Sandra H. Gianturco

Hypertriglyceridemic Very Low Density Lipoproteins Induce Triglyceride Synthesis and Accumulation in Mouse Peritoneal Macrophages

Triglyceride-rich lipoproteins may be responsible for the lipid accumulation in macrophages that can occur in hypertriglyceridemia. Chylomicrons and very low density lipoproteins (VLDL, total and with flotation constant [S(f)] 100-400) from fasting hypertriglyceridemic subjects induced a massive accumulation of oil red O-positive inclusions in unstimulated peritoneal macrophages. Cell viability was not affected. The predominant lipid that accumulated in cells exposed to hypertriglyceridemic VLDL was triglyceride. Hypertriglyceridemic VLDL stimulated the incorporation of [(14)C]oleate into cellular triglyceride up to ninefold in 16 h, but not into cholesteryl esters. Mass increase in cellular triglyceride was 38-fold. The stimulation of cellular triglyceride formation was dependent on time, temperature, and concentration of hypertriglyceridemic VLDL. By contrast, VLDL, low density, and high density lipoproteins from fasting normolipemic subjects had no significant effect on oleate incorporation into neutral lipids or on visible lipid accumulation.(125)I-Hypertriglyceridemic VLDL (S(f) 100-400) were degraded by macrophages in a dose-dependent manner, with 50 and 100% saturation observed at 3 and 24 mug protein/ml (2.5 and 20 nM), respectively. Hypertriglyceridemic VLDL inhibited the internalization and degradation of (125)I-hypertriglyceridemic VLDL (4 nM) by 50% at 3 nM. Cholesteryl ester-rich VLDL from cholesterol-fed rabbits gave 50% inhibition at 5 nM. Low density lipoproteins (LDL) inhibited by 10% at 5 nM and 40% at 47 nM. Acetyl LDL at 130 nM had no effect. We conclude that the massive triglyceride accumulation produced in macrophages by hypertriglyceridemic VLDL is a direct consequence of uptake via specific receptors that also recognize cholesteryl ester-rich VLDL and LDL but are distinct from the acetyl LDL receptor. Uptake of these triglyceride-rich lipoproteins by monocyte-macrophages in vivo may play a significant role in the pathophysiology of atherosclerosis.

Control of 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity in Cultured Human Fibroblasts by Very Low Density Lipoproteins of Subjects with Hypertriglyceridemia

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 mug of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 mug of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively different from the cellular defect found in familial hypercholesterolemia, since the regulation of HMG-CoA reductase activity is normal in Type III fibroblasts. The metabolic defect in hypertriglyceridemia is related to the triglyceriderich lipoproteins which, free of other lipoproteins, have an enhanced ability to interact with cultured fibroblasts to regulate HMG-CoA reductase activity. These studies suggest that, in hypertriglyceridemia, there is a mechanism for direct cellular catabolism of VLDL which is not functional for normal VLDL.

Inhibition of Lipopolysaccharide-Induced Inflammatory Responses by an Apolipoprotein AI Mimetic Peptide

Previous studies suggest that high-density lipoprotein and apoAI inhibit lipopolysaccharide (LPS)-induced inflammatory responses. The goal of the current study was to test the hypothesis that the apoAI mimetic peptide L-4F exerts antiinflammatory effects similar to apoAI. Pretreatment of human umbilical vein endothelial cells (HUVECs) with LPS induced the adhesion of THP-1 monocytes. Incubation of cells with LPS and L-4F (1 to 50 &mgr;g/mL) reduced THP-1 adhesion in a concentration-dependent manner. This response was associated with a significant reduction in the synthesis of cytokines, chemokines, and adhesion molecules. L-4F reduced vascular cell adhesion molecule-1 expression induced by LPS or lipid A, whereas a control peptide (Sc-4F) showed no effect. In contrast to LPS treatment, L-4F did not inhibit IL-1&bgr;- or tumor necrosis factor-&agr;–induced vascular cell adhesion molecule-1 expression. The inhibitory effect of L-4F on LPS induction of inflammatory markers was associated with reduced binding of LPS to its plasma carrier molecule, lipopolysaccharide binding protein, and decreased binding of LPS to HUVEC monolayers. LPS and L-4F in HUVEC culture medium were fractionated by fast protein liquid chromatography and were localized to the same fractions, suggesting a physical interaction between these molecules. Proinflammatory responses to LPS are associated with the binding of lipid A to cell surface receptors. The current studies demonstrate that L-4F reduces the expression of inflammatory markers induced by LPS and lipid A and suggest that apoAI peptide mimetics may be useful in the treatment of inflammation associated with endotoxemia.

