Receptor-mediated catabolism of low density lipoprotein in man. Quantitation using glucosylated low density lipoprotein.

Low density lipoprotein (LDL) catabolism occurs by LDL receptor-dependent and LDL receptor-independent pathways. We have shown previously that nonenzymatic glucosylation of LDL in the presence of cyanoborohydride irreversibly blocks the lysine residues of LDL. Glucosylated LDL (GLC-LDL) was not degraded by the LDL receptor of fibroblasts, and its degradation by macrophages was similar to that of native LDL. This suggested that GLC-LDL should be a good tracer of LDL receptor-independent catabolism, and if combined with a tracer of total LDL catabolism, should enable one to calculate the extent of LDL receptor-dependent catabolism. To determine the contribution of each pathway in man, we prepared (125)I-GLC-LDL and (131)I-control LDL and simultaneously determined the fractional catabolic rate (FCR) of each tracer in four subjects. In preliminary experiments, we showed that the conditions for glucosylation did not affect LDL turnover. In the four subjects, the FCR for total LDL catabolism ranged from 0.345 to 0.724 d(-1) with a mean of 0.57+/-0.16 d(-1). The FCR of GLC-LDL varied from 0.071 to 0.141 d(-1) with a mean of 0.11+/-0.03 d(-1). The latter is similar to the FCR reported for native LDL in subjects with homozygous familial hypercholesterolemia, supporting the interpretation that GLC-LDL traces only the receptor-independent pathway. Despite the wide range of total LDL catabolism in these subjects. LDL receptor-independent catabolism accounted for only 19.5-20.6% of total catabolism. In turn, LDL receptor-dependent catabolism accounted for 80% of total clearance in each person. Furthermore, while the decay curve of LDL showed the usual biphasic pattern, the decay curve of GLC-LDL was monoexponential in each subject even when followed for as long as 48 d. This suggests that LDL receptor activity is responsible for the biphasic nature of LDL decay. These studies emphasize the central role of LDL receptor activity in normal LDL metabolism in man.

Oxidized or acetylated low density lipoproteins are rapidly cleared by the liver in mice with disruption of the scavenger receptor class A type I/II gene.

Oxidized low density lipoprotein (LDL) and acetyl LDL are recognized by the scavenger receptor class A type I/II (SR-AI/II) on macrophages and liver endothelial cells. Several investigators have suggested that there are additional receptors specific for oxidized LDL, but characterization of these alternate receptors for oxidized LDL and evaluation of their quantitative importance in uptake of oxidized LDL has been difficult because of overlapping ligand specificity with SR-AI/II. The purpose of this study was to determine the importance of SR-AI/II in the removal of modified LDL from the bloodstream in vivo. The clearance rate of oxidized LDL from plasma in normal mice was very rapid, and > 90% of injected dose was removed from the blood within 5 min. Clearance rates of oxidized LDL were equally high in SR-AI/II knockout mice, indicating that this receptor is not required for removal of oxidized LDL from plasma. Surprisingly, there was no difference in the clearance rate of acetyl LDL in wild-type and SR-AI/II knockout animals. The plasma clearance of radioiodinated acetyl LDL was almost fully blocked by a 50-fold excess of unlabeled acetyl LDL, but the latter only inhibited oxidized LDL clearance by approximately 5%. Both modified LDLs were cleared mostly by the liver, and there was no difference in the tissue distribution of modified LDL in control and knockout mice. Studies in isolated nonparenchymal liver cells showed that Kupffer cells accounted for most of the uptake of oxidized LDL. Extensively oxidized LDL and LDL modified by exposure to fatty acid peroxidation products were efficient competitors for the uptake of labeled oxidized LDL by SR-AI/II-deficient Kupffer cells, while acetyl LDL and malondialdehyde-modified LDL were relatively poor competitors.

Comparison of glucosylated low density lipoprotein with methylated or cyclohexanedione-treated low density lipoprotein in the measurement of receptor-independent low density lipoprotein catabolism.

