Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.     

Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene

Scavenger receptor type B class I (SR-BI), initially identified as a receptor that recognizes low density lipoprotein (LDL), was recently shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters in liver and steroidogenic tissues. To evaluate effects on atherosclerosis, transgenic mice with liver-specific overexpression of SR-BI (SR-BI Tg mice) have been crossed onto LDL receptor-deficient backgrounds. To induce atherosclerosis in a setting of moderate hypercholesterolemia, heterozygous LDL receptor-deficient mice (LDLR1) were fed a high fat/cholesterol/bile salt diet, and homozygous LDL receptor knock-outs (LDLR0) were fed a high fat/cholesterol diet. LDLR1/SR-BI Tg mice showed decreases in VLDL, LDL, and HDL cholesterol and a significant 80% decrease in mean lesion area in the aortic root compared with LDLR1 mice (female LDLR1 74, 120 micrometers(2) versus LDLR1/SR-BI Tg 12, 667 micrometers(2); male 25, 747 micrometers(2)++ versus 5, 448 micrometers(2), respectively). LDLR0/SR-BI Tg mice showed decreased LDL and HDL cholesterol but increased VLDL cholesterol and no significant difference in extent of atherosclerosis compared with LDLR0 mice. Combined data analysis showed a strong correlation between atherosclerotic lesion area and the VLDL+LDL cholesterol level but no correlation with HDL level. These studies demonstrate a strong anti-atherogenic potential of hepatic SR-BI overexpression. In mice with marked overexpression of SR-BI, the protective effect appears to be primarily related to the lowering of VLDL and LDL cholesterol levels.     

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.     

Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species

Macrophage foam cells of atherosclerotic lesions store lipid in lysosomes and cytoplasmic inclusions. Oxidized low density lipoprotein (oxLDL) has been proposed to be the atherogenic particle responsible for the free and esterified cholesterol stores in macrophages. Currently, however, there is a paucity of data showing that oxLDL can induce much cholesterol accumulation in cells. The present studies compare the ability of mildly oxLDL (TBARS = 5 to 10 nmols/mg LDL protein) with acetylated LDL to induce free cholesterol (FC) and esterified cholesterol (EC) accumulation in pigeon, THP-1, and mouse macrophages. Mildly oxLDL stimulated high levels of loading comparable to acLDL where the cellular cholesterol concentrations ranged from 160 to 420 microg/mg cell protein with EC accounting for 52-80% of the cholesterol. Pigeon and THP-1 macrophages stored most (60-90%) of oxLDL cholesterol (both FC and EC) in lysosomes, and the bulk (64-88%) of acLDL cholesterol in cytoplasmic inclusions. Consistent with lysosomal accumulation, cholesterol esterification was 75% less in THP-1 macrophages enriched with oxLDL cholesterol compared with acLDL. Furthermore, addition of an acyl-CoA:cholesterol acyltransferase inhibitor did not significantly affect either cholesterol loading or the percent distribution of FC and EC in THP-1 and pigeon cells incubated with oxLDL. Surprisingly, mouse macrophages stored most of oxLDL (71%) and acLDL (83%) cholesterol within cytoplasmic inclusions. Also, in mouse macrophages, esterification paralleled cholesterol loading, and was 3-fold more in oxLDL treated cells compared with acLDL treated cells. Inhibition of ACAT led to a 62% and 90% reduction in the %EC in oxLDL and acLDL treated mouse macrophages, respectively. The results demonstrate that mildly oxidized low density lipoprotein (oxLDL) stimulates macrophage foam cell formation and lipid engorgement of lysosomes. However, the fate of oxLDL cholesterol markedly differs in macrophages of different species.     

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.     

