets and IL-17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.

Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice.

Adipose tissue inflammation and reduced pancreatic ß-cell function are key issues in the development of cardiovascular disease and progressive metabolic dysfunction in type 2 diabetes mellitus. The aim of this study was to determine the effect of the DPP IV inhibitor sitagliptin on adipose tissue and pancreatic islet inflammation in a diet-induced obesity model. C57Bl/6J mice were placed on a high-fat (60% kcal fat) diet for 12 wk, with or without sitagliptin (4 g/kg) as a food admix. Sitagliptin significantly reduced fasting blood glucose by 21% as well as insulin by ∼25%. Sitagliptin treatment reduced body weight without changes in overall body mass index or in the epididymal and retroperitoneal fat mass. However, sitagliptin treatment led to triple the number of small adipocytes despite reducing the number of the very large adipocytes. Sitagliptin significantly reduced inflammation in the adipose tissue and pancreatic islet. Macrophage infiltration in adipose tissue evaluated by immunostaining for Mac2 was reduced by sitagliptin (P < 0.01), as was the percentage of CD11b+/F4/80+ cells in the stromal vascular fraction (P < 0.02). Sitagliptin also reduced adipocyte mRNA expression of inflammatory genes, including IL-6, TNFα, IL-12(p35), and IL-12(p40), 2.5- to fivefold as well as 12-lipoxygenase protein expression. Pancreatic islets were isolated from animals after treatments. Sitagliptin significantly reduced mRNA expression of the following inflammatory cytokines: MCP-1 (3.3-fold), IL-6 (2-fold), IL-12(p40) (2.2-fold), IL-12(p35) (5-fold, P < 0.01), and IP-10 (2-fold). Collectively, the results indicate that sitagliptin has anti-inflammatory effects in adipose tissue and in pancreatic islets that accompany the insulinotropic effect.

Role of angiotensin II and free radicals in blood pressure regulation in a rat model of renal hypertension.

One-kidney, 1-clip rats (1K1C) or uninephrectomized controls were treated with either the superoxide dismutase mimetic tempol (0.5 mmol. kg(-1). d(-1)), angiotension type 1 receptor inhibitor losartan (50 mmol. L(-1). kg(-1). d(-1)), or both (n=6 per group) for 2 weeks. At the end of the study, systolic blood pressure (BP) decreased on average by 21% in tempol-treated and 29% in losartan-treated versus untreated 1K1C (217+/-4.4 mm Hg) and was normalized in the losartan plus tempol group. Mean BP also decreased from 159+/-3.7 mm Hg in 1K1C to 93+/-2.8 mm Hg in the losartan plus tempol group. Also, aortic wall area was reduced by 18% in losartan- or tempol-treated 1K1C and by 30% in losartan plus tempol rats compared with untreated 1K1C. Plasma renin activity was increased from 4.8+/-0.3 in untreated 1K1C to 15.9+/-0.9 ng. mL(-1). h(-1) in losartan-treated but not tempol-treated 1K1C. Superoxide generation by the isolated aortic rings assessed by lucigenin chemiluminescence was significantly decreased (by approximately 40%) in all losartan, tempol, and losartan plus tempol groups compared with untreated 1K1C. Nitrotyrosine ELISA in the kidney displayed a significant reduction, from 59+/-13 ng/mg of protein in 1K1C to 12.5+/-5 ng/mg of protein in the losartan plus tempol 1K1C. Western blotting for nNOS in kidney cortex and medulla showed a protein increase in both fractions of 1K1C versus controls and was normalized by losartan plus tempol treatment. Collectively, data show a synergistic effect of losartan and tempol on BP reduction in 1K1C rats. The mechanism may involve reduced superoxide production and nitrotyrosine formation in kidney and decreased kidney neuronal-type NO synthase expression in treated animals. This status in the oxidative balance seems to affect BP in the renal hypertensive rats.

Oxidative stress in a rat model of obesity-induced hypertension.

