Effects of low dose oral contraceptives on very low density and low density lipoprotein metabolism

Oral contraceptives (OC) raise plasma triglyceride and VLDL levels, which may be of concern, since some conditions characterized by elevated triglycerides are associated with atherosclerosis. To identify the responsible mechanism, we studied 11 healthy premenopausal women, 5 of whom were taking OC containing 0.035 mg ethinyl estradiol, and 6 of whom were not. Their rates of VLDL and LDL metabolism were measured by endogenously labeling apoB, the protein component of VLDL and LDL, by an intravenous infusion of deuterated leucine. OC use had the greatest effect on the large, triglyceride-rich VLDL subfraction (Sf 60-400), increasing plasma levels threefold and production rates fivefold (P < 0.05). Among OC users, small VLDL (Sf 20-60) levels were 2.2 times higher, and production rates were 3.4-fold higher (P < 0.05). The fractional catabolic rates of large and small VLDL were similar among OC users and nonusers. LDL levels and metabolic rates were not significantly different between the two groups. Thus, contemporary low dose OC substantially raise VLDL levels by increasing the production rate of large, triglyceride-rich VLDL, and not by slowing VLDL catabolism. Since VLDL catabolism is not impaired, we speculate that the hypertriglyceridemia induced by OC may be less atherogenic than that of hypertriglyceridemia resulting from impaired lipolysis. This may explain why long-term OC use does not appear to promote atherosclerosis.     

Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation

Given the multiple impairments in host defense that occur during HIV infection, patients with AIDS are at risk for a variety of pleural infections and neoplasms. Of infectious causes, bacterial parapneumonic effusions and empyemas and tuberculous pleurisy occur more frequently than effusions caused by P. carinii. In each case, therapy is directed at eradication of the causative organisms. In the setting of systemic Kaposi's sarcoma, pleural involvement is common, although diagnosis is difficult and therapeutic options are limited. Pleural effusions caused by non-Hodgkin's lymphoma often occur in the setting of pulmonary parenchymal disease and can be diagnosed cytologically. The recently described entity of primary effusion lymphoma occurs in the absence of solid-organ involvement. The development of a spontaneous pneumothorax in a HIV-infected individual should prompt a search for P. carinii infection. Although these pneumothoraces often recur and are difficult to manage, recent series suggest that surgical approaches to bronchopleural fistulas are reasonable in selected patients.     

The effects of low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.     

Altered apolipoprotein B metabolism in very low density lipoprotein from lovastatin-treated guinea pigs

Previous studies have shown that treatment of guinea pigs with lovastatin alters the composition and the metabolic properties of circulating low density lipoprotein (LDL). Specifically, LDL isolated from lovastatin-treated animals is cleared from plasma more slowly than LDL isolated from control animals, when injected into the guinea pig. In the present study, we examine whether lovastatin also affects the metabolic properties of very low density lipoprotein (VLDL), the metabolic precursor of LDL. VLDL isolated from lovastatin-treated guinea pigs (L-VLDL) and VLDL isolated from untreated (control) guinea pigs (C-VLDL) were radioiodinated and simultaneously injected into eight untreated guinea pigs. Radioactivity associated with apolipoprotein B-100 (apoB) was measured in four plasma density fractions and analyzed using a compartmental model consisting of fast and slow pools for VLDL, fast and slow pools for intermediate density lipoprotein (IDL), and a single slow pool for LDL. The fractional catabolic rate (FCR) for C-VLDL apoB was 2.8 +/- 1.0 h-1 and for L-VLDL apoB was 5.1 +/- 2.0 h-1 (P < 0.002, paired t test). The fractions of control and lovastatin VLDL apoB converted to LDL averaged 0.15 +/- 0.15 and 0.02 +/- 0.02, respectively (P < 0.05, paired t test). Finally, the FCRs of LDL apoB derived from control and lovastatin VLDL were similar (0.059 +/- 0.007 h-1 and 0.083 +/- 0.038 h-1, respectively; paired t test not significant). These data indicate that L-VLDL was irreversibly removed from the plasma of an untreated guinea pig more rapidly than was C-VLDL. Thus, the metabolic behavior of VLDL apoB is affected by lovastatin. Therefore, changes in lipoprotein particles themselves must be considered in assessing the overall impact of treatment with lovastatin.     

