Beta-very Low Density Lipoprotein Research Articles

The effect of cholesterol overload on mouse kidney and kidney-derived cells

Introduction: Dyslipidemia is one of the onset and risk factors of chronic kidney disease and renal function drop is seen in lipoprotein abnormal animal models. However, the detailed molecular mechanism of renal lipotoxicity has not been clarified. Therefore, the present study aimed to investigate the influence of cholesterol overload using mouse kidney tissue and kidney-derived cultured cells.Methods: C57BL/6 mice were fed normal diet (ND) or 1.25% cholesterol-containing high-cholesterol diet (HCD) for 11 weeks, and we used megalin as a proximal tubule marker for immunohistology. We added beta-very low density lipoprotein (βVLDL) to kidney-derived cells and examined the effect of cholesterol overload on megalin protein and mRNA expression level, cell proliferation and cholesterol content in cells.Results: In the kidney of HCD mice, the gap between glomerulus and the surrounding Bowman’s capsule decreased and the expression level of megalin decreased. After βVLDL treatment to the cells, the protein expression and mRNA expression level of megalin decreased and cell proliferation was restrained. We also observed an increase in cholesterol accumulation in the cell and free cholesterol/phospholipid ratios increased.Conclusions: These findings suggest that the increased cholesterol load on kidney contribute to the decrease of megalin and the overloaded cholesterol is taken into the renal tubule epithelial cells, causing suppression on cell proliferation, which may be the cause of kidney damage.

Alternative Splicing in the Ligand Binding Domain of Mouse ApoE Receptor-2 Produces Receptor Variants Binding Reelin but Not α2-Macroglobulin

LR7/8B and ApoER2 are recently discovered members of the low density lipoprotein (LDL) receptor family. Although structurally different, these two proteins are derived from homologous genes in chicken and man by alternative splicing and contain 7 or 8 LDL receptor ligand-binding repeats. Here we present the cDNA for ApoER2 cloned from mouse brain and describe splice variants in the ligand binding domain of this protein, which are distinct from those present in man and chicken. The cloned cDNA is coding for a receptor with only five LDL receptor ligand-binding repeats, i.e. comprising repeats 1-3, 7, and 8. Reverse transcriptase-polymerase chain reaction analysis of mRNA from murine brain revealed the existence of two additional transcripts. One is lacking repeat 8, and in the other repeat 8 is substituted for by a 13-amino acid insertion with a consensus site for furin cleavage arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4-6. In murine placenta only the form containing repeats 1-3 and 7 and the furin cleavage site is detectable. Analysis of the corresponding region of the murine gene showed the existence of 6 exons coding for a total of 8 ligand binding repeats, with one exon encoding repeats 4-6. Exon trapping experiments demonstrated that this exon is constitutively spliced out in all murine transcripts. Thus, the murine ApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and 4-repeat variants showed that repeats 1-3, 7, and 8 are sufficient for binding of beta-very low density lipoprotein and reelin, but not for recognition of alpha(2)-macroglobulin, which binds to the avian homologue of ApoER2 harboring 8 ligand binding repeats.

Beta-VLDL protects against A beta(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells.

The toxic effects of beta-amyloid (A beta) (1-42), apolipoprotein E (apoE) isoforms, and apoE/A beta complexes were studied in human SH-SY5Y neuroblastoma cells and fibroblasts using MTT reduction. In SH-SY5Y cells, A beta(1-42) gave time-dependent toxicity over 2-48 h, which was reduced by co-incubation with rabbit beta-very low density lipoproteins (beta-VLDL). Human recombinant apoE3 and E4 isoforms were also toxic by themselves and also potentiated A beta effects when used alone, but not when associated with beta-VLDL. None of the treatments were toxic to human fibroblasts. These results suggest that beta-VLDL has a protective role on A beta-induced neurotoxicity and that the status of apoE or the conformation of lipoprotein containing apoE particles may be important for determining the contribution of apoE to neurodegeneration.

Anti-CD69 autoantibodies cross-react with low density lipoprotein receptor-related protein 2 in systemic autoimmune diseases.

