Identification of the proteoglycan binding site in apolipoprotein B48

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.     

Endostatin binds biglycan and LDL and interferes with LDL retention to the subendothelial matrix during atherosclerosis

Retention of lipoproteins to proteoglycans in the subendothelial matrix (SEM) is an early event in atherosclerosis. We recently reported that collagen XVIII and its proteolytically released fragment endostatin (ES) are differentially depleted in blood vessels affected by atherosclerosis. Loss of collagen XVIII/ES in atherosclerosis-prone mice enhanced plaque neovascularization and increased the vascular permeability to lipids by distinct mechanisms. Impaired endothelial barrier function increased the influx of lipoproteins across the endothelium; however, we hypothesized that enhanced retention might be a second mechanism leading to the increased lipid content in atheromas lacking collagen XVIII. We now demonstrate a novel property of ES that binds both the matrix proteoglycan biglycan and LDL and interferes with LDL retention to biglycan and to SEM. A peptide encompassing the alpha coil in the ES crystal structure mediates the major blocking effect of ES on LDL retention. ES inhibits the macrophage uptake of biglycan-associated LDL indirectly by interfering with LDL retention to biglycan, but it has no direct effect on the macrophage uptake of native or modified lipoproteins. Thus, loss of ES in advanced atheromas enhances lipoprotein retention in SEM. Our data reveal a third protective role of this vascular basement membrane component during atherosclerosis.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Arterial retention of apolipoprotein B(48)- and B(100)-containing lipoproteins in atherogenesis

PURPOSE OF REVIEW: The "response to retention" hypothesis of atherosclerosis suggests that the arterial deposition of cholesterol is directly proportional to the concentration of circulating plasma lipoproteins. However, there is increasing evidence to support the concept that specific lipoproteins may be preferentially retained within the arterial wall, possibly as a result of greater affinity for cell surface and extracellular matrices. RECENT FINDINGS: Recently, key studies have provided insight into mechanisms involved in the interaction of apolipoprotein B (apoB)-containing lipoproteins with extracellular matrices. In addition, novel methods and innovative experimental design has enabled us to differentiate between the delivery, retention and efflux of apoB(48)- and apoB(100)-containing lipoproteins. Other studies have demonstrated a relationship between extracellular matrix proteoglycan expression and the development of atherosclerosis. Discussion in the present review also extends to the mechanisms that are involved in the relative intimal retention of apoB(48)- and apoB(100)-containing lipoproteins in order to explain the atherogenicity of these macromolecules. SUMMARY: The perspective of this review is to highlight recent advances in the area of arterial lipoprotein retention and the physiological significance these processes may have in the aetiology of cardiovascular disease. Importantly, an understanding of the mechanisms responsible for the retention of apoB(48)/B(100)-containing lipoproteins will enable new strategies to be developed for the future management of cardiovascular disease.     

CAV1-CAVIN1-LC3B-mediated autophagy regulates high glucose-stimulated LDL transcytosis

ABSTRACT

Diabetes is a recognized high-risk factor for the development of atherosclerosis, in which macroautophagy/autophagy is emerging to play essential roles. The retention of low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Here, we identified that high glucose could promote atherosclerosis by stimulating transcytosis of LDL. By inhibiting AMPK-MTOR-PIK3C3 pathway, high glucose suppresses the CAV-CAVIN-LC3B-mediated autophagic degradation of CAV1; therefore, more CAV1 is accumulated in the cytosol and utilized to form more caveolae in the cell membrane and facilitates the LDL transcytosis across endothelial cells. For a proof of concept, higher levels of lipids were accumulated in the subendothelial space of umbilical venous walls from pregnant women with gestational diabetes mellitus (GDM), compared to those of pregnant women without GDM. Our results reveal that high glucose stimulates LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway.     

