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M A Glukhova

Publications and source records attributed to M A Glukhova.

At least 37 records · Page 2Linked to original sources
PubMed1989

Identification and immunolocalization of a new component of human cardiac muscle intercalated disc.

A new protein was found at the site of interaction of cytoskeletal filaments and the plasma membrane in desmosomes of human cardiac muscle intercalated discs. As revealed by the indirect immunofluorescence method, monoclonal antibody XVE2 was able to stain intercalated discs of cardiac muscle and desmosomes of human skin epidermal cells, whereas it did not react with sections from human uterine smooth muscle, vascular tissue and liver. Western immunoblot analysis of extracts of total human heart and uterus demonstrated that a doublet of 65 kDa and 70 kDa polypeptides were the major proteins recognized by the monoclonal antibody XVE2. These 65 kDa and 70 kDa proteins are immunologically distinct from other known intercalated disc proteins such as vinculin, meta-vinculin, filamin, talin, alpha-actinin, desmin and desmoplakins. The distribution of the XVE2 monoclonal antibody antigens raises the possibility that these polypeptides are involved in linking intermediate filaments to the dense plaque of desmosomes of cardiac muscle intercalated discs.

Antibodies, Monoclonal↗
PubMed1989

Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent.

Different fibronectin (FN) variants arise from the single gene transcript alternatively spliced in a tissue-specific manner (Hynes, R. O. 1985. Annu. Rev. Cell Biol. 1:67-90; Owens, R. J., A. R. Kornblihtt, and F. E. Baralle. 1986. Oxf. Surv. Eurcaryotic Genes. 3:141-160). We used mAb IST-9, specific for extra domain A (ED-A) FN sequence, and cDNA probe to ED-A exon to determine whether ED-A is present in FN synthesized by vascular smooth muscle cells (SMCs) and, if so, whether expression of ED-A is SMC phenotype dependent. ED-A-containing FN (A-FN) was not revealed in tunica media of human arteries and normal rat aorta by immunofluorescence and immunoblotting techniques. A cDNA probe to ED-A exon did not hybridize with RNA isolated from human aortic media. A positive reaction with IST-9 was observed in (a) diffuse intimal thickening and atherosclerotic plaque from human arteries; (b) experimentally induced intimal thickening in rat aorta; and (c) cultured vascular SMCs. A-FN mRNA was present in the RNA preparation from human aortic intima as judged by hybridization with cDNA probe to ED-A. On the other hand, an mAb interacting with an epitope common for all FN variants revealed FN in both intima and media of human arteries and in the normal rat aorta. A cDNA probe to a sequence shared by all FN variants hybridized with RNA from both intima and media of human aorta, though the level of expression was higher in intima. The data suggest that ED-A exon is omitted during splicing of the FN mRNA precursor in medial SMCs while the expression of A-FN is characteristic of "modulated" SMCs--those of intimal thickenings, of atherosclerotic lesions, and growing in culture.

Animals↗
PubMed1988

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells.

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.

Adult↗
PubMed1988

Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression.

Vinculin- and caldesmon-immunoreactive forms and actin isoform patterns were studied in samples of normal and atherosclerotic human aorta. After removal of adventitia and endothelium, the remaining tissue was divided into three layers: media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. In media of normal aorta, meta-vinculin accounted for 41.0 +/- 0.9% (mean +/- SEM) of total immunoreactive vinculin (meta-vinculin + vinculin); 150-kDa caldesmon accounted for 78.2 +/- 5.1% of immunoreactive caldesmon (150-kDa + 70-kDa); the fractional contents of alpha-smooth muscle actin, beta-nonmuscle, and gamma-isoactins were 49.0 +/- 0.6%, 30.4 +/- 0.6%, and 20.8 +/- 0.8%, respectively. Muscular-elastic intima was very similar to media by these criteria. In subendothelial intima, the fractional content of meta-vinculin and 150-kDa caldesmon was significantly lower (6.9 +/- 1.5% and 32.7 +/- 7.0%, respectively) than in muscular-elastic intima and media, whereas the isoactin pattern was identical to that in adjacent layers, demonstrating the smooth muscle origin of subendothelial intima cells. In atherosclerotic fibrous plaque, the fractional content of alpha-actin was decreased in subendothelial intima, rather than in media and muscular-elastic intima. Additionally, the proportion of subendothelial intima cells [i.e., the cells that express low amounts of smooth muscle phenotype markers (meta-vinculin, 150-kDa caldesmon, and alpha-actin)] in the total intima cell population increased dramatically in atherosclerotic fibrous plaque. The results suggest that changes in the relative content of meta-vinculin and 150-kDa caldesmon as well as alpha-actin in human aortic intima are associated with atherosclerosis although, in subendothelial intima of normal aorta, a certain smooth muscle cell population exists that expresses reduced amounts of "contractile" phenotype markers, even in the absence of the disease.

Actins↗
PubMed1988

Immunolocalization of meta-vinculin in human smooth and cardiac muscles.

Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.

Antibody Specificity↗
PubMed1988

Characterisation of connective tissue cells containing factor XIII subunit a.

Paraffin embedded sections of human liver, lymph node, and placenta showed that certain connective tissue cells were positive for factor XIII subunit a. These cells were further characterised by double immunofluorescence labelling and by combined immunofluorescence and enzyme cytochemical staining on frozen sections. They were labelled by the monoclonal antibodies RFD7 and anti-Leu M3 (markers of the macrophage cell line) but gave a negative reaction for the fibroblast marker IIG10 and showed no alkaline phosphatase activity. Immunoblotting detected factor XIII subunit a in macrophages isolated from placenta but not in human fibroblasts. At lower dilutions, the commercially available antibody against the b subunit of factor XIII also positively reacted with the same cell population. The facts that immunoblotting showed that the antiserum crossreacted with the a subunit and that placental macrophages did not stain strongly for the b subunit also indicate that this antigen is not present in adult connective tissue cells.

