PubMed Health⌕ Search

Biomedical subjects

M Simionescu

Publications and source records attributed to M Simionescu.

At least 19 recordsLinked to original sources
PubMed1992

Cardiomyocytes express albumin binding proteins.

We investigated whether cardiomyocytes express specific albumin binding proteins (ABP) which may function in the dissociation of fatty acids from their non-covalent complexes with albumin. The experiments were performed on rat neonatal cardiomyocytes (freshly isolated and up to 3 days in culture) and on an enriched sarcolemmal fraction isolated from adult rabbit ventricular myocardium. Three types of experiments were conducted: (a) identification of ABP on electroblots of cardiomyocytes and sarcolemmal extracts reacted with [125I]-bovine serum albumin ([125I]Alb); (b) kinetic assays of [125I]Alb interaction with cardiomyocytes (at 37 degrees C), and with a sarcolemmal fraction (at 4 degrees C); (c) affinity isolation of ABP from solubilized radioiodinated sarcolemmal proteins interacted with an albumin-agarose matrix. The investigation showed that: first, two pairs of polypeptides (ABP of M(r) 18 and 31 kDa) in either cardiomyocytes or sarcolemmal fraction reacted on electroblots with [125I]Alb; second, the binding of the latter to cardiomyocytes was saturable and competed by unlabeled albumin: 50 microM albumin reduced by approximately 90% the binding of radiolabeled albumin. The sarcolemmal fraction bound [125I]Alb with a Kd of 3.66 x 10(-7) M. Thirdly, among the sarcolemmal proteins retained by the albumin-agarose matrix (18 and 31 kDa), the most prominent was the lower band (approximately 16 kDa) of the 18 kDa pair of ABP. The observations revealed that albumin interacts with relatively high affinity with specific binding sites on cardiomyocyte sarcolemma. This interaction may be a recognition step for subsequent fatty acid dissociation and translocation.

Albumins↗
PubMed1992

Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies.

Studies were conducted to identify and localize albumin binding proteins (ABPs) in endothelial cells using rabbits polyclonal anti-idiotypic antibodies (Ab2) raised against the affinity-purified anti-bovine serum albumin IgG. Ab2 were purified by fast performance liquid chromatography. The anti-idiotypic nature of the IgG was assessed by (i) the capacity to inhibit albumin binding to its specific antibody in a dose-dependent manner, (ii) the lack of interaction with albumin, and (iii) the interaction with anti-albumin antibodies of diverse origins. The latter characteristic indicates that although polyclonal, the purified anti-idiotypic antibodies contain some of the Ab2 beta type. The binding of Ab2 to cultured bovine aortic endothelial cell surfaces was saturable and specific as demonstrated by radioimmunoassay (RIA) and immunofluorescence studies respectively. A competitive RIA was used to test whether Ab2 competed for albumin binding to bovine aortic endothelial cells (BAECs) (presumably to ABPs). It was found that Ab2 inhibited binding of [125I]albumin to BAECs in a dose-dependent fashion. Immunoblot analysis of extracts of BAECs, microvascular endothelial cells, and lung showed that both Ab2 and albumin bind specifically to two polypeptides with an apparent molecular mass of 18 and 31 kDa. In addition, upon radioiodination of BAECs apical membrane proteins, Ab2 bound specifically and immunoprecipitated restrictively two radiolabeled cell surface proteins of 18 and 31 kDa. The results provide direct evidence for the presence of the 18 and 31 kDa peptides (ABPs) on the endothelial cell membrane and/or associated structures, i.e. open plasmalemmal vesicles and uncoated pits.

Animals↗
PubMed1992

Increased adhesion of human diabetic platelets to cultured valvular endothelial cells.

Diabetes is accompanied by impaired platelet function and accelerated vascular disease. To find out whether a correlation exists between these two complications, and if modifications occurring in diabetic platelets influence their relationship with endothelium, we have studied the interaction between platelets isolated from plasma of diabetic patients and bovine valvular endothelial cells (VEC), in culture. For quantitative analysis, normal and diabetic [3H]-adenine-labeled platelets were incubated with confluent VEC grown in Dulbecco's modified Eagle medium, containing 4.5 g/l glucose, for 30 min at 37 degrees C. After extensive washing and solubilization of the monolayer, the calculated adhesion index showed a two-fold increased adherence of diabetic platelets to VEC as compared to normal platelets. Statistical analysis (by Pitman randomization test) indicated that the adhesion was significantly higher (p = 0.0003) than that of normal platelets to VEC. To partially identify the membrane components implicated in the adhesion process, either platelets or VEC were treated with neuraminidase, trypsin or heparinase prior to the adhesion assay. Trypsin or neuraminidase treatment of platelets significantly diminished their adherence to VEC, suggesting a role of platelets sialylated glycoproteins in the adhesion process. Neuraminidase or heparinase treatment of VEC increased the adhesion of both normal and diabetic platelets, indicating that the cell membrane sialyl residues and heparan sulfate participate in the normal thromboresistant properties of VEC. Transmission and scanning electron microscopy revealed a close apposition between platelets and VEC with the formation of an adhesion plaque, characterized by fine fibrillar bridges between the plasma membranes of the two cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Albumin binding proteins are highly expressed in actively proliferating fetal and adult tissues.

