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Biomedical subjects

M Simionescu

Publications and source records attributed to M Simionescu.

At least 37 records · Page 2Linked to original sources
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

Capillary and aortic endothelia interact in situ with nonenzymatically glycated albumin and develop specific alterations in early experimental diabetes.

Diabetic mice (6 weeks duration) were studied to assess the interaction of advanced glycation endproduct-modified albumin (AGE-Alb) with micro- and macrovascular endothelium, and to evaluate the alterations induced in the ultrastructure of the lung, kidney, and aorta. [125I]-AGE-Alb and AGE-Alb-Au were perfused in situ in the vasculature; the total uptake was quantitated by spectrometry, and the endothelial pathways of AGE-Alb-Au and the morphological alterations of the vascular beds were examined by electron microscopy. The results showed that [125I]-AGE-Alb (0.567 microM) was taken up specifically and saturably by all organs studied, and particularly by the lung. AGE-Alb-Au endocytosis and transcytosis occurred in the pulmonary and aortic endothelia, and were enhanced in diabetic animals. Also in diabetic animals, AGE-Alb-Au was detected throughout the kidney glomerular basement membrane (GBM) and within open filtration slits of podocytes, suggesting altered barrier function. The structural modifications progressed, and at the end of the experimental period, in the lung approximately 28% of the capillaries and approximately 25% of the alveoli became compressed or even collapsed, due to the hyperplasia of extracellular matrix and interstitial connective tissue. The presence of adherent intravascular macrophages suggests the development of an inflammatory immune process. The structural modifications observed in kidney glomeruli included thickening (approximately 30%) of the GBM and the disappearance of diaphragms between the cellular processes of podocytes. The aortic endothelium displayed luminal foldings, increased number (2.8-fold) of Weibel-Palade bodies, and proliferation of basal lamina. Together, the results show that in diabetes there is enhanced vascular uptake of AGE-Alb and significant pathomorphological changes of micro- and macrovessels.

Animals↗
PubMed1997

Experimental evaluation of the vascular effects and transport of an iodinated macromolecular contrast medium.

RATIONALE AND OBJECTIVES: For assessment of the tissue blood pool and overall vascularity, macromolecular contrast media have significant advantages over low molecular weight contrast agents. The authors evaluated the vascular effect and transport of a new macromolecular contrast media (MMCM), an iodinated dextran polymer of 32 kDa. METHODS: The new MMCM was obtained from dextran activated by carboxy methylation, followed by linkage with triiodinated aminophtalamid conjugates. To detect whether the tracer induces vascular leakage, MMCM (350 mg I/kg) was administered intravenously in 10 mice, or applied on the cremaster muscle of 26 mice previously injected with carbon particles; after 30 or 45 minutes, the cremaster was fixed and examined by optical microscopy. For investigation of the vascular transport 3, 5, and 15 minutes after MMCM administration, various tissue fragments were processed and examined by electron microscopy. RESULTS: In all vascular examined, MMCM does not induce plasma extravasation and the probe was detected mostly within the vascular lumen. At the ultrastructural level, a small fraction of MMCM was found in endothelial plasmalemmal vesicles (endosome-like structures) and, in time, transcytosed to the subendothelial space. No intercellular junctions were permeated by MMCM. CONCLUSIONS: The MMCM induces no vascular leakage and it is retained mainly in the plasma. Transport of MMCM is restricted to endothelial vesicles, which may explain, in part, its prolonged vascular space retention.

Angiography↗
PubMed1997

Alterations of lung structure in experimental diabetes, and diabetes associated with hyperlipidaemia in hamsters.

