7-Hydroperoxycholesterol and its products in oxidized low density lipoprotein and human atherosclerotic plaque

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (> or = 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (< 0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse-phase method utilizing chemiluminescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 beta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.     

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.     

Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta

Accumulation of extracellular matrix, fibrosis, is regarded to be one of the major manifestations of atherosclerosis. Collagen type I is the predominant matrix component in human atherosclerotic plaques. In this work we have demonstrated procollagen type I expressing cells (PCI-cells) and studied their localization in grossly normal human aorta and atherosclerotic lesions: initial lesions, fatty streaks, fibrolipid lesions (fibrolipid plaque, fibroatheroma), fibrotic lesions (fibrous plaque). PCI-cells were revealed immunocytochemically using SPI.D8 monoclonal antibody against human procollagen type I. We failed to detect PCI-cells in the areas of grossly normal aorta and media underlying atherosclerotic lesions. Positively stained cells were shown in the areas of initial lesions, fatty streaks, fibrolipid and fibrous plaques. The largest amount of PCI-cells was revealed in fatty streaks. These cells were predominantly localized in the preluminal proteoglycan-rich intimal sublayer. Intimal cells in grossly normal regions formed a common cellular network contacting each other with their processes. The cellular network is found to be partly disintegrated in atherosclerotic lesions, which leads to the appearance of isolated cells. The share of isolated PCI-cells localized outside the intimal cellular network was higher in advanced lesions than in the areas of early atherosclerotic lesions. In initial lesions most of PCI-cells were identified as smooth muscle cells using antibodies to smooth muscle alpha-actin. In fatty streaks PCI-expressing smooth muscle cells were fewer in number. Much fewer cells double-stained with anti-alpha-actin and anti-PCI antibodies were found in fibrolipid and fibrous plaques. The proportion of these double stained cells was higher among total number of PCI-cells involved in the cellular network versus PCI-cells outside the network. The results of the study demonstrated that the most active de novo synthesis of interstitial collagen takes place in the regions of atherosclerotic lesions characterized by lipid deposition, which may lead to the further progression of atherosclerotic lesions.     

Isolation and characterization of a proteoglycan variant from human aorta exhibiting a marked affinity for low density lipoprotein and demonstration of its enhanced expression in atherosclerotic plaques

Proteoglycans (PG) are implicated in the pathophysiology of atherosclerosis due to their ability to complex with plasma low density lipoproteins (LDL). Studies were conducted to determine whether human aorta contains PG subclasses that exhibit enhanced LDL binding ability. PG were isolated from normal and atherosclerotic aortas by a combination of dissociative extraction and ion-exchange chromatography. The PG were further subfractionated on an LDL affinity column based on their binding affinity to LDL. Two PG fractions exhibiting high-affinity binding to LDL, as evidenced by their elution at 1.0 and 1.5 M NaCl, respectively, were isolated from both normal and atherosclerotic tissue. Compared with normal tissue, atherosclerotic tissue showed a twofold increase in the high-affinity PG that eluted at 1.5 M NaCl. Gel filtration of the high-affinity PG from normal tissue yielded two peaks (nPG2 and nPG3), while the high-affinity PG from plaque tissue was resolved into three peaks (pPG1, pPG2, and pPG3). pPG1 eluted at the void volume of the column, indicating that it was of very large molecular size. The hydrodynamic size of pPG2 was larger than that of the corresponding nPG2 (Kav = 0.44 versus 0.51), while pPG3 had the same hydrodynamic size as nPG3 (Kav = 0.86). The high-affinity PG subfractions from normal aorta contained varying proportions of chondroitin sulfates, dermatan sulfates, and heparan sulfate. In contrast, the PG subfractions from plaque tissue contained predominantly chondroitin sulfates and heparan sulfate. In vitro complexes of LDL and the high-affinity PG fractions from normal aorta and plaque tissue stimulated cholesteryl ester synthesis in human monocyte-derived macrophages. However, the LDL-plaque PG complex was significantly more potent than the LDL-normal aorta PG complex in this respect. These results indicate that PG subclasses with enhanced binding affinity to LDL occur in the normal human aorta and that their concentration increases significantly in atherosclerotic lesions. In addition, the high-affinity PG in plaque tissue have altered characteristics and increased ability to stimulate LDL-mediated cholesterol ester synthesis in macrophages. This could lead to increased lipid deposition during atherogenesis.     

