A cytofluorometric study of membrane rafts in human monocyte subsets in atherosclerosis.

The peripheral blood monocytes of atherosclerotic patients are pre-activated and have some of the features of tissue macrophages. Their adhesion to the endothelium is 1.5 times higher than that of monocytes from healthy subjects, and they express a number of receptors and antigens typical of tissue macrophages. Additionally, earlier we showed that the biosynthesis of gangliosides, whose main function is the formation of membrane rafts, is significantly activated in blood monocytes from atherosclerotic patients, as well as during the in vitro differentiation of normal monocytes into macrophages. In this study, we investigated the expression of membrane rafts on various monocyte subsets from healthy subjects and atherosclerotic patients. Based on flow cytometry results, the monocytes in the examined atherosclerotic patients were found to differ from those in healthy subjects by a twofold increase in the proportion of the intermediate subset (CD14(++)/CD16(+)) and by enhancement in the expression of the fractalkine receptor CX3CR1 on the intermediate and non-classical subsets (CD14(++)/CD16(+) and CD14(+)/CD16(++)) (2.3 and 1.8 times, respectively). This suggests a pre-activated state of monocytes in atherosclerotic patients. At the same time, the expression of the membrane raft marker on the monocyte subsets was similar in both studied groups. However, a study of the in vitro differentiation of monocytes into macrophages showed that the membrane raft expression increased 2 times as early as on the 1st day of culturing and 3 times on the 7th day compared to that in freshly isolated monocytes. Therefore, it is suggested that monocytes in atherosclerosis accumulate gangliosides that are used to form membrane rafts during the macrophage differentiation after the migration of monocytes into the arterial intima.

[Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis].

Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated activity of GM3 synthase, cellular levels of ganglioside GM3 and its role in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one an order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by incubation in medium containing this ganglioside increased adherence of these monocytes to HUVEC up to the values typical for monocytes from atherosclerotic patients. In addition, an increase in CD1 1b integrin expression was observed that was comparable to that seen in lipopolysaccharide-activated monocytes. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.

Effect of lysophosphatidylcholine on α-adrenoreactivity of rat aorta smooth muscles.

In experiments on rat aortic ring segments, lysophosphatidylcholine in concentrations of 2×10(-6), 2×10(-5), and 2×10(-4) M did not suppress the tonotropic effect of phenylephrine (6×10(-6) and 6×10(-5) M) and in concentration of 2×10(-5) M even potentiated it, which was noted for phenylephrine at a concentration of 6×10(-6) M. It was concluded that the chemomodulating effect of lysophosphatidylcholine depends on the type of receptors and cells.

Effects of LDL lipids on activity of group IIA secretory phospholipase A2.

Effects of phosphatidylcholine, oxidized phosphatidylcholine, sphyngomyelin, cholesterol, and cholesterol esters incorporated in LDL on activity of group IIA secretory phospholipase A2 from human cardiac myxoma were studied. Liposomes containing radioisotope-labeled phosphatidylethanolamine served as the substrate for group IIA secretory phospholipase A2. Oxidized phosphatidylcholine significantly stimulated activity of group IIA secretory phospholipase A2, while phosphatidylcholine in the same concentrations did not modify enzyme activity. Sphyngomyelin incorporated in LDL inhibited group IIA secretory phospholipase A2 activity. Cholesterol and cholesterol esters virtually did not modify enzyme activity. The results indicate that LDL phospholipids and their oxidized forms can be involved in regulation of group IIA secretory phospholipase A2. Study of the mechanisms regulating the proinflammatory group IIA secretory phospholipase A2 can promote the development of new approaches to the diagnosis and treatment of inflammatory processes.

[The effect of LDL on the activity of proinflammatory secretory phospholipase A2 (IIA) depends on the degree of their oxidation].

