Biomedical subjects

M Simionescu

Publications and source records attributed to M Simionescu.

At least 19 recordsLinked to original sources

PubMed2003

Saturated-fat diet induces moderate diabetes and severe glomerulosclerosis in hamsters.

AIMS/HYPOTHESIS: Diets high in saturated fat are thought to be a risk factor for Type 2 diabetes and associated complications. We investigated effects of a medium and high saturated fat diet on the development of diabetes-associated pathologies in Golden Syrian hamsters, an animal that reacts to dietary lipids in a fashion similar to humans. METHODS: Three diets containing 46.5 kcal %, 267.3 kcal %, and 488.2 kcal % as saturated fat respectively, were studied. Metabolic parameters were measured up to 20 weeks. Electron microscopy was used to examine the structure of the pancreas, aorta and kidney. RESULTS: Increased saturated fat consumption was associated with: (i) gradual imbalance of homeostasis, and severe structural alterations of acinar, beta cells and capillaries in the pancreas, and of the kidney glomeruli; (ii) severe hypertriglyceridaemia and augmented creatinine concentrations related to disturbances of the renal function, progressing to nodular glomerulosclerosis and nephropathy; (iii) reduced early insulin secretion in response to glucose; (iv) switch of the aortic endothelium to a secretory phenotype. CONCLUSION/INTERPRETATION: The results show that high-caloric saturated fat intake induced diabetes in hamsters, probably linked to delayed insulin secretion. The model was also associated with the development of a range of pathologies characteristic to human diabetes, including nephropathy and defects in vasculature. Thus, high-fat fed hamsters provide a new model that is likely to be useful in understanding the cellular and molecular mechanisms involved in the pathogenesis of diabetes.

Animals↗

PubMed2003

Investigation of IgG receptors expressed on the surface of human placental endothelial cells.

Fetal passive immunity is acquired by transfer of maternal IgG through the placental syncytiotrophoblast and endothelium; few and contradictory data exist for IgG transcytosis in human placental endothelial cells (HPEC). In this study, we tested the binding and internalization of IgG by cultured HPEC and the expression of FcgammaRs. Biochemical analysis and microscopy revealed that the binding of IgG occurred through the Fc portion of the molecule and was greater on the basolateral than on the apical cell surface. IgG binding and internalization were saturable and the data calculated from Scatchard plot for IgG surface binding indicated a single interaction with an apparent K(d)of 2x10(-7)M. During 3 h of chase, approximately 10 per cent of IgG was released in an intact form in the medium. By electron microscopy, IgG was detected on HPEC surface, internalized in endothelial caveolae and within endosomal compartments. RT-PCR, blotting and microscopy failed to detect the presence of FcgammaRI-III in HPEC. However, the specific radioiodination and affinity chromatography revealed the presence of a 55 kDa-IgG binding polypeptide on cell surface. These findings indicate that HPEC (i) take up and internalize IgG via a receptor mediated process; (ii) bind IgG prevalently on the basolateral surface via the Fc fragment of the molecule and (iii) exhibit a novel FcgammaR of 55 kDa on the cell membrane.

Adult↗

PubMed2002

Endothelial nitric oxide synthase gene polymorphisms in Fabry's disease.

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.

Adolescent↗

PubMed2002

A novel attribute of enoxaparin: inhibition of monocyte adhesion to endothelial cells by a mechanism involving cell adhesion molecules.

Enoxaparin is a low molecular weight heparin, widely accepted as anticoagulant or antithrombotic drug, and is likely to have a role in acute inflammation. To evaluate the anti-inflammatory potential of enoxaparin, we investigated the direct effect of the drug on the activation of endothelial cells. For this purpose we set up an in vitro system in which cultured valvular endothelial cells (VEC) activated by tumor necrosis factor alpha or lipopolysaccharide were exposed to a monocytic cell line; these conditions induced a significant adhesion of monocytes to VEC. Adhesion assays, ELISA, and flow cytometric analysis revealed that pretreatment with enoxaparin, at a relevant plasma concentration (16 microg/ml), acts upon activation of VEC by inhibition of lipopolysaccharide-induced E-selectin expression and tumor necrosis factor stimulated ICAM-1 expression, thus reducing monocyte adhesion to VEC. These results suggest a novel function of enoxaparin, namely to protect VEC from activation and inhibiting the expression of cell adhesion molecules.

Animals↗

PubMed2001

Severity of oxidative stress generates different mechanisms of endothelial cell death.

