Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques.

Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 and the intravascular processing of triglyceride-rich lipoproteins.

Lipoprotein lipase (LPL) is produced by parenchymal cells, mainly adipocytes and myocytes, but is involved in hydrolysing triglycerides in plasma lipoproteins at the capillary lumen. For decades, the mechanism by which LPL reaches its site of action in capillaries was unclear, but this mystery was recently solved. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, 'picks up' LPL from the interstitial spaces and shuttles it across endothelial cells to the capillary lumen. When GPIHBP1 is absent, LPL is mislocalized to the interstitial spaces, leading to severe hypertriglyceridaemia. Some cases of hypertriglyceridaemia in humans are caused by GPIHBP1 mutations that interfere with the ability of GPIHBP1 to bind to LPL, and some are caused by LPL mutations that impair the ability of LPL to bind to GPIHBP1. Here, we review recent progress in understanding the role of GPIHBP1 in health and disease and discuss some of the remaining unresolved issues regarding the processing of triglyceride-rich lipoproteins.

The processing of ligands by the class A scavenger receptor is dependent on signal information located in the cytoplasmic domain.

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.

Enhancement of fatty acid and cholesterol synthesis accompanied by enhanced biliary but not very-low-density lipoprotein lipid secretion following sustained pravastatin blockade of hydroxymethyl glutaryl coenzyme A reductase in rat liver.

A 3-week treatment of rats with pravastatin (PV) augmented biliary cholesterol and phospholipid output 3.6- and 2.2-fold over controls, while bile acid (BA) output and kinetics were unchanged. No major changes were detected in hepatic and serum cholesterol concentrations despite the PV inhibitory property on hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase. To evaluate the mechanisms of this adaptive phenomenon, several parameters of hepatic lipid homeostasis were assessed. Biliary cholesterol changes could not be attributed to an increased influx of lipoprotein cholesterol to the liver and bile. Hepatic low-density lipoprotein (LDL) receptor content, as inferred from Western blot analysis, was unchanged, as was the biliary excretion of labeled cholesterol derived from chylomicron remnants. In vivo 3H2O-incorporation studies showed an 80% increase in hepatic cholesterol synthesis, evidence for bypass of the PV block. Remarkably, fatty acid synthesis was also stimulated twofold, providing substrate for hepatic triglycerides, which were slightly enhanced. However, serum triglycerides decreased 52% associated with a 22% decrease in hepatic very-low-density lipoprotein (VLDL) secretion. Thus, the biochemical adaptation following PV treatment produces complex alterations in hepatic lipid metabolism. An enhanced supply of newly synthesized cholesterol and fatty acids in association with a limited VLDL secretion rate augments the biliary lipid secretion pathway in this experimental model.

Multiple processes are involved in the uptake of chylomicron remnants by mouse peritoneal macrophages.

The processes responsible for the uptake of chylomicron remnants by macrophages were investigated using freshly isolated cells from low density lipoprotein (LDL) receptor, very low density lipoprotein (VLDL) receptor and apolipoprotein E knockout mice. In peritoneal macrophages from normal mice, the metabolism of chylomicron remnants was inhibited 40% by anti-LDL receptor antibody and 60% by a high concentration of receptor-associated protein (RAP). Together they reduced the amount processed by 70%. Digestion of cell proteoglycans decreased remnant degradation by 20% while the addition of acetyl-LDL had no effect. When LDL receptors were absent, the absolute rates of metabolism were less than that of normal cells and were not inhibited by the anti-LDL receptor antibody; the rates, however, were reduced to less than half by RAP. These suggest that the LDL receptor-related protein (LRP) or another LDL receptor family member(s) contributes to chylomicron remnant uptake and becomes the major mechanism of uptake when LDL receptors are absent. In contrast, the VLDL receptor was not involved as its absence did not affect chylomicron remnant metabolism. Similarly, the absence of apoE production did not affect the amount of remnant uptake; however, the proportion that was sensitive to RAP was eliminated. The level of LRP expression was not altered in these cells whereas there was a decrease in LDL receptors. This suggests that the apoE content of chylomicron remnants is sufficient for its recognition by LDL receptors but additional apoE is required for its uptake by the LRP and that there is an up-regulation of a non-LDL receptor family mechanism in apoE deficiency. Together these studies suggest that even in the absence of LDL receptors or apoE secretion, chylomicron remnants could contribute to lipid accumulation in the artery wall during atherogenesis.