We previously showed that glucosylation of lysine residues of low density lipoproteins (LDL) blocks high-affinity degradation by cultured human fibroblasts, and markedly slows LDL turnover in guinea pigs. The present studies were done to evaluate glucosylated (GLC) LDL as a tracer of receptor-independent LDL catabolism, and to compare it with two other modified LDL, methylated (MET) LDL, and cyclohexanedione (CHD)-treated LDL, which have been used previously for this purpose. Glucosylation of LDL did not affect receptor-independent degradation in vivo, as the turnover of GLC-LDL and native LDL were similar in the LDL receptor-deficient, Watanabe heritable hyperlipidemic rabbit. Each modified radiolabeled LDL preparation was injected into eight guinea pigs, and fractional catabolic rates (FCR) determined. The FCR of GLC-LDL (0.024 +/- 0.005 h-1; SD) was similar to that of MET-LDL (0.023 +/- 0.006 h-1), and approximately 22% of that of native LDL (0.105 +/- 0.02 h-1). The FCR of CHD-LDL was greater than that of the other modified LDL, and it varied depending on how soon after preparation the CHD-LDL was injected: when used within 2 h of preparation, the mean FCR was 0.044 +/- 0.007 h-1 (n = 4); when used after overnight dialysis at 4 degrees C, the mean FCR was 0.082 +/- 0.03 h-1 (n = 4). This suggests that CHD-LDL overestimates the amount of LDL degraded by receptor-independent pathways, perhaps because the CHD modification is spontaneously reversible. The present studies indicate that GLC-LDL is a useful tracer of receptor-independent LDL catabolism in animals.

Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma.

Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.

Colonic malakoplakia in a liver transplant recipient.

Malakoplakia is a rare inflammatory condition seen in transplant patients. There are two previously reported cases of malakoplakia involving the gastrointestinal tract in liver transplant patients. The present paper reports a case of colonic malakoplakia in a 58-year-old woman, a liver transplant recipient who was receiving immunosuppressive drugs. She presented with chronic diarrhea while on tacrolimus. There was no history of antecedent infection. Colonoscopy showed patchy mucosal edema, but no discrete yellow plaques or nodules. The diagnosis was made by colon biopsies, which showed chronic inflammation with many histiocytes containing Michaelis-Gutmann bodies. Although rare, malakoplakia is one of many potential causes of diarrhea in a transplant patient. The present case indicates that malakoplakia may be associated with chronic diarrhea, even if there are no macroscopic lesions seen during colonoscopy.

Role of superoxide in endothelial-cell modification of low-density lipoproteins.

Cultured endothelial cells and arterial smooth muscle cells have been shown to modify LDL in a way that leads to rapid uptake by macrophages. Previous studies have demonstrated that this modification involves free radical peroxidation of LDL, and that the role of the cells was to accelerate oxidation under conditions where it otherwise would occur slowly. The objective of the present study was to determine whether the modification was mediated by oxygen-derived free radicals, and whether the ability of a given cell type of line to modify LDL was related to its secretion rate of O2- or H2O2. The results showed that modification required the presence of oxygen, and could be specifically inhibited by superoxide dismutase but not by catalase or by mannitol, a hydroxyl radical scavenger. Rabbit aortic endothelial cells, rabbit arterial smooth muscle cells, monkey arterial smooth muscle cells and human skin fibroblasts were all found to modify LDL, and all of these cell types generated more O2- (superoxide dismutase-inhibitable cytochrome c reduction) than a line of bovine aortic endothelial cells that did not modify LDL. The content of superoxide dismutase and catalase was higher in bovine aortic endothelial cells than in the cell lines that modified LDL, but glutathione peroxidase levels were not different. It was concluded that cells that were capable of modifying LDL produced superoxide or a substance that could be converted to superoxide in the medium, and that superoxide was an important, though possibly indirect, mediator of the modification of LDL by cells.

Receptors for oxidized low density lipoprotein.

An increasing body of evidence indicates that oxidized low density lipoprotein (LDL) is involved in the pathogenesis of atherosclerosis. One of the first biologic actions of oxidized LDL to be identified in vitro was its ability to interact with the 'acetyl LDL receptor' discovered by Goldstein and Brown. Over the past decade, considerable progress has been made in identifying and characterizing cell-surface receptors for oxidized LDL. Most of these receptors are thought to be multifunctional because they interact with several structurally different ligands, and accordingly have been termed 'scavenger receptors'. The objective of this article is to review the most important publications dealing with structure, ligand specificity, regulation, and function of scavenger receptors.