Ligand specificity of LOX-1, a novel endothelial receptor for oxidized low density lipoprotein

Endothelial dysfunction, or activation, elicited by oxidized low density lipoprotein (Ox-LDL) and its lipid constituents has been shown to play a key role in the pathogenesis of atherosclerosis. We recently have identified a novel receptor for Ox-LDL-designated lectin-like Ox-LDL receptor (LOX-1) in vascular endothelial cells. To examine ligand specificity of LOX-1, we established CHO cell lines stably expressing both human and bovine LOX-1 (LOX-1-CHO). LOX-1-CHO bound and degraded 125I-labeled Ox-LDL but did not significantly degrade 125I-labeled acetylated LDL (Ac-LDL). Fucoidin and maleylated BSA (M-BSA), which inhibit 125I-Ox-LDL binding to class A scavenger receptors, did not inhibit 125I-Ox-LDL binding or degradation in LOX-1-CHO. Polyinosinic acid and carrageenan, in contrast, significantly reduced 125I-Ox-LDL binding to LOX-1-CHO by 62% and 60%, respectively. Delipidated and untreated 125I-Ox-LDL were bound and degraded equally in LOX-1-CHO; furthermore, excess amounts of unlabeled, delipidated Ox-LDL inhibited binding and degradation of untreated 125I-Ox-LDL. Taken together, LOX-1 is a receptor for Ox-LDL but not for Ac-LDL. LOX-1 recognizes protein moiety of Ox-LDL, and its ligand specificity is distinct from other receptors for Ox-LDL, including class A and B scavenger receptors.     

The scavenger receptor serves as a route for internalization of lysophosphatidylcholine in oxidized low density lipoprotein-induced macrophage proliferation

We have recently demonstrated that the growth of murine macrophages is induced by oxidized low density lipoprotein (Ox-LDL) and that lysophosphatidylcholine (lyso-PC), a major phospholipid component of Ox-LDL, plays an essential role in its mitogenic effect. The present study was undertaken to further characterize the role of the macrophage scavenger receptor (MSR) in Ox-LDL-induced macrophage growth. The growth-stimulating effect of Ox-LDL on murine resident peritoneal macrophages was inhibited by maleylated bovine serum albumin (maleyl-BSA), a non-lipoprotein ligand for MSR but a poor carrier of lyso-PC, while maleyl-BSA itself failed to induce macrophage growth even in the presence of lyso-PC. Moreover, it competitively inhibited the endocytic uptake of 125I-Ox-LDL and the specific uptake of lyso-PC by MSR, whereas nonspecific lyso-PC transfer to cells was not affected. Furthermore, the Ox-LDL-induced cell growth of peritoneal macrophages obtained from MSR knockout mice was significantly weaker than that of macrophages obtained from their wild-type littermates. Our results suggest that the MSR is an important and efficient internalization pathway for lyso-PC in Ox-LDL-induced macrophage growth.     

Macrophage oxidative modification of low density lipoprotein occurs independently of its binding to the low density lipoprotein receptor

The oxidative modification of low density lipoproteins (LDL) by arterial wall cells is thought to contribute to atherogenesis. Monocyte/macrophages, among other arterial wall cells, oxidatively modify LDL to a form that is recognized by scavenger/oxidized LDL receptors. It has recently been suggested that LDL binding to the LDL receptor (B/E receptor) is essential for macrophage-mediated oxidation of LDL. In the present study, we compared the ability of resident peritoneal macrophages from LDL-R-deficient (LDLR-/-) mice to oxidize LDL with that of resident peritoneal macrophages from C57B6 mice. The LDLR-/- macrophages oxidized LDL at least as rapidly as did the C57B6 macrophages both in F-10 medium and in Dulbecco's modified Eagle's medium supplemented with 1 microM copper (DMEM-Cu2+). Studies were also conducted to examine the effect of preincubation of LDLR-/- and C57B6 macrophages with 10% lipoprotein-deficient serum (LPDS), which up-regulates LDL receptors, or with acetylated LDL (Ac-LDL), which increases cellular cholesterol and down-regulates LDL receptors. Preincubation with 10% LPDS had no significant effect on subsequent LDL oxidation by either type of cells in F10 medium, but the C57B6 cells did show a small (18%) but significant increase in LDL oxidation in DMEM-Cu2+. Preincubation with 50 micrograms/ml Ac-LDL in F10 medium had no effect on LDL oxidation by either LDLR-/- or C57B6 macrophages. Preincubation with 100 micrograms/ml Ac-LDL had no effect on subsequent LDL oxidation by C57B6 cells but, unexpectedly, caused a modest (26%) fall in LDL oxidation by the receptor-negative cells. Using DMEM-Cu2+ medium, preincubation with Ac-LDL reduced LDL oxidation substantially (50-66%) but the effect was just as great in LDL-R negative cells (59-66%) as in C57B6 cells (50-58%), suggesting that the effect is not due to changes in LDL receptor density. It may be related somehow to the Ac-LDL-induced increase in cell cholesterol content. The data demonstrate that the absence of LDL receptors does not reduce the ability of macrophages to oxidize LDL and that LDL binding to LDL receptors is not an essential requirement for macrophage oxidation of LDL.     