The role of reactive oxygen species (ROS) in the pathogenesis of vascular diseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confluent bovine aortic ECs exposed for 30 min to different concentrations of hydroxyl radicals (HO*) generated by hydrogen peroxide (H(2)O(2)) in the presence of 100 microM ferrous sulfate (FeSO(4)). Cell viability assays, Hoechst DNA staining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agarose gel electrophoresis and annexin V assay were used to determine the effect of HO* on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H(2)O(2)/FeSO(4), the large majority of cells are viable, except for approximately 12.5% death, which occurs by apoptosis. At a concentration of 0.2 mM H(2)O(2), the cell viability is reduced to 66%, while EC apoptosis remained at comparable values (14%). At high oxidative stress (0.5 mM H(2)O(2)), the cell viability was drastically reduced (approximately 39%), and the prevalent form of death was necrosis; apoptosis accounted for only approximately 17%. Together, these data indicate that: (1) HO* induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration of HO. Thus, the same insult can cause apoptosis and/or necrosis, as a function of the intensity rather than the nature of the insult.

Animals↗

PubMed2001

Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells.

The mechanism of selective transport of the immunoglobulins G from the placental stroma to the lumen of the fetal blood vessels has not been elucidated yet. It was postulated that the specific transport as well as the regulation of IgG level in the blood, involves the MHC class I related receptor FcRn for the Fc domain of IgG. We questioned whether human placental endothelial cells (HPEC) express FcRn and, if present, whether it is in a functionally active form. The experiments were performed on cultured HPEC and as positive control, human trophoblastic (JEG3) and mouse endothelial cells (SVEC) were used. Expression of FcRn, was demonstrated by indirect immunofluorescence and RT-PCR. The role of FcRn was assessed by quantifying the transcellular transport of [(125)I]-hIgG or [(125)I]-rF(ab')(2) fragments from the apical to basolateral surface, and in the reverse direction of HPEC grown on filters in a double chamber system. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. The results showed that: (a) FcRn is expressed by human placental endothelial cells, in a functionally active form; (b) transcytosis of IgG in HPEC is a time-dependent process that takes place preferentially from the basolateral to the apical compartment; and (c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive. Together these data suggest that the regulation of IgG level by endothelial cells may result from interplay between salvaging, exocytosis, and transcytosis of the molecules. One can assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which IgG homeostasis is maintained.

Animals↗

PubMed2001

Mechanisms of decreased bradykinin- induced vasodilation in experimental hyperlipemia-hyperglycemia: contribution of nitric oxide and Ca2+-activated K+ channels.

Common complications of diabetes are accelerated atherosclerosis and vascular disturbances. We investigated whether the simultaneous insult of hyperlipemia-hyperglycemia affects the reactivity of the resistance arteries to bradykinin (BK), and if so, what are the mechanisms responsible for this disturbance. Experiments were conducted on male Golden Syrian hamsters rendered hyperlipemic (H) by a fat-rich diet, diabetic (D) by streptozotocin injection, or simultaneously hyperlipemic-diabetic (HD). Normal age-matched animals were used as controls (C). At 24 weeks after the induction of disease(s) the vascular reactivity of the mesenteric resistance arteries to BK (10(-8)-10(-4) M) was assayed by the myograph technique. To explore the role of nitric oxide (NO) in modulating the endothelium-dependent BK-induced relaxation, two experimental approaches were employed: (i) in vivo administration of L-arginine (622.14 mg/kg bw) to H, D, and HD hamsters (for 12 weeks); (ii) in vitro blockage of nitric oxide synthase by N(omega)-nitro- L-arginine methyl ester (10(-4) M). To evaluate the contribution of Ca2+-activated K+ channel(s) to BK-induced relaxation, the resistance arteries were exposed to 10(-3) M tetraethylammonium. Comparatively, the endothelium-independent relaxation was assayed using sodium nitroprusside (10(-8)-10(-4) M). The results showed that compared to the H and D groups, the HD hamsters exhibited the most reduced vasodilation of the resistance arteries to BK (34.09 +/- 1.5%). The diminished vasodilation was found to be due to a dual mechanism: an L-arginine:NO pathway and a NO-independent process, mediated via Ca2+-activated K+ channels. In vivo administration of L-arginine had favourable effects especially in the HD group, which manifested (i) an; 30% improvement of attenuated BK relaxation, (ii) an increase in sensitivity of the response to BK, (iii) a 3-fold diminishment of plasma hyperglycemia. Collectively, these data explain in part, the mechanisms and possible ways to correct the arterial endothelial dysfunction when diabetes is complicated with hyperlipemia.