The mechanisms underlying the development of hypertension in obesity are not yet fully understood. We recently reported the development of hypertension in a rat model of diet-induced obesity. When Sprague-Dawley rats (n=60) are fed a moderately high fat diet (32 kcal% fat) for 10 to 16 weeks, approximately half of them develop obesity (obesity-prone [OP] group) and mild hypertension (158+/-3.4 mm Hg systolic pressure), whereas the other half (obesity-resistant [OR] group) maintains a body weight equivalent to that of a low fat control group and is normotensive (135.8+/-3.8 mm Hg). We examined the potential role of oxidative stress in the development of hypertension in this model. Lipid peroxides measured as thiobarbituric acid-reactive substances showed a significant increase in the LDL fraction of OP rats (2.8+/-0.32 nmol malondialdehyde/mg protein) compared with OR and control rats (0.9+/-0.3 nmol malondialdehyde/mg protein). Also, aortic and kidney thiobarbituric acid-reactive substances showed a significant (3- and 5- fold) increase in OP rats after 16 weeks of diet. In addition, superoxide generation by aortic rings, measured by lucigenin luminescence, showed a 2-fold increase in the OP group compared with both the OR and control groups. In addition, free isoprostane excretion and nitrotyrosine in the kidney showed an increase in OP rats only. The urine and plasma nitrate/nitrite measured by the LDH method showed a 1.8-fold decrease in OP rats compared with OR rats. However, endothelial NO synthase expression in the kidney cortex and medulla assessed by reverse transcriptase-polymerase chain reaction showed a strong increase in the OP rats versus OR and control rats (endothelial NO synthase/beta-actin ratio 1.3+/-0.04 in OP rats versus 0.44+/-0.02 in OR rats), suggesting a possible shift toward superoxide production by the enzyme. Collectively, the data show a decreased NO bioavailability in OP animals that is due in part to the increased oxidative stress.

Src tyrosine kinases and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases mediate pressure-induced c-fos expression in cannulated rat mesenteric small arteries.

Chronic hypertension is associated with remodeling of small arteries. There is evidence that the high pressure itself may cause these structural changes, but the responsible mechanisms are not clearly defined. Previously we showed that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries was inhibited by genistein, a general tyrosine kinase inhibitor. The purpose of this study was to further unravel the underlying signal transduction mechanisms, and we particularly tested the involvement of src tyrosine kinases and extracellular signal-regulated kinase (ERK). Rat mesenteric small arteries were cannulated in a dual-vessel chamber. After a 60-minute equilibration period, the pressure in 1 artery was increased to 140 mm Hg, while the other artery remained at 90 mm Hg. Semiquantitative reverse transcriptase-polymerase chain reaction was used to determine c-fos expression, and Western blotting was used to examine levels of ERK phosphorylation. The involvement of src and ERK was tested with the inhibitors herbimycin A (1 micromol/L), PP1 (10 micromol/L), PP2 (10 micromol/L), and PD98059 (30 micromol/L). One-hour exposure to 140 mm Hg increased the c-fos/cyclophilin ratio 3.6-fold, from 0.29+/-0.07 to 1.06+/-0.25. All the tested inhibitors suppressed the pressure-induced increase of c-fos expression. A 5-minute exposure period to 140 mm Hg increased ERK phosphorylation, and this was abolished in the presence of PP1. The results suggest that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries may be mediated, at least in part, by src tyrosine kinases and ERK.

Diabetic state induces lipid loading and altered expression and secretion of lipoprotein lipase in human monocyte-derived macrophages.

Non-insulin-dependent diabetes mellitus (NIDDM) is frequently associated with macroangiopathies and coronary heart diseases. Lipoprotein lipase (LPL), an enzyme known to undergo significant functional alterations in diabetic state, is also a potential atherogenic protein. Since, to the best of our knowledge, there are no data concerning LPL secreted by macrophages of NIDDM patients we conducted a study to assess the expression and activity of LPL secreted by monocyte-derived macrophages from NIDDM patients with cardiovascular complications versus cardiovascular patients without diabetes (controls). Isolated cells from NIDDM patients, after 7 days in culture in the presence of 20% autologous serum, readily exhibit a foam cell phenotype, in contrast to the cells from controls. Macrophages were mainly loaded with triglycerides, whose cellular amount was well correlated to triglyceridemia of NIDDM subjects. Concomitantly, macrophages from NIDDM patients displayed a approximately six-fold decrease of mRNA expression and a approximately two-fold reduction of the activity of secreted LPL, as compared to control cells. These data suggest that in complicated diabetic state, macrophage loading leading to foam cell formation is accelerated, at least in part, due to a diminished expression and activity of LPL. These observations add and extend the data that may explain the occurrence of accelerated atherogenesis and of the atherosclerotic complications associated with diabetes.