Mechanism of uptake of copper-oxidized low density lipoprotein in macrophages is dependent on its extent of oxidation

Several investigators have reported nonreciprocal cross-competition between unlabeled acetyl low density lipoprotein (LDL) and oxidized LDL for the degradation of the corresponding labeled LDLs. The failure of acetyl LDL to compete fully for the degradation of oxidized LDL has been interpreted as evidence for additional receptor(s) specific for oxidized LDL. In the present study, it is demonstrated that the ability of oxidized LDL to compete for the degradation of acetyl LDL is determined largely by its extent of oxidation. Extensively oxidized LDL competed for 90% of acetyl LDL degradation in murine macrophages, and hence there appears to be no pathway in these cells that is specific for acetyl LDL but not oxidized LDL. The reciprocal situation (competition by acetyl LDL for uptake and degradation of oxidized LDL) proved to be more complicated. Oxidized LDL is known to be susceptible to aggregation, and less than half of the aggregates found in the present experiments were large enough to be removed by filtration or centrifugation at 10,000 x g. When oxidized LDL was prepared under conditions that resulted in minimal aggregation, acetyl LDL competed for greater than 80% of oxidized LDL degradation. With more extensive oxidation and aggregation of LDL, acetyl LDL only competed for about 45% of oxidized LDL degradation, while polyinosinic acid remained an effective competitor. Individual preparations of oxidized LDL that differed in degree of oxidation were separated into aggregated and nonaggregated fractions, and it was shown that both fractions were competed to a similar degree by acetyl LDL in mouse peritoneal macrophages and in Chinese hamster ovary cells transfected with human scavenger receptor type I cDNA. Hence, aggregation by itself did not alter the apparent rate of uptake by the scavenger receptor pathway. These results indicate that the extent of oxidation of LDL affects its mechanism of uptake and that about half of the uptake of very extensively oxidized LDL appears to be via a pathway distinct from the scavenger receptor type I/II. The uptake of very extensively oxidized LDL was not affected by cytochalasin D, an inhibitor of phagocytosis. As well, it was not affected by an antibody to CD36 in human monocyte-derived macrophages or in THP-1 cells, suggesting that this alternate pathway does not involve CD36.     

High affinity presentation of an autoantigenic peptide in type I diabetes by an HLA class II protein encoded in a haplotype protecting from disease

Tissue composition and the distribution of body mass are described for four genera of East African Bovidae (Madoqua, Gazella, Damaliscus, Hippotragus) with supporting data from four others (Neotragus, Oryx, Tragelaphus, Connochaetes). These species are high in muscle mass, an adaptation convergent with other high-speed terrestrial cursors, bounders, and saltators. The segments below the elbow/cubitus and knee/stifle/genu joints in small bovids are both lighter in percent of total body mass (8.6% TBM) and less heavily muscled (10-15% of total limb musculature) than those segments in macaques (13.6% TBM, 20-25% of the limb musculature). Bovid species differ from one another in the regional distribution of muscle mass. Madoqua kirkii (4-5 kg) concentrates muscle in the lumbar extensors and hindlimbs; large species such as Damaliscus doreas (50-60 kg) and Hippotragus niger (160-220 kg) distribute it more evenly between the lumbar and cervical regions and between the hindlimbs and forelimbs. Gazella dorcas (10-20 kg) is quantitatively intermediate in those characteristics. The redistribution of muscle mass with increasing size correlates with the loss of axial bending of the vertebral column: in small, hindlimb dominant, 'dorsomobile' species such as Madoqua sagittal mobility increases stride length through 'extended' suspension; in large 'dorsostable' species such as Damaliscus and Hippotragus the vertebral column resists bending, consequently abbreviating or omitting this non-contact phase of the gait cycle. Locomotor adaptation as it is reflected in size, shape, and musculoskeletal structure is the key to habitat choice, dietary specialization, social structure, and male agonistic behavior and, therefore, central to the fabric of behavioral ecology.     

