Receptor-mediated binding and degradation of subfractions of human plasma low-density lipoprotein by cultured fibroblasts.

The receptor-mediated metabolism of human plasma low-density lipoprotein (LDL) subfractions was studied. LDL was isolated from healthy donors and further fractionated by density gradient ultracentrifugation into three subfractions: (I) d = 1.031-1.037, (II) d = 1.037-1.041 and (III) d = 1.041-1.047 g/ml, comprising 24 +/- 7%, 46 +/- 8% and 30 +/- 9% of the total LDL protein, respectively. As assessed by electron microscopy and gradient gel electrophoresis, the LDL particle size decreased and the relative protein content increased from fraction I towards fraction III. Fraction II had the highest (Kd 2.6 micrograms/ml) and fraction I the lowest (Kd 5.8 micrograms/ml) binding affinity to LDL receptors of human fibroblasts at 4 degrees C. The rate of receptor-mediated degradation of fraction II was also higher than that of the other two fractions at 37 degrees C. These results suggest that LDL subfractions have different rates of receptor-mediated catabolism depending on particle size or composition, and therefore their metabolic fate and atherogenic properties may also differ.

Paraoxonase gene polymorphisms and coronary reactivity in young healthy men.

This study examined the relationships between paraoxonase genotypes, coronary artery reactivity, and indices of low-density lipoprotein oxidation in healthy men. Impairment in coronary flow reserve, as assessed by positron emission tomography, is associated with lipoprotein oxidation, which is affected by high-density lipoprotein bound enzyme, paraoxonase. Paraoxonase has two common polymorphisms (M/L55 and R/Q192) that change the activity of the enzyme. Forty-nine healthy men (mean age 35 +/- 4 years) were divided by paraoxonase genotype into low (Q192/Q192, or M55/M55, M55/L55) and high-active (R192/Q192, R192/R192, or L55/L55) groups and related to the myocardial blood flow, to the susceptibility of low-density lipoprotein to oxidation, and the autoantibody titer against oxidized low-density lipoprotein. The blood flow was measured by positron emission tomography at rest and during adenosine infusion. The low-active Q192/Q192 genotype was associated with higher resting blood flow corrected for rate-pressure product compared to the high-active R192/R192 and R192/Q192 genotypes (P=0.011). The blood flow stimulated by adenosine was not significantly different in the low- and high-active genotype groups. Paraoxonase genotypes had no effect on low-density lipoprotein susceptibility to oxidation or autoantibody formation against oxidized low-density lipoprotein. Genotypes of paraoxonase may not clearly contribute to the early changes in coronary reactivity. Coronary vasomotor tone at rest appears to be modulated by paraoxonase R/Q192 polymorphism through mechanism(s) unrelated to low-density lipoprotein oxidation.

HDL enhances oxidation of LDL in vitro in both men and women.

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) is a key event in the oxidation hypothesis of atherogenesis. Some in vitro experiments have previously suggested that high-density lipoprotein (HDL) co-incubated with LDL prevents Cu2+-induced oxidation of LDL, while some other studies have observed an opposite effect. To comprehensively clarify the role of HDL in this context, we isolated LDL, HDL2 and HDL3 from sera of 61 free-living individuals (33 women and 28 men). RESULTS: When the isolated LDL was subjected to Cu2+-induced oxidation, both HDL2 and HDL3 particles increased the rate of appearance and the final concentration of conjugated dienes similarly in both genders. Oxidation rate was positively associated with polyunsaturated fatty acid content of the lipoproteins in that it was positively related to the content of linoleate and negatively related to oleate. More saturated fats thus protected the lipoproteins from damage. CONCLUSION: We conclude that in vitro HDL does not protect LDL from oxidation, but is in fact oxidized fastest of all lipoproteins due to its fatty acid composition, which is oxidation promoting.

Oxidized low-density lipoprotein is chemotactic for arterial smooth muscle cells in culture.

The effects of human native and Cu2(+)-oxidized low-density lipoprotein (LDL) were tested on the migration of cultured bovine aortic smooth muscle cells (SMCs) in blind-well chambers. LDL oxidation was controlled by measuring the formation of conjugated dienes and lipid hydroperoxides, and by agarose gel electrophoresis. Oxidized LDL stimulated SMC migration, and the effect was dose-dependent up to 200 microgram/ml. The stimulation was chemotactic in nature. Native LDL was without significant activity. The results suggest that oxidized LDL may contribute to the migration of medial SMCs into the intima during atherogenesis.

Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of low density lipoprotein.

The effect of a nitric oxide (NO) donor GEA 3162 on the endothelial cell (EC)-mediated oxidation of low density lipoprotein (LDL) was studied. In comparison to LDL incubated with EC without GEA 3162, the presence of GEA 3162 inhibited LDL oxidation by EC, as indicated by the following findings. (a) The degradation rate of LDL in macrophages was reduced to control levels. (b) The electrophoretic mobility of LDL decreased in a dose-dependent manner. (c) The concentrations of thiobarbituric acid-reactive substances and hydroperoxide-derived hydroxy fatty acids were lower. (d) The breakdown of apolipoprotein B was reduced. The results indicate that GEA 3162 prevents EC-mediated oxidation of LDL.

Effect of hypoxia on the synthesis of glycosaminoglycans and collagen by rabbit aortic smooth muscle cells in culture.

This study evaluated the effect of hypoxia on the connective tissue metabolism of rabbit aortic smooth muscle cells (SMCs) in culture. When the oxygen saturation of the incubation medium was lowered from 20% to 2-3%, synthesis of sulphated glycosaminoglycans (GAGs) and hyaluronic acid, as determined from the incorporation of [3H]glucosamine, was stimulated. However, this occurred only after 24 h preincubation of the SMCs in hypoxia. The collagen synthesis of the cells was determined from the incorporation of [3H]proline into protein hydroxyproline and calculated in mass units from the specific intracellular precursor radioactivity. The total protein synthesis was similarly determined from the incorporation of [3H]proline into protein-bound proline. Hypoxia decreased the collagen synthesis, but did not affect the total protein synthesis of the cells. When compared with the control cultures the cell protein of the SMC cultures kept in hypoxia, decreased on the first day in hypoxia whereafter it increased. These results may explain the mechanisms by which hypoxia affects the connective tissue metabolism of the arterial wall in vivo.

Lipoprotein uptake in primary cell cultures of rabbit atherosclerotic lesions. A fluorescence microscopic and flow cytometric study.

To characterize the lipoprotein metabolism of lipid-filled cells of atherosclerotic lesions, uptake of 3,3'-dioctadecylindocarbocyanine (DiI)-labelled low density lipoprotein (LDL), acetylated LDL (Ac-LDL) and beta-very low density lipoprotein (beta-VLDL) was studied by fluorescence microscopy and flow cytometry in primary cultures of enzymatically dispersed aortic cells from cholesterol-fed rabbits. Most of the foam cells were identified as macrophages on the basis of Fc-receptors and high activities of nonspecific esterase and acid lipase, although cholesteryl ester (CE) inclusions were found by filipin staining also in smooth muscle cells (SMCs). During the culture only SMCs proliferated and were confluent in about 1 week. After incubation with DiI-Ac-LDL most macrophage foam cells were brightly fluorescent, but also many SMCs accumulated fluorescence. In SMCs, an excess of LDL inhibited the uptake of DiI-beta-VLDL and DiI-LDL, indicating that these lipoproteins were taken up by the apoB,E receptor; the activity of this receptor was low 2 days after cell isolation but increased considerably during SMC proliferation. DiI-beta-VLDL was not taken up by the macrophage foam cells until after 7 days' culture, when their CE content had decreased, reflecting a feed-back regulation of these receptors as well. Our results indicate that, in primary cultures of enzyme-dispersed cells from rabbit atherosclerotic lesions, most of the foam cells have lipoprotein receptors resembling those described in macrophages and that also many SMCs accumulate Ac-LDL.

The effect of proteoglycans, collagen and lysyl oxidase on the metabolism of low density lipoprotein by macrophages.

