Pluronics suppress association of low-density lipoproteins inducing atherogenesis.

We studied the effect of pluronics P85, L61, and F68 with different hydrophilic-lipophilic characteristics on association of LDL. It was found that pluronics with pronounced hydrophobic properties (P85 and L61) in concentrations close to or surpassing the critical concentration of micelle formation inhibited LDL association, while hydrophilic pluronic F68 in all concentrations had no effect on LDL association.

[Analysis of the inflammatory processes in the diffuse thickening of the intima of human aorta].

It is generally recognized that the accumulation of lipids and immuno-inflammatory cells are early signs of atherosclerosis. In the present study, we have investigated the relationship between the deposition of lipids, of immuno-inflammatory cells and the expression of HLA-DR molecules (a marker of immune activation), the molecules of the class II of major histocompatibility complex (MHC) in diffuse thickening of the intima (DIT). Lipids, including triglycerides, cholesterol esters, free cholesterol and phospholipids were studied by chromatography, Oil Red O histochemisty, as well as by electron microscopic analysis. Immuno-inflammatory cells and the expression of HLA-DR were investigated by immunohistochemistry in consecutive section of the same tissue samples. It has been shown that the lipids were unevenly distributed in DIT. In juxtaluminal sublayer, lipids were detected both in the cytoplasm of intimal cells and extracellularly. In the juxtamedial musculoelastic sublayer of the intima, lipids were present predominantly along elastic fibers. The positive correlation between the presence of lipids and the expression of HLA-DR was revealed (r = 0.79; P < 0.001). Also, a positive correlation was found between the deposition of lipids and the number of immune-inflammatory cells, although correlations was different for different sublayers of the intima. In particular, the correlation between the deposition of lipids and immune-inflammatory cells in the juxtaluminal sublayer of the intima was higher (r = 0.99; P < 0.001) than in the juxtamedial musculoelastic layer (r = 0.28; P < 0.001). These data support the hypothesis that postulates that the accumulation of lipids in the intima is a key factor in the initiation of inflammatory reactions. At the pre-atherosclerotic stage of the development of this disease, earlier pathological processes associated with lipid-dependent activation of immune cells occur mainly in the juxtaluminal portion of the intima.

[Cholesterol of circulating immune complexes as biomarker of atherosclerosis].

In the present 2-years prospective study, the diagnostic and prognostic significance of immune cholesterol was assessed in 98 asymptomatic men aged 40-74 with early atherosclerosis. The rate of carotid atherosclerosis progression was estimated by high-resolution B-mode ultrasonography as the increase in carotid intima-media thickness (IMT) of common carotid arteries. The patients with elevated baseline levels of immune cholesterol were characterized by significantly higher levels of total and LDL (low density lipoproteins) cholesterol as well as significantly increased mean IMT of common carotid arteries. Among all baseline lipid parameters, only immune cholesterol and LDL cholesterol were contingent with the extent of early carotid atherosclerosis (p=0.042 and p=0.049, respectively) and had the highest levels of relative risk and odds ratio. Carotid atherosclerosis progression was characterized by slow IMT increase at a rate of 0.029 +/- 0.011 mm per two years over the mean baseline IMT of 0.939 +/- 0.015 mm (p=0.028). A significant IMT increase was registered in 53.1% (n=52) patients, IMT significant reduction was observed in 21.4% (n=21) patients. The increased level of immune cholesterol along with total serum cholesterol and LDL cholesterol had rather high prognostic significance with the respect of atherosclerosis progression. The normal level of immune cholesterol (below than 16.0 mg/ml) was the only lipid parameter that predicted the absence of carotid atherosclerosis progression for two following years at prognostic value of 78.3%. The results of the study allow assuming that immune cholesterol level may be employed not only as a marker of early atherosclerosis, but also has a sufficient prognostic value for clinical implications.

[Cultured cell systems for revelation of new drugs antiatherosclerotic effects and their mechanisms investigation].

Cultured cell models have been developed to study the cholesterol efflux from the arterial wall as an integral indicator of reverse cholesterol transport. The models and the results of in vivo and ex vivo experiments can be used to propose new antiatherosclerotic drugs and elucidate their mood of action.

[Numbers of cells and cell proliferation in intima of different human arteries].

Increased cell proliferation in early atherosclerotic lesions is recognized as an essential event of atherogenesis but the levels of cell proliferation in different stages of atherosclerotic plague formation in different types of human large arteries are still insufficiently studied. In the present work, we studied intima thickness and proliferation of newly "infiltrates" hematogenous and resident cells in atherosclerotic lesions of the carotid and coronary arteries and compared these parameters with those in the aorta, reported by us in earlier publication. Analysis of intima thickness and proliferation in grossly unaffected intima and in different types pf atherosclerotic lesions (initial lesions, fatty streaks, lipofibrous, plaques, and fibrous plaque) revealed that although there were similar tendencies in the change of the infiltration levels of hematogenous cells and proliferation in different types of arteries, there were significant quantitative differences between different types of arteries. Hematogenous cells in lipofibrous plaques of the coronary and carotid arteries were found to account for a third and almost for a half of the total cell population, respectively, while atherosclerotic lesions in the aorta, as it has been shown by us earlier, to contain no more than 15% ofhematogenous cells. This suggests that the contribution of hematogenous cells to the development of atherosclerosis in the carotid and the coronary artery appears to be more significant than that in the aorta. Despite the differences in numbers of accumulating hematogenous cells in the intima, a similar "bell-shaped" dependence of cell numbers on the lesion type, involved in the following sequence: unaffected intima-initial lesions-fatty streaks-lipofibrous plaques-fibrous plaques, was detected in the coronary and carotid arteries. The visualization of proliferating cells (PCNA-positive) in atherosclerotic and unaffected zones of the coronary and carotid arteries revealed similar patterns. The maximum numbers of PCNA-positive resident cells were identified in lipofibrous plaques. The changes in the total cell numbers were accompanied by the changes in the numbers of both proliferating resident cells and proliferating hematogenous cells.