Since hyperglycaemia is known to affect normal pulmonary physiology and biochemistry and few structure-function correlations have been reported, we designed experiments on hamsters subjected to streptozotocin-induced diabetes or diabetes associated with hyperlipidaemia, and investigated the impact of these conditions on the lung structure. At time intervals ranging 2-24 weeks from the inception of disease (without correcting blood glucose with insulin), the animals were sacrificed, and plasma glucose and cholesterol assayed. The lung was processed for electron microscopy, and the structural changes of the capillary and venular endothelium, of epithelial cells, and interstitium were examined. In diabetic animals, especially after 6 weeks of disease, a gradual narrowing of approximately 35% of the capillaries and approximately 30% of the alveoli, and hyperplasia of the extracellular matrix, rich in collagen bundles, were observed. Frequently, capillaries contained adherent intravascular macrophages suggestive of an inflammatory process. The capillary endothelium was characterized by numerous plasmalemmal vesicles, often fused, well-developed synthesizing apparatus (endoplasmic reticulum and Golgi complex) and cytoskeleton, and an uneven distribution of the anionic sites on the luminal plasmalemma. The venular endothelium was particularly rich in Weibel-Palade bodies. The alveolar epithelium was often collapsed, compressing surfactant within the airspace. The lung interstitium was apparently enlarged, and the fibroblasts and contractile interstitial cells frequently contained lipid droplets. These alterations were more pronounced and occurred at a faster rate (4 weeks) in diabetes associated with hyperlipidaemia. The structural modifications reported in this study support the functional disturbances observed in association with hyperglycaemia, sustaining the conclusion that the lung is an organ affected by diabetes.

Animals↗
PubMed1997

Transcytosis of albumin in endothelial cells is brefeldin A--independent.

To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.

Animals↗
PubMed1997

Expression of transferrin receptors in endothelial cells transfected by electroporation.

Transferrin is the primary iron-binding protein in the plasma. Transferrin receptors (TfR) were detected in brain and liver endothelial cells (EC); however, little information exists about their intracellular routes. To detect the EC structures involved in TfR biosynthetic and endocytotic pathways, cultured aortic EC were transfected with the plasmid pSR alpha containing a construct encoding the human TfR, to which horseradish peroxidase (HRP) was anchored as reporter molecule. Since EC are difficult to be transfected, we tried different techniques, and two forms of the plasmid (circular and linearized), of which the electroporation method and the linearized plasmid were the most efficient in producing stable cell lines. Transfected cells were selected with geneticin, and the expression of TfR-HRP tested by cytochemistry. The stable transformants preserved the general characteristics of EC. At the ultrastructural level, TfR-HRP was associated with the nuclear envelope, rough endoplasmic reticulum, Golgi complex and adjacent secretory vesicles, cytoplasmic vesicles of various sizes (50-130 nm diameter), endosomes, plasma membrane, plasmalemmal pits, and a fraction of plasmalemmal vesicles. The intensity of the reaction product varied, suggesting a different concentration of TfR, in specific organelles. For example, (i) a gradient of HRP-reaction product was found within the Golgi cisternae, (ii) the plasmalemmal pits were more intensely stained than the adjacent plasma membrane, and (iii) the vesicle membrane was decorated stronger than the endosomal membrane (to which it fuses). A striking feature was the coexistence within the same EC of two vesicle populations (or subtypes): some containing TfR-HRP, whereas others lack the receptor. Quantitative data indicated a stronger expression of TfR in confluent cells (approximately 8-fold higher) than in EC at 2 days after plating; a significant decrease (approximately 9-fold) of TfR was found in postconfluent transfectants. Together, the data demonstrate that (i) after electroporation of EC, the stable lines maintain the characteristics of native cells; (ii) the newly synthesized TfR is located in variable concentration within the organelles involved in endocytosis and exocytosis, and (iii) the expression of TfR-HRP is particularly high in confluent cells.

Animals↗
PubMed1997

Pathobiology of the heart in experimental diabetes: immunolocalization of lipoproteins, immunoglobulin G, and advanced glycation endproducts proteins in diabetic and/or hyperlipidemic hamster.

