Accumulation Of Low-density Lipoprotein Research Articles

Oxidation Decreases Low Density Lipoprotein Association with the Subendothelium Extracellular Matrix

Atherosclerosis is initiated by accumulation of low density lipoprotein (LDL) in the subendothelium extracellular matrix (ECM) followed by oxidation and scavenger receptor mediated uptake by the vessel wall recruited macrophages. The effect of oxidation on LDL association with the ECM is not yet clear. In the present study we examined the hypothesis that excessive oxidation of LDL results in decreased LDL association with ECM, thereby increasing its accessibility to the scavenger receptor on macrophages. We have studied the relative association of Cu+2ion oxidized LDL to native LDL with the native or oxidized bovine corneal endothelial cells ECM. Oxidation of LDL decreased its binding to the ECM. The kinetic of this process was characterized by approximately 1 h lag phase followed by a significant decreased binding of 80% after 6.5 h oxidation. This kinetic more closely resembled the pattern of the oxidation product dienals accumulation rather than thiobarbituric acid reactive substance formation. Oxidation of ECM-bound LDL resulted in an increased LDL release from the ECM as a function of Cu+2ion concentration with a maximal increase of 2-fold at 3 μM. ECM oxidation prior to exposure to LDL resulted in a 30% decrease in LDL binding to the ECM. In conclusion, these results suggest that oxidation processes in the vessel wall result in increased dissociation of ECM-bound LDL, which in turn makes this oxidized LDL more accessible for binding and uptake by macrophages leading to foam cell formation.

Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.

To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.

Degranulation of cutaneous mast cells induces transendothelial transport and local accumulation of plasma LDL in rat skin in vivo

In severe hypercholesterolemia and sometimes even in normolipemia, lipoprotein-derived cholesterol accumulates in the skin, forming xanthomas, but the local factors triggering this accumulation are not known. We therefore studied the effect of immunoglobulin E (IgE)-mediated stimulation of cutaneous mast cells on the transport of low density lipoprotein (LDL) from plasma to skin during the passive cutaneous anaphylaxis (PCA) reaction in rats. PCA sites were produced in rats by giving intracutaneous injections of rat serum containing high levels of IgE antibodies against ovalbumin. When ovalbumin was then injected intravenously into the rats, the skin histamine content at the sensitized skin sites decreased, reflecting degranulation of the passively sensitized (IgE-containing) mast cells at these sites, with ensuing release of histamine from the mast cells. Concomitant intravenous injection of increasing amounts of human [14C]sucrose-LDL led to rapid and dose-dependent increases in the skin [14C]sucrose-LDL content. These (maximally about 40-fold) increases were strictly localized to the PCA reaction sites: no such increase in [14C]sucrose-LDL content was observed in nonsensitized skin areas or in any other tissue of the rats. The increase in skin [14C]sucrose-LDL content could be blocked (by about 80%) with a combination of H1 and H2 receptor antagonists, and could be mimicked by intradermal injection of histamine. This study demonstrates an increase in plasma LDL influx into the skin at sites where mast cells are stimulated, and so suggests that mast cells play a role in the accumulation of LDL cholesterol that occurs in the skin when xanthomas form.

Oxidation of Low Density Lipoprotein Particles Decreases Their Ability to Bind to Human Aortic Proteoglycans: DEPENDENCE ON OXIDATIVE MODIFICATION OF THE LYSINE RESIDUES

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products.