5-Aminosalicylate stimulates phospholipase D activity in macrophages.

5-Aminosalicylate, which is considered to be the active moiety of sulfasalazine, is one of the most widely used agents for treatment of inflammatory bowel disease. However, its mechanism of action is unclear. In this report, we provide evidence that the phospholipase D pathway is a target for this drug in macrophages. Activation of phospholipase D leads to the generation of important second messengers such as phosphatidic acid, lysophosphatidic acid and diacylglycerol, all of which can regulate cellular responses involved in inflammation. Murine peritoneal macrophages were labeled with [(3)H]myristate, incubated with various drugs, agonists, or inhibitors, and phospholipase D activity was assayed. 5-Aminosalicylate or sulfasalazine stimulated phospholipase D in a time- and concentration-dependent manner. Chelation of extracellular Ca(2+) inhibited phospholipase D activation by either of these drugs whereas pretreatment of macrophages with the tyrosine kinase inhibitor genistein had no effect. Downregulation of protein kinase C by prolonged incubation with phorbol ester completely blocked the activation of phospholipase D. Pertussis toxin decreased the activation of phospholipase D. The levels of inositol 1,4,5-trisphosphate increased by 260% after treatment of macrophages with 5-aminosalicylate. A phosphoinositide-specific phospholipase C inhibitor U73122 blocked phospholipase D activation completely. Interestingly, long-term preincubation of the macrophages with a relatively low concentration of 5-aminosalicylate that did not stimulate phospholipase D activity by itself, potentiated the effect of phorbol ester-induced activation of phospholipase D. Taken together, these results show that 5-aminosalicylate activates phospholipase D via a pathway involving inositol 1,4,5-trisphosphate generation, calcium fluxes, and Gi/Go. Although the mechanisms by which phospholipase D activation by 5-aminosalicylate or sulfasalazine might attenuate inflammatory responses in the intestine remain to be defined, these results highlight a novel potential mechanism of action for these drugs.

The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity.

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(+/-)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 micrograms/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70-100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 micrograms/ml. Either of these inhibitors when present at 20 micrograms/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.

Glucosylation of low-density lipoproteins to an extent comparable to that seen in diabetes slows their catabolism.

In previous studies we have shown that extensive glucosylation of low-density lipoproteins (LDL) (40% of lysines modified) completely blocks receptor-mediated degradation in animals and in man. Other studies indicated that in some diabetics up to 5% of lysine residues of LDL were glucosylated. The present study was done to determine if the extent of glucosylation of LDL which can occur in diabetics could alter LDL catabolism. We measured degradation by cultured normal human fibroblasts and turnover in guinea pigs of various LDLs with 2-17% of lysine residues glucosylated. Modification of as few as 2-5% of lysines decreased LDL catabolism by 5-25%, and the degree of inhibition of catabolism was linearly related to the extent of LDL glucosylation. These results indicate that the extent of LDL glucosylation that can occur in diabetes may slow LDL catabolism and hence increase plasma LDL levels.

Nonenzymatic glucosylation of high-density lipoprotein accelerates its catabolism in guinea pigs.

Nonenzymatic glucosylation of lysine residues of high-density lipoprotein (HDL) was shown to occur in vitro. Most of the incorporated glucose was localized to apoprotein A-I, but other apoproteins were glucosylated as well. Glucosylated high-density lipoproteins (glcHDL) had enhanced electrophoretic mobility on agarose. With increasing amounts of glucose incorporated there was a proportionate increase in the rate of clearance of glcHDL when injected intravenously into guinea pigs. When 60% of lysines were derivatized, clearance of glcHDL was 60% faster than that of control HDL. When as few as 2% of lysines were glucosylated, there was still an 8% increase in the rate of clearance. Uptake of glcHDL by macrophages was not increased. The accelerated clearance of glcHDL from plasma may be relevant to the decreased HDL levels observed in diabetic subjects.

Serious infection in an adult due to penicillin-tolerant group B streptococcus.