Diabetes is known to be accompanied by atherosclerotic disease and general cardiovascular complications. Hamsters were previously shown to develop hyperlipemia-induced atherosclerosis, similar in many respects to the human atherosclerotic process. To study the effect of hyperglycemia on heart vessels and valves, male Golden Syrian hamsters were rendered either diabetic or hyperlipemic and diabetic; controls were age-matched normal hamsters. At time intervals ranging from 2 to 24 weeks, animals were killed; plasma glucose, cholesterol, and lipid peroxides were measured; and the aortic arch and valves, coronary arteries, and heart microvessels were examined for ultrastructural modifications and for the presence of low-density lipoproteins (LDL), immunoglobulin G (IgG), and advanced glycation endproducts (AGE) proteins. Elevation of plasma glucose, peroxides, and cholesterol were observed in both diabetic as well as hyperlipemic and diabetic animals, along with characteristic diabetic changes: microangiopathy of the myocardium (ie, capillary narrowing, hyperplasia of basal lamina, and proliferation of extracellular matrix) and macroangiopathy of the aortic arch, valves, and coronary arteries (ie, intimal proliferation, fatty-streak formation, and calcification). LDL, IgG, and AGE-proteins were immunolocalized in focal deposits, ie, in the shoulder and cap of the plaques; these antigens were distributed diffusely in the extracellular space or within macrophage-derived foam cells and smooth muscle cells. Our findings indicate that hyperglycemia alone induces atherosclerotic lesions in the coronary arteries, aortic arch, and aortic valves as well as alterations of the extracellular matrix of heart microvessels and cardiomyocytes, changes which together may lead to cardiomyopathy, a common and severe complication of diabetes. In addition, the present study suggests that when hyperglycemia is accompanied by hyperlipemia, detectable amounts of modified LDL (possibly oxidized or glycated) and AGE are present in the intima of atherosclerotic arteries; and also that modified lipoproteins can act as immunoactive components of the atheroscerotic process generated by hyperglycemia.

Animals↗
PubMed1997

The effect of ACE inhibitors on atheroma formation is potentiated by association with a calcium channel blocker. A biochemical and ultrastructural study.

The effect of two angiotensin converting enzyme (ACE) inhibitors, enalapril maleate and captopril, on the progression of atherosclerosis was investigated. Golden Syrian hamsters were divided into five groups: controls (C), fed a standard chow diet; hypercholesterolemic animals (HH) induced by supplementing the diet with 3% cholesterol and 15% butter; HH treated with enalapril (20 mg/kg/day); HH treated with captopril (60 mg/kg/day) and HH treated simultaneously with enalapril and a calcium channel blocker, diltiazem (45 mg/kg/day). The drugs were administered for one month, concomitantly with the atherogenic diet. As compared to controls, in HH group a significant increase in serum cholesterol (approximately 5 fold) and ACE activity (approximately 3 fold) was found. In HH-treated animals, both drugs maintained the serum ACE activity within the normal values. However, the effect upon serum cholesterol was different: enalapril and its combination with diltiazem had a significant hypocholesterolemic effect (128.8 +/- 25 mg/dl), whereas captopril had no effect on the cholesterol values (326.6 +/- 41.48 mg/dl). Electron microscopical examination of the coronary arteries and aortic valve in all experimental groups indicated a good correlation between the high levels of cholesterol, ACE activity and the development of the atherosclerotic lesions. Captopril treatment inhibits the early phases of atherosclerosis at level of the coronary artery but has no influence upon the lesion development in the aortic valve. By comparison, enalapril and enalapril-diltiazem co-administration impede the development of fatty streaks by decreasing the accumulation of lipids and calcium deposits in the lesion-prone areas examined. These data indicate that: 1) captopril does not have a hypocholesterolemic potential and cannot prevent atheroma formation in heart valves; 2) enalapril, especially combined with diltiazem, has a hypocholesterolemic effect and impedes the development of atheromatous plaque; 3) the anti-atherosclerosis therapy may benefit from the co-administration of an ACE-inhibitor with a calcium antagonist.

Angiotensin-Converting Enzyme Inhibitors↗
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↗
PubMed1996

Increased macrophage uptake of irreversibly glycated albumin modified-low density lipoproteins of normal and diabetic subjects is mediated by non-saturable mechanisms.

