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N Simionescu

Publications and source records attributed to N Simionescu.

At least 37 records · Page 2Linked 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

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 sites are expressed on the abluminal plasma membrane of capillary endothelium.

The presence of albumin binding sites on the abluminal front of vascular endothelium was examined on the capillaries of the adipose tissue. The experimental procedure consisted in injecting interstitially within the rat epididymal fat and epicardial fat, albumin (alone or bearing oleic acid) either conjugated with gold particles (Alb-Au or Alb-OA-Au) or radioiodinated [( 125I]-Alb). In controls, polyethyleneglycol-gold complex (PEG-Au) and [125I]-IgG were used as tracers. The results revealed that: a) albumin binding sites are expressed on the abluminal front of endothelium especially concentrated on plasmalemmal vesicles; b) the retrotranscytosis of albumin conjugates from the interstitium to the capillary lumen is a poorly represented process; c) the binding of the tracers used appears to be time and concentration dependent; d) albumin conjugates do not bind significantly to plasmalemmal vesicles of adipocytes, pericytes and smooth muscle cells; e) PEG-Au and [125I]-IgG do not show a binding pattern similar to that of albumin conjugates.

Adipose Tissue↗
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↗
PubMed1990

Experimental obstructive coronary atherosclerosis in the hyperlipidemic hamster.

The evolution of coronary atherosclerotic lesions induced by a hyperlipidemic diet was examined in male hamsters subjected for up to 40 weeks to a standard chow supplemented with 3% cholesterol and 15% butter. Control animals were fed standard chow only. Five to seven hamsters were monthly sacrificed and investigated for serum lipids and coronary artery lesions. As compared with control animals, the hamsters fed the fat diet showed a progressive increase in serum cholesterol which reached maximum values up to 17 fold in the 10th month. The serum of the hyperlipidemic hamster examined by agarose electrophoresis, Laurell immunoelectrophoresis and cross-immunoelectrophoresis showed at most a 14 fold increase in low density lipoproteins after 10 months diet. The examination of coronary arteries revealed morphologic changes already detectable at 2 weeks of diet. The earliest modifications observed were characterized by proliferation of the subendothelial matrix or/and the appearance of liposome-like structures in the intima. After 2-3 weeks of diet, smooth muscle cells appeared occasionally in the intima and monocytes adhered and penetrated through the endothelium. Later on, smooth muscle cells and macrophage displayed lipid deposits. Focally, in areas of intimal proliferation and foam cells, endothelial cells were also lipid-loaded. Like in human atherosclerotic plaque, in the late stages of hamster coronary lesions, there was a progressive accumulation of extracellular unesterified cholesterol, calcium deposition and necrosis. Lesions evolved to a progressive narrowing of the coronary branches affected, with complete obstruction of some small arterial ramifications. Hamster appears to be a suitable model for studying the molecular and cellular events leading to obstructive coronary atherosclerosis.

Animals↗
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

Cytochemical localization of beta-lipoproteins and their components in successive stages of hyperlipidemic atherogenesis of rabbit aorta.

We have investigated cytochemically the correlative distribution of lipoprotein (LP) components in successive lesional stages of plaque formation in hyperlipoproteinemic atherogenesis in the rabbit. Apoprotein B (apo B) was detected by an immunoperoxidase procedure, unesterified cholesterol (UC) by filipin and tomatine and phospholipid lamellae of the extracellular liposomes (EL) as they appear in standard EM. The changes were evaluated in relation to the state of endothelial cells and their transport pathways, and the reaction of the cellular and extracellular components of the intima. Each lesional stage has a relatively characteristic pattern distribution of the LP components. In fatty streaks with no endothelial denudation, apo B reaction product occurs mostly in non-particulate form associated with UC-rich EL; this suggests that transcytosed LP upon partial degradation and interaction with the extracellular components, reassemble as polymorphic EL-UC-apo B complexes. Serofibrinous insudates, although commonly devoid of EL and apo B, may contain UC presumably transported by a carrier other than LP. In advanced fibrolipidic lesions with open endothelial junctions and deendothelialized areas, a bulky intramural insudation of plasma results in the presence of large amounts of apparently little modified LP. This may represent what several investigators have isolated as 'aortic LP', which may be insudated rather than transported plasma LP.

Animals↗
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↗
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↗
PubMed1988

The cerebral microvasculature of the rat: structure and luminal surface properties during early development.

The development of the cerebral microvasculature of the rat was studied during three successive postnatal periods, namely: 1) neonatal period, i.e., 1 to 9 days after birth (capillary sprouting period); 2) myelinization period, i.e., 10 to 20 days; and 3) young adult period, i.e., 2 to 3 months. The survey covered structural aspects and distribution of binding sites for anionic or cationic probes and for albumin-gold complexes on the luminal surface of the microvascular endothelium. The salient results are: a) an extensive development of the endoplasmic reticulum of endothelial cells during the first period (presumably in relation with the production of basement membrane components); b) the high surface density of coated pits and coated vesicles that peaks during the myelinization period; c) the paucity of plasmalemmal vesicle and their differential distribution (their volume density is higher in the endothelium of arterioles than in that of capillaries and venules); d) the existence of an extensive smooth surface tubular system in the cytoplasm of endothelial cells, whose structural connections and functional significance remains to be established; and e) the presence of pericytes with elaborate interactions with endothelia in the early developmental periods. Labeling by perfused tracers indicates an uneven patchy distribution of binding sites for cationic ferritin (generally limited to the plasmalemma proper) and a more even distribution of binding sites for cationic and anionic hemeundecapeptides. Binding patterns did not change during the developmental periods studied. No binding sites were detected for albumin-gold complexes.

Animals↗
PubMed1987

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates.

Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, and Limulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfused in situ into rat pancreatic vasculature previously oxidized with 1 mM sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.

Animals↗
PubMed1987

Endothelial cell plasma membrane obtained by chemically induced vesiculation.

A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.

Animals↗