This report describes a patient with group B streptococcal (GBS) bacteremia with pyelonephritis and septic arthritis whose condition failed to improve after two weeks of therapy with penicillin G sodium. The organism was found to be tolerant to penicillin (minimal inhibitory concentration, 0.06 IU/mL; minimal bactericidal concentration [MBC], 10 IU/mL). Antimicrobial synergy with gentamicin sulfate was demonstrated (MBC of penicillin was 0.07 IU/mL in the presence of 2.5 micrograms/mL of gentamicin). Addition of gentamicin to penicillin therapy was associated with clinical improvement. It is suggested that bactericidal rather than inhibitory susceptibility tests be employed as a guide to therapy in serious GBS infections. Where penicillin tolerance is found in association with a poor clinical response to penicillin, addition of an aminoglycoside should be considered. Antimicrobial synergy studies should be performed to demonstrate that a beneficial effect is possible at clinically attainable antibiotic concentrations.

Torsade de pointes. A cause of syncope with atrioventricular block.

Two patients with syncope due to an atypical ventricular tachycardia are described. In both, undulation of the QRS axis during episodes of tachycardia, a prolonged QT interval, the initiation of the tachycardia by a late ventricular premature depolarization, and the association with high-degree atrioventricular block suggested a diagnosis of "torsade de pointes" (twisting of the points). Therapy with conventional antiarrhythmic drugs did not control the arrhythmia, but increasing the heart rate by transvenous pacing completely suppressed the tachycardia. In one patient, the QT interval became normal with ventricular pacing alone, while in the second, the QT interval remained abnormal until correction of coexisting hypokalemia. Torsade de pointes is a recognized complication of bradyarrhythmias, hypokalemia, and therapy with quinidine-like and psychotropic drugs. The ideal treatment is to recognize and correct the underlying cause, as antiarrhythmic drugs can exacerbate the arrhythmia.

Histoplasmosis and Lennert's lymphoma.

Histoplasma capsulatum was cultured from splenic tissue obtained at diagnostic laparotomy in a patient with "Lennert's lymphoma." The patient had no clinical features suggesting histoplasmosis, and the organism was not seen on histologic specimens. It is suggested that mycologic cultures as well as histopathological examinations of surgical specimens be performed routinely in immunologically compromised individuals.

Role of oxidatively modified LDL in atherosclerosis.

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, including increased electrophoretic mobility, increased density, fragmentation of apolipoprotein B, hydrolysis of phosphatidylcholine, derivatization of lysine amino groups, and generation of fluorescent adducts due to covalent binding of lipid oxidation products to apo B. In addition, oxidation of LDL has been shown to result in numerous changes in its biologic properties that could have pathogenetic importance, including accelerated uptake in macrophages, cytotoxicity, and chemotactic activity for monocytes. The present article summarizes very recent developments related to the mechanism of oxidation of LDL by cells, receptor-mediated uptake of oxidized LDL in macrophages, the mechanism of phosphatidylcholine hydrolysis during LDL oxidation, and other biologic actions of oxidized LDL including cytotoxicity, altered eicosanoid metabolism, and effects on the secretion of growth factors and chemotactic factors. In addition, this review will examine the evidence for the presence of oxidized LDL in vivo and the evidence that oxidized LDL plays a pathogenetic role in atherosclerosis.

Effects of oxidized low density lipoprotein on endothelin secretion by cultured endothelial cells and macrophages.

The aim of this study was to determine how oxidized LDL affects endothelin secretion by endothelial cells, monocytes and macrophages. It was found that different degrees of oxidation of LDL had different effects on endothelin production. Extensively oxidized LDL inhibited endothelin secretion from cultured endothelial cells. It also attenuated endothelin secretion from phorbol ester-activated macrophages. The inhibitory effect on endothelin secretion required a substantial degree of LDL oxidation as reflected by an increase in absorbance at 234 nm (conjugated diene) of 0.7 AU with 125 nM LDL and a two- to three-fold increase in migration distance on electrophoresis. Oxidized LDL inhibited thymidine incorporation in porcine aortic endothelial cells, hence in these cells cytotoxicity may account for at least part of the inhibition of endothelin secretion. Acetyl LDL slightly increased basal endothelin release by endothelial cells, but native LDL or mildly oxidized LDL had no significant effect. Overall, the present findings argue against a stimulatory effect of oxidized LDL on endothelin release as contributing to increased vasoreactivity in atherosclerosis. In fact, the apparent inhibition of endothelin release by extensively oxidized LDL might tend to attenuate vasoreactivity near atherosclerotic lesions.

