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Background: A hallmark of atherosclerosis is its complex pathogenesis, which is dependent on altered cholesterol metabolism and inflammation. Both arms of pathogenesis involve myeloid cells. Monocytes migrating into the arterial walls interact with modified low-density lipoprotein (LDL) particles, accumulate cholesterol and convert into foam cells, which promote plaque formation and also contribute to inflammation by producing proinflammatory cytokines. A number of studies characterized transcriptomics of macrophages following interaction with modified LDL, and revealed alteration of the expression of genes responsible for inflammatory response and cholesterol metabolism. However, it is still unclear how these two processes are related to each other to contribute to atherosclerotic lesion formation. Methods: We attempted to identify the main mater regulator genes in macrophages treated with atherogenic modified LDL using a bioinformatics approach. Results: We found that most of the identified genes were involved in inflammation, and none of them was implicated in cholesterol metabolism. Among the key identified genes were interleukin (IL)-7, IL-7 receptor, IL- 15 and CXCL8. Conclusion: Our results indicate that activation of the inflammatory pathway is the primary response of the immune cells to modified LDL, while the lipid metabolism genes may be a secondary response triggered by inflammatory signalling.

Chronification of inflammation is the process that lies at the basis of several human diseases that make up to 80% of morbidity and mortality worldwide. It can also explain a great deal of processes related to aging. Atherosclerosis is an example of the most important chronic inflammatory pathology in terms of public health impact. Atherogenesis is based on the inflammatory response of the innate immunity arising locally or focally. The main trigger for this response appears to be modified low-density lipoprotein (LDL), although other factors may also play a role. With the quick resolution of inflammation, atherosclerotic changes in the arterial wall do not occur. However, a violation of the innate immunity response can lead to chronification of local inflammation and, as a result, to atherosclerotic lesion formation. In this review, we discuss possible mechanisms of the impaired immune response with a special focus on mitochondrial dysfunction. Some mitochondrial dysfunctions may be due to mutations in mitochondrial DNA. Several mitochondrial DNA mutations leading to defective mitophagy have been identified. The regulatory role of mitophagy in the immune response has been shown in recent studies. We suggest that defective mitophagy promoted by mutations in mitochondrial DNA can cause innate immunity disorders leading to chronification of inflammation.

We have suggested that the molecular mechanism of vascular wall damage in diabetes is not substantially different from that in atherosclerosis. Thus, it can be assumed that aldehyde-modified LDL should be eliminated from the blood stream with much greater speed than non-oxidized LDL. In the available literature there is information about the clearance of native human LDL from the bloodstream, whereas information on the clearance of the aldehyde-modified LDL in animals or humans was not found. Based on this, the present work is devoted to the clearance of aldehyde-modified LDL of rabbits and humans introduced into the bloodstream of rabbits. We investigated the clearance of glyoxal-, methylglyoxal- and MDA-modified LDL from the bloodstream of rabbits. We used biotinylated LDL of rabbit blood plasma and FITC-labeled LDL of human blood plasma. LDL was isolated with preparative ultracentrifugation in NaBr gradient. It was shown that glyoxal- and methylglyoxal-modified LDL of rabbits and humans circulated in the bloodstream for the same time as native LDL while MDA-modified LDL was rapidly eliminated from the bloodstream. The data obtained indicated the possibility of greater atherogenic potential of glyoxal- and methylglyoxal-modified LDL as they circulate in the bloodstream for a rather long time. At the same time, MDA-modified LDL is likely to be exposed to enhanced elimination by macrophages after their "linkage" to blood cells.

Hromaděni lipidů představuje hlavni faktor v rozvoji aterosklerotických změn. Je znamo, že castice lipoproteinů o nizke hustotě (low-density lipoprotein, LDL) jsou hlavnim zdrojem cholesterolu a dalsich lipidů uložených v aterosklerotickem platu. Aterogenni vlastnosti vsak nemaji vsechny LDL castice. Hromaděni lipidů v buňkach tepen vyžaduje modifikaci LDL castic. I když se z těchto modifikaci největsi pozornost věnuje oxidaci, byly popsany i dalsi aterogenni modifikace LDL. Podle řady studii se serem a hodnotami LDL pacientů s aterosklerozou je jednou z prvnich - pokud ne vůbec prvni - aterogenni modifikaci LDL desializace. K te dochazi v krevnim proudu; po ni nasleduje kaskada dalsich modifikaci vcetně zmenseni velikosti LDL castic a jejich zhustěni, ziskani negativniho elektrickeho naboje, oxidace a tvorby vysoce aterogennich komplexů. V tomto minipřehledu se zabývame koncepci vicecetne aterogenni modifikace LDL vedouci k rozvoji a progresi aterosklerozy.

