Biomedical subjects

E Constantinescu

Publications and source records attributed to E Constantinescu.

At least 19 recordsLinked to original sources

PubMed2002

An ex vivo model to study the monocyte-endothelial cell interaction in the prelesional stage of experimentally-induced atherogenesis in hamster.

We imagined an ex vivo atherogenic model consisting in hypercholesterolemic versus normocholesterolemic aortic arch rings or sigmoid valves incubated in cell culture conditions with human monocytes. Normal tissues did not show any attached monocytes. On the atherogenic aortic arch three different aspects were observed: a) there were no monocytes attached on a normal zone; b) many monocytes adhered to the endothelium on a thickened area, with intimal smooth muscle cells infiltration, and c) the greatest number of attached monocytes was seen in mechanically injured zones of the aortic arch, where the subendothelial area was totally exposed. Immunohisto- and immunocytochemistry for LFA-1 revealed the presence of this leukocyte integrin on monocyte plasma membrane. The labelled monocyte had an activated shape, with pseudopodes extended over the endothelial cells and the anti-LFA-1 antibody coupled with colloidal gold decorating areas apposing to a morphologically modified endothelium. In conclusion, the ex vivo model reproduced the in vivo situations where the monocytes adhere to the modified endothelium covering the thickened areas of hypercholesterolemic aortic wall; they express at least one of the adhesion integrins, namely LFA-1. This study intended to contribute, at least in part, to the understanding of some mechanisms governing the monocyte-endothelial cell interactions in hypercholesterolemia.

Animals↗

PubMed2000

Establishment of a pure vascular endothelial cell line from human placenta.

UNLABELLED: Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultures upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying their specific function in the fetoplacental unit, such as in the control of maternofetal traffic. In this paper we report an improved method for isolation, purification and culture of HPEC, that implies an enzymatic perfusion of the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two-step selection procedure, based on differential trypsinization, leading to a pure population of about 8x10(7)cells/placenta, with 2.7-3.4 population doublings. The average population doubling time during eight passages was 60-65 h and the life span of HPEC was approximately 45-50 population doublings. The cell morphology at optical and electron microscopical level revealed a good differentiation of HPEC, which were endowed with numerous plasmalemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm(2). The cultures were mycoplasma free, as revealed by fluorescence microscopy using DNA dyes and the polymerase chain reaction (PCR). The negative immunofluorescent reaction for keratin confirmed that the HPEC were not contaminated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensin converting enzyme. These characteristics substantiate the endothelial nature of cultured cells. The interactions with different lectins (BS-I, SBA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lectin. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC, and at the level of intracellular clefts in post-confluent cultures. IN CONCLUSION: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells have the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, unlike human umbilical vein EC, have a much higher proliferation rate and a long lifespan; (iv) HPEC expressed a characteristic glycosylated coat particularly rich in alpha- L -fucose and beta-GlcNAc containing glycocompounds.

Adult↗

PubMed2000

Endothelial cell-derived foam cells fail to express adhesion molecules (ICAM-1 and VCAM-1) for monocytes.

The purpose of this study was to assess the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cell-derived foam cells. Hamster aortic endothelial cells (HAEC) in culture were exposed to hypercholesterolemic or normal homologous serum for 24 h. At the end of the incubation period, HAEC exposed to hypercholesterolemic serum exhibited numerous lipid droplets and had a general aspect of foam cells. When examined for the expression of ICAM-1 and VCAM-1 (by indirect immunofluorescence) normal HAEC expressed constitutively (to low level) on their surface these adhesion molecules; however HAEC-derived foam cells failed to display any labeling. To further assess these results, HAEC were first incubated with normal or hypercholesterolemic sera (as above) and then exposed to freshly isolated normal hamster blood monocytes. These experiments showed that monocytes adhered in small number to normal cells and failed to adhere to the surface of HAEC-derived foam cells. Together these data indicate that endothelial cell-derived foam cells: a) do not express ICAM-1 and VCAM-1 on their surface; b) have low or no adhesion properties for monocytes and c) may represent an appropriate experimental model to study the cellular alterations that take place in the advanced stages of atherosclerosis.

