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V S Repin

Publications and source records attributed to V S Repin.

At least 109 records · Page 6Linked to original sources
PubMed1986

Adult human aortic cells in primary culture: heterogeneity in shape.

Adult human aortic cells have different shapes in situ. To determine whether populations of cultured aortic cells are also polymorphic, a technique for separation of cells from the intimal and medial layers of the human aorta by enzymatic dispersion of the vascular tissue was employed. It was established that aortic cells are polymorphic in primary culture, at least within the first 7 days after seeding. Four main morphological cell types were identified--elongated, asymmetric, polygonal, and stellate. Polygonal and stellate cells are found only in cultures of grossly normal intima. Elongated and asymmetric cells are present in practically all cultures. The ratio of elongated to asymmetric cells in cultures obtained from healthy aortas and atherosclerotic plaques is more or less the same and is approximately 3:1. In cultures of fatty streaks, the portion of asymmetric cells exceeds 50%. With immunofluorescent staining and ultra-structural analysis, cells of all four types were identified as smooth muscle. Possible reasons for the cells polymorphism in primary culture and the prospects of utilizing this culture method in the investigation of cellular aspects of atherogenesis are discussed.

Adult↗
PubMed1986

Regulation of cholesterol synthesis in isolated epithelial cells of human small intestine.

We have investigated the regulation of cholesterol synthesis in isolated human small intestine epithelial cells (enterocytes). It was established that the amount of cholesterol synthesized increased linearly with the incubation time and the number of cells in the incubation mixture; the synthesis was suppressed by 7-ketocholesterol. Cholic, dehydrocholic, chenodeoxycholic, glycocholic, taurocholic, taurochenodeoxycholic and taurodeoxycholic acids inhibited cholesterol synthesis in enterocytes to different degrees in a dose-dependent manner. Lithocholic acid enhanced the rate of cholesterol synthesis. Deoxycholic acid, methyl ester of cholic acid and cholesterol did not affect the process. No bile acids tested, with the exception of taurodeoxycholic acid, affected fatty acid synthesis in enterocytes. Most bile acids also decreased cholesterol synthesis in cultured human skin fibroblasts. The results obtained make it possible to postulate that cholesterol synthesis in human enterocytes may be subject to a complex regulation by bile acids.

Acetates↗
PubMed1986

Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations.

Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1-3 h after sudden death. At least 35-70% of EC were removed from the intimal surface of aorta, 90-95% of them being viable. Plating efficiency was 70-80%. Monolayer formation was achieved at a seeding density of 5-8 X 10(2) cells/mm2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weigel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher (P less than 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O-positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical vein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC.

Adult↗
PubMed1986

Apolipoprotein B release from activated human platelets.

Apolipoprotein B (apoB) release from activated washed human platelets was measured by enzyme-linked immunosorbent assay (ELISA) using monospecific rabbit antibodies to human low density lipoprotein (LDL). Activation of platelets with thrombin, Ca2+-ionophore A23187 or stable analogue of prostaglandin endoperoxides U46619 stimulated release of approximately 20 ng apoB/10(8) platelets. Thrombin-induced apoB release was inhibited by the prostacyclin analogue carbacyclin. Dose-response curves of thrombin stimulation and carbacyclin inhibition of apoB and beta-thromboglobulin (beta-TG) release were very similar. Treatment of platelets with heparin did not remove significant amounts of apoB or affect the subsequent release of apoB induced by thrombin. The results of density gradient ultracentrifugation indicated that most of the apoB was released in the LDL density range. These data suggest that human platelets contain immunoreactive apoB, which can be released during platelet activation.

Apolipoproteins B↗
PubMed1986

Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine.

The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL3 (density = 1,125 to 1,126 g/cm3) exhibit a high-affinity (Kd = 8.3 X 10(-8). Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL3, the cell surface-bound 125I-HDL3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I-HDL3 binding. The binding is accompanied with internalization and degradation of 125I-HDL3. Chloroquine inhibits the degradation and increases 125I-HDL3 uptake. A threefold excess of HDL3 and HDL2 inhibits the binding and degradation of 125I-HDL3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL3 (20 to 1,000 micrograms/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism.

Acetates↗
PubMed1986

Carrier-directed targeting of liposomes and erythrocytes to denuded areas of vessel wall.

