Deglycosylation of apo B-containing lipoproteins increase their ability to aggregate and to promote intracellular cholesterol accumulation in vitro.

Sub-fractions of all apo B-100 containing lipoproteins (low density lipoproteins, very low density lipoproteins and intermediate density lipoproteins) with reduced contents of sialic acid were found in vivo in human blood. These lipoproteins were inclined to spontaneously form aggregates and were able to stimulate accumulation of cholesterol in cells cultured from human aortic intima. In vitro treatment of apo B-containing lipoproteins with 2,6- and 2,3-specific sialidases, alpha-mannosidase, endoglycosidases F1 or F2 or peptide-N-glycanase F also stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in cells of the intact human aortic intima. So, deglycosylation of various apo B-containing lipoproteins possibly occurs in the blood, decreases their resistance to aggregation and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, thereby increasing their atherogenic potential.

Effect of plant extracts on trans-sialidase activity in human blood plasma.

We studied the effects of plant extracts and products of their biological treatment on trans-sialidase activity in human blood plasma. Extracts of kelp, fucus, yarrow, Saint John's wort, onion, and honey in vitro decreased trans-sialidase activity. Extracts of pollen (beebread) and garlic powder produced the maximum inhibitory effect. trans-Sialidase activity of blood plasma ex vivo decreased 2-fold after peroral administration of pollen and garlic powder. A correlation was found between the decrease in trans-sialidase activity in blood plasma and ability of blood plasma to induce cholesterol accumulation in cultured cells from the intact human aortic intima.

Oxidation-induced aggregation of LDL increases their uptake by smooth muscle cells from human aorta.

Oxidative modification of human blood LDL induced by Cu2+, NaOCl, or 2,2-azobis-(2-aminopropane hydrochloride) was followed by their partial aggregation. Separation of oxidized LDL into aggregates and nonaggregated particles showed that they are characterized by a similar degree of oxidative modification. In contrast to nonaggregated particles, LDL aggregates in the same concentration significantly increased cholesterol content in smooth muscle cells from the intact (no involoved in atherosclerosis) human aortic intima. Our results indicate that atherogenicity of LDL oxidized by various factors is mainly associated with the formation of aggregates, but does not depend on the degree of oxidative modification.

Desialylation decreases the resistance of apo B-containing lipoproteins to aggregation and increases their atherogenic potential.

Subfractions of apo B-containing lipoproteins (VLDL and intermediate-density lipoproteins) with reduced content of sialic acid were found in human blood. These lipoproteins are characterized by high capacity to spontaneous association (aggregation) and stimulated accumulation of cholesterol in smooth muscle cells of human aortic intima. In vitro treatment of apo B-containing lipoproteins with alpha-2,6-sialidase and alpha-2,3-sialidase stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in smooth muscle cells of the intact human aortic intima. Probably, desialylation of various apo B-containing lipoproteins can occur in the blood; this process decreases their resistance to aggregation, and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, i.e. increases their atherogenic potential.

Specificity of human trans-sialidase as probed with gangliosides.

It has been shown that human blood contains a soluble 67 kDa enzyme, belonging by its donor-acceptor properties to trans-sialidases. The enzyme is capable of both cleaving and synthesizing alpha2-3 and alpha2-6 sialosides [Atherosclerosis2001, 159, 103]. In this work the study of donor-acceptor specificity of the new enzyme was extended. It has been demonstrated in vitro that trans-sialidase possesses the ability of transferring Neu5Ac residue to acceptor (asialofetuin) both from alpha2-3- (GM1, GM3, GD1a), and alpha2-8-sialylated gangliosides (GD3 and GD1b, but not GT1b and GQ1b). Transfer of radiolabeled Neu5Ac from fetuin to glycosphingolipids demonstrated that Lac-Cer>mono- and disialogangliosides>GT1b>GQ1b were acceptors for this enzyme. Two methods were used to reveal whether alpha2-8 bond can be formed between Neu5Ac residues during trans-sialylation, that is immunochemical detection using monoclonal antibodies specific to alpha2-8 di- and oligosialic acids, and fluorometric C7/C9 analysis. Both methods demonstrated the formation of Neu5Acalpha2-8Neu5Ac termination by trans-sialidase, for example, in case of the use 3'SL as sialic acid donor and Neu5Ac-PAA or LDL as acceptor. Thus, human trans-sialidase in vitro displays wide substrate specificity: the enzyme is capable of digesting as well as synthesizing alpha2-3, alpha2-6, and alpha2-8 sialosides.

