Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: role of protein tyrosine kinase and cyclic AMP-dependent protein kinase A

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 microg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.     

Molecular fine-specificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays

BACKGROUND: Glomerular monocyte infiltration is an early feature of lipid-mediated renal injury in animal models. Interactions between mesangial and infiltrating mononuclear cells may contribute to the development of glomerular scarring. METHODS: Adherence of U-937 monocytes to low-density lipoprotein (LDL)- or tumor necrosis factor alpha (TNFalpha)-prestimulated human mesangial cells was assessed by colorimetry of nuclear staining with crystal violet. Blocking antibodies were added to examine the mechanisms of binding. Adhesion molecule expression and fibronectin synthesis were measured by ELISA. RESULTS: Preincubation of mesangial cells for 24 hours with LDL (100 micrograms/ml) or mildly oxidized (minimally modified) LDL (MM-LDL) increased monocyte adhesion by 207% and 240%, respectively, compared with control nonstimulated cells (100%). TNFalpha (100 U/ml) enhanced binding by 335% and up-regulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by 505% and 179%, respectively, as compared with MM-LDL (120% and 116%) and LDL, which had no effect. Blocking antibodies to these adhesion molecules inhibited monocyte binding to TNFalpha- and, to a lesser extent, MM-LDL-primed mesangial cells, but had no effect after LDL pretreatment. In contrast to TNFalpha, MM-LDL and LDL increased mesangial cell-associated fibronectin, whereas antibodies to fibronectin inhibited monocyte binding to lipoprotein-stimulated but not TNFalpha-stimulated cells. CONCLUSIONS: Although enhanced monocyte adhesion to TNFalpha- and, to a lesser extent, MM-LDL-stimulated mesangial cells is mediated by changes in ICAM-1 and VCAM-1 expression, both LDL and MM-LDL promote similar cellular interactions as a result of increased fibronectin production.     

Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats

BACKGROUND: Tubulointerstitial fibrosis is the final common pathway to end-stage renal failure. The present study investigated the potential role of tubular epithelial cells (TEC) in progressive fibrosis in the rat remnant kidney model. METHODS: Rats underwent 5/6 nephrectomy or a sham operation (control), and groups of six animals were killed at weeks 1, 3, 5, 9, 13, 17 and 21. RESULTS: Immunohistochemistry staining and in situ hybridization at week 3 after nephrectomy demonstrated de novo expression of alpha-smooth muscle actin (alpha-SMA)--a marker of smooth muscle cells and myofibroblasts--by TEC that was invariably associated with disruption of the tubular basement membrane (TBM). This phenotypic evidence of tubular epithelial-myofibroblast transdifferentiation was supported by ultrastructural studies identifying the presence of characteristic actin microfilaments and dense bodies within TEC with a transformed morphology. In the late stage of this apparent tubular epithelial-myofibroblast transdifferentiation, TEC lost apical-basal polarity and tight junctions, became elongated, detached from the TBM, separated from neighboring cells and appeared to migrate into the peritubular interstitium through the damaged basement membrane. Indeed, focal peritubular accumulation of alpha-SMA+ myofibroblasts and local tubulointerstitial fibrosis was closely associated with alpha-SMA+ tubules, suggesting a tubular epithelial origin for some of these cells. Quantitative analysis found a significant correlation between the number of alpha-SMA+ TEC and the accumulation of interstitial alpha-SMA+ myofibroblasts and the severity of tubulointerstitial fibrosis (both P < 0.001). CONCLUSIONS: This study provides phenotypic and morphological evidence to support the hypothesis that TEC are pro-fibrogenitor cells capable of tubular epithelial-myofibroblast transdifferentiation in progressive renal fibrosis. In addition, we postulate that disruption of the TBM, which facilitates epithelial cell contact with the interstitial matrix, promotes this process of transdifferentiation.     

Inhibition of LPL expression in human monocyte-derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.     