Immunomorphological staining of specimens prepared from human carotid arteries with anti-collagen type I antibodies reveals large amounts of type I collagen in the subendothelium of lipid fibrous plaques. Collagen type I-containing structures, once in direct contact with blood after plaque rupture, can serve as potential targets for selective delivery of liposomes and erythrocytes to these areas. To verify this rationale, [14C]cholesterol oleate-containing liposomes were conjugated with bovine or human anti-collagen type I antibodies or human plasma fibronectin. Biotin derivatives of human anti-collagen type I antibody were coupled to human erythrocytes. Modified liposomes and erythrocytes were perfused in situ through segments of bovine, rabbit, or human arteries partially denuded with a balloon catheter prior to perfusion. After perfusion, the control and denuded areas were excised and subjected to scanning electron microscopic analysis and measurements of associated radioactivity. It was found that conjugates of liposomes or erythrocytes with anti-collagen type I antibodies or fibronectin are selectively bound by endothelium-free zones of arterial segments. Carrier-directed targeting of drug-laden liposomes and erythrocytes to thrombosis-prone areas of arterial lumen is discussed.

Animals↗
PubMed1986

Morphological alterations in endothelial cells from human aorta and umbilical vein induced by forskolin and phorbol 12-myristate 13-acetate: a synergistic action of adenylate cyclase and protein kinase C activators.

The morphological effects on human endothelial cells of phorbol 12-myristate 13-acetate (PMA) and of agents that increase intracellular cAMP concentration were studied. The adenylate cyclase activator forskolin (10 microM), the cyclic nucleotide phosphodiesterase inhibitor methylisobutylxanthine (100 microM), dibutyryl-cAMP (10 microM), histamine (10 microM), and PMA (0.1 microM) significantly altered the morphology of human aortic and umbilical vein endothelial cells in primary cultures. These effects reached a maximum 40-80 min after the effector addition and became negligible 30-60 min after its removal. PMA and forskolin were strongly synergistic in altering endothelial cell morphology. All the effects of cAMP-elevating compounds and of PMA were abolished completely by 1 microM colchicine. In explants taken from human adult or child aortas, forskolin and PMA produced alterations in endothelial morphology qualitatively identical to those observed in endothelial cell cultures. Endothelium in these preparations closely resembled that found in zones of expected altered hemodynamic stresses of human aorta. Our data suggest that the morphology of endothelium in vivo may be regulated by separate or synergistic action of hormone-dependent adenylate cyclase and of inositol phospholipid turnover systems and might be important for maintenance of endothelial monolayer integrity under normal physiological and pathological conditions.

Adenylyl Cyclases↗
PubMed1986

[Quantitative evaluation of the phagocytic activity of endothelial cells in culture].

The phagocytic activity of endothelial cells (EC) from human umbilical vein was analysed quantitatively in primary culture. EC were incubated with fluorescent carboxylated microspheres (FCM) and the intensity of fluorescence was measured on spectrofluorimeter. It was found that EC phagocyted actively only FCM of limited diameter (not more than 0.26 mu). The most intensive phagocytosis occurred in the first 60 min of the experiment, the following incubation with FCM did not influence significantly the number of phagocyted particles but increased nonspecific binding. High doses of FCM stimulated phagocytosis within EC. The phagocytic activity of EC depended on the growth stage: it was maximum in proliferated cells and sharply decreased in confluent cultures. This method may be useful for the comparison of phagocytic activity of different cell types, as well as for drug testing.

Cells, Cultured↗
PubMed1986

Intimal cells and atherosclerosis. Relationship between the number of intimal cells and major manifestations of atherosclerosis in the human aorta.

The subendothelial intima of human aorta is populated by cells of various shapes. Round and ovoid cells which are lymphocyte- and monocyte-like hematogenous cells account for less than 5% of the cell population. The bulk of the intimal population (over 95%) is made up of cells that can be described as elongated, stellate, elongated with side processes, and irregularly shaped. To identify these morphologic forms, the authors have used target electron microscopy. It has been established that elongated cells devoid of side processes possess all the ultrastructural features of differentiated smooth muscle cells: a developed contractile apparatus in the form of microfilament bundles with dense bodies occupying most of the cytoplasm, basal membrane surrounding the whole of the cell, and micropinocytotic vesicles along the plasma membrane. The other morphologic forms have an ultra-structure that allows us to identify them as so-called modified smooth muscle cells. They differ from the typical smooth muscle cells in that they have fewer contractile structures and a more developed biosynthetic apparatus. Some of stellate and irregular shaped cells are utterly devoid of contractile structures. To quantitate the number of cells of different morphologic forms, the authors used alcoholic-alkaline dissociation of prefixed intima. It was established that the intimal population is multiplied at the site of an atherosclerotic lesion, the number of stellate cells being increased much more substantially, compared with other morphologic cell forms. It was found that an increase in the number of stellate cells is related to such sequelae of atherosclerosis in aorta as intimal thickening, deposition of lipids, and an increased amount of collagen. There was a high positive correlation between the alteration in the stellate cell number occurring in the intima and the above-mentioned parameters (correlation coefficients were 0.732, 0.800 and 0.953, respectively). The correlations between these indexes and the total number of intimal cells or the number of cells belonging to each of the other morphologic forms were not so high. A multivariate analysis gave similar results. Thus, it may be suggested that stellate cells are the principal cell type involved in the disease. This report discusses the origin of stellate and other intimal cells and their role in atherogenesis.