Mechanism of lipoprotein retention by the extracellular matrix

PURPOSE OF REVIEW: Considerable evidence suggests that the subendothelial retention of atherogenic lipoproteins is a key early step in atherogenesis. In humans and experimental animals, elevated levels of plasma lipoproteins are associated with increased atherosclerosis, and lipoproteins with higher affinity for arterial proteoglycans are more atherogenic. Here we discuss the molecular mechanisms underlying lipoprotein retention in the arterial wall and how this interaction can be modulated. RECENT FINDINGS: Functional proteoglycan binding sites in lipoproteins containing apolipoprotein B have been identified and shown to have atherogenic potential in vivo. In addition to apolipoprotein B, novel bridging molecules, those that can interact with both proteoglycans and lipoproteins, have been identified that mediate the retention of atherogenic particles in the vessel wall. The interaction between lipoproteins and proteoglycans can be enhanced by the modification of lipoproteins in the circulation and in the arterial wall, by alterations in the subendothelium, and by changes in proteoglycan synthesis that result in a more atherogenic profile. The retention of atherogenic lipoproteins is a potential target for therapies to reverse atherosclerosis, and in-vitro studies have identified compounds that decrease the affinity of proteoglycans for lipoproteins. SUMMARY: Considerable progress has been made in understanding the association between lipoproteins and cardiovascular disease. This review highlights the importance of the interaction between lipoproteins and the arterial matrix.     

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

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

Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells

Elevated levels of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)] have been considered strong risk factors for atherosclerotic cardiovascular disease. Increased production of plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of thrombosis and atherosclerosis. Previous studies by our group and others demonstrated that oxidation enhances LDL- and Lp(a)-induced production of PAI-1 in human umbilical vein endothelial cells (HUVEC). The present study examined the involvement of protein kinase C (PKC) and its isoform in vascular endothelial cells (EC) induced by native or oxidized LDL and Lp(a). Treatment with Lp(a) or LDL transiently increased PKC activity at 15 min and 5.5 h after the start of lipoprotein treatment in EC. Copper-oxidized LDL and Lp(a) induced greater PKC activation in EC compared with comparable forms of those lipoproteins. Additions of 1 microM calphostin C, a PKC-specific inhibitor, at the beginning or > or =5 h, but not > or = 9 h, after the initiation of lipoprotein treatment, blocked native and oxidized LDL- or Lp(a)-induced increases in PKC activity and PAI-1 production. Treatment of LDL, Lp(a), or their oxidized forms was induced in translocation of PKC-beta1 from cytosol to membrane in HUVEC. Treatments with 60 nM 379196, a PKC-beta-specific inhibitor, effectively prevented PAI-1 production induced by LDL, Lp(a), or their oxidized forms in HUVEC and human coronary artery EC. The results suggest that activation of PKC-beta may mediate the production of PAI-1 in cultured arterial and venous EC induced by LDL, Lp(a), or their oxidized forms.     

Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL). Prolonged cell-surface contact during which ldl-cholesteryl ester hydrolysis exceeds ldl protein degradation.

A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins. Although most studies have investigated this interaction by incubating cultured macrophages with monomeric lipoproteins dissolved in media, arterial wall macrophages encounter lipoproteins that are mostly bound to subendothelial extracellular matrix, and these lipoproteins are often aggregated or fused. Herein, we utilize a specialized cell-culture system to study the initial interaction of macrophages with aggregated low density lipoprotein (LDL) bound to extracellular matrix. The aggregated LDL remains extracellular for a relatively prolonged period of time and becomes lodged in invaginations in the surface of the macrophages. As expected, the degradation of the protein moiety of the LDL was very slow. Remarkably, however, hydrolysis of the cholesteryl ester (CE) moiety of the LDL was 3-7-fold higher than that of the protein moiety, in stark contrast to the situation with receptor-mediated endocytosis of acetyl-LDL. Similar results were obtained using another experimental system in which the degradation of aggregated LDL protein was delayed by LDL methylation rather than by retention on matrix. Additional experiments indicated the following properties of this interaction: (a) LDL-CE hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis. In summary, experimental systems specifically designed to mimic the in vivo interaction of arterial wall macrophages with subendothelial lipoproteins have demonstrated an initial period of prolonged cell-surface contact in which CE hydrolysis exceeds protein degradation.     