Modulation of macrophage scavenger receptor transport by protein phosphorylation.

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Relative roles of the LDL receptor, the LDL receptor-like protein, and hepatic lipase in chylomicron remnant removal by the liver.

Studies were carried out in mice utilizing inhibitors of several cell surface molecules to evaluate their relative roles in chylomicron remnant removal. Anti-LDL receptor antibody inhibited approximately 45% of rapid remnant removal from plasma, prolonged their half life (63 s to 115 s) and reduced hepatic uptake by 45%. Receptor-associated protein (RAP) (1 mg/mouse), a high affinity inhibitor of the LDL receptor-related protein (LRP) and a low affinity inhibitor of the LDL receptor decreased remnant removal approximately 55%, prolonged the half life from 63 s to 230 s, and reduced hepatic uptake by 70%. RAP, but not anti-LDL receptor antibody, inhibited splenic uptake. With both injected together, an incremental effect was seen; plasma removal decreased 60%, T1/2 increased to 290 s, and hepatic uptake decreased by 80%. Thus, it is likely that virtually all of the very rapid removal of remnants from the plasma by the liver requires the presence of at least one of these members of the LDL receptor family. Anti-hepatic lipase antibody caused a small but significant delay in remnant removal from plasma and a larger decrease in hepatic uptake (22.5%). It doubled adrenal uptake. The anti-hepatic lipase antibody was not additive with either the anti-LDL receptor antibody or RAP. Anti-rat hepatic lipase antibody did not inhibit lipolysis by mouse hepatic lipase, suggesting that lipolysis is not the way hepatic lipase enhances remnant uptake. Hepatic lipase bound to remnants to a greater degree than it bound to other lipoproteins. Together these data suggest that hepatic lipase may serve as a binding site for chylomicron remnants, thereby enhancing their affinity for the liver surface, and thus removal by the proteins of the LDL receptor family. Other molecules may also play a role in removal from the circulation under conditions where the LDL receptor family receptors are absent or occupied.

Location and regulation of low-density lipoprotein receptors in intestinal epithelium.

The expression, distribution, and some aspects of the regulation of low-density lipoprotein (LDL) receptors in rat intestinal epithelial cells were examined. Cells prepared by a perfusion technique provided a pure preparation of epithelial cells and could be manipulated to produce crypt-villus units or villi alone. On a total protein basis, the abundance of LDL receptors in villus cell membranes was half that in hepatic membranes. The level of receptors in both tissues was reduced by feeding an atherogenic diet but was increased only in the liver by ethinyl estradiol-induced hypocholesterolemia. The level of LDL receptor mRNA in intestinal epithelial cells was somewhat lower than in liver. Regulation of LDL receptor mRNA was similar to that of protein. Judged by the ratio of mRNA in villus cells to the villus-crypt unit and nuclear run-on assay for LDL receptor gene transcription, we conclude that LDL receptor mRNA is produced in the villus cells. The effect of fat feeding was regulated at the level of transcription. Expression in villus cells in ileum was severalfold higher than in jejunum and higher than in the liver. Together the results suggest serum cholesterol level is not the prime determinant of LDL receptor level in intestine, but LDL degradation in this organ may be regulated by factors in the lumen.

Quantitative measurement of 4-hydroxyalkenals in oxidized low-density lipoprotein by gas chromatography-mass spectrometry.

Previously, we reported on the determination of 4-hydroxyalkenals as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry. In this study, an improved method is presented which allows quantitative detection of 4-hydroxyalkenals in tissues by using two stable isotope-labeled internal standards, 9D3-4-hydroxynonenal and 9D3-4-hydroxyhexenal. This assay was used to quantitate 4-hydroxyalkenals in copper-oxidized human low-density lipoprotein, and we found more 4-hydroxynonenal than 4-hydroxyhexenal. This is consistent with the fact that there are more omega-6 than omega-3 fatty acids in human low-density lipoprotein samples, which are the sources of 4-hydroxynonenal and 4-hydroxyhexenal, respectively.