Alteration of the dismal natural history of fibrosing cholestatic hepatitis secondary to hepatitis B virus with the use of lamivudine.

BACKGROUND: Fibrosing cholestatic hepatitis (FCH) is a severe form of hepatitis B virus (HBV) infection occurring as either primary allograft reinfection after liver transplantation for HBV or as severe HBV reactivation induced by immunosuppression in patients with previously latent infection. Without treatment, FCH is universally fatal within a few months of diagnosis. Some improvement has been reported with long-term ganciclovir, with and without foscarnet, but an effective and easily available treatment has not yet been reported. METHODS: We report the prolonged survival of a renal transplant recipient who developed histologically confirmed FCH 6 months after transplantation and was treated with lamivudine. RESULTS: At the time of diagnosis, the patient had jaundice, ascites, and a serum HBV-DNA level of 3868 pg/ml. Lamivudine was instituted 2 months later, and after 6 months of treatment, the HBV-DNA level was undetectable with the serum bilirubin within the normal range. Twelve months after the diagnosis of FCH, the patient remains stable without progression of liver dysfunction. CONCLUSION: Our experience demonstrates that lamivudine therapy can improve the dismal natural history of FCH.

Recurrence of extrahepatic biliary atresia in two half sibs.

Extrahepatic biliary atresia (EHBA) usually is a sporadic disorder. Familial cases, including occurrence in sibs and twins, have been reported. We report the first recurrence of EHBA in two half sibs born to a common father and unrelated mothers and suggest the possibility of gonadal mosaicism for a new dominant mutation.

Scavenger receptors and oxidized low density lipoproteins.

Oxidized LDL has been shown to exhibit a number of potentially proatherogenic actions and properties, including receptor-mediated uptake and lipid accumulation within macrophages. It has been postulated that rapid, unregulated uptake of oxidatively modified LDL could account for the transformation of monocyte-derived macrophages to foam cells in atherosclerotic lesions. In support of this hypothesis, oxidized LDL and lipid peroxidation products have been shown to exist in atheromas in vivo. Furthermore, a number of cell membrane proteins that can bind oxidized LDL with high affinity have been identified on the surface of macrophages, endothelial cells and smooth muscle cells. One characteristic that almost all of these 'scavenger receptors' share is the ability to bind with high affinity to a broad spectrum of structurally unrelated ligands. Of all of the different classes of scavenger receptors that have been identified, the scavenger receptor class A type I/II (SR-AI/II) has received the most attention. Studies with macrophages from mice deficient in the gene for SR-AI/II provide direct evidence that a receptor other than the SR-AI/II is responsible for most of the uptake of oxidized LDL in murine macrophages. This article provides an overview of the characterization and functions of the scavenger receptors that have been shown to interact with oxidized LDL, including SR-AI/II, CD36, SR-BI, macrosialin/CD68, LOX-1, and SREC. Isolation and characterization of these and other scavenger receptors has increased our understanding of their role in the uptake of oxidized LDL and the pathogenesis of atherosclerosis.

Post-surgical shunt hepatopulmonary syndrome in a case of non-cirrhotic portal hypertension: lack of efficacy of shunt reversal.

Hepatopulmonary syndrome, a consequence of significant liver disease and portal hypertension, is thought to be secondary to the effects of vasoactive substances, normally inactivated in the liver, on the pulmonary vasculature. We report a patient with preserved hepatic function who underwent a decompressive surgical porto-systemic shunt for non-cirrhotic portal hypertension. This patient developed hepatopulmonary syndrome with dyspnoea and oxygen desaturation 2 years post-surgical shunt. Over the next 7 years, the patient's respiratory function became increasingly impaired although hepatic function remained preserved. Because of the hypothesized role of porto-systemic shunting in the aetiology of this syndrome, the surgical shunt was successfully reversed angiographically. No improvement in dyspnoea or oxygen saturation occurred and liver transplantation was undertaken. Six months post-transplant, the patient has decreased his oxygen requirements and is free of dyspnoea. Our experience supports the causal role of porto-systemic shunting in the pathogenesis of hepatopulmonary syndrome but suggests that merely decreasing the extent of porto-systemic shunting is not beneficial. Liver transplantation remains the only reliable therapeutic modality available to these patients.