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M Simionescu

Publications and source records attributed to M Simionescu.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1988

Fatty acids binding to albumin increases its uptake and transcytosis by the lung capillary endothelium.

To determine whether uptake and transcytosis of albumin (A) in continuous capillary endothelia are modified when this protein carries fatty acids, the transport of albumin-oleic acid and albumin-palmitic acid complexes was compared with that of defatted albumin. The probes, either radioiodinated or tagged with 5-nm gold particles (Au), or both, were perfused in situ or injected in vivo; after 3 or 30 min lung fragments were radioassayed or examined by electron microscopy. Both in situ and in vivo, the uptake of fatty acid-carrying albumin (A-FA) was consistently 2 to 3 times higher than that of defatted A. Electron microscopy revealed that A-FA complexes tagged with gold were taken up and transported across the endothelium by plasmalemmal vesicles. Morphometric analysis showed that as compared with A-Au, at 3 min the density of (A-FA)Au bound to plasmalemmal vesicles was 2 to 3 times higher, and the extent of transcytosis was increased. Uptake of the iodinated albumin was more effectively competed by A-FA complexes than by defatted A, suggesting a higher affinity of the former for the albumin binding sites of the endothelium. The results indicate that when carrying fatty acids, albumin is taken up specifically and with high affinity by the capillary endothelium, a process that may play a role in the transport of fatty acids from the plasma to the cells where they are metabolized.

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

Cellular events in the development of valvular atherosclerotic lesions induced by experimental hypercholesterolemia.

The onset and evolution of ultrastructural changes in the cardiac valves induced by a cholesterol-rich diet were investigated in rabbit and hamster. In both animal models, the atrioventricular and sigmoid valves were comparably affected by lesions intermediary between fatty streak and fibrous plaque. The earliest detectable modification was the progressive accumulation in the subendothelium of extracellular liposome-like structures rich in unesterified cholesterol, associated with the proliferation of a basal lamina-like material. This was followed by the diapedesis of blood monocytes in the same location, which became macrophages increasingly loaded with lipid deposits. Resident interstitial cells accumulate lipids, as well. In advanced stages, the macrophage-derived foam cells clustered, deforming the valve leaflets. The resident macrophages accumulated lipids later and more slowly, while partly preserving their ultrastructure. The advanced lesions are characterized by marked stromal proliferation, massive intra- and extracellular deposition of lipids and cholesterol crystals and the appearance of a necrotic core. The salient findings of these studies were: (1) the appearance of extracellular liposomes as the earliest event in atherogenesis; (2) the capability of the valvular interstitial cells to accumulate lipids; and (3) the slow response of resident macrophages to the cholesterol-rich diet. The results revealed that hypercholesterolemia produces in the cardiac valves atherosclerotic lesions of an intermediate type, which can deform the leaflets thus altering their normal function.

Animals↗
PubMed1986

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells.

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.

Animals↗
PubMed1986

Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.

Animals↗
PubMed1986

Endothelial cells express a spectrin-like cytoskeletal protein.

Vascular endothelium was investigated by indirect immunofluorescence and immunoautoradiography for the possible presence of spectrin-like molecules. Antibodies were raised against electrophoretically purified rat, rabbit, and bovine red blood cell spectrin and against rabbit brain fodrin. Antibody specificity was assessed by immunoblotting and double-diffusion technique. Homogenates of endothelial cells freshly isolated from heart microvasculature or aorta, as well as cultured aortic endothelial cells, were analyzed by gel electrophoresis. Immunoautoradiograms of gels incubated with spectrin specific antibody, followed by radio-labeled protein A, revealed two bands of electrophoretic mobility similar to that of the alpha- and beta-subunits of spectrin. Indirect immunofluorescence of endothelial cells, both in situ and in vitro, showed the existence of a protein which cross-reacted with the antibodies against spectrin and fodrin. Controls, in which endothelial cells were exposed to spectrin antibody absorbed with pure spectrin or preimmune serum, were negative. These findings indicate that endothelial cells express a protein antigenically related to the spectrin family; both spectrin- and fodrin-like molecules, in various proportions, may coexist. In the endothelial cell, these proteins may play an important role in modulation of the cytoskeleton in response to various stimuli, and in maintaining the biochemically differentiated microdomains of plasmalemma.

Animals↗
PubMed1986

Interstitial cells of the heart valves possess characteristics similar to smooth muscle cells.