To study the interactions of lipoproteins, connective tissue components and cells, mouse peritoneal macrophages were incubated in the presence of human low density lipoproteins (LDL) that had been complexed with pig aortic proteoglycans (PG) or incubated in the presence of soluble collagen and/or lysyl oxidase, which catalyses the formation of cross-linkages in collagen and elastin by oxidising epsilon-amino groups of lysine residues to aldehydes. Soluble and insoluble PG-LDL complexes increased the incorporation of [3H]oleate into cellular cholesteryl esters (CE) 1.6- and 2.8-fold, respectively, while LDL incubated with collagen and lysyl oxidase had no effect compared to control LDL. As judged on the basis of incubations with fucoidin, spermine and 125I-labelled lipoproteins, the mechanism of internalisation of the PG-LDL complexes is different from that of acetylated LDL or dextran sulphate-LDL complexes. The formation of PG-LDL complexes in the arterial intima may lead to an increased uptake of lipoproteins by intimal macrophages during the early phase of atherogenesis.

Macrophage foam cells from human aortic fatty streaks take up beta-VLDL and acetylated LDL in primary culture.

Intimal cells from human aortic fatty streak lesions were isolated with collagenase-elastase digestion and the cellular uptake of lipoproteins fluorescently labeled with 3,3'-dioctadecylindocarbocyanine (DiI) was studied in primary culture. The majority of the cells in primary culture contained lipid droplets and the foam cells consisted of both macrophages and smooth muscle cells (SMC), identified with electron microscopy and the macrophages also using the monoclonal anti-Leu-M3 antibody. The lipid inclusions contained cholesteryl ester, as visualized with filipin staining. Arterial macrophages took up DiI-labeled acetylated low density lipoprotein (DiI-acetyl-LDL) in the same way as did monocyte-macrophages isolated from blood. DiI-labeled beta-very low density lipoprotein (DiI-beta-VLDL) isolated from cholesterol-fed rabbits, was taken up by both macrophages and SMCs. In macrophages DiI-beta-VLDL was internalized also in the presence of excess unlabeled low density lipoprotein (LDL), whereas in SMCs the uptake was partially prevented. DiI-LDL uptake was only seen in SMCs free of lipid inclusions and especially during cell growth. The present results show that, in human aortic fatty streaks, (a) both macrophages and SMCs accumulate cholesteryl ester, (b) macrophage foam cells possess active scavenger receptors capable of mediating the uptake of acetyl-LDL, and (c) macrophages are also capable of accumulating cholesteryl ester by receptor-mediated uptake of beta-VLDL.

Metabolism of glycosaminoglycans and lipids in smooth muscle cells from atherosclerotic rabbit aortas in culture.

Glycosaminoglycan (GAG) and lipid synthesis in smooth muscle cells cultured from normal and atherosclerotic rabbit aortas were studied. The incorporation of [14C]glucosamine into acidic GAGs taken to represent their synthesis rate, was enhanced in cells from atherosclerotic aortas as compared with controls. The synthesis of sulphated glycosaminoglycans was affected most and the percentage of radioactivity incorporated into sulphated GAGs was 48 +/- 3% in cells from atherosclerotic and 38 +/- 4% in cells from healthy aortas. Non-radioactive GAGs secreted into incubation media were fractionated by electrophoresis. There was an elevated ratio of sulphated GAGs to hyaluronic acid in the cultures of cells from atherosclerotic aortas and the fraction corresponding to dermatan sulphate was increased most. The incorporation of [3H]oleate into phospholipids was enhanced in cells from atherosclerotic aortas indicating more rapid synthesis of this lipid fraction in these cells. Concentrations of free and esterified cell cholesterol were similar. The results indicate that the enhanced synthesis of sulphated GAGs typical of proliferative atherosclerosis in vivo is maintained in the third passage cultures of cells from atherosclerotic rabbit aortas. In addition there was an enhancement in the synthesis of phospholipids in these cells.

Growth properties and composition of cytoskeletal and cytocontractile proteins in aortic cells isolated and cultured from normal and atherosclerotic rabbits.