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.     

HepG2 cell LDL receptor activity and the accumulation of apolipoprotein B and E in response to docosahexaenoic acid and cholesterol.

In the present study, the accumulation of apolipoproteins (apo) A-I, B, and E in culture medium was measured after 0, 3, 6, 12, and 24 h of incubation with 150 microM docosahexaenoic acid complexed to 75 microM bovine serum albumin (BSA-22:6), either in the presence or absence of 50 micrograms/ml cholesterol and 4 micrograms/ml 25-hydroxycholesterol (C/25-OH). HepG2 cells incubated with BSA + C/25-OH for 24 h accumulated approximately 2.0-fold greater apoE and B as compared to BSA-treated cells. Moreover, HepG2 cell apoB accumulation after 24 h of BSA-22:6 treatment was approximately 2.0-fold greater than apoB accumulation from cells treated with BSA alone. When BSA-22:6 and C/25-OH were both included in the incubation, apoB accumulation was approximately 5.0-fold greater than BSA-treated cells. Comparative studies using BSA-18:1 were carried out for 24 h and showed similar levels of apoA-I, B, and E accumulation in culture medium as compared to BSA-22:6-treated cells. In addition, apoA-I, B, and E mRNA abundance were found to be unaffected by type of fatty acid treatment or length of incubation, averaging 48.2 +/- 7.5, 222 +/- 33.6, and 17.1 +/- 0.7 pg mRNA/micrograms RNA (mean +/- SEM), respectively. As the accumulation of apoB and apoE in culture medium may be modified by HepG2 cell LDL receptor expression, LDL receptor mRNA abundance and LDL receptor activity were quantified at various times over the course of the study. By 6 h of BSA + C/25-OH treatment, LDL receptor mRNA was reduced approximately 2.3-fold, while receptor activity was reduced approximately 1.5-fold, as compared to BSA controls. In an experiment designed to determine uptake of HepG2 cell lipoproteins, 3H-labeled apoB-containing lipoproteins derived from HepG2 cells were prepared. The 3H-labeled lipoproteins were 1.25-fold more likely to be removed from the media of HepG2 cells treated with BSA than from cells treated with BSA + C/25-OH. From these results, we postulate that HepG2 cell LDL receptor activity mediates the removal of apoB, E-containing lipoproteins from culture medium and contributes to the lower accumulation of apoB and E observed in culture medium from cells treated with BSA as compared to cells treated with C/25-OH.     

Diabetes enhances lectin-like oxidized LDL receptor-1 (LOX-1) expression in the vascular endothelium: possible role of LOX-1 ligand and AGE

Diabetes mellitus accelerating atherosclerosis was associated with the enhanced glycoxidative modification of lipoproteins. LOX-1, the endothelial oxidized LDL receptor might be involved in the pathogenesis of diabetic atherosclerosis. In this study, we examined the vascular expression of LOX-1 in streptozotocin-induced diabetic rats. We found that LOX-1 was significantly increased in diabetic rat aorta compared with nondiabetic control. Immunohistochemistry revealed that the most distinctive staining of LOX-1 was in the endothelial cells, especially in the bifurcations of artery branches from aorta. In cultured aortic endothelial cells, diabetic rat serum and advanced glycation endproducts-BSA induced LOX-1 expression, while control rat serum along with high glucose did not. Applying a competitive inhibition assay, we found that LOX-1 ligand activity was accumulated in the diabetic rat serum, mainly in VLDL/LDL fractions. In addition, VLDL/LDL prominently increased LOX-1 among all the lipoprotein fractions of diabetic rat serum. In conclusion, diabetes markedly upregulated LOX-1 expression in the aortic endothelial cells. The enhanced glycoxidative modification of lipoproteins may contribute to the underlying mechanisms.     

Novel role of ADAMTS-5 protein in proteoglycan turnover and lipoprotein retention in atherosclerosis

Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE(-/-)) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE(441)) and aggrecan ((374)ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE(441) versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.     