The expression of albumin binding proteins (ABP, 31 and 18 kDa peptides) in various organs as a function of their ontogenic development was investigated in fetuses (20 days old), neonates (1 day old) and adult rabbits. At each of these stages, tissue extracts of brain, lung, thymus, heart, skeletal muscle and liver as well as whole embryos (11 days old) were examined by ligand blotting and quantitative immunoblot assays. Blots were either incubated with [125I]albumin followed by autoradiography and radioassay or exposed to a radioiodinated antibody raised against affinity-isolated 31 kDa peptide. Anti-31 kDa IgG cross-reacted with both 31 and 18 kDa peptides. Both methods used revealed that ABP are well expressed in embryos and in all fetal organs investigated. By comparison, in neonates, the ABP expression was diminished (by approximately 2-fold) in brain, heart and skeletal muscle. These changes were even more pronounced in the adult rabbit brain, heart, skeletal muscle and liver; no significant modification was detected in the lung. Prompted by these results, which inferred a high level of ABP in actively proliferating/differentiating tissues, we checked for the presence of ABP in other adult cells and tissues. In bone marrow cells, thymocytes and splenocytes, the 31 and 18 kDa peptides represented the major sodium dodecyl sulfate-urea extracted proteins, whereas in mature circulating white blood cells they were moderately expressed. The results indicate that ABP 1) are present early in embryogenesis, 2) are particularly well expressed in organs (fetal or adult) and cells characterized by active proliferation and differentiation, and 3) are not tissue specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1990

Purification and partial characterization of extracellular liposomes isolated from the hyperlipidemic rabbit aorta.

Extracellular liposomes (EL) that accumulated in the aortic intima of rabbits on 2 weeks (prelesional stage) and 16 weeks (lesional stage) of diet-induced hyperlipidemia were isolated and purified by gel filtration, ultracentrifugation, and affinity chromatography on anti-apoB and anti-albumin Sepharose. The material obtained after each step was examined by negative staining electron microscopy, by protein analysis (SDS-PAGE, immunoblotting, autoradiography, uronic acid), and by lipid analysis for unesterified cholesterol (UC), cholesteryl esters (CE), phospholipids (PL), triglycerides (TG), thiobarbituric acid reactants (TBAR). EL represented the major constituent of intimal lipid deposits; their predominance on particulate beta-lipoproteins (LP) increased with the duration of hyperlipoproteinemia. As compared with serum low density lipoproteins (LDL) and beta-very low density lipoproteins (beta-VLDL), the crude EL fraction obtained after gel filtration and ultracentrifugation had a decrease in CE and TG, with augmentation of UC, PL, and apoB. After removal of apoB and some albumin by immunoadsorption, the purified EL fraction consisted only of UC, PL, and albumin. The albumin was resistant to proteolytic digestion with pronase, and reacted with anti-albumin antibody only after delipidation of EL. This indicated that albumin was trapped in the aqueous core of vesicles, presumably acting as a scavenger of oxygen-free radicals. TBAR was highly associated with intact or degraded beta-LP. The EL that accumulate in the aortic intima of hyperlipidemic rabbits represent the predominant form of lipid deposits, resulting from the transcytosed excess beta-LP, which is degraded and reassembled upon interaction with the extracellular matrix components.

Albumins↗
PubMed1989

Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface.