Diabetes mellitus is known as an independent risk factor in atherosclerosis. Among the prominent biochemical changes that occur in diabetic state, are the enhanced formation of advanced glycosylation end products (AGE) (especially linked to albumin and collagen) and the impaired oxidative-antioxidative balance. Previously, we have shown that AGE-albumin (AGE-Alb) significantly alters the physico-chemical characteristics of low density lipoproteins of normal (nLDL) and diabetic (dLDL) subjects. In this study we tried to establish if incubation of nLDL or dLDL, with AGE-Alb in autoxidative conditions, modifies the rate and/or the pathway of their uptake by macrophages. To this purpose, nLDL and dLDL were exposed to AGE-Alb, and after re-isolation and radiolabeling the lipoproteins were incubated with U937 or peritoneal macrophages (for various time and concentrations), in the absence or presence of different competitors (native LDL, acetylated LDL, AGE-Alb, mannan) or cytochalasin D. As controls, nLDL and dLDL, maintained in similar conditions, but without AGE-Alb, were used. The results showed that preincubation for 24 h and 72 h with AGE-Alb augmented the macrophage uptake for both nLDL and dLDL (1.7-fold). Either pre-incubated or not with AGE-Alb, dLDL was taken up at a constantly higher rate than nLDL; the difference appeared more prominent at 72 h (1.5 vs. 4 micrograms LDL protein/mg cell protein). The increased level of glycation of native dLDL as compared to native nLDL (266 +/- 35 vs. 160 +/- 24 mmol HMF/mol apoB) as well as of the lipid peroxides (1.34 +/- 0.47 vs. 0.3 +/- 0.09 nmol MDA/mg apoB) could account for the greater uptake of dLDL at any preincubation time. Competition experiments indicated that, generally, incubation with AGE-Alb diminished the apo B100,E receptor-mediated uptake in favour of 'scavenger' receptor pathway and phagocytosis. Macrophage uptake of AGE-Alb modified dLDL was reduced approximately 30% by native nLDL, approximately 70% by acetylated LDL and approximately 38% by cytochalasin D. Together, these data suggest that the consequence of the alterations induced by AGE-Albumin on LDL is the increased macrophage uptake, via non-saturable pathways, that ultimately may lead to accelerated formation of atherosclerotic plaques in diabetics.

Adult↗
PubMed1996

The pathomorphological alterations of endocardial endothelium in experimental diabetes and diabetes associated with hyperlipidemia.

The structural alterations of endocardial endothelial cells of the heart right atrium and left ventricle were investigated in Golden Syrian hamsters subjected to streptozotocin-induced diabetes and to a combination of diabetes and diet-induced hyperlipidemia. Animals were examined at time intervals ranging from 2 weeks to 6 months. Anionic sites of the endothelial plasmalemma were visualized by in situ perfusion of cationized ferritin. The results indicated that: (a) both atrial and ventricular endocardial endothelium are affected in streptozotocin-induced diabetes: endothelium converts from continuous into a fenestrated type, (b) although the anionic charge of the plasmalemma decreased in advanced diabetes, the newly formed fenestrae highly bound cationized ferritin, (c) combined diabetes and hyperlipidemia induced more severe alterations of endocardial endothelium: new permeable endothelial structures were formed (transendothelial channels, open intercellular junctions, fused plasmalemmal vesicles), and the cells became particularly enriched in cytoskeleton (intermediate filaments and microtubules), (d) the thick subendocardial layer of connective tissue contained, in the combined experimental model, macrophage derived foam cells indicative for the occurrence of alterations of atherosclerotic type.

Animals↗
PubMed1996

Pathobiochemistry of combined diabetes and atherosclerosis studied on a novel animal model. The hyperlipemic-hyperglycemic hamster.

Because accelerated atherosclerosis is the main complication of diabetes, we devised a new animal model that combines these two diseases, and investigated their joint impact on the main plasma components and organs known to be most affected in each disorder. Male Golden Syrian hamsters were subjected to three experimental conditions: streptozotocin-induced diabetes (D), diet-induced hyperlipemia (H), and a combination of hyperlipemia and diabetes (HD). At time intervals ranging from 2 to 24 weeks, the animals were sacrificed, the appropriate plasma constituents were determined, and the ultrastructural modifications of relevant tissues such as the heart, cardiac valves, coronary arteries, aorta, retina, and kidney were examined. The HD hamsters were characterized by marked alternations of plasma components, ie, increase in circulating glucose, cholesterol and lipid peroxide levels, glycation of albumin, and the appearance of irreversibly glycated albumin (AGE-Alb). These humoral changes coexisted with micro- and macroangiopathic lesions characteristic to both diseases, ie, capillary narrowing, hyperplasia of endothelial basal lamina, proliferation of perivascular extracellular matrix (abnormalities reminiscent of type I diabetes), and concomitant intimal accumulation of modified lipoproteins and macrophage-derived foam cells in the aorta, coronaries, and cardiac valves, leading to accelerated formation of atherosclerotic plaques. These changes eventually appeared in the D hamsters also, but at a much slower rate, whereas the H group showed only modifications characteristic for atherosclerosis. Our findings indicate that, overall, 1) diabetes accelerated the early development and progression of atherosclerotic lesions leading to rapid calcification, and 2) hyperlipidemia associated with diabetes accelerated the rate of development of diabetes-induced microvascular disease. The hamster model may be useful to study the impact of various drugs on the diabetes-related vascular complications.