Phospholipase A2 group IIA which is secreted in inflammation [secPLA2(IIA)] does not always exhibit catalytic activity, although being present in patients' serum. The mechanisms regulating the enzyme activity in peripheral blood have not been studied in sufficient detail. In this study we examined the effects of native and oxidized LDL with varied degree of oxidation on secPLA2(IIA) from human heart myxoma. The degree of LDL oxidation was evaluated from the amount of conjugated dienes and lysophosphatidylcholine. Liposomes containing radio-labelled phosphatidylcholine were used as a substrate in the secPLA2(IIA) activity assay. Native LDL isolated from serum of healthy subjects inhibited secPLA2(IIA) in a dose-dependent manner. Minimally and moderately oxidized LDL in which < 40 % phosphatidylcholine was hydrolysed to lysophosphatidylcholine activated secPL2(IIA). Strongly oxidized LDL in which > 40 % phosphatidylcholine was hydrolysed to lysophosphatidylcholine inhibited the enzyme. Thus, our findings indicate that the characteristics of circulating lipoproteins are changed by their oxidation. Minimal and moderate oxidation of LDL results in activation of secPLA2(IIA), while strong oxidation causes inhibition of the enzyme. Since inflammation leads to an increase in secPLA2(IIA) secretion and LDL oxidation, the results obtained can be used for the diagnostics of inflammatory processes and provide more insight into molecular mechanisms underlying the development of atherosclerosis.

Ganglioside GM3 and its biological functions.

Metabolism, topology, and possible mechanisms for regulation of the ganglioside GM3 content in the cell are reviewed. Under consideration are biological functions of GM3, such as involvement in cell differentiation, proliferation, oncogenesis, and apoptosis.

Lipid second messengers and cell signaling in vascular wall.

Agonists of cellular receptors, such as receptor tyrosine kinases, G protein-coupled receptors, cytokine receptors, etc., activate phospholipases (C(gamma), C(beta), A(2), D), sphingomyelinase, and phosphatidylinositol-3-kinase. This produces active lipid metabolites, some of which are second messengers: inositol trisphosphate, diacylglycerides, ceramide, and phosphatidylinositol 3,4,5-trisphosphate. These universal mechanisms are involved in signal transduction to maintain blood vessel functions: regulation of vasodilation and vasoconstriction, mechanical stress resistance, and anticoagulant properties of the vessel lumen surface. Different signaling pathways realized through lipid second messengers interact to one another and modulate intracellular events. In early stages of atherogenesis, namely, accumulation of low density lipoproteins in the vascular wall, cascades of pro-atherogenic signal transduction are triggered through lipid second messengers. This leads to atherosclerosis, the general immuno-inflammatory disease of the vascular system.

Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing in vitro.

We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 microg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions.

Influence of lysophosphatidylcholine, phosphatidylcholine, and hen egg yolk on contractile effects of acetylcholine on smooth muscles of rat stomach.

Experiments on smooth muscles of rat stomach showed that lysophosphatidylcholine in concentrations of 10(-8) and 10(-7) g/ml does not modulate the tonotropic effect of acetylcholine (10(-6) g/ml), in a concentration of 10(-6) g/ml potentiated this effect (similarly to phosphatidylcholine, 10(-6) g/ml), and reduced it in concentrations of 10(-5)-10(-4) g/ml (similarly to hen egg yolk in dilutions of 1:500, 1:100, and 1:500). These data indicate that lysophosphatidylcholine modifies signal transduction from the receptor to G protein.

[Effect of atorvastatin therapy on the level of secretory phospholipase A2 group IIA and on the modification of low density lipoproteins in patients with ischemic heart disease].

We studied effect of atorvastatin on secretory phospholipase A2 group IIA (sPLA2-IIA) in blood serum of patients with ischemic heart disease (IHD), lipid composition of low density lipoproteins (LDL) and process of modification of LDL induced by sPLA2-IIA in 20 patients taking 20 mg/day of atorvastatin for 3 months. In patients with initially high level of sPLA2-IIA ( > 8 mcg/l) its concentration significantly decreased. Amount of total cholesterol, triglyceride, lecithin, and lysolecithin remained unchanged, however in equimolar relations there occurred decrease of amount of total cholesterol and increase of cholesterol esters. At incubation of LDL, extracted from patient s plasma before initiation of the study, with human sPLA2-IIA from cardiac myxoma, 3.5 nmol of lysolecithin per 1 mg of LDL protein was formed while at incubation of LDL of same patients, extracted after 3 months of atorvastatin administration, amount of lysolecithin was 1.54 nmol/mg LDL protein. Thus atorvastatin therapy causes lowering of sPLA2-IIA in patients with initially high blood level of the enzyme and to a great extent precludes sPLA2-IIA induced LDL modification.