Animals↗

PubMed1998

Diabetes-induced structural changes of venous and arterial endothelium and smooth muscle cells.

The structural alterations of endothelium and smooth muscle cells of the hind limb and heart veins and arteries were investigated in Golden Syrian hamsters subjected to streptozotocin induced diabetes. Animals were examined at 5, 10, and 15 weeks after induction of diabetes. At each time point body weight and plasma glucose concentrations were recorded. Anesthetised animals were washed out of blood, fixed in situ, and the femoral vein and artery, saphenous vein and artery, and heart veins and coronaries were dissected out, and processed for electron microscopical examination. Anionic sites of the endothelial plasmalemma were visualized by in situ perfusion of cationized ferritin. The endothelial localization of von Willebrand factor was carried out by immunocytochemistry. The results showed that induction of experimental diabetes generated morphological changes of the endothelium and smooth muscle cells of both hind limb and heart vessels. The common alterations developed in endothelial cells of venous and arterial origin consisted in: 1) the development of a secretory phenotype, enriched in biosynthetic and degradative organelles; 2) the abundance of cytoskeletal elements, especially intermediary filaments; 3) the increase in number of fused plasmalemmal vesicles and transendothelial channels, and 4) the hyperplasia of the basal lamina. In contradistinction to the arterial endothelium, the peculiarities of the venous endothelium in the diabetic hamsters examined were: 1) the uniform distribution of the anionic sites exposed on the luminal plasma-lemma (as in normal animals), and 2) the increased number of copies of Weibel-Palade bodies (up to 13 copies per endothelial cell in the hind limb). Von Willebrand factor was immunodetected in Weibel-Palade bodies, Golgi cisternae and some vesicles of normal and diabetic hamsters. With time, and especially pronounced at 15 weeks of diabetes, the smooth muscle cells of veins and arteries examined exhibited a characteristic secretory phenotype, and were surrounded by a reticulated basal lamina and a hyperplasic extracellular matrix (especially pronounced in arteries). These data indicate that diabetes affects both heart and hind limb veins and arteries, producing structural changes of the endothelium and smooth muscle cells which may account, at least in part, for the specific vascular complications.

Animals↗

PubMed1997

Exposure to hypercholesterolemic serum modifies the expression of cytoskeletal proteins in cultured endothelia.

Arterial endothelial layer dysfunction is considered to be one of the most important events which initiate the development of the atherosclerotic plaque and the cell cytoskeleton plays an essential role in maintaining the integrity of the endothelium exposed continuously to haemodynamic forces. The aim of this work was to study the modifications of the cytoskeletal proteins in the vascular endothelium exposed to atherogenic conditions. A hamster aortic endothelial cell line (HAEC) grown on glass coverslips was exposed for 24 h to hypercholesterolemic or normal homologous serum. Upon staining with Oil Red O and examination by phase contrast and fluorescence microscopy, HAEC incubated with hypercholesterolemic serum appeared heavily loaded with lipid droplets that showed a yellow autofluorescence in UV light and the general aspect of a foam cell. HAEC were incubated with: a) anti-actin serum; b) anti-vinculin monoclonal antibody (MoAb); c) anti-alpha actinin MoAb, and d) anti-talin MoAb, followed by appropriate secondary antibodies coupled with FITC or rhodamine. As compared to normal HAEC, the cells exposed to hypercholesterolemic serum showed a modified pattern for actin and vinculin localization. Actin appeared as a weakly stained network around the nuclear zone whereas vinculin was distributed as small granules throughout the cell cytoplasm. These experimental data suggest that in advanced atherosclerosis, some of the endothelial cytoskeletal proteins undergo modifications which could represent one of the important factors involved in further development of the atheromatous plaque. In addition they indicate that HAEC exposed to hypercholesterolemic serum could represent an in vitro working model for studying the events occurring in the endothelium at advanced stages of atherosclerosis.