Modified low-density lipoprotein (LDL) is the main source of lipid accumulation in the arterial wall affected by atherosclerosis. We aimed to compare the properties of apolipoprotein B (apoB) from native and modified LDL. Modified (desialylated) LDL and native LDL were extracted from blood of atherosclerotic patients. We characterized apoB structure of LDL particles in total LDL preparation, circulating modified LDL (cmLDL), and native LDL. Intact cmLDL had a twofold lower content of free amino groups than native LDL. Delipidated apoB from cmLDL also had a lower content of free amino groups. The rates of tryptic hydrolysis and elastase digestion of cmLDL were twofold higher in comparison to native LDL. Therefore, cmLDL from atherosclerotic patients had altered apoB properties. Our observations strengthen the hypothesis of multiple modification of LDL in the bloodstream and underscore the importance of desialylated LDL as a possible marker of atherosclerosis.

Atherogenic modification of low-density lipoprotein (LDL) plays a crucial role in the pathogenesis of atherosclerosis, as modified LDL, but not native LDL, induces pronounced accumulation of cholesterol and lipids in the arterial wall. It is likely that LDL particles undergo multiple modifications in human plasma: desialylation, changes in size and density, acquisition of negative electric charge, oxidation, and complex formation. In a total LDL preparation isolated from pooled plasma of patients with coronary atherosclerosis and from healthy subjects, two subfractions of LDL could be identified: desialylated LDL bound by a lectin affinity column and normally sialylated (native) LDL that passed through the column. The desialylated LDL subfraction therefore represents circulating modified LDL. In this work, we performed a careful analysis of LDL particles to reveal changes in the composition of glycoconjugates associated with proteins and lipids. Protein fraction of LDL from atherosclerotic patients contained similar amounts of glucosamine, galactose, and mannose, but a 1.6-fold lower level of sialic acid as compared to healthy donors. Lipid-bound glycoconjugates of total LDL from patients with coronary atherosclerosis contained 1.5-2-fold less neutral monosaccharides than total LDL from healthy donors. Patient-derived LDL also contained significantly less sialic acid. Our results demonstrate that carbohydrate composition of LDL from atherosclerotic patients was altered in comparison to healthy controls. In particular, prominent decrease in the sialic acid content was observed. This strengthens the hypothesis of multiple modification of LDL particles in the bloodstream and underscores the clinical importance of desialylated LDL as a possible marker of atherosclerosis progression.

The review presents the results of the authors' work of many years and the data of foreign studies, indicating the importance of free radical processes in etiology and pathogenesis of atherosclerosis. The justification of the idea that oxidative stress in atherosclerosis develops more often during disorders of carbohydrate metabolism, contributing to emergence of carbonyl stress in diabetes. It is hypothesized that there is a single molecular mechanism of primary pro-atherogenic injuries of vessel walls in atherosclerosis and diabetes, which enhances the formation of carbonyl-modified low-density lipoproteins, accumulating in the foam cells. The results of recent studies indicate that oxidatively modified LDL involved not only the primary injury to the vessel walls, but also provoked the development of endothelial dysfunction, so that once again raises the issue of the need for the use of antioxidants to prevent injuries in vascular walls. It can be assumed that the result of years of intensive research of free radical process mechanisms in atherosclerosis and diabetes will be the development of new drugs for the combined therapy of these diseases that can be disposed not only reactive oxygen species and organic free radicals and reactive carbonyls, which are clearly playing an important role in the development of pro-atherogenic injuries.

Atherosclerosis is associated with acute cardiovascular conditions, such as ischemic heart disease, myocardial infarction, and stroke, and is the leading cause of morbidity and mortality worldwide. Our understanding of atherosclerosis and the processes triggering its initiation is constantly improving, and, during the last few decades, many pathological processes related to this disease have been investigated in detail. For example, atherosclerosis has been considered to be a chronic inflammation triggered by the injury of the arterial wall. However, recent works showed that atherogenesis is a more complex process involving not only the immune system, but also resident cells of the vessel wall, genetic factors, altered hemodynamics, and changes in lipid metabolism. In this review, we focus on foam cells that are crucial for atherosclerosis lesion formation. It has been demonstrated that the formation of foam cells is induced by modified low-density lipoprotein (LDL). The beneficial effects of the majority of therapeutic strategies with generalized action, such as the use of anti-inflammatory drugs or antioxidants, were not confirmed by clinical studies. However, the experimental therapies targeting certain stages of atherosclerosis, among which are lipid accumulation, were shown to be more effective. This emphasizes the relevance of future detailed investigation of atherogenesis and the importance of new therapies development.

