Recombinant gamma interferon induces hypertriglyceridemia and inhibits post-heparin lipase activity in cancer patients.

Animals suffering from malignancy or chronic infection develop characteristic metabolic abnormalities, including a well-defined hypertriglyceridemic state. These abnormalities have been attributed to release of one or more mediators from activated macrophages. We report that cancer patients receiving RIFN-gamma, a potent macrophage activator, at doses of greater than or equal to 0.25 mg/m2/d i.m. show marked increases in triglyceride but not in cholesterol levels (pretreatment triglyceride level of 180 +/- 190 mg/dl [mean +/- SD] vs. a day-14 level of 370 +/- 242 mg/dl, n = 23, p less than 0.001 by the paired t test). This hypertriglyceridemia was characterized by an increase in very low-density lipoproteins and a decrease in plasma post-heparin lipase activity, consistent with defective triglyceride clearance (mean pretreatment lipase level of 2.1 mumol/ml/h vs. a day-14 level of 1.2 mumol/ml/h, n = 6, p = 0.02 by the paired t test). rIFN-gamma did not directly inhibit lipoprotein lipase enzymatic activity in vitro. Other possible mechanisms of action, such as suppression of lipase by an rIFN-gamma-induced mediator released from activated macrophages, or a direct effect of interferon on lipase biosynthesis, require further investigation. Our observations provide evidence that factors produced by the immune system can regulate lipid metabolism in man.

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.

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.

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.

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.

Fibrinolytic and thrombotic factors in atherosclerosis and IHD: the influence of triglyceride rich lipoproteins (TGRLP).

TGRLP interactions with the endothelium may increase the likelihood that a suppressed fibrinolytic capacity and/or an increased procoagulant activity enhances the risk for an ischemic event, that is, for the production of a focal thrombus. The cellular mechanisms and characteristics of TGRLP in hyperlipemia and in the postprandial state that contribute to their potential pathology in IHD are considered.

Expression of LDL receptor binding determinants in very low density lipoproteins.

We have used both proteolysis and reconstitution experiments to characterize the determinants for LDL receptor binding of HTG-VLDL. In these studies, we showed that the removal of approximately one mole of apo E per mole of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100) by thrombin-specific cleavage results in loss of receptor binding and concomitant loss of suppression of HMG-CoA reductase. This is in direct contrast to the lack of effect thrombin cleavage has on the receptor-mediated uptake of LDL, an apo B-mediated process. We were able to reconstitute receptor binding in thrombin-treated HTG-VLDL (Sf 100-400) by the specific reincorporation of one mole of apo E into the VLDL. The incorporation of one mole of apo E into normal non-suppressive VLDL (Sf 60-400) also enables this lipoprotein to bind to the receptor as effectively as LDL. Trypsin, which destroys apo E-mediated, but not apo B-mediated binding to the LDL receptor, abolishes binding of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100), but not that of HTG-VLDL (Sf 20-60), IDL, or LDL to the LDL receptor. Therefore, we conclude that apo E of the appropriate conformation is required for receptor-mediated uptake by the LDL receptor of large TG-rich lipoproteins (Sf greater than 60). This conformation of apo E is probably related to the surface on which it is found (i.e., size of the particle) and the mode of incorporation into the phospholipid surface (i.e., transferred from plasma HDL). In large TG-rich particles, it appears that the intact apo E is necessary for the proper orientation of the molecule on the surface, with the carboxy-terminal one-third needed to anchor the apoprotein to the phospholipid surface. We believe that the binding of apo E to the LDL receptor is a redundant system and is used as a backup system in abnormal pathological states such as hypertriglyceridemia, abetalipoproteinemia, and hypobetalipoproteinemia. In the case of hypertriglyceridemia, where the lipolysis mechanism is overloaded, the abnormal binding of HTG-VLDL (Sf greater than 60) provides an alternate catabolic route for their removal from plasma. In the cases of a beta- and hypobetalipoproteinemia, where the normal particles for cholesterol delivery are either absent or at low levels, apo E-containing particles can serve to deliver cholesterol to cells as has been recently observed in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of HDL subclasses on 3-hydroxy-3-methylglutaryl coenzyme A reductase in human fibroblasts.

Monolayer cultures of normal human fibroblasts were used to study the effects of the main subclasses of high-density lipoproteins, HDL2 and HDL3, on 3-hydroxy-3-methylglutaryl CoA reductase (EC 1.1.1.34) activity. In this system, HDL3 (d = 1.125-1.210 g/cm3) specifically induced HMG-CoA reductase activity. Evaluation of culture dynamics revealed that enzyme induction was restricted to the stationary phase of growth. When the cells were incubated with HDL2 (d = 1.063-1.125 g/cm3), suppression of reductase activity was observed. Mixtures of HDL2 and HDL3 suppressed reductase activity when HDL2 was greater than 35% of the total HDL. The suppressive effects of HDL2 were abolished by treatment with cyclohexanedione and restored by regeneration of the arginyl residues, suggesting an apoprotein-mediated suppressive mechanism. These observations show that the cellular effects of HDL depend upon the stage of cell growth and the ratio of HDL subclasses in HDL as usually isolated.