Effect of serum from cardiovascular patients on catalytic activity of secretory phospholipase A2 (IIA).

Incubation of patients' serum catalytically active by type IIA secretory phospholipase A2 (SP-IIA) with serum containing the enzyme in a high concentration but exhibiting no catalytic activity in 1:1 volume ratio led to a significant inhibition of SP-IIA catalytic activity. donor and patient sera with low levels of SP-IIA had no effect on the serum with SP-IIA activity under these conditions. However, the increase in the content of patients' serum with a low level of SP-IIA in the incubation mixture to 1:2 (v/v) and of donor serum to 1:3 (v/v) also led to blockade of SP-IIA catalytic activity. These results indicate that human serum contains an SP-IIA inhibitor and its concentration decreases significantly in sera with SP-IIA activity.

Development of antibody to human GM3 synthase and immunodetection of the enzyme in human tissues.

Polyclonal antibody was raised to a cloned fragment of human GM3 synthase. Affinity purified R27C1 antibody to the tagged recombinant protein inhibited GM3 synthase activity in human liver and HL-60 cells in a dose-dependent manner. However, the R27C1 antibody did not affect liver sialyltransferase activity towards asialofetuin. We are the first to measure GM3 synthase activity in human liver (194 +/- 60 pmol NeuAc/h per mg protein), which was about 10-fold lower than in phorbol myristate acetate-stimulated HL-60 cells (1353 +/- 573 pmol NeuAc/h per mg protein). On immunoblotting the R27C1 antibody recognized a common protein band in a number of human tissues (liver, brain, atherosclerotic aortic intima, HL-60 cells) with molecular mass of about 60 kD, which is similar to that of the purified GM3 synthase from rat liver. In human liver and aortic intima, the 60-kD band was almost a single band, which makes possible the use of the R27C1 antibody for immunohistochemical studies in these tissues.

Sialidase activity in normal and atherosclerotic human aortic intima.

Sialidase activity has been determined in homogenates of human aortic intima by measuring the amount of GM1 formed during the incubation of ganglioside GD1a with the tissue homogenates. Areas with atherosclerotic lesions as well as adjacent areas without histological evidence of atherosclerosis were taken for comparison. The rate of GM1 formation from GD1a in the presence of homogenates of the atherosclerotic intima was 20 pmol/h per mg protein. Homogenates of the unaffected intima did not desialylate GD1a. Sialidase activity of the atherosclerotic intima was linear for 1.5 h at GD1a content up to 1.5 nmol and at homogenate protein up to 1 micro g. NH4Cl and NeuAc2en, inhibitors of lysosomal function and plasma membrane-bound sialidase, respectively, reduced sialidase activity of homogenates of the atherosclerotic intima by 94%. The results indicate that atherosclerotic lesions and unaffected intima differ in their activity and specificity of sialidases that cleave gangliosides.

[Serum level of secretory phospholipase A2 (sPLA2) as a predictor of restenosis after coronary angioplasty]. / Uroven' sekretornoi fosfolipazy A2 v syvorotke krovi kak prediktor restenoza posle koronarnoi angioplastiki.