Occurrence of the same peroxidative compounds in low density lipoprotein and in atherosclerotic lesions from a homozygous familial hypercholesterolemic patient: a case report

OBJECTIVE: There has been no reference material for T-lymphocyte subsets for normal children in developing countries. We therefore used T-lymphocyte subset determinations among children in three different studies in Guinea-Bissau to construct age-related reference material and to examine possible determinants of T-lymphocyte subset levels. METHODS: A total of 803 healthy West African children younger than 6 years were included in the three community studies of T-lymphocyte subsets among twins and singletons, after measles infection and after measles immunization. We used the immunoalkaline phosphatase method to determine T-lymphocyte subsets. RESULTS: We found differences by age, sex, and season, whereas there were no significant differences by birth order, twinning, or ethnic group. The CD4+ percentage declined from birth to age 2 years, at which time it started to increase to higher levels at age 4 to 5 years. The CD8+ percentage increased gradually from early infancy to age 2 to 4 years. The leukocyte count peaked at age 12 to 23 months and declined thereafter, whereas the lymphocyte percentage peaked at age 1 to 5 months and declined gradually thereafter. Compared with dry-season results, the lymphocyte percentage, the absolute lymphocyte count, the absolute CD4+ T-lymphocyte count, and the CD4+/CD8+ ratio were significantly lower during the rainy season, whereas the CD8+ percentage was increased during the rainy season. Girls had higher CD4+/CD8+ ratios and lower CD8+ percentages than did boys. CONCLUSIONS: Compared with the limited data on T-lymphocyte subsets available from healthy children in developed countries, Guinean children have markedly lower CD4+ percentages and CD4+/CD8+ ratios and higher lymphocyte percentages during the first 2 years of life, when the pressure of infections is particularly high in Africa.     

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis

In response to stimulation with immobilized anti-CD3 antibody, splenocytes from C57BL/6 and BALB/c mice principally produced INF-gamma and IL-4, respectively. However, both splenocytes equally proliferated in response to ConA. We compared the changes after inoculation with BCG (1 mg/mouse) in their capacity to produce IL-4 or IFN-gamma in response to anti-CD3 antibody and to proliferate in response to ConA. Splenocytes from C57BL/6 and BALB/c mice, that had been inoculated with BCG 4 weeks before, produced IFN-gamma with diminished IL-4 production in response to anti-CD3 antibody. Furthermore these splenocytes became anergic to ConA stimulation and died due to cell apoptosis in stead of proliferation. However, we observed the strain difference at 12 weeks after BCG-infection. BCG-primed C57BL/6 splenocytes, that continuously produced IFN-gamma in response to anti-CD3 antibody, failed to proliferate in response to ConA. In contrast, BCG-primed BALB/c splenocytes, that increased IL-4 production but decreased IFN-gamma production when stimulated with anti-CD3 antibody, could proliferate well in response to ConA. Since the splenocytes of BALB/c mice became ConA responsive along with their shifting from Th1 dominant immune response at 4 weeks to Th2 dominant immune response at 12 weeks after BCG-inoculation, IL-4 was assumed to play a crucial role in activation of anergic T cells. Therefore, we stimulated splenocytes from both strains of mice infected with BCG 4 weeks before with ConA in the presence or absence of IL-4. Splenocytes from BCG-infected BALB/c mice showed marked proliferation, while those from BCG-infected C57BL/6 mice failed. We found that IL-4 protected against ConA-induced cell apoptosis in BALB/c splenocytes but not C57BL/6 splenocytes.     

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.     

Isradipine lowers human arterial low density lipoprotein retention in vivo

An 84-year-old woman was admitted to our hospital because of left heart failure of acute onset. Transthoracic echocardiography showed diffuse hypertrophy of the normal sized hyperkinetic left ventricle and chordae-like fluttering echoes attached to the mitral valve with severe mitral regurgitation signals. Mosaic flow signals were seen at the left ventricular outflow tract, but the velocity could not be measured. Emergent transesophageal echocardiography detected no obvious mitral valve prolapse. Cardiac catheterization showed greater than 100 mmHg pressure gradient between the left ventricle and femoral artery. Pressures in the femoral artery and pulmonary capillary wedge changed reciprocally in the intensive care unit; a bisferient narrow pulse pressure of the femoral artery was associated with increased v wave of the pulmonary capillary wedge pressure, and a wide pulse pressure of the femoral artery with absent v wave of the pulmonary capillary wedge pressure. Pressure monitoring in the intensive care unit, catheterization laboratory and transesophageal echocardiography were useful to understand the pathophysiology of the patient.     

Antioxidation of human low density lipoprotein by unconjugated and conjugated bilirubins

We demonstrate here that both unconjugated bilirubin (Bu) and conjugated bilirubin (Bc) can protect human low density lipoprotein(LDL) against oxidation by oxyradicals generated by 2,2'-azo-bis (2 amidinopropane) dihydrochloride at 37 degrees. The oxidation was assessed by agarose gel electrophoresis and was further corroborated by assaying the malondialdehydes and lipid peroxides formed throughout oxidation. On a per mole basis, Bu and less so Bc was more effective than ascorbate in preventing LDL oxidation. Since oxidative modification of human LDL was implicated in plaque formation in blood vessels leading to atherogenesis, the data suggested that either bile pigment may help reduce the risk of atherogenesis.     