We investigated whether autoantibodies to CD69, one of the earliest markers of lymphocyte activation, exist in the sera of patients with systemic autoimmune disease. Serum samples were obtained from patients with rheumatoid arthritis (RA), systemic lupus erythematosus, and Behcet's disease, and were tested for the presence of anti-CD69 autoantibodies by ELISA and Western blotting using rCD69 fusion proteins. IgG-type autoantibodies to CD69 were detected in the sera of 38.3% of the RA patients, 14.5% of the systemic lupus erythematosus patients, and 4.0% of the patients with Behcet's disease. Among those with RA, the anti-CD69 autoantibody-positive patients had a higher serum level of rheumatoid factors and a more accelerated erythrocyte sedimentation rate than the anti-CD69 autoantibody-negative patients. Further, the predominant epitope on the CD69 molecule to which most of the anti-CD69 autoantibody-positive serum samples exclusively reacted, was mapped at the C terminus of CD69. Of interest, this epitope is homologous to a stretch of amino acids in the protein sequence of low-density lipoprotein receptor-related protein 2 (LRP2), which is a receptor for multiple ligands including beta-very low density lipoprotein and is also an autoantigen responsible for Heymann nephritis in rats. The anti-CD69 autoantibody cross-reacted to LRP2 through the homologous amino acid sequence. To our knowledge, this is the first evidence of the existence of anti-CD69 autoantibodies. This autoantibody may modulate the function of CD69- and LRP2-expressing cells.

Low density lipoprotein receptor of macrophages facilitates atherosclerotic lesion formation in C57Bl/6 mice.

Macrophage-derived foam cells play an important role in the initiation and progression of atherosclerosis. To examine the role of the macrophage low density lipoprotein receptor (LDLr) in atherosclerotic lesion formation, bone marrow from LDLr knockout [LDLr(-/-)] mice was transplanted into irradiated wild-type C57Bl/6 [LDLr(+/+)] mice. After 3 months on an atherogenic diet, C57Bl/6 mice, reconstituted with LDLr(-/-) bone marrow, showed a mean lesion area of 34.7 x 10(3)+/-22.4 x 10(3) microm(2) compared with 100. 8 x 10(3)+/-33.0 x 10(3) microm(2) (P<0.001) in control C57Bl/6 mice that were transplanted with LDLr(+/+) bone marrow. There were no significant differences in total serum cholesterol, triglyceride levels, and lipoprotein profiles between the 2 groups. Histochemical analysis of macrophage LDLr expression in the atherosclerotic lesions indicated that C57Bl/6 mice, reconstituted with LDLr(+/+) bone marrow, showed extensive staining of the foam cells in the atherosclerotic lesions, whereas mice reconstituted with LDLr(-/-) bone marrow showed only a few LDLr-positive foam cells. In vitro, peritoneal macrophages isolated from wild-type C57Bl/6 mice were, respectively, 4.7- and 10.7-fold more effective in cell association and degradation of atherogenic (125)I-beta-very low density lipoprotein than were LDLr(-/-) peritoneal macrophages, establishing that the LDLr on macrophages is important for the interaction of macrophages with beta-very low density lipoprotein. It is concluded that the LDLr on macrophages can facilitate the development of atherosclerosis, possibly by mediating the uptake of atherogenic lipoproteins.

A Direct Role for the Macrophage Low Density Lipoprotein Receptor in Atherosclerotic Lesion Formation

To evaluate the contribution of the macrophage low density lipoprotein receptor (LDLR) to atherosclerotic lesion formation, we performed bone marrow transplantation studies in different mouse strains. First, LDLR(-/-) mice were transplanted with either LDLR(+/+) marrow or LDLR(-/-) marrow and were challenged with an atherogenic Western type diet. The diet caused severe hypercholesterolemia of a similar degree in the two groups, and no differences in the aortic lesion area were detected. Thus, macrophage LDLR expression does not influence foam cell lesion formation in the setting of extreme LDL accumulation. To determine whether macrophage LDLR expression affects foam cell formation under conditions of moderate, non-LDL hyperlipidemia, we transplanted C57BL/6 mice with either LDLR(-/-) marrow (experimental group) or LDLR(+/+) marrow (controls). Cholesterol levels were not significantly different between the two groups at baseline or after 6 weeks on a butterfat diet, but were 40% higher in the experimental mice after 13 weeks, mostly due to accumulation of beta-very low density lipoprotein (beta-VLDL). Despite the increase in cholesterol levels, mice receiving LDLR(-/-) marrow developed 63% smaller lesions than controls, demonstrating that macrophage LDLR affects the rate of foam cell formation when the atherogenic stimulus is beta-VLDL. We conclude that the macrophage LDLR is responsible for a significant portion of lipid accumulation in foam cells under conditions of dietary stress.