Acceleration of uptake of LDL but not chylomicrons or chylomicron remnants by cells that secrete apoE and hepatic lipase.

ApoE is a ligand for the low density lipoprotein (LDL) receptor as well as for the LDL receptor-related protein (LRP). The enzyme hepatic lipase (HL) may also affect the uptake of lipoproteins by modifying their composition. We have tested the hypothesis that hepatic lipase and apoE can function as co-factors to alter the rate of lipoprotein uptake. Chinese hamster ovary (CHO) cells were transfected with cDNAs for rat hepatic lipase, human apoE or both HL and apoE. The secreted recombinant proteins were thoroughly characterized and had properties identical to the native proteins. Hepatic lipase and apoE were secreted at 0.17 and 1.25 micrograms/mg cell protein per hour, rates comparable to those in normal liver. 125I-labeled LDL, chylomicron remnants, or chylomicrons were added to media at concentrations near their Kd. In cells that secreted either apoE or hepatic lipase, or both apoE and hepatic lipase, LDL binding was significantly greater than with control cells (2.2-, 2-, 2-fold greater, respectively). Similar enhancement of LDL degradation was observed. In the presence of anti-LDL receptor antibodies, these values were reduced to control levels; thus, the enhanced uptake was mediated by the LDL receptor and not the LRP. The amount of LDL receptor protein, as judged by Western blotting, was similar in the various cell types. Incubation of control CHO cells with media from secreting transfected cells also increased the uptake of 125I-labeled LDL. Kinetic studies indicated that, in apoE-secreting cells, increased LDL binding is associated with a lower Kd and an unchanged Vmax as compared to the control cells; furthermore, when LDL were reisolated by column chromatography (but not by ultracentrifugation) from the incubations where apoE was being secreted, apoE was identified adherent to the LDL particles. Together, these results suggest that the effect is due to alteration of the lipoprotein and not the cell. In contrast, the uptake of 125I-labeled chylomicron remnants, and 125I-labeled chylomicrons was not greater in the transfected cells. Thus, in the amounts secreted by these cells, hepatic lipase and apoE do not convert chylomicrons to chylomicron remnants or alter the uptake of chylomicron remnants by either the LDL receptor or the LRP. The enhancement of LDL removal in cells that secrete hepatic lipase or apoE may help determine the amount of LDL removed by a particular tissue.

Inhibition of the macrophage-induced oxidation of low density lipoprotein by interferon-gamma.

The regulation of the macrophage-induced oxidation of low density lipoprotein (LDL) by cytokines was investigated. As an initial source of cytokines, medium from an activated type 2 helper T-cell clone was tested. This cell-free supernatant inhibited the subsequent oxidation of LDL by mouse peritoneal macrophages. The inhibition was concentration- and time-dependent as measured by changes in thiobarbituric acid (TBA) reactive substances. In addition, there were decreases in conjugated diene formation as well as the generation of LDL particles with an increased net negative charge that were recognized by the scavenger receptor. The inhibition was not due to a decrease in cell viability or to nonspecific antioxidant activity, as assessed by measuring phagocytic activity and metal ion-induced oxidation of LDL, respectively. Using antibodies that inactivate specific cytokines, the role of select individual cytokines in this inhibition was investigated. Addition of antibodies against interleukin-3 (IL-3), granulocyte/macrophage-colony stimulating factor (GM-CSF), or tumor necrosis factor alpha (TNF alpha) to the media had little or no effect on the ability of the cytokines to affect oxidation by macrophages, whereas anti-interferon-gamma (IFN-gamma) antibodies completely reversed the inhibition induced by the T-cell supernatant. A role for this cytokine was confirmed using recombinant IFN-gamma. A concentration-dependent inhibition was produced with a maximum inhibition to 24% of control cells, whereas smooth muscle cell-dependent LDL oxidation was not affected. To examine the cellular basis for the inhibition, the effect of IFN-gamma on oxidant activities (O2- production, lipoxygenase activity, and thiol production) were measured. IFN-gamma at concentrations that maximally inhibit LDL oxidation stimulated the phorbol myristate acetate (PMA)-induced production of O2- 1.4-times greater than control cells after one hour. Similarly, thiol production was increased 29% by IFN-gamma pretreatment. In contrast, macrophage lipoxygenase was inhibited approximately 21%. Based on these in vitro findings, the potential regulation of macrophage LDL oxidation by IFN-gamma in vivo was also investigated. Macrophages from Toxoplasma gondii-infected mice have been shown previously to be activated in situ by an IFN-gamma-dependent mechanism. These were tested for their ability to oxidize LDL. Macrophages from these mice oxidized LDL to a much lesser extent than cells from age-matched control mice, demonstrating that the ability of macrophages to oxidize lipoprotein may also be susceptible to regulation possibly also by IFN-gamma in vivo. Together these studies demonstrate that the cell-mediated oxidation of LDL can be regulated by cytokines, specifically IFN-gamma. This mode of regulation may play a role in regulating this process in the developing atherosclerotic lesion.