Apolipoprotein B-48 or Its Apolipoprotein B-100 Equivalent Mediates the Binding of Triglyceride-Rich Lipoproteins to Their Unique Human Monocyte-Macrophage Receptor

Studies in animals and humans have demonstrated uptake of plasma chylomicrons (triglyceride-rich lipoprotein [TGRLP] of Sf>400) by accessible macrophages in vivo. One potential mechanism is via a unique receptor pathway we previously identified in human blood and THP-1 monocytes and macrophages for the lipoprotein lipase (LpL)- and apolipoprotein (apo) E-independent, high-affinity, specific binding of plasma chylomicrons and hypertriglyceridemic VLDL (HTG-VLDL) to cell-surface membrane-binding proteins (MBP 200, 235; apparent Mr 200, 235 kD on SDS-PAGE) that leads to lipid accumulation in vitro. Competitive binding studies reported here demonstrate that anti-apoB antibodies specifically block the high-affinity binding of TGRLP to this receptor on THP-1 cells and on ligand blots. LpL, which binds to an N-terminal domain of apoB, also inhibits TGRLP binding both to this site on THP-1s and to MBP 200, 235 by binding to apoB. Chylomicrons of Sf>1100 that contain apoB-48, but not apoB-100, bind specifically to MBP 200, 235, and this binding is blocked by anti-apoB IgG. In contrast, lactoferrin and heparin do not inhibit TGRLP binding. We conclude that the receptor-binding domain is within apoB-48 (or an equivalent in apoB-100) near the LpL-binding domain, but not a heparin-binding domain. Uptake of TGRLP by this mechanism could provide essential nutrients or, in HTG, cause excess lipid accumulation and foam cell formation.

Distinct murine macrophage receptor pathway for human triglyceride-rich lipoproteins.

Murine P388D1 macrophages have a receptor pathway that binds human hypertriglyceridemic very low density lipoproteins (HTG-VLDL) that is fundamentally distinct from the LDL receptor pathway. Trypsin-treated HTG-VLDL (tryp-VLDL), devoid of apolipoprotein (apo)-E, fail to bind to the LDL receptor, yet tryp-VLDL and HTG-VLDL cross-compete for binding to P388D1 macrophage receptors, indicating that these lipoproteins bind to the same sites. The specific, high affinity binding of tryp-VLDL and HTG-VLDL to macrophages at 4 degrees C is equivalent and at 37 degrees C both produce rapid, massive, curvilinear (receptor-mediated) triglyceride accumulation in macrophages. Ligand blots show that P388D1 macrophages express a membrane protein of approximately 190 kD (MBP190) that binds both tryp-VLDL and HTG-VLDL; this binding is competed by HTG-VLDL, trypsinized HTG-VLDL, and trypsinized normal VLDL but not by normal VLDL or LDL. The macrophage LDL receptor (approximately 130 kD) and cellular uptake of beta-VLDL, but not MBP 190 nor uptake of tryp-VLDL, are induced when cells are exposed to lipoprotein-deficient medium and decreased when cells are cholesterol loaded. Unlike the macrophage LDL receptor, MBP 190 partitions into the aqueous phase after phase separation of Triton X-114 extracts. An anti-LDL receptor polyclonal antibody blocks binding of HTG-VLDL to the LDL receptor and blocks receptor-mediated uptake of beta-VLDL by P388D1 cells but fails to inhibit specific cellular uptake of tryp-VLDL or to block binding of tryp-VLDL to MBP 190. Human monocytes, but not human fibroblasts, also express a binding protein for HTG-VLDL and tryp-VLDL similar to MBP 190. We conclude that macrophages possess receptors for abnormal human triglyceride-rich lipoproteins that are distinct from LDL receptors in ligand specificity, regulation, immunological characteristics, and cellular distribution. MBP 190 shares these properties and is a likely receptor candidate for the high affinity uptake of TG-rich lipoproteins by macrophages.

