The dynamics of macrophage infiltration into the arterial wall during atherosclerotic lesion development in low-density lipoprotein receptor knockout mice

Atherosclerosis is a progressive disease in which macrophages play an essential role. Macrophage infiltration into the arterial wall induces the development of an early atherosclerotic lesion. However, the dynamics of macrophage infiltration into the arterial wall during lesion progression remain poorly understood. In this study, low-density lipoprotein receptor knockout mice were fed a Western-type diet for 3, 6, 9, and 12 weeks to induce the formation of atherosclerotic lesions with different degrees of complexity. Subsequently, these mice underwent transplantation with bone marrow-overexpressing enhanced green fluorescent protein to track donor-derived cells, including macrophages. After 8 weeks of Western-type diet feeding after transplantation, macrophage infiltration was evaluated by immunohistochemical staining of donor-derived macrophages (enhanced green fluorescent protein-positive F4/80(+)) in the aortic roots. We found that the growth of pre-existing initial lesions was mainly caused by continued recruitment of donor-derived macrophages into the arterial wall. Interestingly, macrophage infiltration into pre-existing more advanced lesions was largely impaired, likely because of the formation of fibrous caps. In addition, interference with the expression of macrophage ATP-binding cassette transporter 1, an ATP-binding cassette transporter involved in cellular cholesterol efflux and macrophage recruitment into tissues, affects the infiltration of macrophages into pre-existing early lesions but not into advanced lesions. In conclusion, our data suggest that the dynamics of macrophage infiltration into the arterial wall vary greatly during atherogenesis and, thus, may affect the efficiency of pharmaceutical interventions aimed at targeting macrophage infiltration into the arterial wall.     

Low-density lipoprotein endocytosis. II. Influence of the multivalent ligand cationized ferritin on acetylated low-density lipoprotein endocytosis in cultured cells

Interactions of the basic multivalent ligand cationized ferritin (CF) with cultured cells markedly alter their endocytic function. In this study, the influence of CF treatment on the binding, internalization, and degradation of chemically modified (acetylated) low-density lipoproteins (Ac-LDL) was examined in aortic smooth muscle cells (SMC); and in normal and FH mutant LDL receptor-negative human skin fibroblasts, which lack the Ac-LDL (scavenger) receptor; and in vascular endothelial cells, which normally express the receptor. Although CF treatment of all three cell types at 37 degrees C resulted in the induction of Pronasesensitive, high-capacity, high-affinity binding (Kd = 12.0 +/- 2.0 nM at 4 degrees C) of labeled Ac-LDL, which at 37 degrees C was accompanied by significant internalization and degradation, these processes were not receptor-mediated. CF-induced high-affinity binding was inhibited by unlabeled Ac-LDL, fucoidan, carrageenan, and dextran sulfate but was unaffected by native LDL and albumin and only partially inhibited by acetylated albumin. However, analysis of membrane preparations of the cells for "scavenger" receptor protein by solid-phase filtration assay and Western blotting identified the receptor in endothelial cells and in granuloma (positive control) macrophages, but not in either CF-treated or untreated SMC. In addition, studies with both glutaraldehyde-fixed cells and CF bound to culture dishes indicated that Ac-LDL avidly binds to CF. Further, ultrastructural studies using colloidal gold-conjugated Ac-LDL showed Ac-LDL preferentially binding to CF aggregates on the cell surface. Thus, these studies indicate that treatment of cells with CF induces an endocytic process which, although remarkably similar to the scavenger pathway, is mediated by Ac-LDL binding to membrane-associated CF. These observations have implications in terms of mechanisms that might regulate the endocytosis of modified low-density lipoproteins.     

Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall

Quantitative immunohistochemical techniques were developed for mapping low density lipoprotein (LDL) oxidation within arterial tissue. Antibodies were raised by immunizing rabbits with Cu(2+)-oxidized rabbit LDL. ELISAs showed that they reacted strongly with oxidized rabbit LDL, weakly with other oxidized lipoproteins, and not at all with native LDL. Using optimized histological procedures, the antibodies were applied to sections of calibration gels containing LDL at various concentrations and levels of oxidation, and to sections of aortas from normal and heritable hyperlipidemic rabbits. Binding was measured with a rhodamine-labeled secondary antibody and carefully calibrated techniques of digital imaging fluorescence microscopy. Values obtained using a nonspecific primary antibody were subtracted. Specific binding to calibration sections increased linearly with respect to the concentration of oxidized LDL and the duration of its exposure to Cu2+, approximately linearly with respect to its modified lysine content, and nonlinearly with respect to its relative electrophoretic mobility. Specific staining was detected in sections of aortas from heritable hyperlipidemic but not normal rabbits. In the former, it was higher in the intima than in the media and was greater downstream than upstream of intercostal branch ostia; the average level was lower in those branches with the least intimal thickening but the difference between upstream and downstream regions was larger. These results correlate with the known pattern of lipid deposition in hyperlipidemic rabbit aortas. A small but significant amount of specific staining was observed in sections which were devoid of intimal thickening, which is consistent with LDL oxidation occurring prior to disease or during its earliest stages.     