AIM: To elucidate whether secretory phospholipase A2 (sPLA2) level in the blood and catalytic activity are significant predictors of restenosis after coronary angioplasty (CAP). MATERIAL AND METHODS: Samples of venous blood were obtained from 24 patients before CAP and 1, 3, 6 days and 6 months after it. sPLA2 was measured with enzyme immunoassay, catalytic activity--using aqueous emulsion of 14C-labelled phosphatidylcholine. Control coronarography was performed in all the examinees 6 months after CAP. RESULTS: Restenosis was detected in 13 patients. In the serum of their blood sPLA2 rose significantly after CAP and persisted for 6 days after it. If restenosis was not registered, this rise was insignificant and disappeared by day 6 after CAP. Catalytic activity of sPLA2 on day 6 after CAP was significantly higher in patients who later developed restenosis. CONCLUSION: Elevated concentrations of sPLA2 in blood serum of patients after CAP may predict restenosis. Moreover, sPLA2 may not only mark inflammation but directly participate in development of restenosis.

Sialyltransferase activity in normal and atherosclerotic human aorta intima.

Sialyltransferase activity has been determined in Golgi membrane fractions isolated from atherosclerotic and normal intima of human aorta by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin-sialyltransferase activity was found to be twofold higher in the atherosclerotic intima than in the normal intima. The mean value of the apparent Michaelis constant (Km) for the sialylating enzyme in both tissues did not differ and was 57 microM. In contrast, the maximal velocity (Vmax) was 2-fold higher for the atherosclerotic intima than for the normal intima. These results suggest that expression of asialofetuin-sialyltransferases of the aortal intima may be increased in atherosclerosis.

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta. Hypothetical role of ganglioside GM3 in foam cell formation.

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.

Ganglioside-mediated metabolic synchronization of protein synthesis activity in cultured hepatocytes.

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.

Plasma sialyltransferase activity in healthy subjects and atherosclerotic patients.

Plasma sialyltransferase activity measured by incorporation of cytidine 5;-phospho[14C]acetylneuraminic acid (CMP-NeuAc) into asialofetuin was twofold higher in patients with documented atherosclerosis than in healthy donors. Kinetic studies showed that the enzyme affinity for CMP-NeuAc is the same in donors and patients. Low activity of plasma sialyltransferase in donors may be due to low blood content of this enzyme.

Autoantibodies to gangliosides in sera of atherosclerotic patients.

Using ELISA we studied the levels and clinical correlation of serum antibodies against gangliosides and 5-hydroxytryptamine (5-HT) in patients with atherosclerosis and clinical manifestations of cardiovascular disease. A range of 70-80% of the patients showed higher titers of anti-GM3(L) and anti-5HT as compared to normal serum. The anti-GM3(L) antibodies appeared to be directed mainly against GM3 present in platelets and were much less reactive against GM3 isolated from the aorta. We concluded that the antigens responsible for the elevated anti-GM3(L) and anti 5-HT levels in atherosclerotic sera are released by vessel-wall activated platelets. These results provide further evidence of on-going autoimmune processes in atherosclerosis. The content of total sialic (TS) and lipid-bound sialic acid (LBS) was measured in sera of patients with IHD and of similar numbers of healthy donors. In the patient groups the average TS and LBS concentration was about 25% higher than in the control group. These changes appeared to be associated with higher degrees of protein sialylation and larger amounts of LDL in the patient sera than in those of healthy controls.

[Determination of lipid composition of high density lipoproteins by thin-layer chromatography on silica gel]. / Opredelenie lipidnogo sostava lipoproteidov vysokoi plotnosti metodom tonkosloinoi khromatografii na silikagele.

Unidimensional chromatography in a thin layer of silica gel with two solvent systems for separation of the lipid extract of high density lipoproteins (HDLP) detected 10 lipid fractions in subjects with normal lipidemia. Four phospholipid fractions were identified: three main (phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin) and one minor, phosphatidylethanolamine. HDLP lipids include free cholesterol and its esters; three glyceride fractions are detected: mono- di, and triglycerides, and free fatty acids. The ratio of individual phospholipid fractions and of free to ester-bound cholesterol in HDLP determined by computer-aided densitometry of chromatograms coincides with the data obtained routinely. Such a method for assessing the HDLP lipid composition is useful in studies of HDLP in patients with disorders of HDLP metabolism and for assessing the changes in HDLP composition during hypolipidemic therapy.