The heterobifunctional, photoactivatable, thiol-cleavable cross-linker sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (SASD) was radioiodinated and used to determine whether endothelial albumin binding proteins (ABP) recently identified (Ghinea, N., Fixman, A., Alexandru, D., Popov, D., Hasu, M., Ghitescu, L., Eskenasy, M., Simionescu, M., and Simionescu, N. (1988) J. Cell Biol. 107, 231-239) are plasma membrane-associated components exposed on the cell surface. Microvascular endothelial cells (MEC) freshly isolated from rat epididymal fat were incubated with 125I-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (ASD)-albumin conjugate which upon photolysis by UV light was cross-linked to the receptor proteins. By cleaving the disulfide linkages of the cross-linker with 5% beta-mercaptoethanol and the ligand-receptor interactions with 0.1% sodium dodecyl sulfate, the radioiodinated ASD moiety remained attached to the receptor peptides which were further detected by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. In parallel, samples were examined by ligand blotting with albumin-gold complex. The results showed that in these experimental conditions ABP are represented by two major peptides of 31 and 18 kDa and two minor bands of 73 and 56 kDa. Densitometric scanning showed that the two major bands constitute more than 70% of the total ABP. The four peptides were not apparent if the samples were not UV-irradiated. The binding of the radioiodinated ligand to ABPs was reduced by approximately 82% in the presence of excess competitive unlabeled albumin. When MEC were incubated with unlabeled SASD and exposed to UV light, the autoradiographic banding pattern obtained was similar to that of either radioiodinated receptor proteins or MEC not treated with SASD. This indicated that the four albumin binding peptides are distinct proteins of the endothelial cell plasmalemma.

Albumins↗
PubMed1989

Transport pathways of beta-VLDL by aortic endothelium of normal and hypercholesterolemic rabbits.

The uptake and transport of beta-VLDL by the aortic endothelium was investigated in normal and hyperlipidemic rabbits fed a cholesterol-enriched diet for 1 week to 5 months. Weekly (in the first month) or every other week afterwards, animals were given one of the following probes: (a) [125I]-beta-VLDL injected in vivo and after 24 h the whole aorta or its intima and media were separately collected and examined by spectrometry and autoradiography; (b) [125I]-beta-VLDL coupled to the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate perfused in situ for 1-2 h and aorta examined by radioassay and fluorescence microscopy; (c) beta-VLDL-gold complex perfused in situ for 10-15 min and aortic fragments examined by electron microscopy. In addition, cryosections of aortic wall were processed for the immunocytochemical detection of apolipoprotein B and apolipoprotein E. The results showed that both in normal and hyperlipidemic rabbits, the aortic endothelium transports plasma beta-VLDL by a dual pathway: (i) endocytosis involving coated pits and vesicles, endosomes, multivesicular bodies and lysosomes, and (ii) transcytosis, the predominant process, carried out by plasmalemmal vesicles. Both processes, and especially transcytosis, are markedly increased in hyperlipidemia leading to progressive accumulation of beta-VLDL or/and its components in the subendothelial extracellular matrix. In prelesional stages of atherogenesis, beta-VLDL-gold complexes or deposits of apo B and apo E were detected in close association with extracellular liposomes. With the appearance of intimal macrophage-derived foam cells, the immunoperoxidase reaction product, revealing the presence of the two apolipoproteins, could also be seen in intracellular lipid inclusions.

Animals↗
PubMed1989

Evidence for thyroxine transport by the lung and heart capillary endothelium.

The uptake and transport of carrier-bound thyroxine by the endothelium were investigated by perfusing through the heart and lung of young rats radiolabeled thyroxine bound to prealbumin ([125I]T4Pa) or serum ([125I]T4S). In addition these complexes were tagged to 5-nm gold particles to obtain quantitative (radioassay) and qualitative (autoradiography) data from the same experiment. The complexes (prewarmed at 37 degrees) were perfused in situ at various concentrations (1 to 50 muCi/ml) for time intervals ranging from 5 to 30 min. After thorough washing of the unbound probe, tissue fragments were either measured for total uptake in a gamma counter or processed for electron microscopy autoradiography. The results showed that both the lung and heart take up [125I]T4 complexes by a process that is saturable at low hormone concentration; uptake is completed by free T4 and Pa. In specimens perfused with double-labeled complexes (iodinated and tagged to gold) autoradiography revealed that silver grains and gold particles colocalize predominantly on endothelial plasmalemmal vesicles. The probe appeared first in vesicles open to the capillary lumen (5 min) and further on in vesicles apparently free within the cytoplasm or open to the abluminal front. At 30 min, only silver grains seem to be present in the pericapillary space, on the alveolar epithelial cells, as well as on the nucleus and mitochondria of heart myocytes. The findings suggest that (1) T4Pa uptake by the endothelial cell (EC) is a specific process (possibly via specific binding sites); (2) T4Pa is taken up and transported across capillary EC by plasmalemmal vesicles; (3) in the pericapillary space T4 seems to dissociate from its carrier.