Three-dimensional triple-quantum-filtered 23Na imaging of the dog head in vivo

Uninephrectomized rats with diet-induced hypercholesterolemia develop interstitial inflammation and fibrosis after 8 to 12 weeks. Fibrosis has been associated with the accumulation of lipid peroxidation products within the tubulointerstitium, along with increased renal mRNA levels for transforming growth factor beta-1 (TCF-beta 1), some matrix proteins, and the tissue inhibitor of metalloproteinases (TIMP-1). However, mRNA levels for urokinase-type plasminogen activator (uPA) have been found to be decreased. The purpose of the present study was to determine whether antioxidant therapy could attenuate interstitial fibrosis in hypercholesterolemic rats and to determine changes in the pattern of renal gene expression induced by antioxidant therapy. Three groups of uninephrectomized rats were studied after 12 weeks of feeding standard rat chow, an atherogenic diet (standard chow plus 4% cholesterol/1% cholic acid), or an atherogenic diet supplemented with high doses of the antioxidants probucol and vitamin E. Rats fed the atherogenic diet developed hypercholesterolemia and a 56% increase in total kidney collagen compared with rats fed standard chow. In comparison, the hypercholesterolemic rats treated with antioxidants had normal levels of renal lipid peroxidation products and a normal kidney collagen content. In contrast, there were no significant differences in urinary albumin excretion rates or the number of interstitial macrophages between the two hypercholesterolemic groups. Compared with the untreated hypercholesterolemic group, antioxidant therapy induced significant reductions in renal mRNA levels for procollagen III (to 60% of untreated levels), collagen IV (60%), and TIMP-1 (20%), while uPA levels were significantly increased (to 210%). Paradoxically, antioxidant therapy was associated with a significant increase in renal TGF-beta 1 mRNA levels (to 150%), although TGF-beta 1 protein expression shifted from interstitial to tubular epithelial cells in predominance. The results of the present study demonstrate the efficiency of antioxidant therapy in preventing renal interstitial fibrosis in hypercholesterolemic rats with a single kidney. Based on changes in renal gene expression at the mRNA level, impaired matrix protein synthesis and increased intrarenal activity of the metalloproteinases and uPA/plasmin may play a role in the attenuation of fibrosis.     

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.     

Lipoprotein lipase (LPL) in the pathogenesis of atherosclerosis

The role of lipoprotein lipase (LPL) in the development of atheromatosis is subject of the increased interest for about 20 years, since then Zilversmit observed that LPL activity is found in greater amounts in atherosclerotic than normal arteries. The general action of this enzyme is hydrolysis of triglycerides in triglyceride rich lipoproteins and thus regulation of metabolism of circulating as well antiatherogenic as proatherogenic lipoproteins. The effect of LPL on the biology of arterial wall seems to be atherogenic. The mechanisms of this effect of LPL is 1) augmentation of the adhesion and aggregation of LDL; 2) influence on the oxygen modification of LDL and increased uptake of oxy-LDL by macrophages; 3) dysfunction of endothelial barrier and retention of atherogenic lipoproteins in the arterial wall and 4) the activity of LPL macrophage origin. Possible atherogenic actions of LPL based on in vitro experimental studies are reviewed.     

CLA-1 is an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins

Lipoprotein metabolism is regulated by the functional interplay between lipoprotein components and the receptors and enzymes with which they interact. Recent evidence indicates that the structurally related glycoproteins CD36 and SR-BI act as cell surface receptors for some lipoproteins. Thus, CD36 has been reported to bind oxidized LDL (OxLDL) and acetylated LDL (AcLDL), while SR-BI also binds native LDL and HDL. The cDNA of human CLA-1 predicts a protein 509 amino acids long that displays a 30% and an 80% amino acid identity with CD36 and mouse or hamster SR-BI, respectively. In this report, we describe the structural characterization of CLA-1 as an 85-kD plasma membrane protein enriched in N-linked carbohydrates. The expression of CLA-1 on mammalian and insect cells has been used to demonstrate that CLA-1 is a high-affinity specific receptor for the lipoproteins HDL, LDL, VLDL, OxLDL, and AcLDL. Northern blot analysis of the tissue distribution of CLA-1 in humans indicated that its expression is mostly restricted to tissues performing very active cholesterol metabolism (liver and steroidogenic tissues). This finding, in the context of the capability of this receptor to bind to both native and modified lipoproteins, strongly suggests that the CLA-1 receptor contributes to lipid metabolism and atherogenesis.     