3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima

Oxidation of LDL may be of pivotal importance in atherogenesis, but the mechanisms that promote oxidation in vivo remain poorly understood. We have explored the possibility that one pathway involves myeloperoxidase, a heme protein secreted by phagocytes. Myeloperoxidase is the only human enzyme known to generate hypochlorous acid (HOCl), a potent oxidizing agent, at physiological halide concentrations. LDL exposed to the complete myeloperoxidase-H2O2-Cl- system underwent chlorination of its protein tyrosyl residues. Treatment of LDL with reagent HOCl resulted in 3-chlorotyrosine formation, implicating HOCl as an intermediate in the enzymatic reaction pathway. In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, hemin, glucose, peroxynitrite, horseradish peroxidase, lactoperoxidase, or lipoxygenase. These results indicate that 3-chlorotyrosine is a specific marker for LDL oxidation by myeloperoxidase. To address the role of myeloperoxidase in promoting LDL oxidation in vivo, we used stable isotope dilution gas chromatography-mass spectrometry to quantify 3-chlorotyrosine in human aortic tissue and in LDL isolated from atherosclerotic lesions. The level of 3-chlorotyrosine in atherosclerotic tissue obtained during vascular surgery was sixfold higher than that of normal aortic intima. Moreover, the level of 3-chlorotyrosine was 30-fold higher in LDL isolated from atherosclerotic intima compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic lesions indicates that halogenation reactions catalyzed by the myeloperoxidase system of phagocytes constitute one pathway for protein oxidation in vivo. These findings raise the possibility that the myeloperoxidase-H2O2-Cl- system plays a critical role in converting LDL into an atherogenic form.     

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.     

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.     

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.     

Tomato lycopene and low density lipoprotein oxidation: a human dietary intervention study

Increase in low density lipoprotein (LDL) oxidation is hypothesized to be causally associated with increasing risk of atherosclerosis and coronary heart disease. In recent epidemiological studies, tissue and serum levels of lycopene, a carotenoid available from tomatoes, have been found to be inversely related to risk of coronary heart disease. A study was undertaken to investigate the effect of dietary supplementation of lycopene on LDL oxidation in 19 healthy human subjects. Dietary lycopene was provided using tomato juice, spaghetti sauce, and tomato oleoresin for a period of 1 wk each. Blood samples were collected at the end of each treatment. Serum lycopene was extracted and measured by high-performance liquid chromatography using an absorbance detector. Serum LDL was isolated by precipitation with buffered heparin, and thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD) were measured to estimate LDL oxidation. Both methods, to measure LDL oxidation LDL-TBARS and LDL-CD, were in good agreement with each other. Dietary supplementation of lycopene significantly increased serum lycopene levels by at least twofold. Although there was no change in serum cholesterol levels (total, LDL, or high-density lipoprotein), serum lipid peroxidation and LDL oxidation were significantly decreased. These results may have relevance for decreasing the risk for coronary heart disease.     

Comparison of the intracellular pathways of immunoglobulin-G and low density lipoprotein in cultured human term trophoblast cells

Trophoblast cells were cultured on microporous membrane filters. After incubation at different times with gold-conjugated ligands, the cells were processed for electron microscopy. Gold particles indicating the presence of both IgG and LDL appeared in a time-dependent manner in coated pits and coated vesicles. LDL-gold appeared primarily within lysosomes whereas approximately 50% of the internalized IgG-gold appeared within vesicles (diameters ranging from 35 to 80 nm) near the basal regions of the cell. These vesicles may be the protective mechanism which prevents IgG breakdown during transcytosis across trophoblast cells, thus allowing transport of the intact molecule to the fetus.     

Colocalization of iron and ceroid in human atherosclerotic lesions

Although clearly a valid alternative to dismembered pyeloplasty for ureteropelvic junction obstruction in adults, endo(uretero)pyelotomy has not been universally accepted in the general urologic community because of the 10% to 30% inferior success rate and the concern regarding hemorrhagic complications. Identification of prognostic factors for success and complications are therefore of primary importance. The length of stricture, the grade of hydronephrosis, and the level of renal function are well recognized prognostic factors. Crossing vessels also play a role, and in association with the grade of hydronephrosis, they seem to be a major prognosticator of outcome. With attention to such prognostic factors, and appropriate patient selection, results can now approach the gold standard of dismembered pyeloplasty.     

