Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages

The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells.     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Modification of copper-catalyzed oxidation of low density lipoprotein by proteoglycans and glycosaminoglycans.

Chondroitin sulfate proteoglycans (CSPG) appear to contribute to retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, CSPG and glycosaminoglycans (GAG) modify LDL structure and increase its uptake by macrophages. This latter effect appears related to increased exposure of arginine- and lysine-rich segments of apoB-100. We explored whether alterations of LDL induced by human arterial CSPG and purified GAG alter the lipoprotein susceptibility to transition metals-catalyzed oxidation. Human LDL was complexed with human arterial CSPG and dissociated by raising the ionic strength. The nonaggregated, CSPG- and GAG-treated LDL was subjected to oxidation by micromolar amounts of Cu+, Cu2+, Fe2+, and Fe3+. This treatment increased LDL susceptibility to Cu2+ oxidation 3- to 5-times, as indicated by the degradation rate of phospholipids and cholesteryl esters and formation rates of dienes and thiobarbituric acid-reacting substances (TBARS). Also, human macrophages degraded the CSPG-treated, Cu2+-oxidized LDL 3- to 6-times faster than native LDL similarly treated. No enhancement of oxidation was observed with Fe2+, Fe3+, and Cu+. Quenching of the LDL intrinsic fluorescence by Cu2+ showed that heparin, CSPG, and chondroitin-6-SO4 pretreatment increased the access of Cu2+ to hydrophobic chromophores, probably tryptophan, 6- to 7-, 3- to 4-, and 2- to 3-fold, respectively. Also, the affinity constant (Ka) of LDL for Cu2+ was increased from 0.12 microM to 0.20 microM by the treatment with CSPG and GAG. These results and evaluation of the fraction of surface-accessible LDL chromophores to acrylamide quenching suggest that the increased susceptibility to oxidation may be associated with an increase in the access of Cu2+ to hydrophobic regions in LDL caused by treatment with CSPG and GAG. This effect was not detected with Cu+, Fe2+, or Fe3+. The phenomenon may contribute to acceleration of the oxidative modifications of LDL in cell culture models and in vivo.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

Cell-induced copper ion-mediated low density lipoprotein oxidation increases during in vivo monocyte-to-macrophage differentiation

Macrophage activation is associated with the production and release of reactive oxygen species (ROS), which are capable of mediating oxidative modification of low-density lipoprotein (LDL). In the present study we questioned whether cellular capacity to oxidize LDL increases during in vivo monocyte/macrophage maturation. We developed a novel model for macrophage maturation in vivo using mouse peritoneal macrophages (MPMs) harvested at increasing intervals after intraperitoneal thioglycollate injection. Macrophage maturation was evidenced by a progressive increase in cellular size, density, granulation, and expression of cell surface markers CD11b and CD36, and by a gradual decrement in myeloperoxidase activity. Cellular capacity to stimulate copper ion-mediated oxidation of LDL increased gradually by up to 2-fold during in vivo macrophage maturation in Balb/C mice, similar to the pattern observed during 1,25-dihydroxyvitamin D3-induced in vitro differentiation of the PLB-985 cell line. These effects were attributed to a gradual increase in production of ROS by up to 9-fold. The mechanism for the increase in cellular oxidative stress during macrophage maturation could be related, at least in part, to NADPH oxidase activation, as demonstrated by a gradual increase over time in p47phox expression (mRNA and protein) and in its translocation to the plasma membrane. In conclusion, in vivo monocyte-to-macrophage differentiation is associated with increased cell capacity to oxidize LDL, which may represent a protective mechanism for rapid removal of atherogenic LDL from extracellular spaces in the arterial wall.     

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.     

人动脉壁蛋白聚糖与血清脂蛋白形成的不溶性复合物

本文报道了人主动脉壁中正常及异常(脂纹,FS)区的三种蛋白聚糖(PG)即:硫酸软骨素PG(CSPG)、硫酸皮肤素—硫酸软骨素PG(DSCSPG)及硫酸乙酰肝素PG(HSPG)与血清极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)所形成的不溶性复合物。在30mmol/L Ca~(2+)对,三种PG都能与这两种脂蛋白形成不溶性复合物,随放置时间的增加,形成的复合物都发生解离,但其复合物形成的曲线及解离程度明显不同。DSCSPG与CSPG比较,前者与两种脂蛋白更易形成不溶性复合物且不易解离。HSPG与两种脂蛋白形成不溶性复合物所需时间远大于CSPG及DSCSPG。FS区及正常区三种PG形成复合物曲线类型相似,异常区CSPG、DSCSPG与VLDL形成的复合物量低于正常区的相应PG,而与VLDL则高于正常区的相应PG。异常区的HSPG与两种脂蛋白形成不溶性复合物的量均高于正常区。     

Red wine administration to apolipoprotein E-deficient mice reduces their macrophage-derived extracellular matrix atherogenic properties

Proteoglycans (PGs) from the arterial extracellular matrix (ECM) contribute to the trapping of LDL and oxidized LDL (Ox-LDL) in the arterial wall, a phenomenon called "lipoprotein retention". Moreover, we have shown that subsequent to their binding to the matrix, LDL and Ox-LDL are taken up by macrophages. Oxidative stress significantly increases macrophage secretion of ECM-PGs, lipoprotein binding to the ECM and the uptake of ECM-retained lipoproteins by macrophages. The aim of the present study was to determine whether red wine administration to atherosclerotic mice would affect their peritoneal macrophage-derived extracellular matrix properties, such as the glycosaminoglycan content and the ability to bind LDL. In addition, we questioned the ability of LDL bound to the mice peritoneal macrophages-derived ECM to be taken up by macrophages. Red wine administration to atherosclerotic mice did not affect the mice peritoneal macrophages-derived ECM glycosaminoglycan content but it significantly reduced the mice peritoneal macrophages-derived ECM ability to bind LDL and the subsequent uptake of ECM-retained LDL by the macrophages. The present study thus clearly demonstrated the inhibitory effect of red wine consumption by E0 mice on their peritoneal macrophage-derived extracellular matrix atherogenic properties.     