Animals↗
PubMed1989

Affinity isolation of albumin-binding proteins using nitrocellulose-bound albumin.

Albumin immobilized on a nitrocellulose membrane was used as an affinity matrix to purify albumin-binding proteins (ABP) from extracts of lung, heart, thymus, and isolated microvascular endothelial cells. Albumin was immobilized onto nitrocellulose either (i) directly (physically adsorbed), (ii) cross-linked by treatment with 0.25% glutaraldehyde, or (iii) covalently coupled to the matrix using NaIO4 and Na-borohydride. The affinity support was incubated with a membrane-enriched fraction (obtained from tissue homogenates) in the presence of protease inhibitors; specific binding of ABP occurred within 30 min of incubation. The adsorbed proteins were eluted with 0.5% sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel electrophoresis and ligand blotting. Analysis of electrophoretic mobility of eluted proteins showed that they consisted exclusively of the two sets of polypeptides of 31 000 Da and 18 000 Da previously identified as ABP (N. Ghinea et al., J. Cell Biol. 107, 231-239 (1988]. As demonstrated by ligand blotting, the ABP purified on nitrocellulose-bound albumin maintain the ability to interact specifically with albumin. Preliminary experiments showed that the method employed may be of a broader use for the isolation of receptor proteins from tissue extracts by incubating the latter with the cognate ligand immobilized on nitrocellulose membranes.

Albumins↗
PubMed1988

Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.

To study the roles played by cardiac valvular endothelium in normal and pathologic conditions, we have established and characterized a system of bovine valvular endothelial cells (VEC) in culture. Viable VEC from calf atrioventricular valves were obtained by a non-enzymatic procedure using 3 mM ethylenediamine-tetraacetic acid (EDTA) as dissociating agent. The cells grown in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, vitamins and 20% fetal calf serum, developed as monolayers of closely apposed polygonal cells which were subcultured for up to seven passages. VEC maintained in culture the general ultrastructure displayed in vivo, expressed von Willebrand factor, presented angiotensin converting enzyme activity and synthesized a rich extracellular matrix. VEC preserved the cell surface anionic sites (detected with cationized ferritin, pI 8.4) and cationic sites (visualized with haemeundecapeptide pI 4.85), and took up, especially by adsorptive endocytosis, albumin-gold conjugate. The cells were coupled by functional communicating (gap) junctions, as demonstrated by microinjection of 6-carboxyfluorescein. VEC in culture produced fibronectin, prostacyclin, hyaluronic acid and heparin-like glycosaminoglycans (identified by electrophoresis, enzyme digestion, and deaminative cleavage of molecules). These properties render cultured VEC a suitable model for investigating their functions and involvement in normal and pathologic heart valves.

Animals↗
PubMed1988

Identification of albumin-binding proteins in capillary endothelial cells.

Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

Adipose Tissue↗
PubMed1988

Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand.

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.

Animals↗
PubMed1988

Differentiated uptake and transcytosis of albumin in successive vascular segments.

The interaction of exogenous albumin with the continuous endothelium of large vessels and microvessels of various organs was investigated in situ in mouse. Bovine serum albumin either tagged with 5 nm gold particles (Alb-Au) or radioiodinated was perfused for 3 to 30 min. The following tissues were processed for electron microscopy: heart (coronaries and microvessels), aorta, vena cava, diaphragm (phrenic arteries, arterioles, capillaries, venules, phrenic veins), and brain cortex. Morphometric analysis showed that in all organs examined, except brain, endothelium of capillaries and postcapillary venules possesses specific binding sites for Alb-Au virtually restricted to plasmalemmal vesicles. The latter contain 1,000 times more particles than the equivalent volume of the perfusate. The Alb-Au binding is saturable and competed by monomeric albumin. Commonly, in these capillary endothelia, coated pits and coated vesicles did not bind Alb-Au. Starting with 3 min and especially at longer time points, tracer-labeled vesicles apparently discharged the ligand into the subendothelial space. At variance, in the endothelium of arteries, arterioles, muscular venules, and veins, usually few vesicles were labeled by rare particles in concentration comparable with that of the perfused tracer. In these endothelia, vesicle Alb-Au content did not increase with time and was not influenced by competition with monomeric albumin. Same differences in albumin uptake between successive vascular segments were found by light microscopy autoradiography with monomeric radioiodinated albumin perfused for 3 or 30 min. The results suggest that among vessels with continuous endothelium, albumin binds and is intensely transported in the capillaries and postcapillary venules of the diaphragm and heart. The rest of the examined vessels perform a nonspecific, low rate uptake, possibly in fluid phase.

Albumins↗