Aortic intima-medias of normal and cholesterol-fed rabbits were studied with EM and cells were isolated by enzyme digestion. The composition of cytoskeletal and cytocontractile proteins was determined with SDS-PAGE and the primary growth and thymidine incorporation rates were assessed after seeding the cells into tissue culture flasks. Ultrastructurally, the SMCs in the thickened atherosclerotic intima differed from the contractile medial SMCs in containing lipid vacuoles, enlarged endoplasmic reticulum and a reduced number of myofilaments, thus showing characteristics of dedifferentiated SMCs. In SDS-PAGE, freshly isolated cells from the atherosclerotic intima-medias had a lower content of myosin and actin, and a higher proportion of vimentin and desmin than SMCs from normal aortas. Enzyme-isolated SMCs from normal aortas did not start to grow and incorporate radioactive thymidine until 5-6 days after seeding, whereas those from atherosclerotic aortas did so within 2 days. After a week in culture, SMCs from both sources resembled each other, and had decreased contents of myosin and actin, and increased concentrations of vimentin in comparison to freshly isolated normal SMCs. The present results indicate (a) that morphological dedifferentiation of SMCs in aortic lesions of cholesterol-fed rabbits is associated with an increased proportion of the proteins of the intermediate filaments and a decrease in those of the thin and thick myofilaments as determined with SDS-PAGE, and (b) that similar changes take place when normal SMCs are cultured in vitro. The results also suggest (c) that enzyme-isolated atherosclerotic SMCs proliferate in a primary culture without the lag period that normal SMCs apparently require for dedifferentiation.

Antibodies to cytoskeletal proteins in sera of patients with angiographically assessed coronary artery disease.

Circulating autoantibodies to various components of the arterial wall have been reported in atherosclerosis. To examine the occurrence of autoantibodies to cytoskeletal proteins in coronary artery disease (CAD) we studied 56 patients with angiographically demonstrable CAD and compared them with 37 controls without CAD. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the serum samples. In coronary patients, antibody absorbance values at least two standard deviations above the mean for the controls were considered positive. The following numbers of positive antibody absorbances were found in the group of 56 patients: actin IgG, 6 (10.7%); cytokeratin-18 IgG, 3 (5.4%), IgA, 2 (3.6%); myosin IgA, 11 (19.6%); desmin IgG, 13 (23.2%), IgM, 3 (5.4%); vimentin IgG, 2 (3.6%), IgM, 7 (12.5%), IgA, 6 (10.7%). The specificity of desmin IgG was tested with Western blotting against extracts of human internal mammary artery. The positive antibody absorbances to one or several cytoskeletal proteins in the patients were not found to correlate with the clinical symptoms of CAD. Our results suggest an association between autoantibodies to cytoskeletal proteins, particularly to those for desmin, with angiographically assessable CAD.

Association of serum MMP-9 with autoantibodies against oxidized LDL.

Monocyte-derived macrophages in atherosclerotic plaques secrete matrix metalloproteinases (MMPs), which may contribute to plaque rupture. There has been much speculation as to which factors precipitate in the arterial inflammation. Oxidized low density lipoprotein (oxLDL) has been suggested to have proinflammatory properties, and it has been shown to increase matrix metalloproteinase-9 (MMP-9) secretion by macrophages in vitro. We determined serum MMP-9 concentration and autoantibodies against oxLDL by ELISA in men with angina pectoris (n=243) and age-matched controls (n=238). The association between serum MMP-9 concentration and autoantibodies against oxLDL was evaluated. Autoantibody level against oxLDL, expressed in optical density units, was significantly higher in subjects with angina pectoris compared to controls (0.100+/-0.064 versus 0.088+/-0.051, respectively, P=0.030), but serum levels of MMP-9 did not differ significantly between these groups (54.2+/-29.9 versus 50.6+/-23.1 microg/l). However, autoantibodies against oxLDL correlated positively with serum MMP-9 (r=0.21, P<0.001). In a multiple regression model (including age, diastolic blood pressure, cholesterol, HDL cholesterol, triglycerides, BMI, smoking and MMP-9) serum MMP-9 (beta=0.200, P<0.001) and smoking (beta=0.179, P<0.001) were significantly associated with autoantibodies against oxLDL. In conclusion, autoantibodies against oxLDL were positively associated with angina pectoris and serum MMP-9. Since autoantibody level against oxLDL could be expected to reflect the degree of oxLDL in the vessel wall, our results suggest that oxLDL is associated with MMP-9 in vivo.

Characteristics of low density lipoprotein isolated from circulating immune complexes.