Animals↗

PubMed1989

A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: an example using immunogold-labeled human erythrocytes.

We designed and built a freeze-drying device that ensures the protection of the specimens against contaminants during mounting on the cold stage of the freeze-fracture machine, transferring into the vacuum chamber and deep etching. The device consists of a copper cap that covers the specimen and a thermal connection that ensures thermal transfer between the microtome arm and the copper cap. This device was used to study the ultrastructural features of the erythrocyte membrane skeleton and the immunocytochemical localization of spectrin in an "in situ" approach, by freeze drying and platinum rotary shadowing. Human erythrocytes adhered to polylysine-coated coverslips and were broken by a stream of buffer that mimics the intracellular ionic environment ("inside buffer"). The samples were prefixed in periodate-lysine-paraformaldehyde fixative, labeled with antispectrin 5-nm gold particles, fixed in glutaraldehyde, mordanted in tannic acid, postfixed in OsO4, repeatedly washed in water, rinsed quickly in 30% ethanol, freeze-dried, and rotary-shadowed. Electron microscopic examination of the replicas revealed the skeletal network on the inner surface of the erythrocyte membrane. Immunocytochemical labeling proved that spectrin represents a fibrillar component of the network. Our data confirm the speculative model of the molecular organization of the erythrocyte skeleton, based on studies on in vitro association of proteic constituents. Both the technique and the device developed by us may lead to a deeper understanding of the spatial organization of the cytoskeletal network of more complex cell types.

Cytoskeleton↗

PubMed1986

Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque.

The assembly of the terminal C5b-9 complement complex is a prime mechanism of complement-induced membrane damage followed by inflammatory response mediation and subsequent extensive tissue damage. In the assembly process the terminal complement components expose neoantigenic determinants which can be recognized by specific antibodies. Using such a specific antibody, affinity-purified rabbit IgG and by means of immunoelectron microscopy, the C5b-9 neoantigens were localized on the structures of the human fibrous plaque from 3 iliac and 3 femoral arteries obtained at surgery. The immunoelectron-dense deposits were localized on the cell debris, enmeshed in the connective tissue matrix, consisting of irregular particles that frequently had the shape and size of intracellular organelles or vesicles with concentric osmiophilic lamellae. No deposits could be found on the intact cells, on the connective tissue matrix or on cholesterol and lipid deposits. The presence of C5b-9 neoantigens deposits in the fibrous plaques frequently associated with other immune-related proteins indicates that complement activation has occurred in situ and could be related to the chronic progression of the atherosclerotic lesion.

Aged↗

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

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↗

PubMed1984

Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine.

The distribution of Man-6-P receptors was determined by immunoperoxidase cytochemistry in Clone 9 hepatocytes cultured in the presence or absence of tunicamycin and chloroquine, agents that perturb lysosomal enzyme sorting and lead to their secretion. In control (untreated) cells, receptors were localized in cis Golgi cisternae, coated vesicles, and in endosomes or lysosomes. After tunicamycin treatment, receptors were found in coated vesicles lined up along the cis cisternae but were not detected in endosomes or lysosomes. After chloroquine treatment, receptors were localized in large vacuolated endosomes or lysosomes but were not usually detected in Golgi cisternae or in coated vesicles. These results demonstrate a redistribution of receptors along the normal Man-6-P-dependent sorting pathway after these treatments. In ligand-deficient (tunicamycin-treated) cells, immunoreactive receptors accumulate at the presumptive sorting site in the cis Golgi and are depleted from endosomes and lysosomes. When the intralysosomal pH is increased (by chloroquine treatment) preventing ligand-receptor dissociation, receptors accumulate at the presumptive delivery site (lysosomes and endosomes) and are depleted from the cis Golgi region. The findings also suggest that (a) ligand binding triggers movement of the receptor to endosomes or lysosomes, and (b) ligand dissociation triggers their movement back to the cis Golgi region.

Animals↗

PubMed1982