Development of hypertension in a rat model of diet-induced obesity.

Although obesity is a risk factor for hypertension, the relationship between these 2 conditions is not well understood. Therefore, we examined some parameters of hypertension and cardiovascular disease in a dietary model of obesity. Male Sprague-Dawley rats were provided either a control diet (C) or a diet containing 32% kcal as fat (similar to a Western diet) for 1, 3, or 10 weeks. Rats in the latter group diverged based on body weight gain into obesity-prone (OP) and obesity-resistant (OR) groups. Systolic blood pressure in OP rats was significantly higher after 10 weeks of the diet (149+/-4. 8 mm Hg) compared with both OR and C groups (131+/-3.7 and 129+/-4.5 mm Hg, respectively). The aortic wall area of OP rats was significantly increased, indicating arterial hypertrophy, and a 2-fold increase in plasma renin activity was found in OP rats compared with OR and C rats. The lipid profile showed a significant increase in plasma and VLDL triglycerides of OP versus OR and C groups as early as 3 weeks on the diet. Plasma and LDL-cholesterol levels were increased in the OP group versus the OR and C groups after 3 weeks of the diet, but the difference was blunted after 10 weeks. Lipid peroxidation (thiobarbituric acid-reactive substances) in OP rats was increased 2-fold in LDL and 1.5-fold in aortic wall compared with OR rats, suggesting an increased oxidative stress in these animals. Periodic acid-Schiff staining of the kidney showed mesangial expansion and focal sclerosis that were more prominent in OP rats than in OR rats. The results suggest that hypercholesterolemia, but not hypertriglyceridemia, is linked to the diet; that hypertension and renin-angiotensin system activation are associated with obesity; and that lipid peroxidation and renal damage are the results of both factors.

AT(1) receptor inhibition does not reduce arterial wall hypertrophy or PDGF-A expression in renal hypertension.

To separate the role of ANG II from pressure in hypertrophy of the vascular wall in one-kidney, one-clip (1K1C) hypertension, experimental and sham-operated rats were given the AT(1)-receptor antagonist losartan (20 mg x kg(-1) x day(-1)) or tap water for 14 days. Mean arterial pressure was elevated in both experimental groups compared with controls. Rats were anesthetized with pentobarbital sodium, and the thoracic aorta and carotid, small mesenteric, and external spermatic arteries were harvested and embedded in paraffin. Tissue sections were used for morphological analysis, immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and platelet-derived growth factor (PDGF)-AA, stereological measurements, and in situ hybridization with a (35)S-labeled riboprobe for PDGF-A mRNA. Elevated cross-sectional areas of thoracic, carotid, and small mesenteric artery in 1K1C rats were not reduced by losartan. The internal diameter of the external spermatic artery and microvascular density of the cremaster muscle were reduced in 1K1C rats. The number of BrdU-positive nuclei per cross section did not differ between 1K1C and control arteries. PDGF-A mRNA was elevated in the arterial walls of 1K1C rats compared with controls and was hardly changed by losartan. PDGF-A protein stained strongly in the media of 1K1C arteries and was not inhibited by losartan; it appeared in the adventitia of all aortas and carotid arteries. These observations demonstrate that effects of ANG II mediated through the AT(1) receptor are not necessary for hypertrophy of the vascular wall during 1K1C hypertension or expression of PDGF-A.

PDGF-A expression correlates with blood pressure and remodeling in 1K1C hypertensive rat arteries.