[Screening of natural compounds with hypolipidemic activity]. / Skrining prirodnykh soedinenii s gipolipidemicheskoi aktivnost'iu.

In the screening programme for natural hypolipidemic compounds 702 strains of soil microorganisms were tested and 25 of them were selected because of their ability to produce compounds inhibiting sterol synthesis in Hep G2 hepatoma cells. The compounds were estimated in the microbiological model with Tolypocladium inflatum 106 as the test microbe. The 2nd stage of the screening resulted in isolation of 13 strains producing compounds with high hypolipidemic activity, analogous to or higher than the activity of lovastatin in the experimental models.

[Screening of natural immunosuppressors by their ability to modify steroid synthesis in hepatocytes]. / Otbor prirodnykh immunosupressorov po sposobnosti k modifikatsii sinteza sterolov kletkami gepatotsitov.

In the programme for screening sterol synthesis inhibitors with the use of actinomycetes and fungi 702 strains were tested. The effect of alcohol extracts of the mycelium of fungi and actinomycetes at a dilution of 1/10(3) on sterol synthesis by the Hep G2 hepatome cells was determined by incorporation of 3H acetate into sterols and proteins. Lovastatin (200 pg/ml) was used as the control: the sterol synthesis was decreased by 49 +/- 4% without inhibiting the protein synthesis. A number of the cultures produced compounds inhibiting under the experimental conditions the synthesis of sterols by 70 to 80% with simultaneous inhibition of the protein synthesis at least by 60 to 70%. Three compounds from that group produced by streptomycetes were subjected to a more detailed investigation. The compounds were demonstrated to be active antifungal antibiotics (MIC 0.1-1 mcg/ml). In a dose of 0.1-1 mcg/ml they showed high immunosuppressive activity in models of lymphocyte transformation in mice, whereas cyclosporin was active in a dose of 1 mcg/ml. Therefore, the model for screening hypolipidemic compounds could be considered useful for screening promising natural immunosuppressors.

Human plasma trans-sialidase donor and acceptor specificity.

Earlier we have isolated from human plasma desialylated low density lipoproteins (dLDL) and showed that, first, dLDL induce cholesterol esters accumulation--the main process accompanying atherosclerosis development. Second, the process of lipoprotein desialylation took place in plasma, and, finally, sialic acids removed from LDL are transferred to other serum glycoconjugates. In this study we have isolated from human plasma an enzyme transferring sialic acid residues (trans-sialidase) by affinity chromatography and studied its donor and acceptor specificity. Isolated enzyme in the presence of saccharide-acceptor can remove sialic acids from different lipoproteins, glycoproteins (fetuin, transferrin), and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Plasma enzyme translocates alpha2-6, alpha2-3 and to a lower extent alpha2-8 bonded sialic acids. Sialoglycoconjugates of human serum erythrocytes, serum lipoproteins, glycoproteins, and gangliosides can serve as donors of sialic acid for trans-sialidase. Desialylated lipoproteins, especially dLDL, are more preferable sialic acid acceptors. Transferred sialic acid is found to be alpha2-6, alpha2-3, and alpha2-8 connected.

The content of lipoperoxidation products in normal and atherosclerotic human aorta.

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 +/- 18 and 92 +/- 18 vs. 70 +/- 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.

Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein.