Influence of native and hypochlorite-modified low-density lipoprotein on gene expression in human proximal tubular epithelium

Inflammatory infiltrates can modify (lipo)proteins via hypochlorous acid/hypochlorite (HOCl/OCl(-)) an oxidant formed by the myeloperoxidase-H(2)O(2)-halide system. These oxidatively modified proteins emerge in tubuli in some proteinuric and interstitial diseases. Human proximal tubular cells (HK-2) were used to confirm the hypothesis of detrimental and differential impact of HOCl-modified low density lipoprotein (HOCl-LDL), an in vivo occurring lipoprotein modification exerting proatherogenic and proinflammatory capacity. HOCl-LDL showed dose-dependent antiproliferative effects in HK-2 cells. Small dedicated cDNA macroarrays were used to identify differentially regulated genes. A rapid increase in the expression of genes involved in reactive oxygen species metabolism and cell stress, eg, heme oxygenase-1, thioredoxin reductase, cytochrome b5 reductase, Gadd 153, amino acid transporter E16, and HSP70 was found after HOCl-LDL treatment of HK-2 cells. In parallel, genes involved in tissue remodeling and inflammation eg, CTGF, VCAM-1, IL-1beta, MMP7, and VEGF were up-regulated. Quantitative RT-PCR verified differential expression of a subset of these genes in microdissected tubulointerstitia from patients with acute tubular damage, progressive proteinuric renal disease, and membranous glomerulonephritis (with declining renal function), but not in stable patients with proteinuria caused by minimal change disease. The demonstration of selective up-regulation of a subgroup of genes if proteinuria is accompanied by the presence of HOCl-modified (lipo)proteins support the potential pathophysiological role of the myeloperoxidase-H(2)O(2)-halide system and HOCl-LDL in renal disease.     

Smoking influences the atherogenic potential of low-density lipoprotein

Summary The possible influence of smoking on the low-density lipoprotein (LDL) and its biological activity was investigated. Plasma LDL was prepared from healthy male smokers and nonsmokers, and oxidized with Cu (11) as prooxidant. Oxidized LDL from smokers generated significantly more lipidperoxidation products, so-called thiobarbituric acid reactive substances (TBARS), when compared to oxidized nonsmoker LDL. Analysis of vitamin E levels in LDL obtained from both smokers and nonsmokers revealed that the vitamin E content of smoker LDL was significantly less than that of nonsmoker LDL. The amounts of cholesteryl esters formed in cultured P388. D.1 macrophages were greater in the presence of smoker LDL than with nonsmoker LDL. The data suggest that some of the proatherogenic effects of smoking may be related to oxidative modification of LDL and alteration of its biological activity.Abbreviations CE cholesterol ester - FC free cholesterol - FCS fetal calf serum - HPLC high performance liquid chromatography - LDL low-density lipoprotein - MDA malondialdehyde - PUFA polyunsaturated fatty acid - SD standard deviation - TBA thiobarbituric acid - TBARS thiobarbituric acid reactive substances - TLC thin-layer chromatography     

The granulovesicular bodies of the arterial wall.

Small, round, membrane-bound electron-dense bodies were observed in the smooth muscle cells and the intimal and medial intercellular spaces of arteries in the dog, rat, and pig under normal and various experimental conditions. These structures, referred to as granulovesicular bodies (GVB), measured from 0.2 to 0.5 mum. in diameter and contained an inner core of granular and vesicular subunits. The intercellular and extracellular forms of the GVB were structurally similar, and in the interstitial spaces they appeared to have a definite spatial relation to the elements of elastic tissue. The GVB were more numerous under experimental than under normal conditions. It is speculated that the GVB are secretory in nature, originating from the smooth muscle cells, and may play a role in the remodeling of arterial elastic fibers.     

Oxidized low-density lipoprotein: a double-edged sword on atherosclerosis

It is well known that atherosclerosis is closely related to lipoprotein metabolism, particularly to the low density lipoprotein (LDL). LDL becomes atherogenic after undergoing oxidation by vascular cells, that transform them into highly bioreactive oxidized LDL (oxLDL). oxLDL is generally though to be involved in foam cell formation, and trigger an array of proatherogenic events. However, there are accumulating evidences that low levels of oxLDL can be atheroprotective through its cytoprotection, modulation of immunity and activation of reverse cholesterol transport. Thus, oxLDL may exert biphasic effects on atherosclerosis, just like a doubled-edged sword.     