Animals↗
PubMed1996

Differential effect of two calcium channel blockers--nifedipine and diltiazem--on atherogenesis in hypercholesterolemic hamster.

We compared the effect of two different calcium channel blockers (CCB), Nifedipine (1,4-dihydropyridine calcium antagonist) and Diltiazem (a benzothiazepine agent) on plasma components and the development of atherosclerotic plaque in experimental hypercholesterolemia. Golden male Syrian hamsters were divided into four groups: atherogenic animals (AT) induced by standard diet supplemented with 3% cholesterol and 15% butter; AT animals treated with Nifedipine (20 or 60 mg/kg/day); AT hamsters treated with Diltiazem (45 mg/kg/day) and controls (C), fed a standard chow diet. For one month, the drugs were administered concomitantly with the atherogenic diet. During the experiment, serum cholesterol, free calcium and angiotensin-converting enzyme (ACE) activity values were determined. Specimens from the lesion-prone areas: aortic valves, coronary arteries, and aortic arch, were collected and processed for light and electron microscopy. The results show that the atherogenic diet induces a significant increase of serum cholesterol (389 +/- 67.47 mg/dl), free calcium (13.44 +/- 0.84 mg/dl) and ACE activity (78.46 +/- 9.25 mU/ml) as compared to controls (cholesterol 73.76 +/- 3.31 mg/dl; calcium 8.9 +/- 0.5 mg/dl; ACE 33.68 +/- 2.6 mU/ml). Administration of Diltiazem reduced significantly these parameters (cholesterol, 196.25 +/- 22 mg/dl; calcium, 8.41 +/- 0.6 mg/dl) while Nifedipine had no effect (cholesterol, 283.03 +/- 44.7 mg/dl; calcium, 11.13 +/- 1.25 mg/dl) and increased the ACE activity (100.28 +/- 36.9 mU/ml). At the structural level, a significant correlation between the apparition and progression of the atherosclerotic lesions and the biochemical parameters detected, was observed. Diltiazem treated animals showed a reduction in the lesion severity, at the level of aortic valves, coronary arteries and aortic arch; we assume that Diltiazem acts on the early phases of atherosclerosis by blocking the lipid transport and accumulation into the subendothelial space. In contradistinction, Nifedipine treatment failed to suppress the atherogenic effect of fat-rich diet, and as in AT hamsters, the plaques developed in all lesion-prone areas. The latter were characterised by numerous lipid-laden cells (in aorta and aortic valves) and calcification and necrotic centres, in all locations, including coronary arteries. The results suggest a different mechanism of action and the ensuing effects of various CCB on atherogenesis.

Animals↗
PubMed1995

Irreversibly glycated albumin alters the physico-chemical characteristics of low density lipoproteins of normal and diabetic subjects.

In diabetic plasma, glycated albumin and glycated LDL coexist with augmented levels of peroxides, conditions frequently associated with the development of accelerated atherosclerosis. The direct interaction between irreversibly glycated albumin, LDL and oxidation have not been explored yet. We tried to elucidate whether irreversibly glycated albumin (AGE-Alb) induces changes in the chemistry and morphology of LDL particle, and if AGE-Alb has the ability to scavenge free radicals, as reported for native albumin. LDL isolated from normal (nLDL) or diabetic human subjects (dLDL) was incubated in vitro with AGE-Alb in conditions of autoxidation (37 degrees C, 24-48 h in the absence of oxidation inhibitors) or of Cu2+ induced-oxidation. The results showed that, especially in the latter condition, AGE-Alb induced marked physico-chemical modifications of both nLDL and dLDL without significant changes in the level of peroxides. Incubation with AGE-Alb decreased the cholesteryl esters/unesterified cholesterol ratio of nLDL by 30% and of dLDL by approximately 50%. Concomitantly, in oxidative conditions a marked increase (approximately 3-fold) in the lysophosphatidylcholine/phosphatidylcholine ratio of dLDL was detected. Apolipoprotein B integrity as well as the morphology of the lipoprotein particles were drastically affected. To a lesser extent, these modifications occurred also in the presence of inhibitors of oxidation at 37 degrees C, but not at 4 degrees C. The above described effects were constantly more pronounced in the case of dLDL. These results indicated that in the absence of other plasma or vascular tissue components (e.g., endothelial cells, extracellular matrix) AGE-Alb by itself induces alterations in the chemistry and morphology of LDL, especially of glycated LDL, modifications that may account for the occurrence of accelerated atherogenesis in diabetes.