We previously demonstrated remodeling of large and small arteries in angiotensin II-treated rats, paralleled by an increased expression of platelet-derived growth factor (PDGF)-A chain mRNA in large arteries. Both remodeling and PDGF-A expression were associated with elevation of blood pressure rather than a direct effect of angiotensin II. To further delineate the role of PDGF-A and elevated blood pressure, we assessed the level of PDGF-A and -B mRNA and protein in the wall of large as well as small arteries in the one-kidney, one-clip (1K1C) hypertensive rat, a non-renin-dependent model of hypertension. Fourteen days after renal artery stenosis, the thoracic aorta and both femoral arteries were collected from 1K1C rats (n = 8) and uninephrectomized controls (n = 8) and immediately processed for morphological measurement, immunohistochemistry, RT-PCR, and Western blotting. Systolic blood pressure was significantly elevated in hypertensive rats (202 +/- 26 mmHg) compared with control rats (122 +/- 7.9 mmHg) and was accompanied by arterial hypertrophy in both aorta and femoral arteries. The mRNA for PDGF-A chain was increased threefold in the thoracic aorta (P < 0.05) of 1K1C rats, whereas the message for PDGF-B was not significantly changed in hypertensive versus control animals. A higher staining of the intima-media was observed by using an anti-PDGF-A chain polyclonal antibody on paraffin-embedded sections. Western blot results indicated an approximately 2-fold increase in PDGF-A protein in aortic and femoral wall of the 1K1C rats. The results showed that both the mRNA and protein for PDGF-A chain are increased and well correlated with the blood pressure and wall area, suggesting a direct effect of elevated pressure on PDGF synthesis, which, in turn, may affect the onset and progression of vascular hypertrophy.

Increased macrophage uptake of irreversibly glycated albumin modified-low density lipoproteins of normal and diabetic subjects is mediated by non-saturable mechanisms.

Diabetes mellitus is known as an independent risk factor in atherosclerosis. Among the prominent biochemical changes that occur in diabetic state, are the enhanced formation of advanced glycosylation end products (AGE) (especially linked to albumin and collagen) and the impaired oxidative-antioxidative balance. Previously, we have shown that AGE-albumin (AGE-Alb) significantly alters the physico-chemical characteristics of low density lipoproteins of normal (nLDL) and diabetic (dLDL) subjects. In this study we tried to establish if incubation of nLDL or dLDL, with AGE-Alb in autoxidative conditions, modifies the rate and/or the pathway of their uptake by macrophages. To this purpose, nLDL and dLDL were exposed to AGE-Alb, and after re-isolation and radiolabeling the lipoproteins were incubated with U937 or peritoneal macrophages (for various time and concentrations), in the absence or presence of different competitors (native LDL, acetylated LDL, AGE-Alb, mannan) or cytochalasin D. As controls, nLDL and dLDL, maintained in similar conditions, but without AGE-Alb, were used. The results showed that preincubation for 24 h and 72 h with AGE-Alb augmented the macrophage uptake for both nLDL and dLDL (1.7-fold). Either pre-incubated or not with AGE-Alb, dLDL was taken up at a constantly higher rate than nLDL; the difference appeared more prominent at 72 h (1.5 vs. 4 micrograms LDL protein/mg cell protein). The increased level of glycation of native dLDL as compared to native nLDL (266 +/- 35 vs. 160 +/- 24 mmol HMF/mol apoB) as well as of the lipid peroxides (1.34 +/- 0.47 vs. 0.3 +/- 0.09 nmol MDA/mg apoB) could account for the greater uptake of dLDL at any preincubation time. Competition experiments indicated that, generally, incubation with AGE-Alb diminished the apo B100,E receptor-mediated uptake in favour of 'scavenger' receptor pathway and phagocytosis. Macrophage uptake of AGE-Alb modified dLDL was reduced approximately 30% by native nLDL, approximately 70% by acetylated LDL and approximately 38% by cytochalasin D. Together, these data suggest that the consequence of the alterations induced by AGE-Albumin on LDL is the increased macrophage uptake, via non-saturable pathways, that ultimately may lead to accelerated formation of atherosclerotic plaques in diabetics.

Intimal thickenings of human aorta contain modified reassembled lipoproteins.

The aim of this study was to determine whether in human aortas early minute changes such as minimal intimal thickenings (MIT), developed in areas known to have a predilection to atherosclerosis, contain modified reassembled lipoproteins (MRLp) such as extracellular liposomes (EL) and lipid droplets (LD). These features have been previously detected in the aortic lesion-prone areas of rabbits and hamsters fed a fat-rich diet. Tissue samples of the aortic arch and thoracic aorta from 12 young subjects who died in accidents were selectively collected from grossly normal regions. By light microscopy, some of these regions were found to contain MIT. The normal areas and the MIT were separately examined by electron microscopy or subjected to fractionation and partial biochemical characterization. The MIT (approximately 25-100 microns thick) were constituted by a pronounced proliferation of extracellular matrix, especially elastin and microfibrils, with interspersed lipid deposits appearing as EL and LD. Commonly, MIT did not contain smooth muscle cells, macrophages, foam cells or cytolytic debris. Such components were only occasionally found in specimens excised from the vicinity of fatty streaks. Saline extracts of MIT or grossly normal aortic regions were subjected to a four-step purification procedure consisting of gel filtration, affinity chromatography on anti-apo B and anti-albumin Sepharose, followed by density gradient ultracentrifugation. The entire procedure was monitored by negative staining, lipid assays, SDS PAGE and immunoblotting. From the initial MRLp mixture, two fractions were obtained: fraction 1 containing multilamellar EL and LD, and fraction 2 composed mostly of unilamellar EL. As compared with serum LDL, the cholesteryl ester/unesterified cholesterol ratio was 4-6-fold lower in fraction 1 and 15-19-fold lower in fraction 2. On SDS-PAGE the fraction 2 displayed a single protein band of 66 kDa, immunochemically identified as albumin. The MRLp isolated from human aortas with minimal intimal thickenings appeared to be similar to those purified from the prelesional stage aorta of hyperlipidemic rabbits and hamsters.

