[Diagnosis of familial hypercholesterolemia by a method of determining the number of low-density lipoprotein receptors on the fibroblasts].
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Cell Count↗
Biomedical subjects
V S Repin
Publications and source records attributed to V S Repin.
[Secretion of very low density lipoproteins by cultured rabbit hepatocytes with hypercholesteremia, induced by purified cholesterol and containing autooxidation products].
To evaluate the impact of oxidized derivatives of cholesterol on the development of hypercholesterolemia in rabbits, the secretion of very low density lipoprotein (VLDL) apoproteins and lipids was studied in cultures of hepatocytes obtained from: i) control rabbits, ii) rabbits fed on purified cholesterol (PCH), and, iii) rabbits fed on old commercial cholesterol (OCH) containing 5% of oxidized cholesterol derivatives. The rabbits fed on OCH for 6 weeks revealed a 5-fold increase in the serum cholesterol level compared with that in PCH-fed rabbits. The secretion of VLDL apoproteins and lipids by hepatocytes of two cholesterol-fed groups was similar, but was 2-3 times as high as that of cells from control rabbits. The cholesterol ester content in hepatocytes and the secretion of VLDL cholesterol esters by hepatocytes from OCH-fed rabbits was dramatically increased in comparison with hepatocytes from control and PCH-fed rabbits. These effects appear to be caused by the activation of cholesterol esterification by oxidized cholesterol derivatives. The rapid development of hypercholesterolemia in OCH-fed rabbits is at least partly associated with the stimulation of hepatic VLDL production.
Animals↗
[Apoprotein B: its plasma level and interaction with cultured fibroblasts].
To study the relationship between the ability of apolipoprotein B (apo B) to interact with LDL receptors and its blood concentration, enzyme immunoassay was used to measure serum apo B levels in 51 healthy donors, 84 patients with coronary heart disease (CHD), and 22 newborns. Employing the same serum samples, the binding of apo B to fibroblasts was determined by the enzyme immunoassay modified for cultured cells. The mean values of apo B concentrations were found to be 0.89 +/- 0.25 mg/ml in the donors, 1.29 +/- 0.51 mg/ml in CHD patients, and 0.57 +/- 0.19 mg/ml in the newborns, while the apo B binding to the cells amounted to 108.9 +/- 16.7, 141.5 +/- 43.6, and 55.0 +/- 24.6 ng/mg, respectively. The mean values of serum apo B binding in the healthy donors and CHD patients were shown to correspond to the concentration relationship of pure LDL interaction with LDL receptors. The findings suggest that higher plasma apo B concentrations in CHD patients cannot be explained by lowered ability of serum apolipoproteins B to interact with LDL receptors of the cells.
Adult↗
A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions.
A monoclonal antibody, designated 10F3, that reacts with an antigen with a molecular weight of 90,000 daltons has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from fetal human aorta (fSMC). In adults the antigen is present on venous, arterial and capillary endothelial cells of heart, kidney, liver, spleen, intestine, skin, uterus, placenta, and arteries only, as shown by immunohistochemical investigation using the PAP technique. The antigen 10F3 is also present on the mesenchymal cells of human fetal tissues (7 and 18-week-old fetuses) and on SMC of 7-week-old fetal aorta, and a subpopulation of cells reacting with 10F3 antibody also has been found in atherosclerotic intima. Double staining using 10F3 antibody and muscle actin-specific monoclonal antibody HHF-35 showed that the antigen-positive cells are smooth muscle cells. In primary culture of adult SMC, antigen-positive cells were detected 2 days after seeding (about 90% positive in medial and intimal cultures). It is suggested that 10F3 is a mesenchymal antigen that, lost during differentiation by cells other than endothelium, but expressed again by the SMC involved in atherogenesis.
Adult↗
Potentiation of platelet interaction with collagen substrates by heparin is insensitive to aspirin.
The effects of two standard, unfractionated heparin preparations on collagen-induced adhesion and aggregation of platelets were studied by Born aggregometry and scanning electron microscopy (SEM). Heparin from porcine intestinal mucosa (HI) and from bovine lung (HL) added to human PRP in the concentration of 2.5 to 5.0 U/ml (1) did not induce platelet aggregation in suspension by itself but stimulated it in combination with the subthreshold doses of fibrillar human collagen type III (CIII); (2) increased by 1.5-2.0 fold the adhesion, but did not affect platelet spreading on a surface coated with human collagen type IV (CIV); and (3) enlarged by 2.5-3.0 fold the area of the CIII-coated surface covered with aggregates, increasing both the number and the size of surface-bound aggregates. Aspirin blocked platelet aggregation in suspension induced by low, near threshold doses, of fibrillar CIII and by subthreshold doses of CIII in combination with heparin, but had no effect on platelet aggregation induced by high (greater than 10 threshold) doses of CIII. Aspirin failed to decrease platelet adhesion to and spreading on CIV substrate, and formation of surface-bound aggregates on CIII substrate in the absence as well as in the presence of heparin.