Mechanisms responsible for the accumulation of low-density lipoprotein (LDL) were investigated in a new model, the perfused hamster aorta. To do this, we developed a method to study LDL flux in real time in individually perfused arteries; each artery served as its own control. Using quantitative fluorescence microscopy, the rates of LDL accumulation and efflux were separately determined. Perfusion of arteries with buffer plus lipoprotein lipase (LpL) increased LDL accumulation 5-fold (0.1 +/- 0.03 mV/min [control] versus 0.5 +/- 0.05 mV/min [LpL]) by increasing LDL retention in the artery wall. This effect was blocked by heparin and monoclonal antibodies directed against the amino-terminal region of apolipoprotein B (apo B). This suggests that specific regions of apo B are involved in LDL accumulation within arteries. Also, the effect of hydrolysis of triglyceride-rich lipoproteins on endothelial barrier function was studied. We compared endothelial layer permeability using a water-soluble reference molecule, fluorescently labeled dextran. When LpL was added to hypertriglyceridemic plasma, dextran accumulation within the artery wall increased > 4-fold (0.024 +/- 0.01 mV/min [control] versus 0.098 +/- 0.05 mV/min [LpL]). Under the same conditions, LpL increased LDL accumulation approximately 3-fold (0.016 +/- 0.003 mV/min [control] versus 0.047 +/- 0.013 mV/min [LpL]). Rapid efflux of LDL from the artery wall indicated that increased endothelial layer permeability was the primary mechanism during periods of increased lipolysis. Our data demonstrate two LpL-mediated effects that may increase the amount of LDL in the artery wall. These findings may pertain to the observed relationship between increased postprandial lipemia and atherosclerosis.

Effect of deoxycorticosterone acetate on blood pressure in relation to accumulation of low-density lipoprotein and fibrinogen by aorta and other tissues of normotensive Wistar rats.

To evaluate the effect of different 4-week doses of deoxycorticosterone acetate (DOCA), together with 0.9% sodium chloride in the drinking water (DOCA-salt) on the blood pressure and on the accumulation of low-density lipoprotein (LDL) and fibrinogen in artery walls ad other tissues in conscious, unrestrained, normotensive Wistar-Kyoto rats. The accumulation of LDL labelled with 125I via the adduct tyramine cellobiose ([125I]-TC-LDL) and of fibrinogen similarly labelled with 131I ([131I]-TC-fibrinogen) was compared in aortic walls, heart, liver, kidney, lung. skeletal muscle, and adrenal gland tissues during the final 24 h of a 4-week administration of DOCA-salt, with vehicle-salt and saline as controls. In control and vehicle rats the blood pressure did not change significantly during the last 5 days of treatment. Administration of DOCA-salt produced a dose-dependent increase in blood pressure during the same period. DOCA-salt administration increased LDL accumulation in the aorta and the heart and decreased LDL accumulation in the adrenal gland compared with those in rats of the control and vehicle groups. DOCA-salt administration did not affect fibrinogen accumulation significantly. DOCA-salt treatment produces an increase in arterial blood pressure accompanied by an increase in LDL accumulation by the aortic wall and heart and a decrease in LDL accumulation by the adrenal gland. These observations raise the possibility that one mechanism by which hypertension affects atherosclerosis is through increased LDL accumulation in arterial walls.

Macromolecular transport in the arterial intima: comparison of chronic and acute injuries

Hypertension is a known risk factor for the development of atherosclerosis, which is characterized by the abnormal accumulation of low-density lipoprotein and other plasma-borne macromolecules. The goal of this study was to measure accumulation of a plasma-borne macromolecular marker, horseradish peroxidase (HRP; 44 kDa), in the aortic intima and media of chronically hypertensive rats. HRP transport in 2-yr-old spontaneously hypertensive rats (SHR) was compared with that in age-matched Wistar-Kyoto rats (WKY) under conditions in which blood pressures were not significantly different during the 15-min HRP circulation. Intimal accumulation and medial HRP concentration profiles were obtained from methacrylate-embedded sections after reaction with 3,3'-diaminobenzidine and H2O2. Data were analyzed using a mathematical model of macromolecular transport to quantify the permeabilities of endothelium and internal elastic lamina (IEL). Chronic hypertension increased endothelial permeability without a change in IEL permeability. An apparent convective flux of HRP into the intima of SHR raised intimal HRP to a concentration higher than that of HRP in the plasma. Our results suggest that the intimal accumulation of plasma-borne macromolecules from pressure-driven convection is normally minimized by an intact endothelium. Similar changes resulted from acute injury by lipopolysaccharide, suggesting endothelial injury could account for transport changes associated with hypertension. After either chronic or acute endothelial damage, transport of macromolecules into the intima increases, but the IEL continues to retard transport of macromolecules beyond the intima, resulting in increased intimal accumulation.