Animals↗

PubMed2001

Structural and transport property alterations of the lung capillary endothelium in diabetes.

There are reports that lung function is altered in diabetes mellitus. Since data are limited concerning the structural--functional correlates in hyperglycemic animals, we designed experiments to assess: (i) whether hyperglycemia induces changes in the structure of the lung capillary endothelial cells (EC), and (ii) the effect of advanced glycation endproducts of albumin (AGE-Alb) on EC. Experiments were conducted on mice and hamsters rendered diabetics by streptozotocin (D-STZ) injection; age-matched animals were used as controls (C). The structure of EC, and the interaction of EC with AGE-Alb adsorbed to 5 nm colloidal gold (AGE-Alb.Au) were examined by electron microscopy; the uptake and cellular distribution of [125I]-AGE-Alb were investigated by spectrometry and autoradiography. The results showed that, compared to C group, in D-STZ animals the alveolar capillary EC exhibited: (i) an uneven distribution of the anionic sites exposed by the luminal plasmalemma, (ii) a metabolicaly active phenotype, (iii) a thickened basal lamina provided with focal nodules, similar in density and distribution to those found in the glomerular basal lamina in diabetes; in addition, (iv) a narrowed or collapsed lumen was found in approximately 30% of the capillaries. The functional alterations of lung EC in diabetes consisted in increased uptake of intravascularly infused [125I]-AGE-Alb and enhanced transcytosis and endocytosis of AGE-Alb.Au. The fusion of plasmalemmal vesicles, and opening of the interendothelial junctions observed, may account for the increased permeability of the alveolar capillary EC. This study asserts that hyperglycemia affects the structure and functional properties of the alveolar capillary EC, and suggests the existence of microangiopathic alterations in diabetic lung.

Animals↗

PubMed2000

Diabetic state induces lipid loading and altered expression and secretion of lipoprotein lipase in human monocyte-derived macrophages.

Non-insulin-dependent diabetes mellitus (NIDDM) is frequently associated with macroangiopathies and coronary heart diseases. Lipoprotein lipase (LPL), an enzyme known to undergo significant functional alterations in diabetic state, is also a potential atherogenic protein. Since, to the best of our knowledge, there are no data concerning LPL secreted by macrophages of NIDDM patients we conducted a study to assess the expression and activity of LPL secreted by monocyte-derived macrophages from NIDDM patients with cardiovascular complications versus cardiovascular patients without diabetes (controls). Isolated cells from NIDDM patients, after 7 days in culture in the presence of 20% autologous serum, readily exhibit a foam cell phenotype, in contrast to the cells from controls. Macrophages were mainly loaded with triglycerides, whose cellular amount was well correlated to triglyceridemia of NIDDM subjects. Concomitantly, macrophages from NIDDM patients displayed a approximately six-fold decrease of mRNA expression and a approximately two-fold reduction of the activity of secreted LPL, as compared to control cells. These data suggest that in complicated diabetic state, macrophage loading leading to foam cell formation is accelerated, at least in part, due to a diminished expression and activity of LPL. These observations add and extend the data that may explain the occurrence of accelerated atherogenesis and of the atherosclerotic complications associated with diabetes.

Adult↗

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

The effects of simultaneous hyperlipemia-hyperglycemia on the resistance arteries, myocardium and kidney glomeruli.