Effect of low density lipoprotein on adenosine receptor-mediated coronary vasorelaxation in vitro

We investigated the effect of low density lipoprotein (LDL) on vasorelaxations and nitric oxide generation induced by the adenosine analogs, 5'-(N-ethylcarboxamide)adenosine, 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamidoadenosine and/or 2-chloroadenosine in porcine coronary artery rings in vitro. Preincubation of tissues with native LDL (100 and 200 microg/ml) for 4 hr in the absence or presence of copper sulfate (5 microM) selectively attenuated the endothelium-dependent relaxations elicited by 5'-(N-ethylcarboxamide)adenosine and 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamideoadenosine+ ++ without altering the response to 2-chloroadenosine which produced endothelium-independent relaxation. The 4-hr exposure of tissues to native LDL (100 microg/ml) also inhibited the production of nitrite induced by 5'-(N-ethylcarboxamide)adenosine in endothelium-intact rings. These effects were associated with enhanced oxidation of the lipoprotein. The inhibitory action of LDL on tissue relaxations and nitrite generation as well as the oxidation of the lipoprotein were all prevented by high density lipoprotein (100 microg/ml). In contrast, a relatively short period (20 min) of tissue incubation with native LDL produced no alterations of the relaxations and nitrite production evoked by 5'-(N-ethylcarboxamide)adenosine and 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamidoadenosine. Under this condition, the oxidation of LDL was not also significantly altered. In conclusion, the results indicate that in coronary artery LDL, with oxidative modification, causes attenuation of nitric oxide-mediated endothelial responses induced by adenosine receptors activation, and this effect is prevented by high density lipoprotein. Such modulation may be of importance in hypercholesterolemia and in the development of atherosclerosis.     

Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance

The oxidation of low density lipoproteins (LDLs) is thought to take place in the arterial intima when the particles have become isolated from circulating water-soluble antioxidants. We hypothesized that isoflavonoid antioxidants derived from soy could be incorporated into lipoproteins and possibly could protect them against oxidation, which is regarded as atherogenic. Six healthy volunteers received 3 soy bars [containing the isoflavonoid antioxidants genistein (12 mg) and daidzein (7 mg)] daily for 2 weeks. LDLs were isolated from blood drawn at the the end of a 2-week dietary baseline period, after 2 weeks on soy, and after discontinuation of soy. Large increases in plasma isoflavonoid levels occurred during soy feeding, but only minute amounts were stably associated with lipoproteins (less than 1% of plasma isoflavonoids in the LDL fraction). The LDLs were subjected to copper-mediated oxidation in vitro. Compared with off soy values, lag phases of LDL oxidation curves were prolonged by a mean of 20 min (P < 0.02) during soy intake, indicating a reduced susceptibility to oxidation. The results suggest that intake of soy-derived antioxidants, such as genistein and daidzein, may provide protection against oxidative modification of LDL. As only very small amounts of these substances were detected in purified LDL, modified LDL particles may have been produced in vivo by circulating isoflavonoids promoting resistance to oxidation ex vivo.     

Effect of estradiol metabolites on the susceptibility of low density lipoprotein to oxidation

The presence of a benign osteoblastoma in the ethmoid sinus is rare and only a few cases have been reported. This is a case of a benign osteoblastoma arising from the perpendicular plate of the ethmoid bone with extension to the nasal cavity. The diagnosis and management of this unusual lesion, as well as the histopathology and the imaging characteristics are reviewed. We also review the previously reported cases of benign osteoblastomas of different origin, with nasal cavity involvement.     