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.     

Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein.

Oxidative modification of low-density lipoprotein (LDL) enhances its uptake by macrophages in tissue culture and in vivo may underly the formation of arterial fatty streaks, the progenitors of atheroma. We investigated the possible protection which high-density lipoprotein (HDL) affords against LDL oxidation. The formation of lipoperoxides and thiobarbituric acid reactive substances when LDL was incubated with copper ions was significantly decreased by HDL. The enzyme, paraoxonase (E.C. 3.1.8.1), purified from human HDL, had a similar effect and thus may be the component of HDL responsible for decreasing the accumulation of lipid peroxidation products.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.     

A proteomic study of the apolipoproteins in LDL subclasses in patients with the metabolic syndrome and type 2 diabetes

The exchangeable apolipoproteins present in small, dense LDL (sdLDL) and large, buoyant LDL subclasses were evaluated with a quantitative proteomic approach in patients with the metabolic syndrome and with type 2 diabetes, both with subclinical atherosclerosis and the B LDL phenotype. The analyses included surface-enhanced laser adsorption/ionization, time-of-flight mass spectrometry, and subsequent identification by mass spectrometry or immunoblotting and were carried out in LDL subclasses isolated by ultracentrifugation in deuterium oxide gradients with near physiological salt concentrations. The sdLDLs of both types of patients were enriched in apolipoprotein C-III (apoC-III) and were depleted of apoC-I, apoA-I, and apoE compared with matched healthy controls with the A phenotype. The LDL complexes formed in serum from patients with diabetes with the arterial proteoglycan (PG) versican were also enriched in apoC-III. In addition, there was a significant correlation between the apoC-III content in sdLDL in patients and the apparent affinity of their LDLs for arterial versican. The unique distribution of exchangeable apolipoproteins in the sdLDLs of the patients studied, especially high apoC-III, coupled with the augmented affinity with arterial PGs, may contribute to the strong association of the dyslipidemia of insulin resistance with increased risk for cardiovascular disease.     

几种哺乳类动物胸主动脉中的蛋白聚糖

本文报道了北京(As高发)、广西南宁(As低发)两地区的人、兔(“易感”As)及狗(“不易感”As)胸主动脉中三种蛋白聚糖(CSPG,DSCSPG及HSPG)的含量。结果表明:a.广西南宁样品的总PGs,HSPG及DSCSPG含量均高于北京,尤以HSPG差异显著。b.兔、狗胸主动脉PG总量均低于人的。其中HSPG,CSPG含量及百分率以及兔的DSCSPG含量亦低于人的相应PG含量,而狗的DSCSPG含量与人的类似。c.南宁人及狗的DSCSPG含量及相对百分率虽高,但其DSCSPG中DS链所占相对百分率低于北京人和兔的。以上结果提示动脉壁PG质与量的差异可能与AS发病有关。     

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte-derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]-oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA-induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal-regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.     

Phospholipase A2 and small, dense low-density lipoprotein

High levels of small, dense LDL in plasma are associated with increased risk for cardiovascular disease. There are some biochemical characteristics that may render small, dense LDL particles more atherogenic than larger, buoyant LDL particles. First, small, dense LDL particles contain less phospholipids and unesterified cholesterol in their surface monolayer than do large, buoyant LDL particles. This difference in lipid content appears to induce changes in the conformation of apolipoprotein B-100, leading to more exposure of proteoglycan-binding regions. This may be one reason for the high-affinity binding of small, dense LDL to arterial proteoglycans. Reduction of the phospholipid content in the surface monolayer LDL by treatment with secretory phospholipase A2 (sPLA2) forms small, dense LDL with an enhanced tendency to interact with proteoglycans. Circulating levels of sPLA2-IIA appears to be an independent risk factor for coronary artery disease and a predictor of cardiovascular events. In addition, in-vivo studies support the hypothesis that sPLA2 proteins contribute to atherogenesis and its clinical consequences. These data suggest that modification of LDL by sPLA2 in the arterial tissue or in plasma may be a mechanism for the generation of atherogenic lipoprotein particles in vivo, with a high tendency to be entrapped in the arterial extracellular matrix.     

Lipoprotein subclass metabolism in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is associated with increased synthesis of triglycerides and cholesterol coupled with increased VLDL synthesis in the liver. In addition, increased cholesterol content in the liver associates with NASH. Here we study the association of lipoprotein subclass metabolism with NASH. To this aim, liver biopsies from 116 morbidly obese individuals [age 47.3 ± 8.7 (mean ± SD) years, BMI 45.1 ± 6.1 kg/m², 39 men and 77 women] were used for histological assessment. Proton NMR spectroscopy was used to measure lipid concentrations of 14 lipoprotein subclasses in native serum samples at baseline and after obesity surgery. We observed that total lipid concentration of VLDL and LDL subclasses, but not HDL subclasses, associated with NASH [false discovery rate (FDR) < 0.1]. More specifically, total lipid and cholesterol concentration of VLDL and LDL subclasses associated with inflammation, fibrosis, and cell injury (FDR < 0.1), independent of steatosis. Cholesterol concentration of all VLDL subclasses also correlated with total and free cholesterol content in the liver. All NASH-related changes in lipoprotein subclasses were reversed by obesity surgery. High total lipid and cholesterol concentration of serum VLDL and LDL subclasses are linked to cholesterol accumulation in the liver and to liver cell injury in NASH.