Effect of Captopril and Losartan on Blood Pressure and Accumulation of LDL and Fibrinogen by Aortic Wall and Other Tissues in Normotensive and Hypertensive Rats

Hypertension, low-density lipoprotein (LDL), and fibrinogen are risk factors for atherosclerosis. We investigated the effect of reducing blood pressure, by blocking the renin-angiotensin system (RAS), on the accumulation of these atherogenic proteins in arterial walls and other tissues in conscious, unrestrained, normotensive and hypertensive rats. The accumulation of LDL and fibrinogen, labeled respectively with 125I and 131I via the adduct tyramine cellobiose ([125I]-TC-LDL and [131I]-TC-fibrinogen) was compared in aortic walls, heart, lung, skeletal muscle, liver, kidney, and adrenal gland during the final 24 h of treatment with either the angiotensin-converting enzyme (ACE) inhibitor captopril or the angiotensin II-receptor I (AT1) antagonist losartan. In normotensive rats, the blood pressure was decreased only by losartan. In spontaneously hypertensive rats (SHRs), the blood pressure was decreased by both losartan and captopril. Captopril had no significant effect on the accumulation of LDL or fibrinogen by the aortic wall. Losartan significantly increased the accumulation of LDL by the aortic wall of SHRs. Neither agent produced any change in LDL or fibrinogen accumulation in any of the other tissues. These results indicate that although blocking the RAS at either the enzymic or receptor level produces significant decrease of blood pressure in hypertensive animals, only losartan has any affect on LDL accumulation by the aortic wall.

Metabolism of Native and Naturally Occurring Multiple Modified Low Density Lipoprotein in Smooth Muscle Cells of Human Aortic Intima

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.

Effects of Diets Containing Tallow and Soybean Oil with and Without Cholesterol on Hepatic Metabolism of Lipids and Lipoproteins in the Preruminant Calf

The effects of long-chain fatty acids (230g/kg of dietary DM) from tallow and from soybean oil, with or without cholesterol (10g/kg of dietary DM), on hepatic lipid contents and on in vivo hepatic production rates of lipids and lipoproteins were investigated in 22 preruminant male calves fitted with chronic catheters and with electromagnetic blood flow probes implanted in the hepatic vessels. Diets containing soybean oil and soybean oil with cholesterol led to the development of triglyceride infiltration in the liver and to higher apparent hepatic secretion of very low density lipoproteins than did diets containing tallow or tallow with cholesterol. Addition of cholesterol to diets favored accumulation of low density lipoproteins in plasma and the net apparent secretion of these particles by the liver, especially for the diet containing soybean oil with cholesterol. Regardless of the diet, calf liver clearly removed large high density lipoproteins of type 1 that were rich in cholesteryl esters but secreted heavy high density lipoproteins that were rich in proteins. The intensity of removal of high density lipoproteins of type 1 by the liver depended on the plasma concentration of these particles, probably by mass action. This removal did not prevent the accumulation of high density lipoproteins of type 1 in plasma, such as it did in calves fed soybean oil.

Effects of pressure-induced stretch and convection on low-density lipoprotein and albumin uptake in the rabbit aortic wall.