Macrophage oxidative modification of low density lipoprotein occurs independently of its binding to the low density lipoprotein receptor

The oxidative modification of low density lipoproteins (LDL) by arterial wall cells is thought to contribute to atherogenesis. Monocyte/macrophages, among other arterial wall cells, oxidatively modify LDL to a form that is recognized by scavenger/oxidized LDL receptors. It has recently been suggested that LDL binding to the LDL receptor (B/E receptor) is essential for macrophage-mediated oxidation of LDL. In the present study, we compared the ability of resident peritoneal macrophages from LDL-R-deficient (LDLR-/-) mice to oxidize LDL with that of resident peritoneal macrophages from C57B6 mice. The LDLR-/- macrophages oxidized LDL at least as rapidly as did the C57B6 macrophages both in F-10 medium and in Dulbecco's modified Eagle's medium supplemented with 1 microM copper (DMEM-Cu2+). Studies were also conducted to examine the effect of preincubation of LDLR-/- and C57B6 macrophages with 10% lipoprotein-deficient serum (LPDS), which up-regulates LDL receptors, or with acetylated LDL (Ac-LDL), which increases cellular cholesterol and down-regulates LDL receptors. Preincubation with 10% LPDS had no significant effect on subsequent LDL oxidation by either type of cells in F10 medium, but the C57B6 cells did show a small (18%) but significant increase in LDL oxidation in DMEM-Cu2+. Preincubation with 50 micrograms/ml Ac-LDL in F10 medium had no effect on LDL oxidation by either LDLR-/- or C57B6 macrophages. Preincubation with 100 micrograms/ml Ac-LDL had no effect on subsequent LDL oxidation by C57B6 cells but, unexpectedly, caused a modest (26%) fall in LDL oxidation by the receptor-negative cells. Using DMEM-Cu2+ medium, preincubation with Ac-LDL reduced LDL oxidation substantially (50-66%) but the effect was just as great in LDL-R negative cells (59-66%) as in C57B6 cells (50-58%), suggesting that the effect is not due to changes in LDL receptor density. It may be related somehow to the Ac-LDL-induced increase in cell cholesterol content. The data demonstrate that the absence of LDL receptors does not reduce the ability of macrophages to oxidize LDL and that LDL binding to LDL receptors is not an essential requirement for macrophage oxidation of LDL.     

The role of a triplet repeat sequence of the very low density lipoprotein receptor gene in plasma lipid and lipoprotein level variability in humans

The biological role of the very low density lipoprotein receptor (VLDL-R) in humans is not yet elucidated. This cellular receptor binds apolipoprotein E (apoE)-containing lipoparticles and is mainly expressed in peripheral tissues. The VLDL-R gene contains a polymorphic triplet (CGG) repeat located 19 bp upstream of the initiation codon. We explored the allelic distribution of this repeat in 1384 subjects of European Caucasian origin, 609 of them surviving a myocardial infarction. Six alleles corresponding to 5, 6, 7, 8, 9, and 11 repeats were detected in this population. The alleles 5, 8, and 9 were the most frequent, with frequencies of 0.413, 0.275, and 0.292, respectively. No association was found between the VLDL-R polymorphism and myocardial infarction. In controls without lipid lowering treatment, a statistically significant interaction between VLDL-R genotype and apoE phenotype was found for plasma triglycerides (P < .04), suggesting a gene-gene interaction. There was also a main effect of the VLDL-R polymorphism on LpE:B and LpA-I. The VLDL-R 9 allele was associated with lower levels of plasma LpE:B (P < .05) and higher concentrations of plasma LpA-I (P < .01) than the other alleles. These results suggest that VLDL-R has a modest influence on circulating lipoproteins in humans.     