Background The size and receptor-binding abilities of plasma lipoproteins are closely related with their structure/functions. Presently, the sizes of native lipoproteins have been measured by various methods including atomic force microscopy (AFM) whereas the sizes of modified lipoproteins are poorly determined and the receptor-binding ability of lipoproteins is less detected and compared at the nanoscale. Methods Here, AFM was utilized to detect/compare the size and scavenger receptor-binding properties of three native human lipoproteins including high-density lipoprotein, low-density lipoprotein (LDL), and very low-density lipoprotein, and two modified human lipoproteins including oxidized and acetylated LDL, as well as bovine serum albumin and their antibodies as negative and positive controls, respectively. Results AFM detected that the sizes of these lipoproteins are close to the commonly known values and the previously-reported AFM-detected sizes and that native and modified LDL have different height/size. AFM also revealed that the CD36-binding abilities of the five lipoproteins are different from one another and from their SR-B1-binding abilities and that the anti-CD36/SR-B1 antibodies as positive controls have strong CD36/SR-B1-binding abilities. Conclusions The data provide important information on lipoproteins for better understanding their structures/functions. Moreover, the data certify that besides size measurement AFM also can visualize receptor-lipoprotein binding at the nanoscale, as well as antigen–antibody (scavenger receptors and their antibodies) binding.

Aggregation of LDL is considered the initial key event in atherogenesis and apoJ is an extracellular chaperone part of the quality control system against protein aggregation. A minor fraction of apoJ in blood is transported by LDL, but its role is poorly understood. Considering both the importance of LDL aggregation in atherosclerosis and the modulatory role of apoJ on protein aggregation, the function of apoJ bound to LDL could be relevant. Recent studies have shown that apoJ prevents the LDL aggregation and inhibits the cytotoxic potential of modified LDL. Other studies have reported increased content of apoJ in atherogenic LDL fractions. These observations point to apoJ as a key modulator of LDL atherogenicity.

Cardiovascular disease of atherosclerotic origin is the main cause of death in diabetes mellitus and metabolic syndrome. One of the mechanisms involved in such increased risk is the high incidence of lipoprotein modification in these pathologies. Increased glycosylation, oxidative stress or high non-esterified fatty acid levels in blood, among other factors, promote the modification and subsequent alteration of the properties of lipoproteins. Since the modification of low-density lipoprotein (LDL) is the triggering factor in the development of atherosclerosis, considerable research has been focused on the quantification of modified LDLs in blood to be used as biomarkers of cardiovascular risk. The present review deals with the main molecular mechanisms involved in the modification of LDL in diabetes and metabolic syndrome and briefly describe the atherogenic effects that these modified LDLs exert on the arterial wall. The possibility of using the high levels of modified LDLs or their immunocomplexes as a predictive tool for cardiovascular risk in diabetes-related pathologies is also discussed.

Objectives: Modified LDL uptake by macrophages (MΦ) leading to foam cell formation is an early event in atherosclerosis. Recent studies suggest that MΦ polarization contributes to the progression of atherosclerosis. Therefore, we investigated the hypothesis that macrophage plasticity affects modified LDL uptake and subsequent foam cell formation. Approach & Results: Bone marrow-derived MΦ were treated with LPS and IFNγ or IL-4 to induce MΦ polarization to M(LPS/IFNγ or M1) and M (IL4 or M2) phenotypes. Foam cell formation was determined after incubating M(LPS/IFNγ) and M(IL4) MΦ with oxidized-LDL. Our results show that formation of foam cells was marginally increased in M(IL4) MΦ compared to un-polarized M(-) MΦ. However, accumulation of neutral lipids was higher in M(LPS/IFNγ) MΦ compared to M(IL4) MΦ indicating that foam cell formation is enhanced by polarization to pro-inflammatory M(LPS/IFNγ) MΦ. Interestingly, mRNA and protein expression of CD36, one of the major scavenger receptors, were lower in M(LPS/IFNγ) MΦ compared to un-polarized MΦ. To assess the specific contribution of CD36, foam cell formation was assessed in MΦ isolated from CD36-deficient mice. Notably, oxidized-LDL uptake and foam cell formation in CD36-deficient M(LPS/IFNγ) MΦ was similar to M(LPS/IFNγ) MΦ from wild type mice. This indicates that CD36 does not mediate the increased foam cell formation in M(LPS/IFNγ) MΦ. We therefore examined expression of LOX-1, another scavenger receptor implicated in foam cell formation. We found that both mRNA and protein expression for LOX-1 increased several fold in M(LPS/IFNγ) MΦ. We also showed that TLR4-mediated NF-κB activation is the primary mechanism for the increased LOX-1 expression and foam cell formation in M(LPS/IFNγ) polarized MΦ. Conclusion: Macrophage plasticity, specifically polarization of MΦ to pro-inflammatory M(LPS/IFNγ) phenotype promotes foam cell formation, which has implications for the progression of atherosclerosis.