Induction of neonatal tolerance to oxidized lipoprotein reduces atherosclerosis in ApoE knockout mice

BACKGROUND: In the course of atherosclerosis, humans and apolipoprotein (apoE) Knockout (KO) mice exhibit an active cell-mediated and humoral immune process, both at the systemic level and within atheromata. Low density lipoproteins (LDL) infiltrate the vascular wall, where they are oxidatively modified. This oxidative modification may generate new epitopes for which tolerance is not achieved during ontogenesis. Such epitopes could constitute new targets for autoreactive immune responses that may have a physiopathological role in disease development. MATERIALS AND METHODS: Exposing mice to high dose of antigens during thymic T-cell education induces immunological tolerance to the administered antigens. We injected newborn apoE KO mice with oxidized LDL. They were fed a cholesterol-rich diet and aortic atherosclerosis, cell-mediated immune response, and T-cell repertoire were analyzed after 5 months. RESULTS: Injection of oxidized LDL at birth reduced not only the immune response to oxidized LDL, but also susceptibility to atherosclerosis in apoE mice. Injection of oxidized LDL induced T-cell tolerance due to clonal deletion, rather than anergy of the reactive T cells. The T-cell repertoire of apoE KO mice was affected by the development of the disease, whereas tolerization normalized it. CONCLUSIONS: This study demonstrates that the immune response against oxidized LDL has a deleterious role in atherogenesis and that a fine-tuning of this response could modify the course of the disease.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells.

alpha-tocopherol, the most potent antioxidant form of vitamin E, is mainly bound to lipoproteins in plasma and its incorporation into the vascular wall can prevent the endothelium dysfunction at an early stage of atherogenesis. In the present study, the plasma phospholipid transfer protein (PLTP) was shown to promote the net mass transfer of alpha-tocopherol from high density lipoproteins (HDL) and alpha-tocopherol-albumin complexes toward alpha-tocopherol-depleted, oxidized low density lipoproteins (LDL). The facilitated transfer reaction of alpha-tocopherol could be blocked by specific anti-PLTP antibodies. These observations indicate that PLTP may restore the antioxidant potential of plasma LDL at an early stage of the oxidation cascade that subsequently leads to cellular damages. In addition, the present study demonstrated that the PLTP-mediated net mass transfer of alpha-tocopherol can constitute a new mechanism for the incorporation of alpha-tocopherol into the vascular wall in addition to the previously recognized LDL receptor and lipoprotein lipase pathways. In ex vivo studies on rabbit aortic segments, the impairment of the endothelium-dependent arterial relaxation induced by oxidized LDL was found to be counteracted by a pretreatment with purified PLTP and alpha-tocopherol-albumin complexes, and both the maximal response and the sensitivity to acetylcholine were significantly improved. We conclude that PLTP, by supplying oxidized LDL and endothelial cells with alpha-tocopherol through a net mass transfer reaction may play at least two distinct beneficial roles in preventing endothelium damage, i.e., the antioxidant protection of LDL and the preservation of a normal relaxing function of vascular endothelial cells.     

Induction of antioxidant stress proteins in vascular endothelial and smooth muscle cells: protective action of vitamin C against atherogenic lipoproteins.

Elevated levels of lipid peroxidation and increased formation of reactive oxygen species within the vascular wall in atherosclerosis can overwhelm cellular antioxidant defence mechanisms. Accumulating evidence implicates oxidatively modified low density lipoproteins (LDL) in vascular dysfunction in atherosclerosis and oxidized LDL have been localized with in atherosclerotic lesions. We here report that human oxidatively modified LDL induce expression of 'antioxidant-like' stress proteins in vascular cells, involving increases in the activity of L-cystine transport, glutathione synthesis, heme oxygenase-1 and the murine stress protein MSP23. Moreover, treatment of human arterial smooth muscle cells with the dietary antioxidant vitamin C markedly attenuates adaptive increases in endogenous antioxidant gene expression and affords protection against smooth muscle cell apoptosis induced by moderately oxidized LDL. As vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque 'necrotic' core, cap rupture and thrombosis, our findings suggest that the cytoprotective actions of vitamin C could limit plaque instability in advanced atherosclerosis.     