Keishi-bukuryo-gan prevents the progression of atherosclerosis in cholesterol-fed rabbit.

In this study, we examined whether in vivo keishi-bukuryo-gan (a Kampo formulation) could prevent the progression of atherosclerosis in cholesterol-fed rabbits, an animal model for hypercholesterolaemia. Sixteen male Japanese white rabbits (2 kg body weight) were divided into two groups. Group A (n = 8) was fed standard rabbit chow containing 1% cholesterol for 8 weeks. Group B (n = 8) was fed standard rabbit chow containing 1% cholesterol and 1% keishi-bukuryo-gan for 8 weeks. At the end of the experiment, average plasma concentrations of total-cholesterol and IDL-cholesterol were 2055.9 +/- 201.8 mg/dL and 408.1 +/- 62.6 mg/dL in group A and 1950.5 +/- 126.3 mg/dL and 407.6 +/- 56.6 mg/dL in group B, respectively. The percentage of the surface area of the total thoracic aorta with visible plaque was significantly reduced by keishi-bukuryo-gan administration; group A was 33.2% +/- 5.3% and group B was 14.3% +/- 2.9%. beta-very low density lipoprotein (VLDL) and low density lipoprotein (LDL) isolated from cholesterol fed rabbits treated with keishi-bukuryo-gan (group B) were shown to be highly resistant to oxidative modification by cupric ion. Sera isolated from rabbits administered keishi-bukuryo-gan had reduced lipid peroxide formation compared with those from rabbits without keishi-bukuryo-gan. Thus, keishi-bukuryo-gan prevents the progression of atherosclerosis in cholesterol-fed rabbits in vivo by limiting oxidative LDL modification.

Recycling of Apolipoprotein E in Mouse Liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.

Beta-VLDL accumulation in familial dysbetalipoproteinemia is associated with increased exchange or diffusion of chylomicron lipids to apo B-100 containing triglyceride-rich lipoproteins.

To gain more insight into the accumulation of beta-very low density lipoprotein (beta-VLDL) in familial dysbetalipoproteinemia (FD), we followed the courses of the levels of retinyl palmitate (rp), alpha-tocopherol (alpha-T) and apolipoprotein (apo) B-48 in various lipoprotein fractions for up to 48 h in eight patients with FD and six normolipidemic control subjects after an oral fat load (50 g fat/m2 containing 150000 IU of rp and 5000 IU of alpha-T). Alpha-T was added because of its rapid transfer to other lipoproteins. Fasting apo B-48 concentration in FD was normal to strongly elevated, dependent on the fasting lipid concentrations. 3 h after fat loading, total apo B-48 content did not abnormally increase; while the apo B-100 content in the triglyceride-rich lipoprotein fraction remained stable. The levels of both vitamins increased considerably, especially in the remnant fraction (Sf 15-100), which in due course exclusively contained apo B-100 in most hyperlipidemic patients. This, together with the observation that peaks for rp and alpha-T were observed 3-6 h later than for apo B-48 strongly suggests that both vitamins transfer or diffuse rapidly towards the apo B-100 containing VLDL. RP is thus more a marker for this process, which also comprises chylomicron lipids, than a specific marker for chylomicrons. This process, first described here, appears decisive in the pathogenesis of FD.