Arteriosclerosis↗
PubMed1995

Intimal thickenings of human aorta contain modified reassembled lipoproteins.

The aim of this study was to determine whether in human aortas early minute changes such as minimal intimal thickenings (MIT), developed in areas known to have a predilection to atherosclerosis, contain modified reassembled lipoproteins (MRLp) such as extracellular liposomes (EL) and lipid droplets (LD). These features have been previously detected in the aortic lesion-prone areas of rabbits and hamsters fed a fat-rich diet. Tissue samples of the aortic arch and thoracic aorta from 12 young subjects who died in accidents were selectively collected from grossly normal regions. By light microscopy, some of these regions were found to contain MIT. The normal areas and the MIT were separately examined by electron microscopy or subjected to fractionation and partial biochemical characterization. The MIT (approximately 25-100 microns thick) were constituted by a pronounced proliferation of extracellular matrix, especially elastin and microfibrils, with interspersed lipid deposits appearing as EL and LD. Commonly, MIT did not contain smooth muscle cells, macrophages, foam cells or cytolytic debris. Such components were only occasionally found in specimens excised from the vicinity of fatty streaks. Saline extracts of MIT or grossly normal aortic regions were subjected to a four-step purification procedure consisting of gel filtration, affinity chromatography on anti-apo B and anti-albumin Sepharose, followed by density gradient ultracentrifugation. The entire procedure was monitored by negative staining, lipid assays, SDS PAGE and immunoblotting. From the initial MRLp mixture, two fractions were obtained: fraction 1 containing multilamellar EL and LD, and fraction 2 composed mostly of unilamellar EL. As compared with serum LDL, the cholesteryl ester/unesterified cholesterol ratio was 4-6-fold lower in fraction 1 and 15-19-fold lower in fraction 2. On SDS-PAGE the fraction 2 displayed a single protein band of 66 kDa, immunochemically identified as albumin. The MRLp isolated from human aortas with minimal intimal thickenings appeared to be similar to those purified from the prelesional stage aorta of hyperlipidemic rabbits and hamsters.

Adolescent↗
PubMed1995

Ultrastructural evidence of differential solubility in Triton X-100 of endothelial vesicles and plasma membrane.

Endothelial plasmalemmal vesicles (EV) are distinct membrane-bound structures characteristic for all vascular endothelia, being involved in transcytosis of plasma macromolecules. EV are considered to be similar to the caveolae (characterized by a specific peptide called caveolin) found in other cell types. Caveolin-rich membrane domains were recently isolated from whole lung and chicken gizzard as a Triton X-100 (TX)-insoluble membrane fraction. However, ultrastructural data on the localization of these domains within cells have not yet been reported. We have examined whether EV are TX-insoluble structures. Cultured bovine aortic endothelial cells (BAEC) briefly fixed in paraformaldehyde (10 min, 37 degrees C) were exposed to 0.1% TX for 5 min at 22 degrees C and further subjected to standard electron microscopy procedure. The results showed an extensive solubilization of endothelial plasmalemma as well as other intracellular membranes. Individual or clusters of EV were not affected by TX extraction, retaining their trilaminar unit membrane appearance and dimensions. Moreover, a crude membrane fraction prepared from unfixed BAEC was also extracted with 1% TX for 20 min at 4 degrees C and the insoluble material was examined by electron microscopy. In this fraction clusters of about 10 membranous vesicles were found. These data suggest that EV and plasma membrane have a different lipid composition; the low TX solubility is a characteristic common to caveolin-rich domains (caveolae) of other cells types and EV, whereas the ultrastructural complexity and intracellular localization of the latter are specific for endothelia.

Animals↗
PubMed1994

The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.

Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane.

Amino Acid Sequence↗