Early induction of LDL receptor gene during rat liver regeneration.

After partial hepatectomy in the rat, there is substantial lipid accumulation in the liver. No information is available on the possible role of receptor-mediated endocytosis in this process. Since the low density lipoprotein (LDL) receptor is stimulated as a part of an early growth response in cell culture (Ellsworth et al.: Biochem. J. 279:175-187, 1991), the metabolism of this receptor during liver regeneration was studied. The mRNA and membrane protein level of the receptor were measured in the liver and in the adrenal glands at different times after partial hepatectomy, corresponding to different phases of the cell cycle. A discontinuous pattern of receptor expression is detectable in the regenerating liver; a large increase of mRNA and membrane protein occurs at an early time (2-4 h), suggesting that there is induction of LDL receptor gene transcription shortly after partial hepatectomy. This response seems specific for the liver following injury since the adrenal receptor does not show a different pattern in partially hepatectomized rats and sham-operated controls. After returning to control levels, the LDL receptor again increases slightly above control at 24 h, a time when cell replication begins.

Inhibition of lipopolysaccharide-induced interleukin-1 beta mRNA expression in mouse macrophages by oxidized low density lipoprotein.

Recent studies have demonstrated the expression of messenger RNA (mRNA) for several cytokines within atherosclerotic arteries. Since cytokines have been shown to modulate functions of cultured arterial wall cells in a manner that could influence atherogenesis, this suggests that factors that modulate cytokine production would influence the atherosclerotic process. To examine whether lipoproteins can modulate cytokine production, the effect of lipoproteins on mouse macrophage interleukin-1 beta (IL-1 beta) mRNA expression was examined by dot blot and Northern blot analyses. Low density lipoprotein (LDL), acetylated-LDL, or malondialdehyde-LDL did not induce IL-1 beta mRNA expression or affect the expression in response to lipopolysaccharide (LPS). Similarly, copper ion-oxidized LDL did not stimulate the production of IL-1 beta mRNA. However, oxidized LDL inhibited the LPS-induced expression in a concentration- and time-dependent manner with a maximum inhibition (greater than 90%) observed after a 2.5 h preincubation with 25 micrograms protein/ml. These conditions did not affect protein synthesis or phagocytosis and the inhibition was partially reversible after 24 h, which together suggest that the inhibition was not due to cell death. An inhibition of IL-1 alpha and IL-6 mRNA expression was also observed while there was no change in gamma-actin mRNA levels. The level of inhibition of IL-1 beta mRNA was dependent upon the extent of LDL oxidation, but did not correlate with recognition by the scavenger receptor. A non-receptor pathway was supported by two lines of evidence: 1) the inhibition could be reproduced with a lipid extract, and 2) oxidized LDL also inhibited scavenger receptor negative THP-1 cell IL-1 beta mRNA expression. Finally, oxidized LDL had no effect on the turnover of IL-1 beta mRNA, suggesting that the decreased accumulation of IL-1 beta mRNA is due to a decrease in gene transcription. Together these studies suggest that as macrophages become foam cells their immune responsiveness is attenuated.

Use of an anti-low density lipoprotein receptor antibody to quantify the role of the LDL receptor in the removal of chylomicron remnants in the mouse in vivo.