Cymbopogon citratus (lemon grass) is commonly used in traditional folk medicine. The essential oil extracted from C. citratus has been reported as a potential anti-oxidant and anti-inflammatory agent. This study has been designed to explore the protective effect of C. citratus (lemon grass) against modified LDL (OxLDL and H 2 O 2 LDL) induced cytotoxicity in Peripheral Blood Mononuclear Cells (PBMC). The essential oil extracted from C. citratus (EOC) was subjected to FT-IR spectroscopic analysis for the identification of functional groups. In vitro antioxidant assays were carried out to assess the electron donating capability of EOC as compared with a known standard L -ascorbic acid. The cytoprotective effects of EOC were determined in PBMC induced with modified LDL. Spectra obtained from FT-IR analysis showed the presence of functional groups in EOC such as H-bonded, O H stretching, N H stretching, aldehyde C H stretching, aldehyde/ketone C O stretching, C C-stretching, CH 3 bending, C H in plane bending. EOC has greater antioxidant property when compared with the standard L -ascorbic acid. EOC at all test concentrations demonstrated free radical scavenging activity and cytoprotective effect when challenged against modified LDL in PBMC. The above results show EOC as a promising antioxidant and cytoprotective agent.

We have earlier found that blood sera of patients with coronary heart disease (CHD) increase lipid levels in cells cultured from subendothelial intima of human aorta. We have also revealed that the ability of blood sera to raise intracellular cholesterol; that is, their atherogenicity is caused by at least modified low density lipoprotein (LDL) circulating in the blood of patients and autoantibodies to modified LDL. In the present work we have demonstrated significant impact of nonlipid factor(s) to blood atherogenicity. We have developed an approach to removal of nonlipid atherogenicity factor(s) from blood serum based on the use of immobilized LDL. This approach was used for extracorporeal perfusion of patient’s blood through the column with immobilized LDL. Pilot clinical study confirmed the efficacy of this approach for prevention of coronary atherosclerosis progression.

Lipid accumulation in the arterial wall is a crucial event in the development of atherosclerotic lesions. Circulating low-density lipoprotein (LDL) is the major source of lipids that accumulate in the atherosclerotic plaques. It was discovered that not all LDL is atherogenic. In the blood plasma of atherosclerotic patients, LDL particles are the subject of multiple enzymatic and non-enzymatic modifications that determine their atherogenicity. Desialylation is the primary and the most important atherogenic LDL modification followed by a cascade of other modifications that also increase blood atherogenicity. The enzyme trans-sialidase is responsible for the desialylation of LDL, therefore, its activity plays an important role in atherosclerosis development. Moreover, circulating modified LDL is associated with immune complexes that also have a strong atherogenic potential. Moreover, it was shown that antibodies to modified LDL are also atherogenic. The properties of modified LDL were described, and the strong evidence indicating that it is capable of inducing intracellular accumulation of lipids was presented. The accumulated evidence indicated that the molecular properties of modified LDL, including LDL-containing immune complexes can serve as the prognostic/diagnostic biomarkers and molecular targets for the development of anti-atherosclerotic drugs.

In this mini-review, the role of macrophage phenotypes in atherogenesis is considered. Recent studies on distribution of M1 and M2 macrophages in different types of atherosclerotic lesions indicate that macrophages exhibit a high degree of plasticity of phenotype in response to various conditions in microenvironment. The effect of the accumulation of cholesterol, a key event in atherogenesis, on the macrophage phenotype is also discussed. The article presents the results of transcriptome analysis of cholesterol-loaded macrophages revealing genes involved in immune response whose expression rate has changed the most. It turned out that the interaction of macrophages with modified LDL leads to higher expression levels of pro-inflammatory marker TNF-α and anti-inflammatory marker CCL18. Phenotypic profile of macrophage activation could be a good target for testing of novel anti-atherogenic immunocorrectors. A number of anti-atherogenic drugs were tested as potential immuno-correctors using primary macrophage-based model.