First direct evidence for lipid/protein conjugation in oxidized human low density lipoprotein

It has been postulated that lipids incorporated in atherosclerotic plaques are derived from the uptake of oxidized low density lipoprotein (LDL) by a macrophage-bound receptor. In vitro studies of LDL oxidation have established that reactive lipids are formed and that the exposure of native LDL to these products leads to modified protein with physical properties similar to oxidized LDL. Here we describe the application of highly specific tandem mass spectrometric techniques to the first characterization of lipid-modified LDL by demonstrating the addition of 4-hydroxy-2-nonenal to histidine residues of apolipoprotein B-100, following oxidation of LDL. The modified residues have been assigned to specific locations that have been previously shown to reside on the surface of the LDL particle.     

Both hypothyroidism and hyperthyroidism enhance low density lipoprotein oxidation

Hypothyroidism is frequently associated with hypercholesterolemia and an increased risk for atherosclerosis, whereas hyperthyroidism is known to precipitate angina or myocardial infarction in patients with underlying coronary heart disease. We have shown previously that L-T4 functions as an antioxidant in vitro and inhibits low density lipoprotein (LDL) oxidation in a dose-dependent fashion. The present study was designed to evaluate the changes in LDL oxidation in subjects with hypothyroidism and hyperthyroidism. Fasting blood samples for LDL oxidation analyses, lipoprotein determinations, and thyroid function tests were collected at baseline and after the patients were rendered euthyroid. The lag phase (mean +/- SEM hours) of the Cu+2-catalyzed LDL oxidation in the hypothyroid state and the subsequent euthyroid states were 4 +/- 0.0.65 and 14 +/- 0.68 h, respectively (P < 0.05). The lag phase during the hyperthyroid phase was 6 +/- 0.55 h, and that during the euthyroid phase was 12 +/- 0.66 h (P < 0.05). The total and LDL cholesterol levels were higher in hypothyroidism than in euthyroidism and were lower in hyperthyroidism than in the euthyroid state. We conclude that LDL has more susceptibility to oxidation in both the hypothyroid and hyperthyroid states. Thus, the enhanced LDL oxidation may play a role in the cardiac disease process in both hypothyroidism and hyperthyroidism.     

Effect of homocysteine on copper ion-catalyzed, azo compound-initiated, and mononuclear cell-mediated oxidative modification of low density lipoprotein

Homocysteine is an independent risk factor for cardiovascular diseases. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. To examine whether homocysteine is implicated in atherogenesis through the modification of low density lipoprotein (LDL), the effect of homocysteine on the oxidation of LDL was studied by three different oxidation systems. Thus, LDL was subjected to Cu(2+)-catalyzed, azo compound-initiated, and peripheral blood mononuclear cell-mediated oxidative modification. The extent of modification was assessed by measuring the formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances, and the relative electrophoretic mobility. Homocysteine at a normal plasma concentration (6 microM) showed no effect, whereas a concentration corresponding to moderate hyperhomocysteinemia (25 microM) or to concentrations seen in homocystinuria patients (100, 250, and 500 microM) protected LDL from modification of the lipid as well as of the protein moiety. One exception was observed: when the oxidation was initiated by copper ions, homocysteine at concentrations 6 and 25 microM stimulated the lipid peroxidation of LDL to a small, but statistically significant extent. High concentrations of homocysteine showed antioxidative properties as long as the thiol groups were intact, thereby delaying the onset of the oxidation. The 1,1-diphenyl-2-picrylhydracyl radical test demonstrated that homocysteine at concentrations > or = 50 microM possessed marked free radical scavenging capacity. Finally, LDL isolated from two patients with homozygous homocystinuria showed similar extent of Cu(2+)-catalyzed oxidation as LDL from a group of healthy control subjects. Taken together, our data suggest that low concentrations of homocysteine in the presence of copper ions may enhance the lipid peroxidation of LDL, whereas high concentrations of homocysteine may protect LDL against oxidative modification in the lipid as well as in the protein moiety. Thus, homocysteine-induced atherosclerosis may be explained by mechanisms other than oxidative modification of low density lipoprotein.     

Protein kinase calpha regulates human monocyte O-2 production and low density lipoprotein lipid oxidation

Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O-2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O-2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The conventional PKC group includes PKCalpha, PKCbetaI, PKCbetaII, and PKCgamma. With the exception of PKCgamma, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCalpha, PKCbetaI, and PKCbetaII protein levels were induced upon monocyte activation, and PKCalpha and PKCbetaII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O-2 production and LDL oxidation, PKCalpha or PKCbeta isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCalpha expression inhibited both monocyte-mediated O-2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCbeta expression (including both PKCbetaI and PKCbetaII) did not affect O-2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O-2 production remained functionally intact in monocytes with depressed levels of PKCalpha because O-2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCalpha, and not PKCbetaI or PKCbetaII, is the predominant isoenzyme required for O-2 production and maximal oxidation of LDL by activated human monocytes.