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M Simionescu

Publications and source records attributed to M Simionescu.

At least 55 records · Page 3Linked to original sources
PubMed1994

A method for selective radiolabeling of lung endothelium plasmalemmal vesicles, in situ.

Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these findings, as well as on the ability of lactoperoxidase (LPO) to mediate the radioiodination of proteins, we have prepared a complex of gold particles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO). The complex had a pI of 5.8, largely preserved aLPO enzymatic activity (approximately 74%), and was able to catalyze protein radioiodination. Upon washing out the blood, the complex was perfused in the mouse lung, the excess tracer removed, and a Na125I/H2O2 solution was introduced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membrane-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further radioiodinated as described for Alb-Au-aLPO. By EM, it was found that both in tissue and in the isolated membrane fraction, only Alb-Au-aLPO labeled markedly and preferentially some uncoated pits and most plasmalemmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane-enriched fraction prepared from lungs perfused with Alb-Au-aLPO had some major identified 125I-labeled polypeptides of apparent molecular masses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypeptides was labeled in lungs perfused with LPO, whereas after administration of LPO-Affi Gel the major radiolabeled polypeptides had a molecular mass of 33, 55 kDa and several peptides in the range of 77 to 160 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

In vitro formation of oxidatively-modified and reassembled human low-density lipoproteins: antioxidant effect of albumin.

In early atherogenesis, excess plasma lipoproteins accumulate into the arterial lesion-prone areas as modified and reassembled lipoproteins (MRLp) appearing mostly as lipid droplets and vesicles. In the present study we produced such MRLp, in a cell-free system, devoid of any component of extracellular matrix, by subjecting in vitro human low-density lipoproteins (LDL) to autoxidation or copper-induced oxidation, for up to 96 h. As visualized by negative staining electron microscopy, a large number of lipoprotein particles (Lp) were progressively transformed into aggregates (ALp), fused particles (FLp) and vesicles (VLp). These modifications were paralleled by peroxidation of the samples as revealed by chemical analysis of each MRLp fraction isolated by a three-step purification procedure. LDL peroxidation in the above conditions was inhibited by the presence of albumin as assessed by TBARS and lipid analysis, and by the lack of MRLp formation. This protective effect was independent of albumin source (bovine, human, rabbit) and occurs at an albumin/LDL ratio of 1 when Cu2+ was present, and at a ratio of 0.25 in autoxidative conditions. The results show that: (i) in vitro LDL autoxidation or copper-induced peroxidation in a cell-free system can generate modified and reassembled lipoproteins similar to those detected in vivo in the arterial intima at the inception of atherogenesis; (ii) Lp particles appear to be sequentially transformed in self-aggregates, droplets and vesicles; (iii) serum albumin can completely prevent these LDL alterations.

Antioxidants↗
PubMed1993

Proatherosclerotic events: pathobiochemical changes occurring in the arterial wall before monocyte migration.

The inception of experimentally induced atherogenesis is marked by subtle biochemical and ultrastructural changes of the arterial intima, modifications that precede the monocytes migration within the vessel wall. Long before any recognizable endothelial denudation, hypercholesterolemia induces a sequence of prelesional events that start with the enhanced transport of excess plasma lipoproteins especially by transcytosis, followed by intima accumulation of chemically (oxidatively) modified and reassembled lipoproteins (MRLp). Exposure of endothelial cells concomitantly to hypercholesterolemia on the luminal side, and to the MRLp cytotoxic effects on the abluminal side, generates endothelial dysfunctions with altered biosynthetic activities. The latter are manifested initially by the hyperplasia of basal lamina and its disjunction from endothelium, and by the proliferation and reorganization of a modified extracellular matrix that traps MRLp. The cytotoxic effects of MRLp contribute to the endothelial production of chemoattractants and adhesion molecules that are instrumental in monocyte recruitment and migration in the subendothelium, where activated and differentiated as macrophages, they avidly ingest MRLp and their complexes to form foam cells. The latter are the hallmark of fatty streaks that marks the transition from the prelesional to the lesional stage of atherogenesis. Understanding the earliest biochemical alterations of the artery wall is a prerequisite for designing more successful means of prevention and treatment of atherosclerosis.

Animals↗
PubMed1993

Albumin-binding proteins function in the receptor-mediated binding and transcytosis of albumin across cultured endothelial cells.

The functional significance of the endothelial albumin-binding proteins (ABP) was tested on cultured bovine aortic endothelial cells (BAEC) incubated with radiolabeled albumin ([125I]Alb) alone or carrying fatty acids (oleic acid (OA) or arachidonic acid (AA)) or triiodothyronine. The [125I]Alb binding was estimated on BAEC grown on 96-well plates, and the transport was evaluated on BAEC cultured in a dual chamber system. The probe interaction with the monolayer was monitored as a function of concentration and temperature in the presence or absence of either unlabeled Alb or an anti-albumin anti-idiotypic antibody (Ab2), which was previously demonstrated to specifically recognize the ABP of endothelial cell surface. Cultured BAEC bound specifically and with high affinity [125I]Alb. The binding of fluorescein isothiocyanate (FITC)-Alb to endothelial cells was inhibited by Ab2 in immunofluorescence studies. As compared to albumin, the binding of albumin carrying either OA or AA was higher and was diminished by Ab2. Transport of [125I]Alb across BAEC grown on gelatin-coated filters increased with time, and after 60 min, approximately 30% of [125I]Alb was transported from the upper to the lower compartment; unlabeled Alb or Ab2 reduced this process by approximately 75%. Colchicine decreased transcytosis of [125I]Alb by approximately 80%, whereas chloroquine by approximately 27%. Transendothelial transport of [125I]Alb carrying fatty acids was 40% and 20% higher for OA and AA, respectively, as compared to that of defatted albumin. The results suggest the coexistence of a receptor-mediated and a receptor-independent transcytosis of albumin across cultured endothelial cells; ABP of the endothelial cell surface appear to be involved in the specific binding and transport of albumin.

Animals↗
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↗