Aspirin↗
Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition.
The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.
Apolipoprotein A-I↗
7-ketocholesterol inhibits VLDL secretion by cultured human and rabbit hepatocytes.
7-ketocholesterol, one of the major product of autoxidation of dietary cholesterol, was found to inhibit secretion of very low density lipoprotein [14C]cholesterol, [14C]triacylglycerol and [35S]apoprotein B,E,C by cultured human and rabbit hepatocytes. A parallel inhibition (about 35%) of cholesterol synthesis but not of triacylglycerol formation was observed. Incubation with 10 micrograms/ml of oxysterol also reduced the total apo-B secretion measured by ELISA and increased intracellular apo-B mRNA level. These results seem to indicate that 7-ketocholesterol decreases secretion of very low density lipoprotein (VLDL) particles and exerts inhibitory effects on apo-B production at the co-translational or posttranslational level.
Adult↗
Substances that polymerize or depolymerize cytoskeletal proteins affect platelet spreading and thrombus-formation on surfaces coated with human collagen isotypes I, IV, and V.
The effect of substances that affect platelet cytoskeleton on the interaction of gel-filtered platelets with surfaces coated with human monomeric type I, IV, and V collagen was studied. The sulfhydryl group oxidizing agent azodicarboxylic acid-bis-dimethylamide (diamide) which causes disulfide-linked polymer formation of certain cytoskeletal proteins, the actin-polymerization inhibitor, cytochalasin B, and 2-mercaptopropionylglycine (2-MPG), a cell-permeable SH-reagent, completely abolish adhesion-induced platelet spreading and mural platelet aggregate formation on collagen-coated surfaces. Extrusion of pseudopods was inhibited by cytochalasin B and 2-MPG as well as by diamide, but only the latter caused spherulation of platelets, whereas cytochalasin B and 2-MPG left the discoid shape of resting platelets intact. These effects are dose-dependent and are not accounted for by a chemical modification of the collagenous substrates by the cytoskeletal perturbing substances. The present data indicate that (i) cytoskeletal rearrangements are essential in adhesion-induced platelet spreading and aggregate formation on surfaces coated with collagen, but not in supporting the initial attachment of native platelets to the substrate; (ii) both, polymerization and depolymerization of actin filaments affect platelet activation; (iii) the sulfhydryl-disulfide status of the platelet seems to be a possible target for anti-platelet drugs, since chemical modification of platelets by the GSH-GSSG-active substances, diamide and 2-MPG, leads to a reversible inhibition of adhesion-induced platelet activation.
Chromatography, Gel↗
VLDL apoprotein secretion and apo-B mRNA level in primary culture of cholesterol-loaded rabbit hepatocytes.
Incubation of cultured rabbit hepatocytes with beta very low density lipoproteins (beta-VLDL) induces a dose-dependent increase in cell cholesterol (CH) content and VLDL apoprotein (apo) B and E secretion without change in apo-B mRNA level. These data suggest that beta-VLDL may exert a stimulatory effect on hepatic apo-B production at the co-translational and/or posttranslational level.
Animals↗
Stimulation of platelet adhesion and activation by ganglioside GD3 adsorbed to plastic.
Platelet interaction with gangliosides GD3, GM3, GM1, GD1a and GT1b has been investigated. These gangliosides were previously identified in the vessel wall and ganglioside GD3 was found to accumulate selectively in the intima of atherosclerotic vessels. Gangliosides were adsorbed to plastic and incubated with 51Cr-labeled platelets. The adhesion of gel-filtered platelets to ganglioside GD3 was 3-4 times higher than to other immobilized gangliosides and to albumin-treated plastic. As was shown by scanning electron microscopy, GD3 stimulated intensive spreading of adherent platelets and formation of surface-bound aggregates, while only single unspread platelets were present on the surfaces coated with other gangliosides. GD3 isolated from milk and from human aorta possess the same stimulating activity. Platelet adhesion to GD3 decreased significantly in the presence of the stable prostacyclin analogue, carbacyclin.
Adsorption↗
Comparison of different lasers in terms of thrombogenicity of the laser-treated vascular wall.