Abnormal effects of hypertriacylglycerolemic very low-density lipoproteins on 3-hydroxy-3-methylglutaryl-CoA reductase activity and viability of cultured bovine aortic endothelial cells

Abstract Our previous studies showed that hypertriacylglycerolemic very low-density lipoproteins (VLDL) are functionally abnormal. Hypertriacylglycerolemic VLDL, but not normal VLDL, suppress 3-hydroxy-3-methylglutaryl-CoA reductase in fibroblasts cultured from normal human subjects. To determine if hypertriacylglycerolemic VLDL also differ from normal VLDL in their interaction with vascular cells, their effects on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase in cultured subconfluent bovine endothelial cells were quantified. All hypertriacylglycerolemic VLDL subclasses (S f 100–400, S f 60–100 and S f 20–60) from a Type III hyperlipoproteinemic subject were even more effective than normal low-density lipoproteins ( d 1.006−1.063) (LDL) in suppression; 50 and 100% suppression by S f 100–400 hypertriacylglycerolemic VLDL occurred at 0.5 and 5 μg protein/ml, respectively. The VLDL subclasses from two subjects with Type V hyperlipoproteinemia were comparable to LDL in suppression. By contrast, under experimental conditions when normal LDL gave 50% suppression at 1–5 μg protein/ml, neither normal S f 100–400 VLDL nor high-density lipoproteins (HDL) ( d 1.063−1.21) suppressed, even at lipoprotein concentrations of 35 and 200 μg protein/ml, respectively. The smaller S f 20–60 VLDL subclass from normal plasma suppressed, but with less than 20% of the potency of LDL. LDL and hypertriacylglycerolemic VLDL also suppressed the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase in confluent endothelial cell cultures. Moreover, exposure to low levels of hypertriacylglycerolemic VLDL, but not normal VLDL or LDL, reduced the number of viable endothelial cells up to 2-fold. We suggest that cytotoxic effects of hypertriacylglycerolemic VLDL on endothelial cells could impair the normal function of the endothelium in vivo.

Interaction of low density lipoproteins with human aortic elastin.

Interaction between lipoproteins and elastin in the arterial wall may play an important role in atherosclerotic lipid deposition, but binding affinities and other characteristics of the interaction have not been determined previously. Elastin was isolated by hot alkali treatment of human aortic tissue. At 4 degrees C, radioiodinated human low density lipoprotein (LDL) bound to more than one class of binding sites on elastin. Sites of highest affinity had an apparent dissociation constant of 3.6 x 10(-8) M. Total binding at an LDL concentration of 50 micrograms/ml ranged from 4 to 50 ng LDL protein/mg elastin. The binding was relatively specific, since binding was competitively inhibited by LDL and apo E-containing high density lipoprotein (HDL) but only modestly by HDL3. Atherosclerotic elastin exhibited a twofold to fourfold higher capacity for binding LDL, but a reduced affinity. At 37 degrees C, normal elastin exhibited an initial rapid binding of LDL, with a slower linear phase of binding over a 15-hour period, indicating an additional complex process at this temperature. Consideration of the expected LDL concentrations in the arterial intima, in comparison with binding affinities, suggests that LDL binding to elastin probably occurs in the intima and may foster atherosclerotic lipid deposition.

Apolipoprotein-E degradation in human very low density lipoproteins by plasma protease(s): Chemical and biological consequences

Serine proteases coisolate with human very low density lipoproteins (VLDL) which degrade apolipoprotein E and cause hypertriglyceridemic VLDL to lose the ability to interact with the LDL receptor of human skin fibroblasts. We identified proteolytic fragments of apolipoprotein-E in isolated VLDL which can be produced by the action of thrombin on purified apoE. There are two major thrombin cleavage products: Mr ∼ 22,000 (E-22) and Mr ∼ 12,000 (E-12), the N- and C-terminal fragments, respectively, of apoE. We conclude that the structural integrity and the ability of VLDL to interact with cell receptors are a function of not only VLDL constituents but also of the extent to which VLDL apoprotein E has been degraded.

Stimulation and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in normal human fibroblasts by high density lipoprotein subclasses.