Circulating immune complexes (CIC) containing low density lipoprotein (LDL) were recently found in the blood of patients with coronary atherosclerosis. In the present study, we investigated the chemical composition and physical characteristics of the lipoprotein constituents of these CIC. CIC were isolated from the blood of atherosclerotic patients by affinity chromatography using anti-human immunoglobulin G-agarose. Low density lipoprotein of these complexes (CIC-LDL) was obtained by ultracentrifugation. CIC-LDL was compared with free circulating LDL isolated from the blood plasma of the same patients. Plasma LDL was fractionated by lectin-chromatography on RCA120-agarose to obtain desialylated LDL (atherogenic) and sialylated LDL (nonatherogenic). Both CIC-LDL and desialylated LDL, but not native (sialylated) lipoprotein, induced a 1.8- to 3-fold increase in the intracellular contents of free and esterified cholesterol of cells cultured from grossly normal areas of human aorta. The sialic acid level in CIC-LDL was 1.3- and 2.1-fold lower than in desialylated or native LDL, respectively. The neutral lipid and phospholipid contents of CIC-LDL and desialylated LDL were reduced as compared to native LDL. The levels of lipid-oxidation products, thiobarbituric acid-reactive substances and hydroperoxides, were similar in all lipoprotein preparations. However, desialylated LDL and CIC-LDL had an elevated oxysterol content. Gradient ultracentrifugation revealed that CIC-LDL particles had a higher density than native LDL. The mean diameters of native, desialylated and CIC-LDL accounted for 24.0, 21.3 and 19.5 nm, respectively. Like desialylated LDL, CIC-LDL displayed a higher electrophoretic mobility compared with that of native LDL. Thus, LDL obtained from circulating immune complexes appears to be a multiple-modified lipoprotein possessing many similarities to desialylated LDL. It was also found that the LDL content of circulating immune complexes correlates well with the desialylated LDL level in human plasma but not with the total LDL concentration. We believe that desialylated LDL predominately interacts with antibodies forming immune complexes. Taken together, our findings suggest that multiple-modified desialylated LDL is the circulating autoantigen for anti-LDL autoantibodies.

The relation of oxidized LDL autoantibodies and long-term hormone replacement therapy to ultrasonographically assessed atherosclerotic plaque quantity and severity in postmenopausal women.

BACKGROUND: In epidemiologic studies, the incidence of atherosclerosis rises soon after menopause in women, and hormone replacement therapy (HRT) has proved to be useful in preventing onset of clinical manifestations of the disease. However, it is not known how HRT affects sonographically determined atherosclerotic severity (AS) and number of atherosclerotic plaques (NAP) in large arteries. Furthermore, it is not clear how HRT affects oxidation of low density lipoproteins (LDL), which obviously has an important role in the pathogenesis of atherosclerosis. OBJECTIVES: The purpose of the study was to determine whether HRT has a beneficial effect on sonographically determined AS and NAP in large arteries of 101 postmenopausal women compared to 40 controls without HRT. We also studied the interaction of HRT and antibodies against oxidized LDL on AS and NAP progression. RESULTS: Estradiol valerate alone, combined estradiol valerate-levonorgestrel and combined estradiol valerate-medroxyprogesterone acetate therapy are each associated with lower NAP and AS as compared to controls without HRT. In a multiple regression model explaining NAP in the whole study population, the strongest predictors were HRT (P=0.0006) and copper-oxidized LDL cholesterol autoantibodies (P=0.0491). DISCUSSION: Our findings indicate that postmenopausal HRT is associated with a lower total number of atherosclerotic plaques and less severe atherosclerotic lesions, as compared to controls without HRT, and that this outcome may be associated with the effect of HRT on LDL cholesterol oxidation.

The level of autoantibodies against oxidized LDL is not associated with the presence of coronary heart disease or diabetic kidney disease in patients with non-insulin-dependent diabetes mellitus.

Oxidation of low-density lipoprotein (LDL) may be an important factor in the development of diabetic macrovascular and renal complications. The level of autoantibodies against oxidized LDL (oxLDL-Ab) can be used as an index of LDL oxidation in vivo. The purpose of this study was to investigate the association between the level of oxLDL-Ab and the presence of coronary heart disease and renal dysfunction in patients with non-insulin-dependent diabetes mellitus (NIDDM). We determined the plasma levels of oxLDL-Ab in 46 NIDDM patients and 48 well matched nondiabetic control subjects. NIDDM patients had a moderately higher level of oxLDL-Ab than control subjects (0.083 +/- 0.051 vs. 0.062 +/- 0.045, p = 0.04). However, there was no difference in the level of oxLDL-Ab between subjects with and without coronary heart disease, and the level of oxLDL-Ab was not associated with indices of glomerular filtration rate or urinary albumin excretion.