Phosphatidylinositol 3-kinase is involved in the induction of macrophage growth by oxidized low density lipoprotein

Early atherosclerotic lesions are characterized by the presence of cholesterol-rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the development of early lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this induction of macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization and degradation, interaction with integral plasma membrane proteins coupled to signaling pathways, or direct or indirect activation of growth factor receptors on the cell surface (e.g. GM-CSF receptor) through an autocrine/paracrine mechanism. The present study was undertaken to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. Extensively oxidized LDL increased protein-tyrosine phosphorylation and caused a 2-fold increase in phosphatidylinositol (PI) 3-kinase activity in phorbol ester-pretreated THP-1 cells (a human monocyte-like cell line). Similar concentrations of native LDL had no effect. Oxidized LDL also stimulated growth of resident mouse peritoneal macrophages, and this effect was reduced by 40-50% in cells treated with PI 3-kinase inhibitors (100 nM wortmannin or 20 microM LY294002). These results suggest that PI 3-kinase mediates part of the mitogenic effect of oxidized LDL, but parallel pathways involving other receptors and signal transduction pathways are likely also involved.     

Attenuation of plasma low density lipoprotein cholesterol by select 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in mice devoid of low density lipoprotein receptors

Low density lipoprotein (LDL) reduction independent of LDL receptor regulation was investigated using HMG-CoA reductase inhibitors in LDL receptor-deficient mice. In males, LDL cholesterol dose-dependently decreased with atorvastatin treatment after 1 week. As untreated mice grew older, their LDL cholesterol progressively rose above basal levels, but was quelled with atorvastatin treatment. In females, atorvastatin treatment time-dependently decreased LDL cholesterol levels and induced hepatic HMG-CoA reductase activity. Unlike males, cholesterol-lowering effects of the drug were sustained in females. Lovastatin, simvastatin, and pravastatin also reduced total and LDL cholesterol; however, additional studies in females demonstrated that atorvastatin caused the greatest dose-dependent and sustained effect after 2 weeks. In females, hepatic HMG-CoA reductase mRNA inversely correlated with LDL cholesterol lowering, with atorvastatin showing the greatest increase in mRNA levels (17.2-fold), followed by lovastatin (10.7-fold), simvastatin (4.1-fold), and pravastatin (2.5-fold). Atorvastatin effects on lipoprotein production were determined after acute (1 day) or chronic (2 week) treatment prior to intraperitoneal injection of Triton WR1339. Acute treatment reduced cholesterol (-29%) and apoB (-16%) secretion, with no change in triglyceride secretion. In contrast, chronic treatment elevated cholesterol (+20%), apoB (+31%), and triglyceride (+57%) secretion. Despite increased cholesterol and apoB secretion, plasma levels were reduced by 51% and 46%, respectively. Overall, under acute or chronic conditions, apoB paralleled cholesterol secretion rates, and triglyceride to cholesterol secretion ratios were elevated by 38% and 32%, respectively. We propose that atorvastatin limits cholesterol for lipoprotein assembly, which is compensated for by triglyceride enrichment. In addition, with either acute or chronic atorvastatin treatment, apoB-100 secretion was blocked, and compensated for by an increased secretion of apoB-48. The apoB-48 particles produced are cleared by LDL receptor-independent mechanisms, with an overall effect of reducing LDL production in these mice. These studies support the idea that HMG-CoA reductase inhibitors modulate lipoprotein levels independent of LDL receptors, and suggest they may have utility in hyperlipidemias caused by LDLreceptor disorders.     