Interstitial cells of heart atrioventricular and sigmoid valves were examined in several laboratory animals (rabbit, hamster, rat, and mouse) and in humans. These cells constitute a large fraction of the total cell population of the valve; in mouse atrioventricular valves, they amount to approximately 30% of the volumetric density. By their ultrastructural features and functional properties, valvular interstitial cells are intermediate between fibroblasts and vascular smooth muscle cells. Like fibroblasts, valvular interstitial cells lack a basal lamina establishing direct and extensive contacts with collagen fibers, elastin microfibrils, and proteoglycans of the matrix. The cells have numerous slender and long processes, connected to one another, forming a complex cellular framework spanning the entire valve. Similar to smooth muscle cells, valvular interstitial cells are extensively coupled by communicating junctions as shown by thin sections, freeze-fracture, lanthanum staining, and carboxyfluorescein microinjection. The cells contain numerous bundles of actin filaments, which are decorated by the S1 fragment of heavy meromyosin. Valvular interstitial cells also express cyclic guanosine-monophosphate-dependent protein kinase, as detected by immunofluorescence and immunoperoxidase histochemistry. Motor nerve endings are located closely apposed to valvular interstitial cells: structurally most of them appear to be of the adrenergic type. Valvular interstitial cells contract on epinephrine or angiotensin II stimulation as shown both in culture and in situ (valvular strips). Taken together these observations suggest that VIC may have contractile properties, which can account for a controlled tonus, actively correlated with the cyclically changing forces acting on valves during diastole and systole.

Actin Cytoskeleton↗
PubMed1986

Uptake of low density lipoproteins by the hamster lung. Interactions with capillary endothelium.

The mechanism by which the circulating low density lipoproteins (LDL) contribute to the lung surfactant cholesterol was investigated by perfusing the hamster lung in situ with LDL either radiolabeled or coupled to gold, or both. Part of [125I]-LDL and [3H]-cholesterol LDL were taken up by a specific process which was time- and concentration-dependent and reached saturation within 20 to 30 min of perfusion. Competition experiments and removal of receptor-bound LDL by heparin suggested that about 50% of LDL uptake is receptor-independent. Experiments using double labeled LDL showed a preferential uptake of 3H-cholesterol versus 125I by the lung both in situ and in vivo. LDL-gold particles (LDL-Au), recirculated through the isolated lung, bound to the endothelial luminal plasma membrane and to features potentially involved in receptor-mediated endocytosis (coated pits, coated vesicles, lysosomelike structures) and in transcytosis (plasmalemmal vesicles). The results suggest that LDL uptake by the lung takes place by both receptor-mediated and receptor-independent mechanisms. Cholesterol may be in part transferred to the lung without the apoprotein moiety; the alveolar capillary endothelium appears to be the first monitor of this complex process.

Animals↗
PubMed1986

Prelesional events in atherogenesis. Accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit.

Biochemical, physiologic, and ultrastructural modifications which appear in the aortic intima and atrioventricular valves before monocyte diapedesis and foam cell formation were investigated in rabbits fed a cholesterol-rich diet. In the first 2 weeks of the diet, while plasma beta-VLDL cholesterol was increased up to 15-fold, the intima showed an enhanced uptake and deposition of dietary 3H-cholesterol, 125I-beta-VLDL, and the fluorescent beta-VLDL-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine conjugate. beta-VLDL-gold complex perfused in situ was transcytosed across endothelium by plasmalemmal vesicles. Concomitantly, within the intima, a progressive accumulation of extracellular densely packed uni- or multilamellar vesicles took place. These commonly occurred in cell-free subendothelial spaces and were not associated with any sign of cytolysis. In freeze-fracture preparations, these vesicles appeared as smooth surfaces, suggesting the absence of translamellar proteins. Upon incubation with filipin, these extracellular liposomes (EL) displayed characteristic approximately 20 nm filipin-sterol complexes, revealing the presence of preparations unesterified cholesterol in the phospholipid lamellas. EL deposition was paralleled by proliferation of basal lamina-like material, microfibrils, and proteoglycans, and continued to increase during foam cell formation. For the entire period of our experiments, the endothelium was morphologically intact, and no platelet involvement was detected. The results show that an early prelesional ultrastructural change in lesion-prone aortic and valvular areas is the accumulation of extracellular phospholipid liposomes rich in unesterified cholesterol.

Animals↗
PubMed1985

Interactions of endogenous lipoproteins with capillary endothelium in spontaneously hyperlipoproteinemic rats.