The effects of pressure-driven convection and vessel wall stretching in the pressure-related changes in low-density lipoprotein (LDL) and albumin transport across the arterial wall were studied in vitro in freshly excised rabbit thoracic aorta held at in vivo length and pressurized at 70, 120, or 160 mm Hg for 30 minutes. External rigid polyester sleeves of various diameters (4, 5, or 6 mm) were passed around half of the arterial segments in order to prevent vessel distension during pressurization. The intraluminal solution contained 131I-LDL and 125I-albumin. The transmural distribution of relative concentrations of LDL (CLDL) and albumin (Calb) across the wall was determined in wrapped and unwrapped segments using a serial frozen-sectioning technique. In the unwrapped segments, Calb increased uniformly between 70 and 120 mm Hg (P < .0001) but did not change significantly between 120 and 160 mm Hg (0.0063 +/- 0.0009 [n = 4], 0.0520 +/- 0.0055 [n = 9], and 0.0620 +/- 0.0071 [n = 12], respectively). In contrast, CLDL increased markedly both between 70 and 120 mm Hg (P < .001) and between 120 and 160 mm Hg (P < .05) (0.0025 +/- 0.0005 [n = 4], 0.0234 +/- 0.0029 [n = 9], and 0.0393 +/- 0.0056 [n = 12], respectively), with the increase being much more pronounced in the inner than in the outer media. In the segments wrapped with the 4-mm sleeves, both CLDL and Calb did not vary significantly between 70, 120, and 160 mm Hg. In the segments wrapped with the 5-mm sleeves, CLDL increased significantly between 120 and 160 mm Hg, whereas Calb did not vary significantly with increasing pressure. Our results demonstrate that (1) pressure-induced stretching of the arterial wall is a major determinant of arterial mass transport, and (2) pressure-driven convection accentuates LDL accumulation in the inner media, which may explain enhanced atherosclerosis in hypertension.

Benefit/risk of estrogen therapy in cardiovascular disease: current knowledge and future challenges.

In the United States, cardiovascular disease represents the leading cause of death among women. A majority of the deaths are due to coronary disease. In addition, the incidence of heart attacks increases with age. Among those who are 65 years of age or older, the estimated heart attack rate is 374,000 per year for women, compared with 440,000 per year for men. In the past three decades, a number of observational studies have suggested that estrogen therapy can reduce the risk of coronary disease in postmenopausal women. This protective effect appears to be much greater in women who have existing coronary disease. These observational data point to the potential usefulness of estrogen therapy in preventing cardiovascular death among women. Although large, well-controlled, clinical trials are needed to confirm the benefit of estrogen therapy, several important findings strongly support the cardioprotective effect of estrogen therapy. For example, in monkeys estrogen prevents the accumulation of low-density lipoprotein (LDL) cholesterol (a known risk factor for heart disease) in coronary arteries, and estrogen has also been shown to increase high-density lipoprotein (HDL) cholesterol (a known cardioprotective factor). Estrogen also possesses a vasodilating property, which can improve cardiac performance in ischemic heart disease. In addition, recent studies have demonstrated that estrogens (especially equilin) exhibit a high antioxidant effect, which may also be related to cardioprotectivity. Although estrogen therapy has been observed to decrease the risk of coronary disease, long-term estrogen therapy has also been found to increase the risk of uterine carcinoma; the addition of progesterone to estrogen therapy may lessen this undesirable risk, however. On the other hand the addition of progesterone to estrogen therapy may decrease estrogen's beneficial effect on HDL cholesterol. What should be the present position on estrogen therapy in postmenopausal women? What is the best dosage regimen? Should it be used alone or in combination with a progesterone? These important issues are discussed, as are several current clinical trials addressing the issue of estrogen therapy in postmenopausal women.