Induction of murine macrophage growth by oxidized low density lipoprotein is mediated by granulocyte macrophage colony-stimulating factor

We have examined whether certain secreted factor(s) is involved in oxidized low density lipoprotein (Ox-LDL)-induced murine macrophage growth. An antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) effectively inhibited Ox-LDL-induced macrophage growth by >80%. Ox-LDL as well as phospholipase A2-treated acetylated LDL enhanced mRNA levels and protein release of GM-CSF from macrophages, while neither acetylated LDL nor lysophosphatidylcholine (lyso-PC) showed such effects. The maximal induction of GM-CSF by Ox-LDL was noted at 4 h, followed by a time-dependent decrease to a basal level within 24 h. Ox-LDL-induced macrophage growth was inhibited by 75% by replacement of the culture medium at 24 h by a fresh medium containing the same concentration of Ox-LDL, when GM-CSF had already returned to the basal level. Thus, a cytokine(s) other than GM-CSF is also expected to participate in Ox-LDL-induced macrophage growth in a later phase. The Ox-LDL-induced GM-CSF release was inhibited by calphostin C, a protein kinase C inhibitor, and was significantly reduced in macrophages from the knockout mice lacking class A, type I and type II macrophage scavenger receptors (MSR-AI/AII). These results taken together indicate that effective endocytosis of lyso-PC of Ox-LDL by macrophages through MSR-AI/AII and subsequent protein kinase C activation have led to GM-CSF release into the medium which may play a priming role in conjunction with other cytokines in Ox-LDL-induced macrophage growth.     

The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock

During gram-negative bacterial infections, lipopolysaccharide (LPS) stimulates primed macrophages (Mphi) to release inflammatory mediators such as tumor necrosis factor (TNF)-alpha, which can cause hypotension, organ failure, and often death. Several different receptors on Mphi have been shown to bind LPS, including the type A scavenger receptor (SR-A). This receptor is able to bind a broad range of polyanionic ligands such as modified lipoproteins and lipoteichoic acid of gram-positive bacteria, which suggests that SR-A plays a role in host defense. In this study, we used mice lacking the SR-A (SRKO) to investigate the role of SR-A in acquired immunity using a viable bacillus Calmette Guérin (BCG) infection model. We show that activated Mphi express SR-A and that this molecule is functional in assays of adhesion and endocytic uptake. After BCG infection, SRKO mice are able to recruit Mphi to sites of granuloma formation where they become activated and restrict BCG replication. However, infected mice lacking the SR-A are more susceptible to endotoxic shock and produce more TNF-alpha and interleukin-6 in response to LPS. In addition, we show that an antibody which blocks TNF-alpha activity reduces LPS-induced mortality in these mice. Thus SR-A, expressed by activated Mphi, plays a protective role in host defense by scavenging LPS as well as by reducing the release by activated Mphi of proinflammatory cytokines. Modulation of SR-A may provide a novel therapeutic approach to control endotoxic shock.     

The attenuated elevation of cytoplasmic calcium concentration following the uptake of low density lipoprotein in type C Niemann-Pick fibroblasts

In Niemann-Pick disease type C fibroblasts, the deficiency of cholesterol esterification has been reported. In this experiment, we detected the attenuated elevation of cytoplasmic calcium concentration following low density lipoprotein uptake in the fibroblasts. Moreover, we administered calcium channel agonist (0.5 microM YC-170) and calcium channel antagonists to the fibroblasts. YC-170 improved the attenuated elevation of calcium concentration and the deficient cholesterol esterification by 40% and 90% respectively. Calcium channel antagonists decreased the cholesterol esterification in normal and affected fibroblasts. These data indicated that the attenuated elevation of cytoplasmic calcium concentration was strongly related to the etiology of this disease.     

A nonexchangeable apolipoprotein E peptide that mediates binding to the low density lipoprotein receptor