Differential Cellular Accumulation/Retention of Apolipoprotein E Mediated by Cell Surface Heparan Sulfate Proteoglycans: APOLIPOPROTEINS E3 AND E2 GREATER THAN E4

Isoform-specific effects of apolipoprotein E (apoE) on neurite outgrowth and the cytoskeleton are associated with higher intracellular levels of apoE3 than apoE4 in cultured neurons. The current studies, designed to determine the mechanism for the differential intracellular accumulation or retention of apoE, demonstrate that apoE3- and apoE4-containing beta-very low density lipoproteins (beta-VLDL) possess similar cell binding and internalization and delivery of cholesterol to the cells. However, as assessed by immunocytochemistry, analysis of extracted cellular proteins, or quantitation of 125I-apoE-enriched beta-VLDL, there was a 2-3-fold greater accumulation of apoE3 than apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes (HepG2) after 1-2 h, and this differential was maintained for up to 48 h. ApoE2 also accumulated in Neuro-2a cells to a greater extent than apoE4. The differential effect was mediated by the apoE-enriched beta-VLDL and not by free apoE. Neither the low density lipoprotein receptor nor the low density lipoprotein receptor-related protein was responsible for the differential accumulation of apoE3 and apoE4, since cells deficient in either or both of these receptors also displayed the differential accumulation. The effect appears to be mediated primarily by cell surface heparan sulfate proteoglycans (HSPG). The retention of both apoE3 and apoE4 was markedly reduced, and the differential accumulation of apoE3 and apoE4 was eliminated both in mutant Chinese hamster ovary cells that did not express HSPG and in HSPG-expressing cells treated with heparinase. The data suggest that cell surface HSPG directly mediate the uptake of apoE-containing lipoproteins, that the differential accumulation/retention of apoE by cells is mediated via HSPG, and that there is a differential intracellular handling of the specific apoE isoforms.

Immunolocalization of Acyl-Coenzyme A:CholesterolO-Acyltransferase in Macrophages

Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.

A Minimally Lipidated Form of Cell-derived Apolipoprotein E Exhibits Isoform-specific Stimulation of Neurite Outgrowth in the Absence of Exogenous Lipids or Lipoproteins

Within the central nervous system, apolipoprotein E (apoE) synthesis is increased in response to nerve injury, a finding that may reflect a role for apoE in neuronal remodeling. Recent studies show that apoE3 promotes and apoE4 inhibits neurite outgrowth in cultured neuronal cells. Interestingly, these isoform-specific effects are observed only when apoE is presented to cells in the presence of an exogenous lipid source such as rabbit beta-very low density lipoprotein (beta-VLDL), making it difficult to discern the biologically active form of apoE or to understand the role of the lipid source. In the present study we tested whether a cell-derived lipidated form of apoE can alter neurite outgrowth in the absence of beta-VLDL by constructing Neuro-2a cell lines expressing high levels of apoE. Our results showed that endogenous apoE3 stimulated neurite outgrowth, whereas the endogenous apoE4 isoform was neutral. Furthermore, beta-VLDL antagonized the stimulatory effects of the endogenous apoE3. Characterization of the secreted apoE3 indicated that the neurite outgrowth-stimulating activity could be recovered from culture medium with an anti-apoE immunoaffinity column and was present in a poorly lipidated particle with a density between 1.19 and 1.26 g/ml. These results indicated that the biological activity of apoE3 in stimulating neurite outgrowth was inherent in the cell-derived apoE particle and was not dependent on either (a) an interaction of apoE3 with an artificial lipid source or (b) independent actions of apoE3 and beta-VLDL.

Plasma clearance and biodistribution of oxidatively modified 99mTc-beta-VLDL in rabbits.

The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled beta-very low density lipoprotein (99mTc-beta-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of beta-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized beta-VLDL (99mTc-ox-beta-VLDL) was cleared from the circulation faster (0.362 +/- 0.070/h) than native beta-VLDL (99mTc-nat-beta-VLDL, 0.241 +/- 0.070/h). In contrast, the FCR of 99mTc-ox-beta-VLDL in HC rabbits was lower (0.100 +/- 0.048/h) than that of 99mTc-nat-beta-VLDL (0.163 +/- 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 +/- 0.071% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.116 +/- 0.057% injected dose/g tissue for 99mTc-ox-beta-VLDL) than in C rabbits (0.301 +/- 0.113% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.305 +/- 0.149% injected dose/g tissue for 99mTc-ox-beta-VLDL). The uptake of 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-beta-VLDL: 0.033 +/- 0.012% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.039 +/- 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-beta-VLDL: 0.023 +/- 0.010% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.019 +/- 0.010% injected dose/g tissue). However, 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-beta-VLDL than 99mTc-nat-beta-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-beta-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-beta-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-beta-VLDL.