Lipoproteins are removed from the plasma by LDL receptor-dependent and -independent pathways. The relative contribution of these has been established for LDL by using modified lipoproteins, but this has not been possible for apoE-rich lipoproteins, such as chylomicron remnants. To do this, we used a monospecific antibody to the rat LDL receptor. The antibody was injected intravenously into mice followed by 125I-lipoproteins. Blood samples were obtained sequentially and radioactivity measured to determine the plasma clearance of the lipoproteins. The animals were then sacrificed and the tissues removed, dried, and the radioactivity measured to determine tissue uptake. An albumin space was also measured to correct for blood trapping. With 125I-human LDL, approximately 50% of the injected dose was cleared in 180 min. This was reduced to 30% by the antibody and this was identical to the disappearance of reductively methylated LDL. This is a lower estimate of LDL-mediated uptake (40%) than in other species. LDL uptake per gram tissue was similar for the liver and the adrenal gland and was approximately 50% LDL receptor-dependent in both tissues. With 125I-chylomicron remnants, clearance was much more rapid with approximately 50% cleared in 5 min. By agarose gel electrophoresis, radioactivity was not transferred from chylomicron remnants to other lipoprotein classes. Chylomicron remnants with label on only apoB or in 3H-cholesterol esters showed a similar pattern. Combining the estimates of the three labeling procedures, approximately 35% of the 30 s and 25% of the 5 min chylomicron remnant disappearance was LDL receptor dependent. The liver, per gram tissue, took up five times as much radioactivity as the adrenal gland. At 5 min, at least 50% of this was LDL receptor-dependent in liver and 65% in adrenal gland. We conclude that the LDL receptor plays a major, and somewhat similar quantitative role in the clearance of both LDL and chylomicron remnants in the mouse. However, at least in the mouse, non-LDL receptor-mediated lipoprotein clearance is quantitatively important and is also very rapid for chylomicron remnants. Thus, for chylomicron remnants, it can easily compensate for LDL receptors if they are blocked or absent. Further, the tissue distribution of lipoprotein uptake may be directed by factors other than LDL receptor density.

Dose response of fish oil versus safflower oil on graft arteriosclerosis in rabbit heterotopic cardiac allografts.

With the advent of cyclosporin A, accelerated coronary arteriosclerosis has become the major impediment to the long-term survival of heart transplant recipients. Due to epidemiologic reports suggesting a salutary effect of fish oil, the dose response of fish oil on graft coronary arteriosclerosis in a rabbit heterotopic cardiac allograft model was assessed using safflower oil as a caloric control. Seven groups of New Zealand White rabbits (n = 10/group) received heterotropic heart transplants from Dutch-Belted donors and were immunosuppressed with low-dose cyclosporin A (7.5 mg/kg/day). Group 1 animals were fed a normal diet and served as control. Group 2, 3, and 4 animals received a daily supplement of low- (0.25 mL/kg/day), medium- (0.75 mL/kg/day), and high- (1.5 mL/kg/day) dose fish oil (116 mg n-3 polyunsaturated fatty acid/mL), respectively. Group 5, 6, and 7 animals were supplemented with equivalent dose of safflower oil (i.e., 0.25, 0.75, and 1.5 mL/kg/day). Oil-supplemented rabbits were pretreated for 3 weeks before transplantation and maintained on the same diet for 6 weeks after operation. The extent of graft coronary arteriosclerosis was quantified using computer-assisted, morphometric planimetry. When the animals were killed, cyclosporin A was associated with elevated plasma total cholesterol and triglyceride levels in the control group. While safflower oil prevented the increase in plasma lipids at all dosages, fish oil ameliorated the cyclosporin-induced increase in total cholesterol only with high doses. Compared to control animals, there was a trend for more graft vessel disease with increasing fish oil dose, as assessed by mean luminal occlusion and intimal thickness. A steeper trend was observed for increasing doses of safflower oil; compared to the high-dose safflower oil group, animals supplemented with low-dose safflower oil had less mean luminal occlusion (16.3% +/- 5.9% versus 41.4% +/- 7.6%, p less than 0.017) and intimal thickness (7.9 +/- 1.9 microns versus 34.0 +/- 13.0 microns, analysis of variance: p = 0.054). Low-dose safflower oil also had a slight, but nonsignificant, beneficial effect on graft vessel disease when compared to control rabbits. The same trends were observed in the degree of histologic rejection (0 = none to 3 = severe) in fish oil- and safflower oil-treated animals. Rejection score correlated weakly but significantly (p = 0.0001) with mean luminal occlusion (r = 0.52) and intimal thickness (r = 0.46). Therefore allograft coronary disease in this model appeared to exhibit an unfavorable, direct-dose response to fish oil and safflower oil, independent of effects on plasma lipids.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxidative modification of LDL: comparison between cell-mediated and copper-mediated modification.