Voice prostheses mechanisms in total laryngectomy

Familial combined hyperlipidemia is a common inherited disorder characterized by a hepatic overproduction of apo B particles and an elevated risk for the development of atherosclerosis. LDL particles are smaller and denser and are more prone to oxidation. The exact pathogenesis of familial combined hyperlipidemia is unclear at present. Treatment should aim to reduce the synthesis of atherogenic lipoproteins and to increase the clearance of triglyceride-rich lipoproteins.     

Diurnal heterogeneity in structure and function of low density lipoproteins of normolipidemic males

Structural changes in low density lipoproteins (LDL) have been shown to alter their metabolism and atherogenic potential. We investigated the diurnal changes in size and composition of LDL in seven healthy, non-obese, normolipidemic male volunteers consuming a standard diet (14.5% protein, 31.9% fat, 53.6% carbohydrate and 383 mg cholesterol/day) and continuing their daily routine. The food was divided into three meals and three snacks, and blood samples were obtained at 7 AM (after 12 h fasting), noon, 8 PM, midnight and 3 AM. LDL were isolated by both sequential and density gradient ultracentrifugation (d = 1.019 - 1.050 g/ml), and analyzed for lipids, apolipoproteins, size, and affinity to LDL receptors. Diurnal LDL preparations differ from fasting LDL in both chemical and physical parameters. The former get richer in triglyceride (TG/cholesterol weight ratio 0.23 vs. 0.16), larger in diameter (21.2 +/- 0.2 vs. 22.4 +/- 0.1 nm), and enriched in a more buoyant fraction (74.0 +/- 4.6 vs. 41.9 +/- 3.8% of LDL cholesterol in d = 1.019 - 1.035 g/ml). These structural changes in LDL were associated with enhanced affinity to LDL receptors in both human skin fibroblasts and HepG2 cells, as demonstrated by competition experiments with fasting human 125I-LDL. The observed diurnal heterogeneity in both the structure and the function of LDL may be attributed to the absorptive state as it did not occur during prolonged fasting. These diurnal changes may be important for better understanding LDL metabolism in vivo and for the elucidation of the atherogenic process.     

Uptake and metabolism of lipoproteins from patients with diabetes mellitus type II by glomerular epithelial cells

BACKGROUND: Recent studies suggest that dyslipidaemia accelerates the progression of diabetic nephropathy, but the various pathomechanisms underlying such abnormalities are not completely delineated. METHODS: We isolated, radiolabelled, and characterized very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) from eight diabetic patients with moderate impairment of renal function and dyslipidaemia and studied their interaction with LDL receptors in human glomerular epithelial cells. RESULTS: While diabetic VLDL showed no compositional changes, LDL particles contained a higher proportion of triglycerides at the expense of cholesterol in comparison with healthy controls. Despite differences in composition, both VLDL and LDL from patients exhibited reduced receptor affinity and cellular uptake capacity by glomerular epithelial cells. Since LDL composition was altered intracellular cholesterol homeostasis was investigated. Due to reduced cholesterol content and lower uptake capacity, diabetic LDL were less effective in suppressing intracellular sterol synthesis and in activating acylcholesterol acyltransferase than LDL from controls. Electrophoretic mobility of apoB from diabetic patients was enhanced as compared to controls, most probably due to the higher degree of glycation (17 + 1.7 versus 11 + 1%, P < 0.05) but not to oxidation (TBARS 0.5 + 0.2 versus 0.2 + 0.1 mumol/1). Oxidized LDL was not taken up in significant amounts, indicating no scavenger receptor activity in glomerular epithelial cells. CONCLUSION: The receptor-specific uptake of diabetic VLDL and LDL by glomerular epithelial cells is impaired. Compositional changes of the LDL particle and glycation of the protein moiety may contribute to altered glomerular uptake. However, glycation of the protein moiety may be superior to compositional changes. Because glomerular structures like mesangial matrix and endothelial cells are known for preferential binding of modified lipoproteins, further studies are required to elucidate their potential role in the progression of diabetic glomerulosclerosis.     