The experimental model of Golden Syrian hamster subjected to concomitant hyperlipemia (diet-induced) and diabetes (by streptozotocin injection) for 24 weeks is characterised by the prevalence of micro- and macroangiopathies. We have used the hyperlipemic-diabetic (HD) hamsters to investigate: a) whether there is an alteration in the reactivity of the resistance arteries (mean internal diameter: 210-250 microm), b) if present, which are the structural and biochemical changes that accompany the functional modifications, and c) to examine the pathomorphological changes induced by the association of hyperlipemia and diabetes on vital organs such as myocardium and kidney glomeruli. To these aims, biochemical assays of plasma components, light- and electronmicroscopy, myographic, morphometric and spectrofluorimetric techniques were used. The mesenteric resistance arteries of HD hamsters exhibited (as compared to similar arteries in normals) a decreased contractile response to noradrenaline (1.86+/-0.35 vs. 2.43+/-0.21), and an impeded endothelium dependent relaxation to acetylcholine (approximately 61.40% vs. approximately 79.80%). The association of hyperlipemia with diabetes induced changes in morphology of the resistance arteries consisting in approximately 10% increase of the intima plus media cross-sectional area, approximately 20% decrease of the vascular lumen area, and approximately 2.85 fold augmentation of the wall to lumen ratio. The resistance arteries exhibited structural modifications of the endothelium (up to 8 copies of Weibel-Palade bodies/endothelial cell), and smooth muscle cells (secretory phenotype), and in the vessels media small calcification cores appeared embedded in a hyperplasic extracellular matrix. The vascular mesenteric bed of the HD hamsters contained approximately 2.30 and approximately 1.30 fold increased concentrations of AGE-collagen and pentosidine, respectively, above the normal values. The HD hamsters displayed also modifications that may be dependent on or may lead to an increase in blood pressure, such as: a) approximately 2 fold increase in the activity of serum angiotensin converting enzyme; b) approximately 4.8 fold enhancement of erythrocytes fragility (as a measure of the oxidative stress); c) left ventricular hypertrophy associated with a progressive disarray of cardiomyocyte contractile fibers, interruptions of the Z bands, and accumulation of collagen-rich extracellular matrix indicative of interstitial fibrosis; d) the kidney glomerular capillaries appeared partially or totally collapsed, with a thickened basement membrane which appeared polymorphic, and in some locations made up of successive layers connected by fine bridges and intercalated nodules; in addition, an increase (approximately 1.50 fold) of the mesangial volume was indicative of glomerulosclerosis.

Acetylcholine↗

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↗

PubMed1999

The effects of low density lipoproteins modified by incubation with chondroitin 6-sulfate on human aortic smooth muscle cells.

One of the first changes that take place within the artery intima at the inception of atherosclerosis is the accumulation of LDL-derived modified lipoproteins which appear as subendothelial lipid droplets and vesicles. With time, the LDL retention and interaction with intimal chondroitin sulfate-proteoglycans may induce further structural and functional modification of the lipoproteins. The aim of this study was to produce 'in vitro' modified lipoproteins by LDL incubation with chondroitin 6-sulfate (CS, at 37 degrees C, for 48 h, in the absence of antioxidants) and to test their effects on cultured human aortic smooth muscle cells (SMCs). CS induced LDL modification (CS-mLDL) consisted in formation of a mixture of fused particles (up to 150 nm diameter) and monomers with a small content of lipid peroxides and a partially degraded apo B-100, corresponding to a mild oxidation. Upon incubation with SMCs, CS-mLDL produced a concentration-dependent stimulation of 3H-thymidine incorporation, that, at low concentration (25 microg/ml), was 2-3-fold higher than that obtained when native LDL was used; this increase correlates well with the level of CS-mLDL uptake at the same concentration. Besides the mitogenic effect, CS-mLDL induced a significant stimulation of SMCs migration, comparable with that reported for oxidized LDL. Upon incubation with CS-mLDL, SMCs accumulated lipid droplets of various number and dimension, as revealed by Nile red staining and electron microscopy. Competition studies performed in the presence of 20-fold excess of native LDL and acetyl LDL showed that 125I-CS-mLDL were taken up both by LDL receptor and scavenger receptor. At high concentration (200 microg/ml), CS-mLDL had a cytotoxic effect that was not significantly different from that of native LDL. Together these results provide evidence of (i) the direct alteration produced by CS on LDL and (ii) the effect of CS-mLDL on SMCs migration, proliferation and transformation in lipid-laden cells, events that are crucial in the development of fibro-muscular atherosclerotic lesions.

Aorta↗

PubMed1999

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.

Acetates↗

PubMed1999

High glucose induces enhanced monocyte adhesion to valvular endothelial cells via a mechanism involving ICAM-1, VCAM-1 and CD18.