Homogenous low-density lipoprotein assay in type III hyperlipoproteinemia

To investigate whether homogenous analysis of serum low-density lipoprotein cholesterol(LDL-C) was applicable in type III hyperlipoproteinemic subjects, 3 reagents were used to estimate LDL-C in 2 cases with apoE 2/2 phenotype. Each measurements were compared to LDL-C levels by ultracentrifugation(density: 1.019-1.063). LDL-C levels by homogenous analysis with any of the reagents were higher than those by ultracentrifugation, and the difference was varied between the reagents: +14-24% by LDL-EX, +29-55% by Cholestest LDL, and +89-115% by Determiner LDL-C. The cross-reaction by the reagents to intermediate density lipoprotein(IDL) and very low density lipoprotein(VLDL) cholesterol was studied in a case; 22% of cholesterol in these fractions was measured by LDL-EX, 32% by Cholestest LDL, and 110% by Determiner LDL-C. A part of the sample serum was stored at -60 degrees C for several days and melted for re-analysis of homogenous LDL-C levels. After the freezing process, IDL fraction pattern on electrophoresis had been changed and homogenous LDL-C levels by all reagents were different from the measurements of native serum. These results indicated that using reagents with low cross-reaction to IDL and VLDL fractions and careful sample handling are important in homogenous LDL-C analysis in type III hyperlipoproteinemia.     

In vitro glycated low-density lipoprotein interaction with human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) was glycated in vitro (5 days, glucose 50 mmol/l), labelled with 125I, and its binding and uptake by human monocyte-derived macrophages studied. Glycation produced lower binding and lower uptake. Competition experiments using unlabelled LDL (control, glycated, and acetyl-LDL) showed that most glycated LDL was taken up by the apolipoprotein-B100: E receptor pathway. Results suggest that less of the glycated LDL may enter the cells via scavenger receptors, and very minute amount via non-saturable receptor-independent pathways.     

Increased expression of high-affinity low-density lipoprotein receptors on human T-blasts

Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL). We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25-5,000 micrograms/ml LDL. Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes. The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 microgram LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts. The LDL receptor activities of individual blood donors' resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells. Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data. Both methods yielded essentially identical dissociation constants (Kd) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1-8.9 nM). In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation, morphology, and low-density lipoprotein metabolism of arterial endothelial cells cultured from normal and diabetic minipigs

Aortic endothelial cells from control and streptozotocin diabetic minipigs were cultured. Both groups of cells exhibited the typical cobblestone-like appearance and gap junction formation. Endothelial cells derived from diabetic minipigs differed, however, from those from control animals by a higher rate of proliferation and a higher percentage of large and often multinucleated cells. In these cells the specific binding of low-density lipoproteins (LDL) to coated pits on the cell surface, the LDL uptake, and the intracellular transport of LDL to lysosomes were visualized by gold-labeled LDL complexes. The binding, internalization, and degradation of LDL by subconfluent, non-contact-inhibited endothelial cells was quantified using 125I-labeled LDL. The LDL metabolism of endothelial cells derived from diabetic animals was increased by about 40% compared to endothelial cells derived from nondiabetic animals.     

Effect of low-density lipoprotein buoyant density and cholesterol content on the formation of lipoprotein(a) particles

Lipoprotein(a) [Lp(a)] is a unique lipoprotein which resembles low-density lipoprotein (LDL) both in lipid composition and the presence of apolipoprotein B-100 (apo B-100). Lp(a) is, however, distinguishable from LDL by the presence of an additional glycoprotein apolipoprotein(a) [apo(a)], which is covalently attached to apo B-100 by a single disulfide bond. It is now generally accepted that Lp(a) assembly is a two-step process in which the initial non covalent interaction between apo(a) and apo B-100 is mediated by the weak lysine binding sites present in kringle IV types 6, 7 and 8 of apo(a). In the present study, we have investigated the effect of LDL heterogeneity on Lp(a) assembly in a group of 111 individuals. The three parameters of LDL composition assessed in this study were the cholesterol content, the apo B content, and the relative flotation rate (a measure of LDL buoyancy and thus size). We found no correlation between the size of LDL particles and the extent of Lp(a) formation; a weak negative correlation was observed between cholesterol content of LDL and Lp(a) formation (P=0.042). This may suggest a role for free (i. e., surface-associated) cholesterol in the ability of LDL to form Lp(a) particles. Received: 1 June 2001 / Accepted: 2 July 2001     

Distribution of magnesium in the arterial wall

1. The Mg, Ca, Na and K content were determined in the rat tail artery in vitro under various experimental conditions.2. The arterial Mg could be varied selectively at a constant [Mg](o) by altering the content of intracellular compounds capable of forming complexes with Mg(2+) such as nucleoside-5'-triphosphates or citrate.3. The release of tissue Mg during incubation in Mg-free solutions was found to consist of a fast and a slow component. The size of the fast component was dependent on the original [Mg](o). The size of the slow component was dependent on the content of intracellular compounds forming complexes with Mg(2+). The rate of efflux of the slow component could be increased by increasing [Mg(2+)](i), by removing external Ca, or by making the artery metabolically inactive.4. The results seem to indicate that [Mg(2+)](i) in metabolically active vascular smooth muscle cells is maintained at a level of ca. 10(-4)M.