Pathophysiology of triglyceride-rich lipoproteins in atherothrombosis: cellular aspects.

Elevated plasma levels of triglyceride-rich lipoproteins (TGRLP), including very low-density lipoproteins (VLDL), chylomicrons, and their remnants, are now acknowledged as risk factors for cardiovascular disease. Interactions of TGRLP with lipoprotein receptors on monocytes, macrophages, and endothelial cells may be mechanistically linked to this risk. Triglyceride-rich lipoproteins from hypertriglyceridemic (HTG) subjects have the abnormal ability to bind to low-denisty lipoprotein receptors via apoE, and plasma chylomicrons from all subjects bind to a new, distinct receptor for apoB48 that is expressed specifically by monocytes, macrophages, and endothelial cells. Receptor binding and uptake of TGRLP by these cells are likely mechanisms involved in the formation of lipid-filled, macrophage-derived "foam cells" of atherosclerotic lesions and for defective fibrinolysis due to endothelial dysfunction. Recognition of the atherothrombogenic potential of TGRLP may lead to improved interventions to lessen or prevent the often fatal sequelae of coronary atherosclerosis and thrombosis associated with elevated plasma triglyceride levels.

Hypertriglyceridemia: lipoprotein receptors and atherosclerosis.

We have shown first, that apoB mediates the binding of small VLDL Sf 20-60 and IDL, as well as LDL, to the LDL receptor. Second, apoE of an appropriate, accessible conformation is required for the binding of large VLDL to the LDL receptor; HTG-VLDL Sf greater than 60 but not normal VLDL Sf greater than 60 have this apoE population. Third, the same population of apoE that mediates binding of HTG-VLDL Sf greater than 60 to the LDL receptor modulates its binding to the beta-VLDL receptor, but it is not required for the latter interaction. Fourth, a domain of processed apoB or apoB-48 in association with a domain of the inaccessible apoE is required for binding to and uptake by the beta-VLDL receptor. Fifth, our observations suggest that the abnormal catabolism of VLDL that occurs in hypertriglyceridemia may be explained by the abnormal uptake of HTG-VLDL by either the LDL or the beta-VLDL receptor pathway. Finally, we suggest that plasma proteases may route apoB/E-containing lipoproteins to macrophages for disposal, and this results in foam cell formation.

Triglyceride-rich lipoproteins and atherosclerosis: pathophysiological considerations.

The potential role of triglyceride-rich lipoproteins (TGRLP) in the pathogenesis of atherosclerosis is briefly reviewed. Structural attributes of TGRLP are related to functional cellular interactions relative to their ability to interact with macrophage receptors and produce foam cells. Unlike low-density lipoproteins (LDL), no prior modification (oxidation or acylation) is necessary with TGRLP from certain hypertriglyceridaemic subjects and certain postprandial-TGRLP before rapid, receptor-mediated lipid engorgement occurs. In addition, arguments are examined that challenge the differing views that this lipoprotein class is not important in atherogenesis.

Interactions of triglyceride-rich lipoproteins with receptors: modulation by thrombin.

Large VLDL from subjects with HTG but not normal humans bind with high affinity to both the classic LDL receptor present on all cells and the beta-VLDL receptor of macrophages. Binding of HTG-VLDL to the LDL receptor is mediated by a thrombin-accessible conformation of Apo E that is absent in normal VLDL. Binding of HTG VLDL to the beta-VLDL receptor appears to be mediated by one or more apo B species. We find that thrombin-accessible apo E is required for uptake of HTG-VLDL via the LDL receptor but not by the beta-VLDL receptor. Domains within apo B that are present in native chylomicrons and HTG-VLDL or created in vitro with thrombin appear to be required for binding to the beta-VLDL receptor but not the LDL receptor. Triglyceride-rich lipoproteins could be processed in vivo by thrombin or other proteases for preferential uptake and disposal by the beta-VLDL receptor pathway. This process may be involved in the foam cell accumulation and atherosclerosis associated with some types of hypertriglyceridemia.

ApoE is necessary and sufficient for the binding of large triglyceride-rich lipoproteins to the LDL receptor; apoB is unnecessary.

Large triglyceride-rich very low density lipoproteins (VLDL) Sf 60-400 from hypertriglyceridemic (HTG) patients, but not VLDL from normal subjects, bind to the LDL receptor of human skin fibroblasts because they contain apolipoprotein E (apoE) of the correct conformation, accessible both to the LDL receptor and to specific proteolysis by alpha-thrombin. Trypsin treatment of HTG-VLDL Sf 60-400 causes extensive apoB hydrolysis (fragments less than 100,000 mol wt), total degradation of apoE, and thus complete loss of LDL receptor binding. The reincorporation of apoE (1 mol/mol VLDL) into trypsin-treated HTG-VLDL completely restored the ability of HTG-VLDL to interact with the LDL receptor, suggesting that apoE probably does not induce a conformational change in apoB which results in receptor recognition, nor is intact apoB necessary to maintain the appropriate conformation of apoE for LDL receptor binding. As a model of large triglyceride-rich VLDL Sf greater than 60, we fractionated Intralipid by the Lindgren method of cumulative flotation and prepared apoE-Intralipid complexes. Competitive binding studies demonstrated that apoE-Intralipid is at least as effective as LDL for uptake and degradation of 125I-labeled LDL. Control Intralipid complexes containing apoA-I instead of apoE do not compete with iodinated LDL. Since these TG-rich complexes contain no apoB, apoB is, therefore, not only not sufficient for receptor-mediated uptake of large particles, it is not necessary. ApoE of the correct conformation is not only necessary but is sufficient to mediate receptor binding of large triglyceride-rich particles to the LDL receptor.