Oxidation of LDL in the early stages of the development of atherosclerosis and antioxidant supplements to prevent it have been the basis of public healthcare to prevent cardiovascular disease (CVD) for over 60 years. With the limited success of antioxidant therapy in countering CVD, it is appropriate to consider if there are nonoxidative mechanisms driving atherosclerosis in the early stages. Modification of LDL by the reactive dicarbonyl metabolite methylglyoxal (MG) is nonoxidative. It is nevertheless a proatherogenic transformation of LDL – forming small, dense LDL (sdLDL) with increased binding to arterial proteoglycans. Methylglyoxal modified LDL formation is increased in diabetes, renal failure and aging. New clinical interventions are required to counter this nonoxidative atherogenicity, including dicarbonyl scavengers and glyoxalase 1 inducers.

Highlights ► OxLDL promotes induction of pATM and p21 in fibroblasts/endothelial cells. ► OxLDL activates ATM-kinase by a doubled-stranded DNA break-independent mechanism. ► ATM-deficient fibroblasts exhibit low cell viability but increased oxidative stress towards oxLDL. ► ATM expression exerts protective effects against ox-LDL-induced cellular toxicity., Chronic oxidative stress is involved in the pathogenesis of multiple inflammatory diseases, including cardiovascular disease and atherosclerosis. The rare autosomal recessive disorder Ataxia-telangiectasia (A-T) is characterized by progressive cerebellar ataxia secondary to Purkinje cell death, immunodeficiency, and increased cancer incidence. ATM, the protein mutated in A-T, plays a key role in cellular DNA-damage responses. A-T cells show poor cellular anti-oxidant defences and increased oxidant sensitivity compared to normal cells, and ATM functions, in part, as an oxidative stress sensor. The oxidation of low-density lipoprotein (oxLDL) and its uptake by macrophages is an initiating step in the development of atherosclerosis. We demonstrate that oxLDL activates ATM and downstream p21 expression in normal fibroblasts and endothelial cells. In ATM-deficient fibroblasts oxLDL induces DNA double-strand breaks, micronuclei formation and causes chromosome breaks. Furthermore, oxLDL decreases cell viability and inhibits colony formation in A-T fibroblasts more effectively as compared to normal controls. Formation of oxLDL-induced reactive oxygen species is significantly higher in A-T, than normal fibroblasts. Last, pre-treatment of cells with ammonium pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of transcription factor nuclear factor κB, reduces oxLDL-induced reactive oxygen species formation. Our data indicates that ATM functions in the defence against oxLDL-mediated cytotoxicity.

There has been increasing interest in investigating the role of glycation as an atherogenic modification of lipoproteins; especially after the failure of antioxidant therapy to suppress the incidence of atherogenic cardiovascular diseases in high-risk individuals. Glycation of lipoproteins impairs their function. In the case of LDL, it also leads to rapid scavenger-receptor uptake by monocyte/macrophages. Glycation also diminishes the antiatherogenic properties of HDL. The concentration of circulating glycated LDL, even in nondiabetics, is higher than that of oxidatively modified LDL. Glycated small dense LDL is significantly higher in nondiabetic individuals as well as in Type 2 diabetic patients. Lowering the level of small dense LDL by using statins lowers glycated LDL in Type 2 diabetic patients. This may be one of the reasons for their effect in decreasing cardiovascular risk in these patients. Treatments that prevent glycation of lipoproteins may prevent atherosclerosis and other diabetic complications.

Oxidized l-α-1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10–50 μg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2−· production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2−· by xanthine oxidase-lumazine and H2O2by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species.

Despite a long history of studies on lipid abnormalities, numerous problems in laboratory technologies and techniques remain unresolved. One of the most commonly tested molecules is low-density lipoprotein (LDL) cholesterol, and a homogenous assay technique for measurement of this molecule has recently been introduced. Although the method represents remarkable technological breakthroughs with great potential for improving lipoprotein analysis, some discrepancies exist among assay protocols. Even for direct measurement of high-density lipoprotein (HDL) cholesterol, which has widely been accepted, there are still large discrepancies among data obtained by different protocols. Oxidatively modified LDL is an independent factor that is considered to directly contribute to the pathogenesis of early atherosclerosis. Lipid peroxidation products, surface charge, and spectrophotometric patterns are all applicable to the evaluation of in vitro oxidation. Only enzyme-linked immunosorbent assays using monoclonal antibodies have a potential for clinical use, but such methods are not yet standardized. There is also increasing evidence for the presence of anti-oxidized LDL autoantibodies in human sera, but the diagnostic utility remains controversial. In addition, small dense LDL has recently attracted much attention as an independent risk factor. Although this is a potential target of oxidization, a robust and simple analytical method does not yet exist. This review presents the current state of laboratory technologies for testing lipid abnormalities.