Irreversibly glycated albumin alters the physico-chemical characteristics of low density lipoproteins of normal and diabetic subjects.

In diabetic plasma, glycated albumin and glycated LDL coexist with augmented levels of peroxides, conditions frequently associated with the development of accelerated atherosclerosis. The direct interaction between irreversibly glycated albumin, LDL and oxidation have not been explored yet. We tried to elucidate whether irreversibly glycated albumin (AGE-Alb) induces changes in the chemistry and morphology of LDL particle, and if AGE-Alb has the ability to scavenge free radicals, as reported for native albumin. LDL isolated from normal (nLDL) or diabetic human subjects (dLDL) was incubated in vitro with AGE-Alb in conditions of autoxidation (37 degrees C, 24-48 h in the absence of oxidation inhibitors) or of Cu2+ induced-oxidation. The results showed that, especially in the latter condition, AGE-Alb induced marked physico-chemical modifications of both nLDL and dLDL without significant changes in the level of peroxides. Incubation with AGE-Alb decreased the cholesteryl esters/unesterified cholesterol ratio of nLDL by 30% and of dLDL by approximately 50%. Concomitantly, in oxidative conditions a marked increase (approximately 3-fold) in the lysophosphatidylcholine/phosphatidylcholine ratio of dLDL was detected. Apolipoprotein B integrity as well as the morphology of the lipoprotein particles were drastically affected. To a lesser extent, these modifications occurred also in the presence of inhibitors of oxidation at 37 degrees C, but not at 4 degrees C. The above described effects were constantly more pronounced in the case of dLDL. These results indicated that in the absence of other plasma or vascular tissue components (e.g., endothelial cells, extracellular matrix) AGE-Alb by itself induces alterations in the chemistry and morphology of LDL, especially of glycated LDL, modifications that may account for the occurrence of accelerated atherogenesis in diabetes.

In vitro formation of oxidatively-modified and reassembled human low-density lipoproteins: antioxidant effect of albumin.

In early atherogenesis, excess plasma lipoproteins accumulate into the arterial lesion-prone areas as modified and reassembled lipoproteins (MRLp) appearing mostly as lipid droplets and vesicles. In the present study we produced such MRLp, in a cell-free system, devoid of any component of extracellular matrix, by subjecting in vitro human low-density lipoproteins (LDL) to autoxidation or copper-induced oxidation, for up to 96 h. As visualized by negative staining electron microscopy, a large number of lipoprotein particles (Lp) were progressively transformed into aggregates (ALp), fused particles (FLp) and vesicles (VLp). These modifications were paralleled by peroxidation of the samples as revealed by chemical analysis of each MRLp fraction isolated by a three-step purification procedure. LDL peroxidation in the above conditions was inhibited by the presence of albumin as assessed by TBARS and lipid analysis, and by the lack of MRLp formation. This protective effect was independent of albumin source (bovine, human, rabbit) and occurs at an albumin/LDL ratio of 1 when Cu2+ was present, and at a ratio of 0.25 in autoxidative conditions. The results show that: (i) in vitro LDL autoxidation or copper-induced peroxidation in a cell-free system can generate modified and reassembled lipoproteins similar to those detected in vivo in the arterial intima at the inception of atherogenesis; (ii) Lp particles appear to be sequentially transformed in self-aggregates, droplets and vesicles; (iii) serum albumin can completely prevent these LDL alterations.