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.

Characteristics of low-density lipoprotein subfractions from patients with coronary artery disease.

BACKGROUND: The low-density lipoprotein (LDL) of patients with coronary artery disease (CAD) has been reported to enhance cholesterol ester accumulation in aortic cells. The LDL of patients with CAD also has a higher mean density than the LDL of healthy subjects, indicating a different subspecies distribution. Because these density differences may be associated with altered metabolism and atherogenicity of LDL, we studied the properties and effects of isolated LDL subfractions on cell lipid metabolism and cholesterol accumulation. METHODS: Plasma pools of patients with angiographically proven CAD (A) and healthy controls (C) were used to isolate and fractionate LDL into five subfractions (1 to 5, from the lowest to the highest density) by gradient ultracentrifugation. Each of the LDL subfractions was analyzed for particle diameter, chemical composition, sialic acid content, and their effect on lipid content and cholesterol esterification in arterial cell and macrophage cultures, respectively. RESULTS: The chemical compositions of the respective subfractions revealed no differences between patients and controls, except that the sialic acid content was reduced in dense LDL subfractions, especially in the samples from patients with CAD (fractions A3, A4, A5, and C5). LDL subfractions with reduced sialic acid content also enhanced the incorporation of [14C]-oleate into cholesterol esters in mouse peritoneal macrophage cultures (fractions A4, A5, and C5) and increased the cholesterol ester content in primary cultures of human aortic intimal cells (fractions A3, A4, A5, and C5). CONCLUSIONS: The results suggest that the dense LDL subfractions of patients with CAD are atherogenic by promoting intracellular cholesterol ester accumulation. The results also suggest that the atherogenicity is associated with reduced sialic acid content of the LDL subspecies.

Suppression of viability and acetyl-LDL metabolism in RAW 264 macrophage-like and smooth muscle cells by bisphosphonates in vitro.

Etidronate and clodronate are bisphosphonates that inhibit the development of experimental atherosclerosis. Etidronate decreases the intimamedia thickness of carotid artery even in man. Liposome-encapsulated bisphosphonates inhibit the cellular metabolism of atherogenic, modified low-density lipoprotein (acetyl-LDL) by cultured macrophages. In the present study, the effects of new bisphosphonate tiludronate and nitrogen-containing bisphosphonate alendronate on cell viability and cellular uptake and degradation of acetyl-LDL were investigated in vitro with macrophages and arterial smooth muscle cells, which have a significant role in atherogenesis. Tiludronate and alendronate decreased the viability of RAW 264 macrophages at high concentration (1,000 microM; p < 0.05), while liposome-encapsulated drugs suppressed the viability at concentrations of 30-300 microM. At concentrations greater than or equal to 10 microM, tiludronate and alendronate inhibited the uptake and degradation of acetyl-LDL by RAW 264 cells in a concentration-dependent manner (p < 0.001). None of the bisphosphonates affected the viability of smooth muscle cells, and none but alendronate at a high concentration (1,000 microM) inhibited the uptake and degradation of acetyl-LDL by smooth muscle cells. The results show that tiludronate and alendronate inhibit the atherogenic activity of macrophages in vitro, as shown previously with etidronate and clodronate, providing further evidence for the antiatherogenic effects of bisphosphonates.

D-Amino acids of the amino acid pool and occurrence of racemase and D-amino acid oxidase activities in Escherichia coli B.

Less than 20% of the amino acid content of the amino acid pool of Escherichia coli B exists in the D-form. Alanine, glutamic acid, and valine were shown by gas- chromatography to be partially in the D-form. Only D-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles but D-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid--D-amino acid--oxo acid which exists in the oxidation of L-alanine does not exist with other L-amino acids. It is likely that other D-amino acids in the pool are formed in the presence of D-amino acid oxidase or D-amino acid aminotransferase.