Vitamin K-dependent proteins bind to very low-density lipoproteins.

It has been demonstrated that the surface of large VLDL Sf 100-400 can bind both prothrombin and Factor X(Xa) and that on VLDL Factor Xa can convert prothrombin to thrombin, which degrades apo B and apo E. It has been reported also that the VLDL kinetically supports the conversion of prothrombin to thrombin. The binding of vitamin K-dependent proteins to phospholipid is partially Ca2+-dependent and probably involves their Gla residues. The complex of VLDL, prothrombin, Factor Xa, and Ca2+ lacks only Factor Va, a lipid associating, non-Gla residue containing 330 kd protein, to complete the "prothrombinase complex." Factor V (Va) is found at very low concentrations in the circulation, but is localized on platelets, monocytes, and the endothelium. VLDL can bind both to monocytes and to the endothelium, for example, through both receptor and non-receptor pathways. When carrying this complement of the prothrombinase complex, this subpopulation of VLDL, in the presence of Factor Va on cell surfaces, could conceivably upset the local balance of pro- and anticoagulant activities. Thus, directly or indirectly the increased triglyceride levels, reflected in increased VLDL in patients, may alter this balance, and thereby produce a "hypercoagulable state." This is a simplistic view of the potential role of VLDL in the interplay of cells, coagulation proteins, and the regulatory systems involved in vivo. To realize the degree of complexity that we may need to address, we need only look at the work of Booyse et al in this issue of Seminars, in which they demonstrate that hypertriglyceridemic VLDL, in contrast to normal VLDL, do not support the early release of t-PA from endothelial cells, an antifibrinolytic event.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein-mediated cellular mechanisms for atherogenesis in hypertriglyceridemia.

Structurally and functionally abnormal VLDL exist in hypertriglyceridemic humans. These large abnormal VLDL, in contrast to large normal VLDL, interact with cell surface receptors. One large VLDL particle carries far more total cholesterol than one LDL particle. Receptor-mediated uptake of abnormal VLDL is injurious to endothelial cells and converts macrophages into foam cells in vitro. These observations lead to the hypothesis that if similar phenomena occur in vivo, abnormal VLDL, which have prolonged residence times and increased opportunity to interact with arterial cells, are atherogenic by promoting endothelial injury and initiating foam cell formation. Since premature atherosclerosis is associated with some forms of hypertriglyceridemia and foam cells accumulate in certain hypertriglyceridemic subjects, similar events may indeed occur in vivo.

Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.

Triglyceride- and cholesterol-rich foam cells derived from monocyte-macrophages are commonly associated with some forms of hypertriglyceridemia. In this report, direct binding studies at 4 degrees C demonstrate that human monocyte-macrophages (HMM) 1-6 days after isolation from blood and human THP-1 monocytic cells, before and up to 7 days after differentiation with phorbol ester, exhibit a high affinity (Kd 3-6 nM), saturable, specific, and apolipoprotein (apo) E-independent binding site for the uptake and degradation of certain triglyceride-rich lipoproteins (TGRLP). Ligand blotting analysis identified two membrane binding proteins (MBP) of apparent molecular weights of 200 and 235 kDa (MBP 200 and MBP 235) in both cell types that share the same ligand specificity as the cellular site and bind hypertriglyceridemic (HTG) VLDL, trypsinized VLDL devoid of apoE (tryp-VLDL), and dietary plasma chylomicrons from normal subjects but not LDL, acetyl LDL, or normal VLDL with high affinity. Neither lipoprotein lipase nor apoE are required for TGRLP binding to the cells or the isolated MBPs. The cellular binding site and the MBPs are expressed at similar levels at all stages of differentiation, unlike the LDL or the acetyl LDL receptor. TGRLP that bind to the MBPs induce rapid, saturable, cellular triglyceride accumulation in monocytes as well as macrophages; normal VLDL does not. In addition, the cellular high affinity binding site and MBP 200 and 235 are not affected by the media sterol content, unlike the LDL receptor. Taken together, these data indicate that human monocyte-macrophages exhibit a high affinity, saturable, specific, apoE- and lipoprotein lipase-independent binding site and membrane binding proteins for TGRLP that differ in expression, specificity, and molecular size from receptors of the LDL receptor gene family or the acetyl LDL receptor. The shared characteristics of the cellular binding site with MBP 200 and MBP 235 suggest that they are candidates for the receptor-mediated, apoE-independent uptake of HTG-VLDL and chylomicrons by monocytes and macrophages and therefore may be involved in foam cell formation.