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.

Antiatherogenic effect of grape flavonoids in an ex vivo model.

The effects of grape phytoestrogens on cholesterol accumulation were studied in primary culture of human blood monocytes incubated with blood serum from postmenopausal women obtained before and 2, 4, and 6 h after single intake of plant components of grapes. Phytoestrogens from grape seeds, pressed out grapes, and fermented grape ridges prevent cholesterol accumulation in cells and can be regarded as prospective components for the development of natural preparations for the prevention of atherosclerosis in postmenopausal women.

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.

[A comparative study of the lipid phase in native and circulating multiple-modified low-density lipoproteins of human blood by the use of the spin probe method].

Different approaches based on the spin probe method were used to compare the physical state of the surface lipid monolayer in subfractions of low-density lipoproteins: in native low-density lipoproteins constituting the bulk of human blood low-density lipoproteins and in circulating multiple-modified low-density lipoproteins whose portion is minor in healthy persons but significantly increases in atherosclerotic patients. The data obtained in in vitro experiments suggest that circulating multiple-modified low-density lipoproteins possess atherogenic properties. The order parameter S, rotational correlation time tau, and hydrophobicity parameter h were calculated from electron spin resonance spectra of a series of spin probes whose paramagnetic groups are located at different depths of the lipid monolayer. These parameters characterize the molecular packing, fluidity, and polarity in the microenvironment of paramagnetic groups. The kinetics of the reduction of paramagnetic groups by ascorbate and oxidation by hypochlorite were obtained for the spin probe whose paramagnetic group is located deeply in the lipid monolayer at the level of the terminal segments of phospholipid acyl chains. No difference between native low-density lipoproteins and circulating multiple-modified low-density lipoproteins was revealed in respect of the physical properties of the lipid domain of surface proteolipid layer, as sampled by spin probes.

Proteolysis of apoprotein B-100 impairs its topography on LDL surface and reduces LDL association resistance.

Serine proteinases (trypsin and chymotrypsin) cause destruction of apolipoprotein B-100 on the surface of human blood LDL. Incubation of LDL with these enzymes increases the mean size of LDL particles. Proteolysis of apolipoprotein B-100 induces changes in surface structure, destabilizes LDL particles, and reduces their association resistance. Presumably, this proteolytic modification of LDL with subsequent association of these particles plays an important role in accumulation of cholesterol in the vascular wall and in the development of early stages of atherosclerosis.

Phospholipid hydrolysis with phospholipases A2 and C impairs apolipoprotein B-100 conformation on the surface of low density lipoproteins by reducing their association resistance.

Modification of apolipoprotein B-100 conformation on the surface of LDL isolated from human blood was demonstrated by enzyme immunoassay with a panel of monoclonal antibodies to this protein. The study by the light transmission fluctuation method showed that incubation of LDL with phospholipases A2 or C led to association of LDL particles. This lipolytic modification seems to impair LDL surface properties inducing association of these particles, which can play an important role in lipid accumulation in the vascular wall and at early stages promote the development of atherosclerosis.

Antigenic differences between apo-B in native and circulating modified low-density lipoproteins.

The state of apo-B in native and circulating modified low-density lipoproteins was studied by solid-phase enzyme immunoassay. We studied the interaction of these particles with monoclonal antibodies to apo-B of low-density lipoproteins. Native and circulating modified low-density lipoproteins had different affinity for the studied antigens. Our results illustrate conformational changes in apo-B of circulating modified low-density lipoproteins compared to native low-density lipoproteins. These changes probably contribute to increased accumulation of particles in vascular cells and their transformation into foam cells giving way to atherosclerotic vascular lesions.

Low ionic strength promotes association of circulating modified LDL in human blood.

The resistance to association of circulating multiply-modified low-density lipoproteins (LDL) isolated from human blood and characterized by a decreased content of sialic acids in comparison with native LDL was studied by analysing light transmission fluctuations. LDL association was stimulated by decreasing environmental ionic strength. It is established that circulating modified LDL are less resistant to association than native LDL. Association of LDL in a medium with low ionic strength was irreversible. Probably, increased capacity to irreversible association determines the atherogenic properties of circulating modified LDL subfraction.

Resistance of native and circulating modified low-density lipoproteins in human blood to association.

The resistance of native and circulating modified low-density lipoproteins from human blood to spontaneous and polyethylene glycol-induced association was studied by recording light transmission fluctuations. Circulating modified low-density lipoproteins were less resistant to association than native low-density lipoproteins. Polyethylene glycol-induced association of low-density lipoproteins was irreversible. Our results suggest that atherogenic activity of circulating modified low-density lipoproteins is associated with their increased predisposition to irreversible association.