ApoE is a 34-kDa apoprotein that mediates lipoprotein binding to the low density lipoprotein (LDL) receptor and to the LDL receptor-related protein. Receptor binding is mediated by a highly basic, alpha-helical sequence of approximately 15 amino acids that interacts with cysteine-rich repeat regions of the receptor. To determine the relationship between the receptor binding and lipid associating properties of apoE, we have synthesized a series of apoE peptides containing all (residues 129-169) or part (residues 139-169, 144-169, and 148-169) of the receptor-binding domain. The lipophilicity of these peptides was increased by modification of their N termini by acylation with either palmitic acid (C16-apoE peptide) or the N,N-distearyl derivative of glycine (diC18-Gly-apoE peptide). The unmodified peptides demonstrated low affinity for lipid surfaces (Kd > 10(-5) M) and moderate alpha-helicity in the presence of lipid (40%) and had no effect on LDL uptake by fibroblasts. N-Palmitoyl peptides had increased affinity for lipid (Kd approximately 10(-6) M) and increased alpha-helicity (55%) in the presence of lipid. The addition of the C16-apoE-(129-169)-peptide to 125I-LDL enhanced its uptake and degradation by fibroblasts 8-10-fold; however, < 50% of the degradation was mediated by the LDL receptor. By contrast, the diC18-Gly-apoE-(129-169)-peptide was essentially nonexchangeable (Kd < or = 10(-9) M) and highly helical (78%) in the presence of lipid. The addition of the diC18-Gly-apoE-(129-169)-peptide to 125I-LDL enhanced the specific uptake and degradation of LDL by both LDL receptor-mediated and non-LDL receptor-mediated mechanisms. Uptake and degradation of methylated LDL containing diC18-Gly-apoE-(129-169) revealed that the lipoprotein-bound peptide is the active agent. In agreement with this finding, a mutant diC18-Gly-apoE peptide (Arg142-->Gln) was much less effective than the wild-type peptide in potentiating binding, uptake, and degradation of 125I-LDL. Complexes of diC18-Gly-apoE-(129-169), apoA-I, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine containing four to six copies of the peptide/particle displayed an affinity for the LDL receptor similar to that of apoE-L-alpha-dimyristoylphosphatidylcholine discs containing four copies of apoE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.     

Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A

The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.     

Chronic administration of dexamethasone results in Fc receptor up-regulation and inhibition of class I antigen expression on macrophages from MRL/lpr autoimmune mice

OBJECTIVE: To assess the influence of hypertensive disorders in pregnancy on the subsequent risk of placental abruption and uterine bleeding of unknown aetiology, and to examine the combined effects of hypertensive disorders and cigarette smoking during pregnancy on the risk of uteroplacental bleeding disorders. DESIGN: Retrospective cohort study. SETTING: Data for this study were derived from the Nova Scotia Atlee Perinatal database, Canada, comprising of women who were delivered in the province between 1980 and 1993. POPULATION: 120,666 pregnancies resulting in singleton births, of which 13,360 pregnancies were complicated by pre-eclampsia and/or chronic hypertension. MAIN OUTCOME MEASURES: Risks and relative risks of placental abruption and uterine bleeding of unknown aetiology in pregnancies complicated by chronic hypertension, mild and severe pre-eclampsia, and chronic hypertension with superimposed pre-eclampsia, each compared with normotensive patients. Adjusted relative risks were obtained through the fit of multivariable logistic regression models based on the method of generalised estimating equations. RESULTS: Chronically hypertensive women had no increased risk of abruption (RR 1.4; 95% CI 0.5-3.6), while women whose pregnancies were complicated by severe pre-eclampsia (RR 3.8; 95% CI 2.1-6.9), and chronic hypertension with superimposed pre-eclampsia (RR 2.8; 95% CI 1.2-6.3) showed strong associations with placental abruption. However, none of the hypertensive disorders were associated with uterine bleeding of unknown aetiology. The association between placental abruption and hypertensive disorders varied by parity. Parous women with chronic hypertension and superimposed pre-eclampsia were at greater risk of placental abruption (aRR 3.8; 95% CI 1.9-7.8) than nulliparous women with chronic hypertension and superimposed pre-eclampsia (aRR 1.6; 95% CI 0.5-4.9). The joint effects of smoking and hypertension had a greater effect on the risk of placental abruption than would have been expected based on their individual effects. CONCLUSIONS: The pattern of association between placental abruption and hypertension varied in relation to the specific type of hypertensive disorder. However, uterine bleeding of unknown aetiology was not associated with hypertension. Findings from this study suggest that placental abruption and uterine bleeding of unknown origin are aetiologically distinct obstetric complications with respect to hypertensive disorders during pregnancy.