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acalpha2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le(x) and Le(a), oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: alpha2,6>alpha2,3>>alpha2,8. Transferred molecule of sialic acid can form alpha2,6, alpha2,3 and to a lesser degree alpha2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.

Optimization of the assay for sialic acid determination in low density lipoprotein.

Sialic acid level in blood plasma and circulating glycoproteins is considered to be a marker for a number of pathologic conditions, including atherosclerosis, cancer, etc. The precise measurement of sialic acid level is an important laboratory procedure to allow correct interpretation of results. Colorimetric methods commonly used for the measurement of sialic acid are not highly specific, as interfering substances may alter the results. Among these, malondialdehyde and other aldehydes play the decisive role. In the circulation, aldehydes are commonly produced during lipid peroxidation in the lipid core of lipoprotein particles, especially low density lipoprotein (LDL). To establish the impairment to the sialic acid determination in LDL introduced by interfering substances, the optimized assay based on Warren's traditional method was developed and tested in 606 LDL samples. The optimization implies the comparison of color developed using the standard Warren procedure with that due to contaminating agents, mainly thiobarbituric acid-reactive substances (TBARS). In LDL stored at 4 degreesC, the estimates obtained by the modified procedure were 41.5% or 30.1 nmol/mg lower, on average, compared to the standard procedure (n = 45, P < 0.0001). Even in LDL stored at -70 degreesC, sialic acid estimates obtained by the modified procedure were 6.6% or 3.6 nmol/mg lower, on average, compared to the standard measurement (n = 561, P < 0.005). Thus, the modified procedure avoids significant distortion of the measurement induced by the presence of interfering agents.

Antioxidant content in low density lipoprotein and lipoprotein oxidation in vivo and in vitro.

UNLABELLED: Human blood contains naturally occurring multiple-modified low density lipoprotein (nomLDL) capable of inducing the accumulation of cholesteryl esters in the cells of human aortic intima. NomLDL is desialylated particles of small size with an increased electronegative charge which can be separated from native low density lipoprotein (LDL) by lectin chromatography. The purpose of this study was to determine the content of antioxidants in native and nomLDL obtained from healthy subjects and from patients with coronary heart disease as well as to elucidate a possible relationship between the level of antioxidants and the degree of in vivo and in vitro LDL oxidizability. The apoB-bound cholesterol level in native and nomLDL of healthy subjects was 0.25 +/- 0.08 and 0.28 +/- 0.05 mol/mol apoB, respectively. The level of apoB-bound cholesterol in native LDL of coronary atherosclerosis patients showed no significant difference from that in healthy subjects' native lipoprotein. At the same time, the level of apoB-bound cholesterol in patients' nomLDL was 7-fold higher than in native LDL. The average duration of the lag phase of native LDL oxidation did not show a significant difference between the lipoprotein of healthy subjects and coronary atherosclerosis patients. The lag phase of nomLDL obtained from healthy subjects and patients was significantly shorter (3- and 6-fold, respectively) than for their native LDL. The latter finding points to their increased susceptibility to in vitro oxidation. Oxidizability of total LDL preparations correlated positively with their nomLDL content. The content of all the antioxidants studied (coenzyme-Q10, alpha- and gamma-tocopherols, beta-carotene and lycopene) in nomLDL was 1.5- to 2-fold lower than in native LDL. The level of apoB-bound cholesterol in nomLDL, correlated positively with the ubiquinone-10 content and showed negative correlation with ubiquinol-10 and beta-carotene levels. On the other hand, the content of apoB-bound cholesterol in native LDL correlated positively with the ubiquinol-10 level. Susceptibility of nomLDL to in vitro oxidation exhibited negative correlation with alpha-tocopherol and beta-carotene levels and a positive correlation with the ubiquinone-10 content. On the contrary, oxidizability of native LDL correlated positively with the ubiquinone-10 level. CONCLUSIONS: (a) elevated apoB-bound cholesterol level in nomLDL of coronary atherosclerosis patients indicates that peroxidation of lipids occurs in vivo; (b) in vivo lipoperoxidation in nomLDL is corroborated by increased proportion of oxidized form of coenzyme-Q10; (c) content of lipid-soluble antioxidants in nomLDL is lower than in native lipoprotein; (d) nomLDL has a higher susceptibility to in vitro oxidation than native LDL; (e) it is necessary to use isolated subfractions of native LDL and nomLDL, but not total lipoprotein preparations, to study the mechanisms of lipid peroxidation.