コレステロール分画・トリグリセライド分画同時解析法を用いて, 虚血性心疾患, 動脈硬化を起こしやすい糖尿病・腎疾患, 脂質異常を高頻度に認める肝疾患・悪性腫瘍・膠原病の計6疾患群の脂質分画と変性LDLを疾患別に比較検討した. 変性LDLの出現率は, 腎疾患で最も高率で, 高脂血症のみならず正脂血症や低脂血症でも高率であった. 脂質項目の平均値を, 高脂血症と正脂血症に分けて変性LDL陽性例と陰性例を比較すると疾患による特徴を認めた. 変性LDL陽性例では, 高TG・低HDL-C・IDLの出現は高率であったが, 高LDL-Cの出現では陰性例と差を認めなかった. CHDでは, 低HDL-Cが高率で, 陽性例では93%出現した. 糖尿病では, TCおよびTGが高値で, IDLの出現が高率であった. 高脂血症表現型は, 変性LDL陽性例でII b型・IV型が多くII a型は少なかった. 以上の結果, 脂質分画と変性LDLの出現は疾患により異なり, 本法により脂質分画の詳細を把握することは臨床的に有用であった.

糖尿病においてコレステロール分画とトリグリセライド分画の同時解析法を用いて脂質分画の詳細と変性LDLを荷電変性度数 (CMF) として測定し, 変性LDL陰性例と陽性例にわけて比較した. 空腹時血糖・ヘモグロビンA1c値では差を認めなかったが, 高脂血症表現型, IDLの出現, 各項目の平均値, HDL分画・LDL分画の組成比に差を認めた. 高脂血症表現型では対照群・DM-CMF(-)・DM-CMF(+)の順にIIa型が減少し, IIb型・IV型が増加した. HDL分画・LDL分画では対照群・DM-CMF(-)・DM-CMF(+)の順に Chol の減少, TGの増加が認められた. 糖尿病性合併症の有無で比較すると, 腎症では病期の進行に伴って変性LDLの出現率が増加し, CMFも上昇した. 糖尿病患者において脂質分画と変性LDLを測定することは, 高脂血症・腎症のモニターとして臨床的に有用と考えられた.

PAG電気泳動のLDL分画像から変性LDLを捉え, 加齢に伴う変化や糖尿病や動脈硬化性疾患との関係について検討した. LDLの分画像の形状を, S, A, N, Dの4型に分類し, IDLやレムナントを含むN型とD型を重視した. また, LDL分画像のピークおよびLDL分画の最も速い移動部位の相対移動度から small LDLと fast β-Lpの有無を捉え, この3点を変性LDLの所見とした. LDL分画像で捉えるものとRLP-Cとは異質であり, 酸化LDLはN型・D型と fast β-Lp の異常分画に存在した. N型・D型と fast β-Lp の増加には加齢に伴う変化が観察され, 特に40歳代と50歳代との間に大きな有意差を認めた. 加齢に伴う同様の変化はT-CやLDL-Cにおいてもみられるが, その変化よりも顕著であった. 若年者 (中学2年生) のLDL分画像は, N型とD型が少ないのに対し, fast β-Lp は50歳代と同等以上の高い陽性率を示し, 酸化LDLも fast β-Lp と同様の傾向を示した. 若年者の酸化LDLは主として fast β-Lp に存在し, 成人で増加するN型・D型に存在する酸化LDLとは異質であろうと考えられた. 糖尿病のLDL分画像は, 蛋白尿 (+) 群では, N型とD型が多く, 健常者に対しても, 蛋白尿 (-) 糖尿病群に対しても有意差を認めた. 50歳代健常者と比べ, 動脈硬化性疾患群にはN型・D型と small LDLが有意に多く, fast β-Lp 陽性も増加していた.

Benoit Vlaminck,1 Damien Calay,1 Marie Genin,1 Aude Sauvage,1 Noelle Ninane,1 Karim Zouaoui Boudjeltia,2 Martine Raes,1 Carine Michiels1 1Laboratory of Biochemistry and Cellular Biology (URBC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium; 2Laboratory of Experimental Medicine (ULB 222 Unit), Universite Libre de Bruxelles, CHU de Charleroi, Charleroi, Belgium Abstract: Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia. Keywords: Ox-LDL, myeloperoxidase, hypoxia, UPR, apoptosis, autophagy, macrophages