Oxidized LDL bind to nonproteoglycan components of smooth muscle extracellular matrices

Arterial wall lipid retention is believed to be due primarily to ionic interactions between lipoproteins and proteoglycans. Thus, oxidized low density lipoproteins (LDL), with decreased positive charge relative to native LDL, should have decreased interaction with negatively charged proteoglycans. However, oxidized LDL does accumulate within arterial lesions. Therefore, this study investigated the binding of native and oxidized LDL to a complex smooth muscle extracellular matrix and the role of ionic charge interactions in their binding. LDL was modified with 2,2-azo-bis(2-amidinopropane)-2HCl, hypochlorite, soybean lipoxygenase, and phospholipase or copper sulfate. The extracellular matrix had 15- to 45-fold greater binding capacity for the different forms of oxidized LDL than for native LDL. However, the affinity of binding for all forms of oxidized LDL was high (K(a) = approximately 10(-9) M) and was similar to that for native LDL. Preincubation of the lipoproteins with chondroitin sulfate decreased the binding of native LDL, but had no effect on the binding of oxidized LDL. Digestion of matrices with chondroitin ABC lyase and heparinase decreased the binding of native LDL, but increased the binding of oxidized LDL; matrix digestion with pronase or trypsin markedly reduced the binding of both native and oxidized LDL.Thus, the binding of native LDL involves matrix proteoglycans, whereas the binding of oxidized LDL involves a nonproteoglycan component(s) of the matrix. The markedly enhanced retention of oxidized LDL compared with native LDL may play an important role in the progression of atherosclerosis.     

The role of complement in atherosclerosis

PURPOSE OF REVIEW: Although it has long been recognized that atherosclerotic lesions show evidence of complement activation, the functional roles of the complement system in atherogenesis are not yet fully resolved. This article highlights recent publications on the complement system in the atherosclerosis field. RECENT FINDINGS: There have been a number of recent papers reporting on the association of complement proteins and complement regulators with high density lipoproteins, complement activation by enzymatically-modified LDL, signalling pathways downstream of C3a and C5a receptors and membrane C5b-9 assembly, and the prevention of C5b-9 assembly on endothelial cells via upregulation of CD59 expression in response to arterial laminar flow. C1q has been found to play a protective role in early lesion formation in LDL receptor deficient mice, and Crry-Ig and soluble C1 inhibitor have both been shown to have therapeutic effects in models of vascular injury in ApoE deficient mice. The possibility that the Y402H Factor H polymophism influences atherosclerosis has been supported in a recent paper showing increased risk in white hypertensive individuals. SUMMARY: The articles that have emerged over the last year highlight the relevance of the complement system to the atherosclerosis field.     

Induction of antioxidant stress proteins in vascular endothelial and smooth muscle cells: Protective action of vitamin C against atherogenic lipoproteins

Elevated levels of lipid peroxidation and increased formation of reactive oxygen species within the vascular wall in atherosclerosis can overwhelm cellular antioxidant defence mechanisms. Accumulating evidence implicates oxidatively modified low density lipoproteins (LDL) in vascular dysfunction in atherosclerosis and oxidized LDL have been localized with in atherosclerotic lesions. We here report that human oxidatively modified LDL induce expression of ‘antioxidant-like’ stress proteins in vascular cells, involving increases in the activity of l-cystine transport, glutathione synthesis, heme oxygenase-1 and the murine stress protein MSP23. Moreover, treatment of human arterial smooth muscle cells with the dietary antioxidant vitamin C markedly attenuates adaptive increases in endogenous antioxidant gene expression and affords protection against smooth muscle cell apoptosis induced by moderately oxidized LDL. As vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque ‘necrotic’ core, cap rupture and thrombosis, our findings suggest that the cytoprotective actions of vitamin C could limit plaque instability in advanced atherosclerosis.