In vivo oxidized low density lipoprotein: degree of lipoprotein oxidation does not correlate with its atherogenic properties.

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.

Low-density lipoprotein modification occurring in human plasma possible mechanism of in vivo lipoprotein desialylation as a primary step of atherogenic modification.

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.

The effects of antihypertensive agents on atherosclerosis-related parameters of human aorta intimal cells.

Four antihypertensive agents - amlodipine, verapamil, propranolol and perindoprilat - were studied in human cell cultures. Antiatherogenic activity was investigated using uninvolved human aortic smooth muscle intima cells and atherogenic serum obtained from patients with coronary atherosclerosis. Amlodipine and verapamil significantly inhibited serum-induced increases in cholesterol content, cell-proliferative activity and protein synthesis in the cultured cells. Propranolol increased all three parameters, while perindoprilat had no effects. In addition, amlodipine and verapamil significantly lowered the intracellular cholesterol content of smooth muscle cells derived from atherosclerotic plaque and inhibited cell proliferation and protein synthesis. Propranolol increased all of these parameters, while perindoprilat produced no effects. The antiatherogenic and antiatherosclerotic actions of verapamil and amlodipine were confirmed in an ex vivo model. These studies demonstrated a beneficial antiatherosclerotic effect of amlodipine that was greater than that of verapamil. Perindoprilat had a neutral effect on atherosclerotic parameters, while the action of propranolol appeared to be potentially detrimental.

In vitro effect of garlic powder extract on lipid content in normal and atherosclerotic human aortic cells.

In the present study, the mechanism of the in vitro effect of garlic powder extract (GPE) on lipid content of cultured human aortic cells was investigated. The addition of GPE abolished atherogenic blood serum-induced accumulation of free cholesterol, triglycerides, and cholesteryl esters in smooth muscle cells derived from uninvolved (normal) intima. In cells isolated from atherosclerotic plaque, GPE lowered these lipids. GPE inhibited lipid synthesis both in normal and atherosclerotic cells. It inhibited acyl-CoA:cholesterol acyltransferase activity that participates in the cholesteryl ester formation and stimulated cholesteryl ester hydrolase that degrades cholesteryl esters. This may explain the lipid reduction caused by GPE in atherosclerotic cells. GPE inhibited the uptake of modified low density lipoprotein and degradation of lipoprotein-derived cholesteryl esters, thus considerably reducing the intracellular accumulation of cholesteryl esters. This suggests the mechanism responsible for the prevention of lipid accumulation in aortic cells caused by atherogenic blood serum.

Lack of correlation between degree of human plasma low density lipoprotein oxidation and its atherogenic potential.

We have recently found that adducts of lipids, particularly cholesterol, with apolipoprotein B (apoB) are stable markers of human plasma low density lipoprotein (LDL) oxidation [7]. In this study we attempt to assess the relationship between the degree of plasma LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intima smooth muscle cells, i.e., LDL atherogenic potential. LDL samples of 32 out of 39 healthy subjects did not increase cholesterol content in cells cultured from grossly normal intima of human aorta. Most of LDL preparations isolated from coronary atherosclerosis patients with (34 out of 43) or without (35 out of 45) hypercholesterolemia stimulated intracellular cholesterol accumulation by 32-302%. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.10, n = 127). These results suggest that atherogenicity of LDL circulating in human plasma does not depend on the degree of lipid peroxidation in LDL particles.

Metabolism of native and naturally occurring multiple modified low density lipoprotein in smooth muscle cells of human aortic intima.

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