Plasma concentrations of high density lipoproteins, (HDL3), a subclass of HDL, are relatively constant, while those of HDL2 are variable. We report that HDL2 suppress, while HDL3 stimulate 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) activity in normal human fibroblasts. HDL3, which contained no detectable HDL2 or low density lipoproteins, stimulated 3-hydroxy-3-methylglutaryl-CoA reductase activity 2-fold from 60 to 120 pmol/mg per min. The induction, which exhibited saturation kinetics with maximum stimulation at 150 microgram HDL phospholipid/ml medium, occurred only in the late-log and stationary phases of cell growth and was abolished by 0.1 mM actinomycin D or cycloheximide.l Apoliproprotein HDL3 did not stimulate enzyme activity, whereas the total lipid extract of HDL3 was about 1.7 times more potent than were the native HDL3 in stimulating enzyme activity. HDL2 consistently suppressed 3-hydroxy-3-methylglutaryl-CoA reductase in normal fibroblasts by 20-50% at 80 microgram HDL2 protein/ml. Mixtures of HDL2 and HDL3 suppressed when HDL2 were greater than 35% of the total HDL. The suppressive effects of HDL2 were abolished by treatment with 0.1 M cyclohexanedione and restored by regeneration of arginyl residues, suggesting an apolipoprotein-mediated suppressive mechanism. The total lipid extract of HDL2 stimulated 3-hydroxy-3-methylglutaryl-CoA reductase 2-fold at 3 microgram lipid phosphorus/ml. Moreover, HDL2 and HDL3 both stimulated 3-hydroxy-3-methylglutaryl-CoA reductase activity in receptor-negative fibroblasts. HDL2 have both the ability to suppress 3-hydroxy-3-methylglutaryl-CoA reductase activity in cells which possess low density lipoproteins receptors and to activate the enzyme in receptor-negative cells. These results show that variations in culture conditions and differences in the proportions of HDL subclasses must be considered in the interpretation of studies investigating cellular responses to HDL.

Abnormal suppression of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by hypertriglyceridemic very low density lipoprotein subclasses

Our previous studies showed that hypertriglyceridemic very low density lipoproteins (HTG VLDL) are functionally abnormal. HTG VLDL, but not normal VLDL, suppress HMG-CoA reductase in cultured normal human fibroblasts. To determine if the suppression by HTG VLDL resulted from a subpopulation of smaller suppressive particles, more homogeneous subclasses of VLDL-VLDL1 (Sf 100–400), VLDL2 (Sf 60–100), and VLDL3 (Sf 20–60) were obtained from the d<1.006 (g°ml−1) fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient and tested for suppression in normal human fibroblasts. VLDL1 and VLDL2 from each of the 12 normolipemic subjects tested failed to suppress HMG-CoA reductase activity in normal fibroblasts. Eleven out of 12 preparations of normal VLDL3 suppressed HMG-CoA reductase, but only one-third as effectively as LDL. By contrast, the VLDL1, VLDL2 and VLDL3 from 15 out of 17 hypertriglyceridemic patients (hyperlipoproteinemia Types IIb, III, IV and V) were highly effective in suppression, with half-maximal suppression at 0.1–2.0 μg VLDL protein/ml. The VLDL abnormality is apparently associated with hypertriglyceridemia and not hypercholesterolemia, since VLDL from a homozygous familial hypercholesterolemia patient with a Type IIa pattern did not suppress whereas each of the VLDL subclasses from a Type IIb patient suppressed. Suppression by HTG VLDL in normal cells is apparently a consequence of interaction of the protein portion of the VLDL with the specific LDL cell surface receptor since HTG VLDL1 treated with 0.1 M 1,2-cyclohexanedione to block arginyl residues failed to suppress the enzyme. Moreover, hypertriglyceridemic Sf 60–400 VLDL failed to suppress HMG-CoA reductase activity in LDL receptor-negative fibroblasts. There were no consistent major compositional differences between comparable normal and hypertriglyceridemic VLDL subclasses which could account for differences in suppression. All VLDL subclasses from Type III subjects were enriched in cholesteryl esters and depleted in triglyceride, relative to the corresponding normal VLDL subclasses. However, Type IV and Type V VLDL subclasses were normal in this repect. We conclude from these studies that small particle diameter is not required for suppression, since HTG VLDL1 and VLDL2 which contained few, if any, small particles were effective in suppression.