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.

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.

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.

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.

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.

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.

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.

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.

Binding of vascular anticoagulant alpha (annexin V) to the aortic intima of the hypercholesterolemic rabbit. An autoradiographic study.

Qualitative ultrastructural autoradiography was used to study the binding of the vascular anticoagulant alpha (annexin V) to normal and atherosclerotic (AS) rabbit aortic intima. Recombinant annexin V was labelled with 125I by the Iodogen method. Rabbits were fed a hypercholesterolemic (HC) diet up to 10 months. After laparotomy and exsanguination, the aortae were perfused with dilutions of 125I-annexin V (I-AV) for 10-15 min, either on ice or at 22 degrees C and then perfusion-fixed with aldehydes. Fragments of the labelled aortae were used for en face contact autoradiography, followed by Sudan Black staining of intimal lipid. Specimens were also included in Epon and sectioned for light- and electron-microscopic autoradiography. The binding of I-AV was increased on the AS aortae as compared with the normal ones, with an apparent preference for the lesioned areas. Microscopically, I-AV was found at the luminal front of aortic intima, on endothelial cells (EC), on macrophage foam cells, and on their disrupted remnants. The presence of the AV binding sites (reportedly known to interact with high affinity with phosphatidylserine) in the rabbit AS aortic intima, together with other known procoagulant conditions, may contribute to the initiation of coagulation events into the lesioned vascular wall, and may offer a rationale for the use of annexin V as an anticoagulant drug.

4-Hydroxynonenal induces membrane perturbations and inhibition of basal prostacyclin production in endothelial cells, and migration of monocytes.

Cultured bovine aortic endothelial cells (BAEC) were incubated for 5 days with 10(-5) 4-hydroxynonenal (HN). HN treated BAEC and controls were either (i) further incubated with 125I-polymyxin B (IPxB) or with radioiodinated, inactivated coagulation factor Xa (IFXai) as markers of membrane phospholipid perturbation, or (ii) assayed for the synthesis of prostacyclin (PGI2) and thromboxane A2 (TXA2). Rabbit blood mononuclear cells enriched in monocytes (MC) were isolated and assayed for chemotactic response to HN. The results showed six - fold increases of IPxB and IFXai binding to BAEC treated with HN, as compared to untreated controls. We also found in HN treated cells a marked inhibition of PGI2 synthesis, but an unmodified TXA2 production. In addition, HN in the 10(-5)-10(-10) M range induced oriented migration of MC.

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)

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.

Intrinsic procoagulant surface induced by hypercholesterolaemia on rabbit aortic endothelium.

The effect of hyperlipidaemia on endothelial cell haemostatic properties was examined using ex vivo studies on aortic segments obtained from fat-fed Chinchilla rabbits, mounted in a template device which exposed the luminal surface. Exposure of arterial endothelium to lipids resulted in marked enhancement of externally exposed anionic phospholipids, detected using either fluorescence microscopy with the probe merocyanine 540 or by binding of 125I-polymyxin B and 125I-Annexin V. Consistent with the known procoagulant properties of anionic phospholipid, following the lipid and cholesterol-rich diet intake, intact endothelial cells demonstrated enhanced binding of radioiodinated factors IX/IXa and Xa, and enhanced factor IXa/VIII-dependent factor X activation and factor Xa-factor Va-mediated prothrombin activation. Both factor Xa and thrombin formation were blocked, in large part, by polymyxin B, suggesting dependence of the reaction on anionic phospholipids. Consistent with these results, evidence of increased activation of the coagulation mechanism in vivo was observed in hyperlipidaemic animals, as assessed by a three-fold increase in levels of circulating antithrombin-protease complexes, compared with normolipidaemic controls.

Posterior fossa extradural hematomas in children: report of nine cases.

Extradural post-traumatic posterior fossa hematoma is a rare condition estimated to complicate about 0.13% of all craniocerebral injuries, and represents 5.8% of the entire group of extradural hematomas in our records. Nine cases of posterior fossa extradural hematomas are presented. The clinical picture was dominated by headache, vomiting, and gait ataxia. An occipital fracture was seen in 77.7% of the patients. In all cases, the diagnosis was made by computed tomography. The postoperative evolution was good.

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.

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.

Some Major Plasmalemma Proteins of Human Diabetic Platelets are Involved in the Enhanced Platelet Adhesion to Cultured Valvular Endothelial Cells.

To extend our investigations on the interaction between diabetic platelets and endothelium, we tried to identify the molecular components involved in the increased adhesiveness of diabetic platelets to cultured valvular endothelial cells (VEC). Platelets from diabetic patients were radiolabeled with ((3)H]-adenine, incubated for 30 min at 37°C with confluent VEC grown in medium containing 4.5 g/L glucose, and the monolayer-associated radioactivity was used to calculate the adhesion index. To identify the plasmalemma proteins involved in the adhesion process, platelets were incubated for 30 min prior to (the) adhesion assay with one of the following monoclonal antibodies: AP-2 (anti GP IIb-IIIa), AP-5 (anti GP IIIa), TM 83 (recognizes an epitope other than the fibrinogen binding site in GP IIIa), PECAM 1.2 (anti PECAM-1) or a polyclonal anti-fibronectin receptor (anti FnR). In addition, two synthetic peptides, RGDS and GPRP, applied alone or together, were used. The effect of paraformaldehyde fixation of diabetic platelets on their adhesion was also tested. The results showed that except for TM 83, all antibodies reduced significantly (∼45%) the adhesion index of diabetic platelets to VEC. The synthetic peptides also decreased the adhesion by ∼30%. Paraformaldehyde-fixed diabetic platelets fail to adhere to VEC. Taken together these observations suggest that: (1) platelet GP IIb-IIIa complex, PECAM-1 and FnR may be instrumental in the increased adhesion of diabetic platelets to VEC; (2) fibrinogen binding sites in the GP IIb-IIIa complex and fibrinogen/fibrin are important contributors to the adhesion process and (3) impairment of diabetic platelets adhesion by chemical fixation, supports the role of cytoskeletal proteins reorganization and redistribution of some plasmalemma components during adhesion.

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)