The thrombogenic properties of the laser-treated vascular wall are reported as quantitatively assessed on the basis of 33 experiments with peripheral canine vessel segments. Three types of surfaces, namely, 1) intact, 2) mechanically de-endothelized, and 3) postlaser, have been simultaneously exposed to platelet-enriched plasma in a sequentially organized artificial circulation system. Then the adherent platelets have been counted on the treated surfaces, and the relative thrombogenicity index has been calculated according to the equation T = (A1 - Aint)/(Ad - Aint), where Aint, Ad, A1 = the adherent platelet counts on intact, mechanically de-endothelized, and laser-treated surfaces, respectively. The following lasers have been evaluated: 1) Nd-YAG, 1,060 nm, continuous wave, 4W; 2) argon-ion, 480 and 514 nm and argon-ion, 350 nm, continuous-wave, 1 W and 400 mW [corrected], respectively; 3) excimer XeCl, 308 nm, pulsed, 30 mJ per pulse, repetition rate 10 Hz [corrected]; and also 4) the laser-heated metal probe (2 mm diameter, Trimedyne, Nd-YAG) 1,060 nm, 8 W. The thrombogenicity index values obtained were 83 +/- 7, 72 +/- 8, 57 +/- 9, 63 +/- 7, and 82 +/- 9%, respectively. The differences between these values were statistically insignificant. The data are suggestive of the essential requirement of, at least, anticoagulant therapy after laser angioplasty irrespective of the laser type.
Animals↗
Use of enzyme label for quantitative evaluation of liposome adhesion on cell surface: studies with J774 macrophage monolayers.
A method for quantitation of cell surface-bound liposomes utilizing J774 macrophage monolayers is developed. Surface-bound biotinyl-containing and 125I-labeled liposomes were quantified with avidin-peroxidase in an ELISA-like assay. Peroxidase substrate absorbance values were recalculated into the absolute amount of liposomal lipid using a special calibration plot. Total liposome uptake by macrophages was determined following the binding of 125I radioactivity. The approach suggested allows quantitative evaluation of the changes in the content of surface-adhered liposomes during their interaction with cells in vitro.
Adsorption↗
Identification of intimal subendothelial cells from human aorta in primary culture.
Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.
Aorta, Thoracic↗
Effects of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of cAMP increase by collagens type I, III, and IV.
We investigated the effects of a stable prostacyclin analogue, carbacyclin, on the interaction of platelets with collagen substrates differing in their ability to activate platelets: human collagens type I, III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhesion was measured using 51Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelet cAMP did not affect the adhesion of platelets to weak substrates, CV and CSC, but reduced the adhesion to strong substrates, CIV (by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-bound aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhesion: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of agonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.
Animals↗
Isolated cells in suspension for biological research--Part III. Structure and functional properties of enterocytes and adipocytes.
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Adipose Tissue↗
Effects of an extract of feverfew on endothelial cell integrity and on cAMP in rabbit perfused aorta.
Extracts of feverfew inhibit platelet aggregation and secretion of granular contents from platelets and other cells. They also modify the interaction of platelets with collagen substrates: feverfew extracts inhibit both platelet spreading and formation of thrombus-like platelet aggregates on the collagen surface. We have now investigated the effect of an extract of feverfew on the vessel wall using rabbit aortas that were perfused with a physiological salt solution in-situ. Addition of feverfew extract to the perfusion medium protected the endothelial cell monolayer from perfusion-induced injury and led to a reversible increase in the cAMP content of aorta segments. The results indicate that feverfew may have a vasoprotective effect in addition to its effects on platelets.
Animals↗
Cholesterol synthesis in the small intestine of patients with malabsorption syndrome.
Cholesterol synthesis was studied in intestinal biopsies obtained from children with different forms of malabsorption syndrome. It was demonstrated that intestinal cholesterol synthesis is enhanced in celiac disease and several other forms of malabsorption syndrome. Mevinolin inhibited intestinal cholesterol synthesis in all groups of patients. No correlation was found between cholesterol synthesis and age and sex of the patients, clinical manifestation of the disease and plasma cholesterol level.
Child, Preschool↗
Feverfew--an antithrombotic drug?
The effects of an extract of the plant feverfew on the interaction of platelets with surfaces coated with human collagens of type III and IV (CIII, CIV), and on the integrity of the endothelial cell (EC) monolayer in perfused rabbit aorta were studied. It was shown that feverfew extract (FE) inhibited the deposition of [51Cr]-labelled platelets on both CIII and CIV in a dose-dependent way. Similar concentrations of FE were needed to inhibit formation of surface-bound aggregates in CIII and platelet spreading on CIV in both platelet-rich plasma and GFP. When aorta segments were perfused in situ with a physiological salt solution, the addition of FE to the solution protected the EC monolayer from spontaneous injury. The results indicate that feverfew may have antithrombotic potential in addition to its claimed benefit in fever, migraine and arthritis.
Animals↗