917-97 Decreased Resistance Against Oxidation of LDL from Patients with Homozigous Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) was the first genetic disorder recognized to cause myocardial infarction. Homozigotes (H) inherit two mutant genes at the low density lipoprotein (LDL) receptor locus, and as a result of the increased levels and prolonged residence time of LDL in plasma, there is a strong tendency toward accumulation of LDL in the arterial wall, causing early atherogenesis. It has been shown that LDL might undergo oxidation before it can be taken up by macrophages and it become foam cells. Thus, one additional explanation for atherogenesis in FH may be the extent to which LDL is susceptible to oxidation. We selected 8 homozigous FH pts (mean total-cholesterol 825 ± 70 mg/dl) matched with 8 healthy subjects to investigate the LDL oxidizability. Skin fibroblast cultures showed that one patient was receptor negative, while others were receptor-defective. LDL were isolated from serum by ultracentrifugations in KBr. Purified LDL was exposed to oxygen radicals generated by the xanthine/xanthine oxidase reaction (2 mM and 100 mU, respectively for 18 hs at 37°C). Malonildihaldehyde (MDAI content was evaluated by the thiobarbiturate method. LDL analysis was carried on polyacrylamide (PAGE; 5 to 16% gradient) and agarose gel electrophoresis (0.8% in Tris-HCL buffer). No significant increase was observed in the basal concentration of MDA between LDL from H and controls (0.8 ± 0.12 and 0.9 ± 0.15 nmoles of MDAlmg of protein, respectively). Instead, afteroxidation MDAwas 35.1 ± 4.5* nmoles/mg of protein LDL from H, and 23.5 ± 4.1 in controls (*p &lt; 0.05). PAGE confirmed the purity of LDL, present as an intact apolipoprotein B 100 (apo-B 100 ). When oxidized LDL was run on PAGE an extensive apo-B 100 fragmentation, replaced by lower fragments ranging from 97.400 to 205.000 m.w., was only observed in LDL from H but not in controls in our experimental conditions. MDA content after oxidation of LDL correlated well with the loss of intact apo-B 100 . Finally, the relative LDL mobility on agarose gel was evaluated. This assay allows to detect changes in electric charge induced by oxidation. Basal LDL from H and controls migrated as homogeneous bands to 1.2 ± 0.2 and 1.1 ± 0.2 cm from the origin. In contrast, oxidized LDL from H migrated to 2.1 ± 0.3* cm from the origin while those of controls migrated to 1.5 ± 0.2 (*p &lt; 0.05). Thus, in FH LDL appear to be more susceptible to oxidationin vitro; the indices for LDL oxidizability were all significantly different from those of controls. This phenomenon may be an important additional mechanism of atherogenesis in homozigous FH.

Binding and uptake of oxidized low density lipoprotein (LDL) by macrophage scavenger receptors are enhanced by substrate-bound fibronectin.

The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages and the resulting accumulation of low density lipoprotein (LDL) lipids within the cells has been implicated in the pathogenesis of atherosclerosis. The effect of fibronectin (FN) on the binding and uptake of ox-LDL by macrophages was investigated using thioglycollate-induced mouse peritoneal macrophages. The ability of the macrophages to bind ox-LDL was assessed by the binding of mouse red blood cells (RBC) pre-coated with ox-LDL (ox-LDL-RBC) prepared in vitro to macrophages at 37 degrees C. The binding of ox-LDL-RBC to macrophages was significantly enhanced when the macrophages were plated on a FN-coated substrate. Similar enhancement was observed when the macrophages were plated on a substrate pre-coated with Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide, an adhesive sequence of FN involved in binding to the cells, but not with the control Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptide. The effect of FN was inhibited when GRGDSP, but not GRGESP, was present during the macrophage attachment to the FN-coated substrate, suggesting that the specific interaction of this sequence and the FN-receptor is responsible for the effect of FN. The addition of FN or GRGDSP in solution to the macrophage layers on an uncoated substrate was ineffective. Thus, attachment to a substrate is necessary for FN to be effective on the macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiologic mechanisms of action of lovastatin in nephrotic syndrome

The effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on the metabolism of apolipoprotein (apo) B-containing lipoproteins appear to differ according to the predominant lipoprotein profiles present and the condition being treated. In familial hypercholesterolemia, with isolated low density lipoprotein (LDL) elevations, the LDL-apoB elimination rate is increased by up-regulated LDL-receptors. In familial combined hyperlipidemia where very low density lipoprotein (VLDL) and LDL both may be increased and enhanced production of LDL-apoB may be present, HMG-CoA reductase inhibitors seem to diminish increased LDL-apoB production. The drug-induced decreases in LDL-apoB production could be due to decreased production of precursor VLDL-apoB or due to decreased conversion of VLDL-apoB to LDL-apoB after enhanced removal of VLDL by up-regulated LDL-receptors. To distinguish between these possibilities, we assessed the effects of HMG-CoA reductase inhibitors in another condition in which there is both apoB overproduction and accumulation of VLDL and LDL in plasma, the nephrotic syndrome. We used endogenous labeling of apoB with [13C]leucine and a multicompartmental model to calculate the metabolic parameters of apoB-containing lipoproteins. Only subjects with focal segmental glomerular sclerosis (FSGS) were included, as FSGS is a chronic, very slowly progressive form of nephrotic syndrome. A double-blind, randomized, placebo-controlled, crossover design was used. Treatment periods of 6 weeks were separated by a 2-week washout period. Of the four men studied, three had high triglyceride levels and four had high cholesterol levels. Lovastatin (20 mg/day) significantly decreased cholesterol (27.6 +/- 6%), LDL-cholesterol (27.6 +/- 9%) and plasma apoB (17.9 +/- 2.9%) (P < 0.01 for all). During the placebo period, calculation of kinetic parameters revealed VLDL-, intermediate density lipoprotein (IDL)-, and LDL-apoB overproduction and decreased VLDL-apoB fractional catabolic rate. Lovastatin significantly decreased LDL-apoB production rate in all cases (34.1 +/- 14%, P = 0.03). The decreased LDL-apoB was mainly due to a channelling of LDL precursors away from conversion to LDL (conversion of VLDL to LDL decreased from 80.6 +/- 8.3% to 55.9 +/- 17.2%, P = 0.05). Thus, lovastatin decreased LDL-cholesterol in nephrotic subjects mainly by inhibiting LDL-apoB production from VLDL.

Inhibition by Nifedipine of the Efflux of Plasma Low-Density Lipoprotein and Albumin from the Vasculature of the Mouse Mesentery in Vivo

Efflux and extravascular accumulation of intravenously administered fluorescent-labeled low-density lipoprotein (LDL) and albumin were measured in superfused mouse mesenteric preparation in anesthetized mice. Pretreatment of animals with nifedipine (1 and 3 mg/kg sc) significantly reduced both the extravascular accumulation of LDL (measured by fluorescence densitometry) and the efflux (measurement of the tracers in the superfusate) of both LDL and albumin. These effects may help to explain the possible anti-atherogenic effect of nifedipine.

Localization of lipoprotein in pre- and post-transition atherosclerotic lesions following short-term incubation with [125I]LDL

Ultrastructural autoradiography has been used to test the hypothesis that atherosclerotic regions of vessels differ with respect to lipoprotein uptake and localization. White Carneau pigeons, in which the prevalence and localization of aortic lesions are highly predictable, were fed a 0.25% cholesterol-supplemented diet to accelerate atherosclerosis. One hour prior to necropsy the birds were given a single intravenous injection of homologous [125I]LDL (low-density lipoprotein). Plasma die-away and tissue distribution of label were determined, and after the birds had been killed, the aortas, spleen and liver were processed for electron microscope autoradiography. Initial [125I]LDL uptake was rapid, with 35% of the label removed within 30 min. Predominant accumulation was in the liver, followed by the lung, kidney, the spleen and the aorta, in which the [125I]LDL level was approximately 4% that of the liver. Autoradiographic analysis documented hepatocyte (33%) and Kupffer cell (19.9%) localization in the liver and reticuloendothelial cell (57.4%) localization in the spleen. The aortic analysis involved serially sectioned lesions for direct comparison of non-lesion, lesion/non-lesion interface (edge) and deep lesion regions. Analysis of 2275 silver grains documented a ten-fold increase in LDL accumulation at the lesion edge (as compared to adjacent non-lesion) where macrophage foam cells contained more than 70% of the label. The other 30% was distributed equally among endothelium, the intimal matrix and smooth muscle cells. This distribution changed with more complex (deeper) lesions, although grain density in the complex lesions was comparable to the edge. In the complex regions, macrophage foam cell grains were reduced to 37%, whereas smooth muscle cell (22%) and the extracellular matrix (24%) label were both increased. These studies substantiate enhanced accumulation of lipoprotein specifically at lesion sites in the aorta and demonstrate a shift from macrophage localization at the developing edge to smooth muscle cell and the extracellular matrix in more complex deeper lesions.