Composition of plasma and nascent very low density lipoprotein from perfused livers of hypercholesterolemic squirrel monkeys

The composition of circulating very low density lipoprotein (VLDL) was compared with the composition and secretion of nascent VLDL from perfused livers of squirrel monkeys that were fed unsaturated or saturated fat diets to elicit different degrees of plasma hypercholesterolemia. All squirrel monkeys studied had cholesteryl ester-rich plasma VLDL, although greater enrichment occurred in hypercholesterolemic animals fed saturated fat. Livers from hypercholesterolemic animals were capable of secreting VLDL particles enriched in cholesteryl ester, suggesting hepatic origin for a portion of this circulating lipid moiety. Total VLDL lipid, but not protein output by perfused livers of hypercholesterolemic monkeys, was greater than that by livers from hypocholesterolemic animals. These results indicate that saturated fat-induced hypercholesterolemia is associated with changes in the composition of hepatic VLDL in the squirrel monkey.     

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.     

Fatty acid metabolism and very low density lipoprotein secretion in liver slices from rats and preruminant calves

Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.     

Inhibitory action of silibinin on low density lipoprotein oxidation

The purpose of this study is to demonstrate a method of how to remove epithelium grown beneath the hinge area after laser in situ keratomileusis (LASIK) without affecting the refractive part of the lenticule. In three cases, an incision was made at the base of the hinge by RK diamond knife to free the lenticule from the stroma. The lenticule was lifted from the nasal edge. The epithelium grown along the interface beneath the hinge area was removed with a Bard-Parker No. 15 knife. The lenticular flap was repositioned with interrupted sutures using 10-0 nylon. No further epithelial ingrowth was observed. The central cornea remained clear leaving a peripheral ring-shaped opacity without affecting the preoperative naked visual acuity. In conclusion, epithelial ingrowth along the interface after LASIK can be removed safely without affecting the refractive part by the incision of the hinge area with a RK diamond knife, removal of the epithelium, and suturing of the lenticule to the stromal bed.     

Charge properties of low density lipoprotein subclasses

Measurements of electrophoretic mobility and particle size of low density lipoproteins (LDL) allowed use of standard electrokinetic theory to quantitate LDL charge characteristics from subjects with predominance of large LDL (pattern A, n = 9) or small LDL (pattern B, n = 8). Pattern A LDL was found to have significantly lower (P < or = 0.001) mobility (-0.22 +/- 0.01 micron s-1 cm V-1), surface potential (-4.2 +/- 0.3 mV) and charge density (-500 +/- 34 esu/cm2) than pattern B LDL (-0.25 +/- 0.01 micron s-1 cm V-1, -4.9 +/- 0.3 mV, and -580 +/- 30 esu/cm2), but no significant difference in particle valence (-22.0 +/- 1.4 for pattern A vs. -21.8 +/- 1.9 for pattern B). Thus, the greater mobility of pattern B LDL is due to similar net charge residing on a smaller particle. Comparison of subfractions in pattern B relative to pattern A LDL revealed greater surface potential in all pattern B subfractions and greater charge density in fractions of d > or = 1.032 g/ml. In a subset of subjects incubation with neuraminidase produced significant reductions in all LDL charge parameters for all subfractions, but did not abolish the differences between pattern A and B. Thus increased surface potential and charge density of unfractionated pattern B LDL is due both to charge properties of particles across the size and density spectrum as well as enrichment of pattern B LDL with smaller, denser particles that have higher surface charge density.     

Determinants of the kinetics of very low-density lipoprotein apolipoprotein B-100 in non-obese men