Macrophage-derived foam cells are hallmarks of early atherosclerotic lesions. Oxidatively modified LDL has been suggested to be a more atherogenic form than native LDL. Oxidized LDL--but not native LDL--is chemotactic to monocytes and is avidly degraded by macrophages, resulting in their conversion to foam cells. Incubation of LDL with any of several different types of cells, or with copper ion even in the absence of cells, results in the oxidative modification of LDL. While the cell and the copper systems generate oxidatively modified LDL with similar properties, the two systems differ in their sensitivity to inhibition by superoxide dismutase and by several lipoxygenase inhibitors. In cultured endothelial cells, inhibitors of lipoxygenase, some of them without non-specific antioxidant activity, inhibited cell-mediated modification by 50-80%. In contrast, superoxide dismutase inhibited the process by 20% or less. Moreover, we have shown that soybean lipoxygenase in a cell-free system can modify LDL directly to a form recognized and degraded specifically and rapidly by macrophages. Lipoxygenase-modified LDL is also chemotactic for human monocytes and is cleared rapidly from the circulation, properties shared by cell- or copper-modified LDL. Thus, it is suggested that cellular lipoxygenase(s) may play an important role in cell-mediated oxidative modification of LDL.

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages.

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.

Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma.

In vitro, metabolism of modified forms of low density lipoprotein (LDL) by macrophages via the acetyl-LDL receptor pathway promotes the massive cellular accumulation of lipid. It has been postulated that in vivo this contributes to foam cell formation in the atherosclerotic lesion. Recent studies have shown that arterial wall cells in vitro can secrete a number of cytokines, several of which have been reported to modulate macrophage cell function. Thus, cytokines have the potential to modulate the acetyl-LDL receptor pathway and to influence the rate of foam cell generation. To study the regulation of this pathway by cytokines, the effect of cytokines on the degradation of acetyl-LDL protein by mouse peritoneal macrophages was examined. Initially, supernatant from stimulated lymphocytes was used as a source of cytokines. Macrophages preincubated with supernatants obtained after the stimulation of T-cell helper type 1 (Th1) clone HDK-1 or BALB/c spleen cells degraded acetyl-LDL at a slower rate, whereas supernatant from stimulated T-cell helper type 2 (Th2) clone D-10 had no effect. Comparison of the lymphokine profiles showed that spleen and HDK-1 cells secreted several lymphokines in common including significant levels of interferon-gamma. Interferon-gamma was then directly shown to be inhibitory; an anti-interferon-gamma monoclonal antibody blocked the HDK-1-mediated inhibition by 70% and the addition of recombinant interferon-gamma (IFN-gamma) to macrophages inhibited the specific degradation of acetyl-LDL in a dose- and time-dependent manner with a maximum suppression to approximately 40% of control. The inhibition was not accompanied by an increase in the amount of cell-associated acetyl-LDL and was not due to cell death nor could it be accounted for by the presence of endotoxin. To study the mechanism of the inhibition, the effects of IFN-gamma on the itinerary of acetyl-LDL and its receptor were examined. IFN-gamma decreased specific acetyl-LDL binding only to a small degree, and the rate of lysosome-mediated degradation was not affected. The principal alteration was in the rate of transport to the lysosome which was markedly slowed. Since the receptors eventually returned to the surface to maintain a steady state, and there was not an increase in cell-associated lipoprotein, there must be other changes in the itinerary that were not identified with the techniques used. Thus, the receptor cycle is being regulated at a discrete point. IFN-gamma also suppressed the LDL receptor pathway in macrophages, but this pathway was not affected by IFN-gamma in mouse fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein.

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.