Adult↗
PubMed1986

[Effect of bezafibrate on lipoprotein secretion by cultured human hepatocytes].

The rate of synthesis and secretion of lipid and apoprotein components of lipoprotein fractions and of proteins of the nonlipoprotein fraction d greater than 1.21 g/ml by human hepatocytes in culture was determined. The cells were incubated for 18 hours in the media containing [14C] acetate or [3H]-marked leucine in the presence of bezafibrate or without it. VLDL, HDL and d greater than 1.21 g/ml fractions were separated by differential ultracentrifugation. The lipids were divided using thin-layer chromatography, and the apoprotein-using SDS-electrophoresis in polyacrylamide gel. Bezafibrate in the therapeutic concentration of 10 microM specifically inhibits apoprotein and VLDL lipid secretion. Lowered secretion of all VLDL apoproteins was revealed, which points to a decrease in the number of secreted particles. The effect of bezafibrate was found to be associated with the inhibition of the intracellular synthesis of lipids. The secretion of HDL proteins and of d greater than 1.21 g/ml fraction proteins was unchanged with 10 microM of bezafibrate.

Adult↗
PubMed1985

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes.

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.

Adult↗
PubMed1985

Enzyme-linked immunoreceptor assay of low-density-lipoprotein receptors.

A new modification of enzyme immunoassay-enzyme-linked-immunoreceptor assay (ELIRA) was used to study low-density lipoprotein (LDL) binding by cultured human skin fibroblasts. Time-course and concentration curves obtained by this method were typical of LDL binding by cultured fibroblasts. According to ELIRA, fibroblasts bound native LDL 10-20 times more effectively than reductively methylated LDL or native LDL in the presence of heparin. Receptor-specific Km and maximum binding capacity calculated from these data were 5.4 micrograms/ml and 177.5 ng/mg of cell protein, respectively. Receptor-specific Km and maximum binding capacity for surface 125I-LDL binding measured in the same experiment were 11.0 micrograms/ml and 138.0 ng/mg of cell protein. Incubation of cells with isolated LDL or with unfractionated serum containing the same amount of apoB yielded similar concentration curves for lipoprotein binding. These data indicate that the ELIRA can be used for investigation of receptor-mediated lipoprotein binding without purification of lipoproteins.

Apolipoproteins B↗
PubMed1985

Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins.

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of malondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified LDL.

Antibodies↗
PubMed1985

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture.

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL (125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to complete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells.

Adult↗
PubMed1985

[Cellular mechanisms of human atherosclerosis].

Structural features of the endothelial integument of human arteries, not recorded in animals, have been revealed with scanning electron microscopy. Lipid streaks and atherosclerotic plaques areas were found to be covered with polymorphous endothelium, over 50% of the total surface being occupied by multinuclear endotheliocytes. These areas are characterized by increased absorption of low-density lipoproteins (LDL) due to non-specific binding. An analogous effect was observed in an endothelial culture from atherosclerotic aorta of an adult. Modified LDL are, on the contrary, absorbed in normal and atherosclerotic portions of the intima predominantly, or exclusively, through scavenger receptors. Human hepatocytes in the primary culture were found to contain receptors both for native and for modified LDL, the latter being absorbed 4 times as effectively as native LDL. It was shown that there are about 2500 binding sites of high-density lipoproteins on the surface of isolated human enterocytes. The cellular cultures obtained might be used in search for pharmacologic compounds correcting lipoprotein and cholesterol metabolism disorders.

Arteries↗
PubMed1985

Catecholamine-mediated injury to endothelium in rabbit perfused aorta: a quantitative analysis by scanning electron microscopy.

Perfusion of aortic segments of intact narcotized rabbits by medium 199, containing 10(-6)-10(-4) M adrenalin or noradrenalin, induced morphological injuries to endothelium: local oedema and cell losses, emergence of argyrophilic cells or cells with the widen argyrophilic cell borders. Regional changes of the mean cell density in situ were found by the quantitative scanning electron microscopy. The data obtained supports the possibility of direct injury to endothelium of aorta and large arteries as a result of intravascular infusion of catecholamines or their local therapeutic application.

Animals↗