Effects of supplementing with vitamin E on the uptake of low density lipoprotein and the stimulation of cholesteryl ester formation in macrophages

Vitamin E supplementation has been reported to protect low density lipoprotein (LDL) from copper-induced oxidation and macrophage-mediated oxidation. We investigated the effect of in vitro vitamin E enrichment of LDL on the accumulation of [ 3H]cholesteryl ester (CE)-LDL and stimulation of cholesteryl ester formation in J774 macrophages. Vitamin E supplementation prolonged lag time (2.9-fold) before the initiation of copper-induced LDL oxidation. LDL, preincubated with 5 μM copper or with macrophages in Ham's F10 medium, accumulated in macrophages much more than did native LDL. However, following vitamin E enrichment, LDL accumulation was significantly reduced following oxidative stress. Vitamin E-enriched LDL also reduced the stimulation of cholesteryl ester formation in macrophages. Moreover, vitamin E enrichment of macrophages reduced the ability of the cells to oxidize LDL. The present results indicate that vitamin E supplementation protects LDL against copper-induced and macrophage-mediated oxidation, inhibits oxidation-dependent accumulation of LDL in macrophages, and prevents stimulation of cholesteryl ester formation in macrophages. Additionally we have provided evidence that intra-cellular enrichment with vitamin E prevents oxidative modification of LDL by macrophages.

Chylomicrons or their remnants penetrate rabbit thoracic aorta as efficiently as do smaller macromolecules, including low-density lipoprotein, high-density lipoprotein, and albumin.

The aortic accumulation of chylomicrons, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and albumin were compared in normal New Zealand White rabbits. Lipoproteins and albumin were labelled with radioiodinated tyramine cellobiose (TC) to avoid potential oxidative modification of lipoproteins and as a marker of intracellular degradation. In preliminary experiments it was established that TC labelling did not alter the kinetic properties of lipoproteins in vivo. Importantly, radiolabelled apolipoproteins did not transfer significantly between plasma lipoproteins. Therefore, aortic radioactivity following infusion of TC-radiolabelled lipoproteins was considered to be indicative of lipoprotein accumulation. In conscious rabbits, net aortic accumulation of chylomicrons or their remnants was similar to those of LDL, HDL and albumin up to 2 h after infusion, despite rapid clearance from plasma. When accumulation was calculated on the basis of mean arterial exposure to allow for the differences in plasma clearance, the accumulation of aortic chylomicrons/remnants was substantially greater than that of LDL, HDL or albumin. Qualitatively similar results were obtained in rabbits that were functionally eviscerated to slow clearance of chylomicron remnants. Chylomicrons/remnants did not appear to efflux from aortic tissue as rapidly as did LDL or other plasma lipoproteins. Autoradiographic analysis showed that the primary site of lipoprotein accumulation was within medial smooth muscle cells. Our data demonstrate that chylomicrons/remnants accumulate in arterial blood vessels more rapidly than does LDL, suggesting that dietary lipoproteins may be directly involved in the pathogenesis of atherosclerosis.