Apolipoprotein E-containing High Density Lipoprotein Promotes Neurite Outgrowth and Is a Ligand for the Low Density Lipoprotein Receptor-related Protein

Presence of the epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease (AD), although the mechanism(s) by which it confers this risk is unknown. ApoE may play a direct role in AD neuropathology by modulating neuronal structure. We previously showed that apoE3-containing beta-very low density lipoprotein (beta-VLDL) can stimulate neurite outgrowth to a significantly greater extent than apoE4-enriched beta-VLDL in a central nervous system-derived neuronal cell line and that this effect is mediated by interaction with the low density lipoprotein receptor-related protein (LRP). To determine whether similar differences exist when apoE is associated with other lipoprotein particles, the effects of high density lipoprotein (HDL) derived from plasma and cerebrospinal fluid were defined. ApoE3-enriched HDL significantly enhanced neurite outgrowth as compared with apoE4-enriched HDL, and the majority of this stimulation was blocked in the presence of the receptor-associated protein or a neutralizing antibody to LRP. We also found that cholesterol esterification in the presence of apoE-containing plasma HDL was attenuated in fibroblasts lacking LRP. Therefore, apoE-containing HDL can serve as an LRP ligand, and apoE isoform-specific effects on neurite outgrowth are observed when HDL is the carrier particle.

The Distal Pathway of Lipoprotein-induced Cholesterol Esterification, but Not Sphingomyelinase-induced Cholesterol Esterification, Is Energy-dependent

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.

Components of the protein fraction of oxidized low density lipoprotein stimulate interleukin-1 alpha production by rabbit arterial macrophage-derived foam cells.

Oxidized low density lipoproteins (oxLDL) (0.5-50 micrograms/ml) generated from both rabbit and human LDL stimulated the production of interleukin-1 alpha (IL-1 alpha) by as much as 2- and 6-fold, respectively, as compared to native LDL after a 2-h incubation with macrophage-derived foam cells isolated from the balloon-injured arteries of cholesterol-fed rabbits. Northern blot analyses confirmed that there was also an increase in the mRNA for IL-1 alpha and IL-beta in response to oxLDL in the isolated foam cells. The stimulation of IL-1 expression and production was not due to the contamination of the oxLDL preparations with endotoxin as neither the amount of endotoxin found to be associated with the lipoproteins nor amounts up to 1 ng/ml stimulated IL-1 alpha production to the same degree as oxLDL. Neither oxidized beta-very low density lipoprotein (VLDL) nor oxidized high density lipoprotein (HDL) stimulated IL-1 alpha production by the foam cells. Furthermore, acetyl-LDL had a very limited stimulatory effect, but other known ligands of the scavenger receptor such as maleylated-BSA, polyinosinic acid, and fucoidin elicited maximal IL-1 alpha responses. Fractionation of the oxLDL into lipid- and protein-soluble fractions showed that there was some stimulatory activity in the lipid phase but that known products of lipid peroxidation such as 9- and 13-HODE had no effect when added independently of lipoproteins. When added in combination with native LDL, only 13-HODE stimulated IL-1 alpha production. The delipidated apolipoprotein fragments of oxLDL that had been solubilized in beta-octylglucoside stimulated the production of IL-1 alpha by the foam cells to a greater degree than the lipid extract, while reductively methylated oxLDL did not. These data suggest that interactions of components of both the lipid- and protein-soluble fractions of oxLDL with scavenger receptors or potentially with surface proteins that bind oxLDL may induce production of IL-1 by arterial macrophages.

Lipid-regulating action of gemfibrozil in the stroke-prone spontaneously hypertensive rat.