Lipoprotein receptors and atherogenic receptor-mediated mechanisms

The liver plays a decisive role in the regulation of the plasma levels of atherogenic lipoproteins. The primary liver interaction site of chylomicron remnants and VLDL remnants (beta-VLDL) is still unidentified, whereas the subsequent cellular uptake is likely to be mediated in concert by the LDL receptor-related protein and the LDL receptor. The nature of the primary interaction site of remnants (remnant receptor) might be a liver-specific proteoglycan or a liver-specific protein. Atherogenic modified LDL can be recognized by a family of scavenger receptors. A newly identified 95 kDa protein forms the most likely candidate for mediating the in-vivo uptake of oxidized LDL from the circulation and may, therefore, protect the body against the presence of oxidized LDL in the blood compartment.     

Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats

The 5/6 nephrectomy model is used to study pathogenetic mechanisms underlying chronic renal failure. We previously demonstrated that increased mesangial cell proliferation and glomerular PDGF B-chain expression precede glomerulosclerosis in this model. In the present study we have assessed the concomitant changes in the cortical tubulointerstitium. A wave of tubular and interstitial cell proliferation (as determined by immunostaining for PCNA) occurred at week 1 after 5/6 nephrectomy. This wave preceded the peak glomerular cell proliferation by one week. Tubulointerstitial cell proliferation decreased thereafter and reached control values by week 10. In situ hybridization and immunostaining for PDGF B-chain and beta-receptor in sham-operated controls showed labeling of distal tubules and collecting ducts, while no signal was present in the interstitium. PDGF B-chain mRNA and protein expression was markedly increased in tubules at weeks 2 and 4 after 5/6 nephrectomy and in the interstitium (particularly in areas of inflammatory infiltrates) at weeks 2 to 10. Similar changes occurred with PDGF receptor beta-subunit immunostaining. Interstitial expression of desmin and alpha-smooth muscle actin (markers of myofibroblasts) progressively increased after week 1. Interstitial influx of monocytes/macrophages with focal accentuation started at week 2. Counts of lymphocytes, neutrophils and platelets showed only minor changes. In parallel to the monocyte/macrophage influx, progressive interstitial accumulation of collagens I and IV, laminin, and fibronectin occurred. All of these changes were correlated with the increase in serum creatinine, proteinuria and an index of tubulointerstitial damage. We conclude that tubulointerstitial changes after 5/6 nephrectomy show similarities with those observed in the glomeruli. Tubular and interstitial overexpression of PDGF B-chain and its receptor may play a role in mediating fibroblast migration and/or proliferation in areas of tubulointerstitial injury.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Troglitazone decreases the proportion of small, dense LDL and increases the resistance of LDL to oxidation in obese subjects

OBJECTIVE: Insulin resistance is associated with a predominance of small, atherogenic LDL particles that are more prone to oxidative modification. Treatment with the insulin-sensitizer troglitazone may improve LDL composition and resistance to oxidation. RESEARCH DESIGN AND METHODS: In a randomized double-blind crossover design, 15 obese subjects were treated with either 400 mg troglitazone daily or placebo for 8 weeks. Insulin sensitivity (clamp), (apo)lipoproteins, LDL subclass pattern, plasma TBARS, and ex vivo LDL oxidation were determined. RESULTS: Troglitazone treatment improved insulin sensitivity. LDL cholesterol increased from 2.58 +/- 0.18 to 2.77 +/- 0.20 mmol/l (P = 0.03) because of an increase in large (buoyant) LDL1 (from 0.45 +/- 0.04 to 0.62 +/- 0.09 mmol/l, P = 0.008). Because small (dense) LDL3 decreased, LDL1:LDL3 ratio increased (P = 0.02). Plasma TBARS concentration declined significantly, and the lag time of ex vivo LDL oxidation showed a small but significant increase. CONCLUSIONS: In obese subjects, treatment with troglitazone improves insulin sensitivity, increases the ratio of large buoyant to small dense LDL, and appears to enhance the resistance of the LDL particle to oxidation. These qualitative changes in lipoproteins may have a beneficial effect on cardiovascular risk profile and compensate for a small increase in LDL cholesterol.     

Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier

We previously found in human blood a fraction of low-density lipoprotein (LDL) that is characterized by a reduced content of sialic acid. Desialylated LDL also has a low neutral carbohydrate level, decreased content of major lipids, small size, high density, increased electronegative charge and altered tertiary apolipoprotein B structure. Unlike native LDL, this fraction of desialylated (multiple-modified) LDL induces the accumulation of lipids in smooth muscle cells cultured from unaffected human aortic intima, i.e. it exhibits atherogenic properties. In this study, we attempted to elucidate the mechanism of desialylation and other changes in the multiple-modified LDL by investigating the possibility of LDL modification by different cells and the blood plasma. A 24-h incubation at 37 degrees C of lipoprotein with intact endotheliocytes, hepatocytes, macrophages and smooth muscle cells or cell homogenates did not cause alterations either in the physical properties or in the chemical composition of native LDL. On the other hand, a significant fall in the lipoprotein sialic acid level was observed already after a 1-h incubation of native LDL with an autologous plasma-derived serum. While LDL sialic acid level continuously decreased, LDL became capable of inducing the accumulation of total cholesterol in the smooth muscle cells cultured from unaffected human aortic intima after 3 h of incubation. Starting from the sixth hour of LDL incubation with serum, a steady decrease in the lipoprotein lipid content was observed as well as the related reduction of LDL size. Following 36 h of incubation, an increase in the negative charge of lipoprotein particles was also seen. Prolonged incubation of LDL with plasma-derived serum (48 and 72 h) leads to the loss of alpha-tocopherol by the LDL as well as to an increase in LDL susceptibility to copper oxidation and to accumulation of cholesterol covalently bound to apolipoprotein B, a marker of lipoperoxidation. Degradation of apolipoprotein B starts within the same period of time. Hence, desialylation of LDL particles represents one of the first or the primary act of modification which is, apparently, a sufficient prerequisite for the development of atherogenic properties. Subsequent modifications just enhance the atherogenic potential of LDL. The loss of sialic acid by LDL occurred at neutral pH and was not inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The [3H]sialic acid removed from LDL was not found in free form, but in the plasma fraction precipitated by trichloroacetic acid. These data along with the fact that cytidine-5'-triphosphate inhibited LDL desialylation suggest that enzymes close to sialyltransferases play a role in this process. Thus, this study demonstrated that the LDL modification processes imparting atherogenic properties to this lipoprotein can take place in human blood plasma. Multiple modification of LDL is a cascade of successive changes in the lipoprotein particle: desialylation, loss of lipids, reduction in particle size, increase of its electronegative charge and peroxidation of lipids.     

Cholesteryl ester accumulation in macrophages treated with oxidized low density lipoprotein

The ability of CuSO4- and hypochlorite-oxidized LDL to promote cholesterol accumulation in macrophages was examined. Both CuSO4- and hypochlorite-oxidized LDL were rapidly metabolized by mouse peritoneal macrophages to a level approximately 10 times that observed for native LDL and both modified lipoproteins increased the accumulation of unesterified cholesterol. However when each modified lipoprotein was incubated with macrophages for 40h, only hypochlorite-oxidized LDL produced significant accumulation of cholesteryl esters, with levels approaching 85 micrograms/mg cell protein. This finding was verified by nile red staining. The cholesteryl ester content of cupric sulfate-modified LDL was found to be significantly decreased when compared to either native or hypochlorite-modified LDL promotes massive cholesteryl ester accumulation because the cholesteryl ester content of the LDL particle is preserved.