L’LDL electronegativa (LDL(-)) és una LDL modificada present en circulació amb propietats inflamatòries incloent la inducció de l’alliberament de citoquines en cèl·lules relacionades amb l’arteriosclerosi, tals com monòcits i cèl·lules endotelials. Es coneix poc sobre les vies intracel·lulars activades per l’LDL(-), en especial en monòcits. En aquesta tesi s’han estudiat els primers passos a través dels quals l’LDL(-) indueix l’alliberament d’IL-6, IL-10 i MCP-1 en monòcits humans. Concretament, s’han estudiat els components inflamatoris de l’LDL(-) i la seva interacció amb receptors cel·lulars de monòcits. En relació amb els components inflamatoris, es va observar que la ceramida (CER) és, en part, responsable dels efectes inflamatoris de l’LDL(-). La CER es relaciona amb un augment en la susceptibilitat a l’agregació de l’LDL(-) i a la seva inducció de citoquines en monòcits. Aquestes propietats de l’LDL(-) augmenten a 37ºC i disminueixen per la incubació d’aquesta lipoproteina amb l’HDL. El contingut en CER en l’LDL(-) augmenta per acció de l’activitat tipus fosfolipasa C (PLC), intrínseca en l’LDL(-) i que també es veu inhibida per l’HDL. Aquest fet suggereix que l’activitat PLC de l’LDL(-) participa en les modificacions que tenen lloc en l’LDL nativa (LDL(+)) per a la formació de LDL(-). L’enriquiment d’LDL en CER (CER-LDL)) i el tractament d’LDL amb PLC promouen una LDL que mimetitza l’alliberament de citoquines de l’LDL(-). Tanmateix, l’efecte de la CER-LDL és menor al de l’LDL(-), proposant la possible acció d’altres components en l’LDL(-) que podrien contribuir al seus efectes inflamatoris. En l’estudi dels receptors involucrats en l’alliberament d’IL-6, IL-10 i MCP-1 induït per l’LDL(-) en monòcits es va determinar que CD14 i TLR4 juguen un paper central. L’adició dels anticossos antiTLR4 i antiCD14 va disminuir un 70%-80% l’alliberament de citoquines, mentre que antiTLR2 va disminuir l’alliberament sols en un 15-25%. L’alliberament de citoquines també va disminuir al tractar les cèl·lules amb un inhibidor específic de TLR4. Aquest alliberament de citoquines va augmentar en la línia de monòcits THP1 que sobreexpressen CD14 (THP1-CD14). Aquests resultats es van confirmar per silenciament gènic de TLR4 i CD14. Experiments d’unió van mostrar que l’LDL(-) presenta una gran afinitat per CD14 i TLR4; la neutralització d’aquests receptors va disminuir la quantitat d’LDL(-) unida a monòcits. Per immunoassaig es va demostrar que CD14 és el principal receptor involucrat en la unió de LDL(-). L’LDL(-) uneix CD14, es forma un complex amb TLR4 i d’aquesta manera s’activa la senyalització intracel·lular que donarà lloc a l’alliberament de citoquines per aquestes cèl·lules. L’LDL(-) pot, d’altrabanda, interaccionar directament amb TLR4 per activar la senyal intracel·lular. CD14 i TLR4 són receptors que mitjancen l’alliberament de citoquines induït pel lipopolisacàrid (LPS) present en la membrana bacteriana. Els resultats mostren una competència entre LDL(-) i LPS tant per l’efecte inflamatori com per la unió al receptor CD14 de monòcits i de plaques multipou recobertes de CD14. Aquesta competència podria representar una acció compensatòria de l’LDL(-) en casos d’excessiva inflamació. El contingut augmentat en CER present en l’LDL(-) sembla ser el responsable de l’activació del sistema CD14-TLR4, el qual provoca l’alliberament de citoquines en monòcits. L’alliberament de citoquines induït per la CER-LDL en monòcits va disminuir per l’adició de l’inhibidor de TLR4. La secreció de citoquines induïda per la CER-LDL és similar a la de l’LDL(-), excepte pel fet que la CER-LDL no pot activar l’alliberament de citoquines directament per TLR4 i necessita CD14 per a aquest efecte. En conclusió, l’augment del contingut en CER per l’LDL(-) exerceix un paper central en l’alliberament d’IL-6, IL-10 i MCP-1 a través de CD14- TLR4 en monòcits., Electronegative LDL (LDL(-)) is a minor modified LDL present in circulation with pro-inflammatory effects, including the induction of cytokine release in cells involved in atherosclerosis, such as endothelial and mononuclear cells. However, the cellular pathways activated by LDL(-) are scarcely understood, particularly in monocytes. In this thesis, we aimed to determine the first steps of the mechanisms by which LDL(-) induces IL-6, IL-10 and MCP-1 release in human monocytes. We focussed the study on the inflammatory components of LDL(-) and their interaction with cell receptors in monocytes. Regarding the inflammatory components, we found that ceramide (CER) is, in part, responsible for the inflammation promoted by LDL(-). CER has been related to the increased LDL(-)-susceptibility to aggregation and the induction of cytokine release in monocytes. These properties of LDL(-) are enhanced at 37ºC and diminished by the incubation of this lipoprotein with HDL. CER levels increase in LDL(-) because of the LDL(-)-intrinsic PLC-like activity, which is also counteracted by HDL. This suggests that PLC-like activity of LDL(-) participates in the modifications undertaken in native LDL (LDL(+)) to form LDL(-). The enrichment of LDL with CER (CER-LDL) and its treatment with PLC-like activity mimics the cytokine secretion of LDL(-), although the effect of CER-LDL does not reach that of LDL(-). This suggests that other components linked to LDL(-) could contribute to its inflammatory effects. The study of the receptors involved in the IL-6, IL-10 and MCP-1 release induced by LDL(-) in monocytes revealed that CD14 and TLR4 were pivotal. The addition of antiTLR4 and antiCD14 antibodies decreased cytokine release by 70%-80%, whereas that of TLR2 inhibited it by 15%-25%. The cytokine release was also diminished when cells were treated with a specific TLR4 inhibitor, but this release increased in a monocytic THP1 cell line overexpressing CD14 (THP1-CD14). These results were confirmed by TLR4 and CD14 gene silencing studies. Binding studies showed that LDL(-) presents high affinity to CD14, and in a lesser extent to TLR4; the neutralisation of these receptors decreased the amount of LDL(-) bound to monocytes. Immunoassay techniques elucidated that CD14 is the main receptor involved in LDL(-) binding. LDL(-) binds to CD14 and, then, form a complex with TLR4 to activate the intracellular signalling leading to cytokine release in these cells. However, LDL(-) can alternatively interact with TLR4 to activate the intracellular signalling. CD14 and TLR4 are receptors that mediate the cytokine release induced by the lipopolysaccharide (LPS) present in the bacterial membrane. The results showed a competition between LDL(-) and LPS for both the inflammatory effect and the binding to CD14 receptor in monocytes and in CD14-coated microtiter wells. This competition could represent an LDL(-)-counteracting role in cases of overwhelming inflammation. The increased content of CER present in LDL(-) seems to be responsible for the activation of the CD14-TLR4 system which leads to cytokine secretion in monocytes. The cytokine release induced by CER-LDL in monocytes decreased with the addition of the TLR4 inhibitor. The CER-LDL-induced cytokine secretion in THP1-CD14 cells was much greater than in THP1 cells, which showed almost null cytokine release. This behaviour of CER-LDL is similar to LDL(-), excepting the fact that CER-LDL is not able to induce its cytokine release directly through TLR4 and needs CD14 for this effect. To sum up, the increased content of CER present in LDL(-) plays a key role in the induction of IL-6, IL-10 and MCP-1 release through CD14-TLR4 in monocytes.