Upon induction of experimental hyperglycemia (i.e. diabetes) pathological modifications are early detected (approximately 7 days) at the level of the cardiac valves leading rapidly to the development of valvular atheroma. Monocyte adhesion to the vascular endothelium is one of the initial event at the onset of atherosclerosis. We questioned whether high glucose enhances monocyte adhesion to the valvular endothelial cells (VEC) so as to explain, in part, the accelerated atheroma formation that occur in diabetic conditions. To this purpose we compared the adhesion of monocytes to VEC cultured in 5.5 mM (normal) glucose (NG) or in 33 mM (high) glucose (HG) or in high mannitol (HM) (27.5 mM mannitol plus 5.5 mM glucose), a concentration known to simulate the hyperosmolar effect of high glucose. After incubation for 30 min at 37 degrees C, the adhesion of monocyte cell line (U937 cells) to VEC was quantitated by a fluorimetric assay or by direct counting. Statistical data showed a significant increased adhesion of monocytes to VEC grown in HG (up to 4 fold) or in HM (up to 2.7) when compared to normal conditions. Using a battery of specific monoclonal antibodies molecules it was found that the increased adhesion of monocytes to VEC grown in high glucose was specifically inhibited (p < 0.05) by anti-ICAM-1, anti-VCAM-1 and anti-CD18 monoclonal antibodies. Together, the results indicate that high glucose induces enhanced monocyte adhesion to VEC via a mechanism involving in part an osmotic effect and mainly the cell adhesion molecules: ICAM-1, VCAM-1 and CD18.

Animals↗

PubMed1998

Protein and fatty acid composition of caveolae from apical plasmalemma of aortic endothelial cells.

In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) represent a structurally and biochemically specialized membrane microdomain. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC by using cationic colloidal silica (silica-ap), or the EC were surface-radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fraction and cell homogenate followed by ultracentrifugation on a sucrose gradient gave detergent-soluble and detergent-insoluble membranous fractions. The lowest density TX-insoluble fraction appeared morphologically as distinct vesicles (caveolae; 60 nm average diameter; PVs fraction). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentration, of specific caveolar markers, viz., caveolin (both isoforms, the 24-kDa form being conspicuously more abundant) and Ca2+-ATPase. By contrast, angiotensin-converting enzyme and alkaline phosphodiesterase were present almost exclusively in the TX-soluble fraction. The glycoproteins in the PVs fraction were of apparent molecular weights 52, 68, 95, and 114 kDa. Analysis of the fatty acid composition revealed more palmitoleic and stearic acid in the PVs fraction then in the TX-soluble fraction. Thus, in comparison with the plasmalemma proper, the PVs fraction (1) is detergent-insoluble; (2) contains caveolin in two isoforms; (3) contains Ca2+-ATPase at high concentration; (4) contains a set of specific glycoproteins; and (5) is enriched in palmitoleic and stearic acids.

Animals↗

PubMed1998

Heart microvessels and aortic endothelial cells express the 15 kDa heart-type fatty acid-binding proteins.

Due to their hydrophobic nature, free fatty acids require carriers for transport across and within the cells. The endothelial layer is the first barrier to be traversed by the fatty acids, from the plasma to the underlying cells and tissues. We tried to find out whether cytosolic fatty acid-binding proteins (FABPs) are present in the endothelium of large vessels (aortic endothelial cells) and small vessels (myocardial capillaries) using the following experimental approaches: (i) loading the delipidated aortic endothelial cell (EC) homogenate and the heart cytosolic proteins and membrane proteins with [14C]palmitate or [14C]oleate, respectively, followed by autoradiographic detection of electrophoretically separated bands; (ii) detection by immunoprecipitation of heart-type FABP (H-FABP) using an affinity-purified antibody raised against bovine H-FABP (anti-H-FABP), and (iii) localization of FABP by indirect immunofluorescence and gold-immunocytochemistry applied to cultured EC and to thick and thin frozen sections of mouse heart. The results showed that: (i) within the EC homogenate proteins that express affinity for [14C]palmitate have an apparent Mr of 15000, and 40000-45000, that correspond as molecular mass to cytosolic and membrane FABPs, respectively. Similar affinity was found by incubation with [14C]oleate, that binds to a protein of Mr 15000 in the heart cytosol, and to a 40-45 kDa protein in the membrane fraction; (ii) anti-H-FABP immunoprecipitated specifically a cytosolic 15 kDa peptide (H-FABP); (iii) by indirect immunofluorescence, cytosolic H-FABP was localized on heart microvessels and myocytes and also in cultured aortic EC where intense spotted fluorescence characteristic for cytosolic antigens was present; (iv) by immunocytochemistry, H-FABP was detected in the EC cytoplasm, and in close proximity to the cytoplasmic aspect of plasmalemma and vesicle membranes. Together the data attest the presence of the 15 kDa, heart-type FABP in the endothelium of aorta and heart microvessels.

Animals↗

PubMed1998

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.

Amino Acid Sequence↗