In spontaneously hyperlipoproteinemic old Sprague-Dawley rats, endogenous lipoproteins (LP) in the size range of 15 to 40 nm were directly visualized within the blood vessels due to specimen mordanting with tannic acid. LP morphometric analysis at the level of the endothelium of diaphragm capillaries revealed that particles of the dimensions of low-density lipoproteins, high-density lipoproteins (HDL1), and very low density lipoproteins occur in endothelial structures involved in receptor-mediated endocytosis coated pits-vesicles, endosomes, lysosomes) and transcytosis (plasmalemmal vesicles and transendothelial channels). No such particles could be detected in the intercellular junctions. Intravenously injected cationized ferritin (CF) of pI 8.4 bound uniformly to LP forming an CF-LP complex. Examined at 5, 20, and 60 min after CF administration, the CF-LP complex was found to be taken up by endothelium only by endocytosis (adsorptive via coated pits-vesicles, and fluid phase through a fraction of plasmalemmal vesicles). CF-LP complexes are progressively accumulated within lysosomes. These findings reveal the importance of the net surface charge of macromolecular complexes for their intracellular sorting and fate.

Animals↗
PubMed1985

Differentiated microdomains of the luminal plasmalemma of murine muscle capillaries: segmental variations in young and old animals.

We investigated the luminal surface of the continuous endothelium of the microvasculature of the murine heart and diaphragm to find out whether it has differentiated microdomains. The probes were ferritin molecules, cationized to pI's 6.8, 7.15, 7.6, 8.0 and 8.4, which were introduced by retrograde or anterograde perfusion through the aorta or vena cava after the blood was removed from the vasculature. The pattern of labeling was analyzed by electron microscopy and assessed quantitatively by morphometry in arterioles, capillaries, and venules identified in bipolar microvascular fields in the diaphragm. The results showed that the plasmalemma proper was heavily but discontinuously labeled by all cationized ferritins (CF) used, the labeling being less extensive on the venular endothelium. CF had access as individual molecules to a fraction of the vesicular population opened on the luminal front of the endothelium. Plasmalemmal vesicle labeling increased from approximately 10 to approximately 25% as the pI decreased from 8.4 to 6.8. Vesicle labeling also increased with CF concentration in the perfusate. All CF binding sites were removed by pronase and papain. Heparinase and heparitinase caused only a slight reduction in CF labeling. Neuraminidase decreased the extent and density of labeling, especially on the plasmalemma proper of the venular endothelium; this decrease was particularly pronounced in old animals.

Aging↗
PubMed1985

Organization of the intercellular junctions in the endothelium of cardiac valves.

The intramembranous organization of valvular endothelial cells and the structure of their intercellular junctions were studied using both thin section and freeze fracture electron microscopy. Using the double replica method, large areas of fractured endothelial cell plasma membrane are exposed. On both faces, the intramembranous particles are randomly distributed, but they are 2-3 times more frequent on the P face than on the E face: on the former, their number varies from 340 to 1700 particles/micron 2. The density of vesicular openings seems to be slightly higher on the tissue front (29-43 openings/micron 2) than on the blood front (24-34 openings/micron 2). The vesicular stomata are absent in parajunctional areas. The intercellular junction structure appears as a variation to that described for arteries. The occluding junctions appear as a network of 1-6 (most frequently 3-4) interconnected ridges on P faces or grooves on E faces. Occasionally, the presence of strands formed by association of short bars can be observed on the P face ridge. In addition, there are a small number of occluding junctions with low profile ridges, free or marked by few particles, similar to those described for the venules. These junctions are probably involved in the inflammatory reaction occurring during clinically manifested valvular disease. The communicating (gap) junctions, small or large, are free, partially or completely associated. In all valves examined we observed a special kind of communicating junction the particles of which are disposed in 1-4 rows, forming branched or circular patterns. Intracellular injection of 6-carboxyfluorescein shows transfer of the dye to the neighboring cell, suggesting that the cells are coupled. In both atrioventricular and sigmoid valves, the endothelial junctions have a similar pattern with some differences in the degree of complexity. The ventricular aspect of the valves contains junctions with a larger number of junctional strands than the atrial or arterial aspect. This suggests a possible relationship between the number of strands and the stress factors (i.e. blood hydrostatic pressure). The presence of functionally communicating junctions of various dimensions and shapes suggests that the endothelial cells of valvular endocardium are metabolically coupled. At short exposure times (5-10 min), filipin-incubated valves exhibit characteristic filipin-sterol complexes (FSC) around the vesicular stomata of the endothelium. After a longer exposure (30-90 min) FSC labeled randomly the rest of plasma membrane except for coated pits, gap junction regions and area boundering tight junctional strands.

Animals↗