The oxidation of low density lipoprotein (LDL) is a key event in the development and progression of atherosclerosis because it generates molecular epitopes that are more atherogenic than parent LDL. We found previously that patients with carotid atherosclerosis have a significantly higher titer of autoantibodies against oxidatively modified LDL than normal subjects. The aim of this study is to correlate biological markers of in vivo LDL oxidation with the degree of carotid stenosis and of plaque ulceration (PU) in a series of patients undergoing carotid endoarteriectomy (CEA). Ninety-four consecutive patients (68M and 26F, aged 67.3 +/- 8.2 years) who underwent CEA at our institution between June 1993 and January 1994 were included in the study. The degree of carotid stenosis and the presence and extent of PU were correlated with the level of autoantibodies (IgG) against oxidatively modified LDL (Cu++-oxidized [oxLDL] or malondialdehyde derivatized LDL [MDA-LDL]), that consistently mirrors the occurrence of oxidative modifications in vivo. A statistically significant correlation (r = 0.23, p = 0.039) was found between the degree of carotid stenosis and antiMDA-LDL specific ratio (a parameter that describes the specificity of LDL towards other proteins as target for oxidative modification). A statistically significant correlation was also found between the PU score and antioxLDL IgG (r = 0.32, p = 0.011), antiMDA-LDL IgG (r = 0.25, p = 0.045) and antiMDA-LDL IgG specific ratio (r = 0.38, p = 0.002). None of the classical biochemical parameters (total, LDL and HDL cholesterol and triglycerides) correlated with the above-mentioned plaque characteristics. The results shown, support the use of biological markers of in vivo LDL oxidation (antioxidatively modified LDL autoantibody titers) to evaluate the clinical setting of high-risk carotid atherosclerosis both in screening and in follow-up studies.