1. Apolipoprotein B-100 (ApoB) is the principal structural and functional protein of the pro-atherogenic lipoproteins. Elevated plasma apoB is an independent risk factor for coronary artery disease. In the present study we aimed to assess the factors that determine the kinetics of apoB in the very low-density lipoprotein (VLDL) in healthy men. 2. We studied 17 non-obese men who were consuming an ad libitum diet and had the following characteristics: mean (+/-SD) age 45.5 +/- 9.7 years, body mass index (BMI) 25.1 +/- 1.4 kg/m2, waist:hip ratio 0.91 +/- 0.04, serum cholesterol 5.2 +/- 0.6 mmol/L, triglycerides 1.08 +/- 0.53 mmol/L and high-density lipoprotein-cholesterol 1.24 +/- 0.31 mmol/L. Daily dietary intake was as follows: total fat 76 +/- 26 g, carbohydrate 238 +/- 67 g, protein 103 +/- 33 g and alcohol 20 +/- 16 g. 3. The kinetics of VLDL ApoB were studied using a primed, constant infusion (1 mg/kg per h) of 1-[13C]-leucine over 8 h with measurement of isotopic enrichment of ApoB using gas chromatography/mass spectrometry. The fractional turnover rate of VLDL ApoB was estimated using a monoexponential function. The mean (+/-SD) absolute hepatic secretion rate (ASR) of ApoB was 8.5 +/- 4.6 mg/kg per day and the fractional catabolic rate (FCR) was 7.9 +/- 5.6 pools/day. The ASR was significantly correlated with the waist:hip ratio (r = 0.60; P = 0.04), but not with age, BMI, weight or nutrient intake. The FCR was significantly and inversely correlated with plasma triglycerides (r = -0.53; P = 0.03) and alcohol intake (r = -0.48; P = 0.05). 4. In conclusion, the hepatic secretion of VLDL ApoB in nonobese, healthy men is primarily determined by the waist:hip ratio, a measure of visceral fat. This is consistent with the hypothesis that the rate of lipid substrate supply in the liver regulates the output of ApoB. The fractional catabolism of VLDL ApoB may, however, be inversely related to alcohol intake and appears to determine the plasma concentration of triglycerides.     

Physico-chemical properties of copper-oxidized very low density lipoproteins

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.     

The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells

In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL. A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (-BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.     

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.     

Identification of oxidized low density lipoprotein in human renal biopsies

BACKGROUND: Intraglomerular lipid deposition is frequently observed in routine renal biopsies, and it has been suggested that lipid peroxidation of low density lipoprotein (LDL) may be implicated in the pathogenesis of progressive glomerulosclerosis. We have examined whether oxidized LDL (Ox-LDL) is present in the glomeruli of patients with renal disease and whether intrinsic human glomerular cells express NADPH-oxidase (a superoxide-generating enzyme found in professional phagocytes). METHODS: Immunocytochemical study was performed on 939 renal biopsy specimens, using monoclonal antibodies (mAbs) OL-10, 48 and 449, and polyclonal antibody against human apolipoprotein (apo) B. Mouse mAb OL-10 recognizes malondialdehyde (MDA)-modified peptide epitope, and mAbs 48 and 449 react with alpha and beta subunits of cytochrome b558, an essential component of NADPH-oxidase. RESULTS: Sixty-two (6.6%) of the 939 patients with renal disease exhibited a staining for MDA-altered protein or Ox-LDL in the glomeruli, mainly in the sclerotic segments or mesangial areas. Group 1 patients with heavy Ox-LDL deposition mainly in the sclerotic segments showed a higher frequency of renal insufficiency and heavy proteinuria and a greater degree of glomerulosclerosis, compared to those in group 2 with mesangial Ox-LDL staining. The distribution of MDA protein epitopes, in general, paralleled the deposition of apo B epitopes. Immunoelectron microscopy of ultrathin frozen sections showed the presence of immunogold particles for mAbs 48 and 449 in the cytoplasm of resident glomerular cells of both normal and diseased kidneys. When immunoblotted with mAb OL-10, one band from the IgA nephropathy and focal segmental glomerulosclerosis groups at approximately 260 kD was labeled, whereas immunostaining of normal control samples revealed no staining. CONCLUSIONS: These results indicate that Ox-LDL is present mainly in the lesions of glomerulosclerosis and mesangial areas in human renal biopsies. They also suggest that patients with heavy Ox-LDL accumulation in the sclerotic segments of glomeruli have more advanced renal disease than those with mesangial Ox-LDL and that resident glomerular cells generate cytochrome b558, the potential of which may not suffice to induce peroxidation of LDL in the diseased glomeruli.