Connective Tissue↗
PubMed1987

Immunoreactive forms of caldesmon in cultivated human vascular smooth muscle cells.

150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.

Aorta↗
PubMed1987

Association of low-density lipoprotein with particulate connective tissue matrix components enhances cholesterol accumulation in cultured subendothelial cells of human aorta.

Low-density lipoproteins (LDL) were incubated with elastin particles, collagenase-resistant debris isolated from human aorta, and latex beads of 1.13 microns in diameter. As a result of incubation, insoluble LDL-associates were formed. These associates, as well as LDL-heparin-fibronectin-gelatin complexes described by other workers, were added to a 7-day primary culture of enzyme-isolated cells of human aortic subendothelial intima. The culture contained a mixed cell population made up mostly of typical and modified smooth muscle cells. 24 h later, total cholesterol, phospholipid, triacylglycerol, free cholesterol and cholesteryl ester levels were measured. Addition of insoluble LDL-complexes as well as LDL-associates to culture brought about a substantial accumulation of intracellular lipids; primarily, cholesteryl esters. The total cholesterol level in cultured cells was raised 3- to 8-fold. Addition of free LDL or LDL-free particles had no effect on the content of intracellular lipids. The results obtained allow the assumption that the occurrence of the LDL-mediated accumulation of intracellular lipids is due mainly to the LDL penetration inside the cell via 'nonspecific' phagocytosis and not through a regulated receptor-dependent pathway.

Adult↗
PubMed1987

Identification of smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta with monoclonal antibody IIG10.

We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.

Antibodies, Monoclonal↗
PubMed1987

[Effect of components of the extracellular matrix on the accumulation of lipids in human cells].

The effect of extracellular matrix components on cholesterol accumulation in different human cells was studied. Insoluble LDL-Heparin-Fibronectin-Gelatin complexes were incubated with human cells: fibroblasts, monocytes, peritoneal macrophages, cells of the aortic wall--endothelial, subendothelial intimal, medial; and total cholesterol content in these cells was determined. It has been demonstrated that components of extracellular matrix being complexed with LDL enhance total cholesterol accumulation in all cell types studied: the highest amount of cholesterol was accumulated by subendothelial intimal cells and peritoneal macrophages. It is suggested that components of extracellular matrix can play an important role in the development of lipid-laden foam cells that are accumulated in the arterial wall in atherosclerotic lesions.

Endothelium, Vascular↗
PubMed1986

Meta-vinculin distribution in adult human tissues and cultured cells.

Meta-vinculin distribution in adult human tissue was studied by immunoblotting technique. Meta-vinculin was found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, kidney and cultured cells - macrophages, foreskin fibroblasts, peripheral blood lymphocytes and vascular endothelial cells. In the primary culture of smooth muscle cells from human aorta the meta-vinculin/vinculin ratio was reduced, and on the onset of cell division meta-vinculin could hardly be detected. Subcultured smooth muscle cells from human aorta did not contain meta-vinculin. The data show that the presence of meta-vinculin is characteristic of 'contractile' smooth muscle cells rather than of proliferating in vitro.

Aorta↗
PubMed1986

Isolation and localization of filamin in heart muscle.

High-molecular-mass protein was isolated from chicken heart muscle. The apparent molecular mass of a single polypeptide chain is similar to that of chicken gizzard filamin: 250-270 kDa. The protein interacts with antibodies against chicken gizzard filamin and induces F-actin gelation in a concentration-dependent manner. Immunofluorescent staining of cardiomyocytes and chicken heart sections with antifilamin antibody demonstrates two types of filamin localization: filamin was located on the sarcomere border in the periphery of the Z-disk; filamin was found in intercalated disks between cardiomyocytes.

Animals↗
PubMed1986

Red blood cell targeting to smooth muscle cells.

Monoclonal antibody discriminating between endothelial and smooth muscle cells is suggested to be used as a vector for directed transport of drugs to injured (denuded) areas of blood vessel wall. An in vitro model system was used in the studies: vascular smooth muscle or endothelial cells grown on plastic surface were treated with specific mouse monoclonal antibody recognizing an antigen localized on the surface of smooth muscle rather than endothelial cells; then erythrocytes coated with secondary (rabbit antimouse) antibodies were added. The results were analyzed spectrophotometrically or with scanning electron microscopy. Under the experimental conditions, erythrocytes, possible 'containers' for carrying the drugs, were found to bind only to smooth muscle cells. The data show that antibody provides absolute discrimination between endothelial and smooth muscle cells and, thus, may be used as a vector for drug targeting.

Animals↗
PubMed1986

[Targeted delivery of erythrocytes to human aortic smooth muscle cells].

Monoclonal antibodies specific for surface antigens of target cells are supposed to be good vectors for drug transport. It is suggested using monoclonal antibodies that distinguish between smooth muscle and endothelial cells as vectors for directed drug transport to injured (denuded) areas of the blood vessel wall. The following in vitro model was used: monoclonal antibodies were added to cultured vascular smooth muscle or endothelial cells, this was followed by the addition of erythrocytes conjugated with rabbit antimouse antibodies. Spectrometry and scanning electron microscopy were used to assess the results. The erythrocytes, possible containers of drugs, under the experimental conditions were found to bind only to smooth muscle cells. The data obtained suggest that antibody IIG10 discriminating between smooth muscle and endothelial cells provides a specific tool for erythrocyte delivery to smooth muscle cells.

Animals↗
PubMed1985

Monoclonal antibodies that distinguish between human aorta smooth muscle and endothelial cells.

A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor immunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract.

Antibodies, Monoclonal↗