1. Gemfibrozil (Lopid) is extensively used as lipid-regulating agent in the Western World, and its beneficial effect is demonstrated in human studies such as the Helsinki Heart Study. However, the mechanism of its hypolipidaemic action is not fully understood. In the present paper, to elucidate the hypolipidaemic mechanism, we examined the effects of gemfibrozil on lipid metabolism in the normocholesterolaemic and hypercholesterolaemic stroke-prone spontaneously hypersensitive rat (SHRSP). 2. Gemfibrozil effectively increased high density lipoprotein (HDL) subfraction rich in apoE (apoE-HDL) and significantly decreased very low density lipoprotein (VLDL) in normocholesterolaemic SHRSP. In the liver of normocholesterolaemic SHRSP, gemfibrozil significantly reduced the activity of microsomal acyl-CoA:cholesterol acyltransferase. 3. Gemfibrozil markedly reduced atherogenic beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) in hypercholesterolaemic SHRSP fed a high-fat and high-cholesterol diet (HFC diet). On the other hand, it significantly increased the contents of apoA-I, A-IV and E in the HDL fraction compared with the control group, suggesting that gemfibrozil effectively increases anti-atherogenic HDL subfractions rich in apoA-I, A-IV or E. In the liver of hypercholesterolaemic SHRSP, gemfibrozil markedly prevented lipid accumulation.

Alpha 2-macroglobulin receptor mediates binding and cytotoxicity of plant ribosome-inactivating proteins.

It has been proposed that unconjugated type I ribosome-inactivating proteins (RIP) enter cells through passive mechanisms such as fluid-phase pinocytosis. However, some observations, such as the difference in sensitivity to type I RIP among different cell types, and the organ-specific toxicity of type I RIP, indicate a specific mechanism for the entry of these proteins into target cells. The alpha 2-macroglobulin receptor (alpha 2MR) is responsible for the binding and endocytosis of several ligands, including alpha 2-macroglobulin/proteinase complexes, plasminogen-activator-inhibitor complexes, apoE-enriched beta-very low density lipoproteins, and lipoprotein lipase. Here we demonstrate that saporin, a potent type I RIP, binds specifically to purified alpha 2MR and the binding is prevented by some alpha 2MR ligands. Moreover, the occupancy of specific ligand-binding sites on cell surface alpha 2MR decreases the cytotoxicity of saporin. The A chain of ricin, a type II RIP, also interacts with alpha 2MR. This, and the fact that saporin and ricin A chain both interact also with alpha 2-macroglobulin, indicates a general mechanism of complex interactions between RIP and cellular membranes that is mediated by alpha 2-macroglobulin and the alpha 2MR system.

Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans.

Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta-VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo.

Effects of Efonidipine Hydrochloride on Cholesterol Estérification Mediated by Beta-Very Low Density Lipoprotein in J774 Macrophages

The effects of efonidipine hydrochloride (efonidipine), a dihydropyridine calcium antagonist, on the cholesterol ester metabolism induced by beta-migrating very low density lipoprotein (β-VLDL) in J774 macrophages were studied. The cholesteryl ester content in the macrophages was increased by incubation with β-VLDL, and the increase was inhibited by efonidipine. Oleic acid incorporation into cellular cholesteryl ester was increased by β-VLDL in J774 macrophages. The incorporation at an early phase of β-VLDL induction (0-3 hr) was inhibited by efonidipine. This inhibitory effect of efonidipine was greater at an early phase of β-VLDL induction (0–3 hr) than at a late phase of the induction (8–11 hr). Pretreatment of the cells with efonidipine enhanced the inhibitory effect. Efonidipine also inhibited β-VLDL degradation but not the binding and association in macrophages without pretreatment. β-VLDL binding and association to macrophages were decreased by pretreatment of the cells with efonidipine. β-VLDL metabolism was also decreased by dibutyryl cyclic AMP pretreatment. The decrease of β-VLDL metabolism by efonidipine was prevented by co-treatment with efonidipine and HA1004, a protein kinase A inhibitor. Furthermore, efonidipine increased the intracellular cyclic AMP content in J774 macrophages. These findings suggest that efonidipine suppresses cholesterol ester deposition in atherosclerotic foam cells by inhibiting the modified lipoprotein metabolism and cholesterol esterification mainly through elevation of the cellular cyclic AMP level.