concanavalin-a and Arteriosclerosis

Topics: Animals; Aorta; Arteriosclerosis; Cholesterol, Dietary; Concanavalin A; Guinea Pigs; Microscopy, Electron, Scanning; Rabbits; Serum Albumin, Radio-Iodinated

Although several lines of evidence support the role of calcifiable vesicles in dystrophic vascular calcification, the mechanisms whereby vesicles promote aortic calcification remain incompletely understood. Previous reports indicate that ATP promotes in vitro vesicle calcification. Whether ATP-initiated calcification is simply mediated through increased Pi concentrations or by other unknown mechanisms related to ATP hydrolysis is unclear. To determine whether high Pi levels resulting from ATP hydrolysis may cause CaxP ion products to surpass the threshold for calcium phosphate precipitation, 3 mM Pi instead of 1 mM ATP was added to calcifying media. The inclusion of 1 mM ATP in calcifying media with an initial serum level of Ca2+ (1.45 mM) and Pi (2.3 mM) was much more effective in promoting calcification than the addition of 3 mM Pi. The higher effectiveness of ATP over Pi in promoting calcification was consistent throughout various incubation periods and vesicle protein ranges. To minimize the effect of CaxPi ion products on calcification, the ion product was kept within the physiological ranges throughout the incubation period by reducing initial Pi or ATP concentrations in calcifying media. At these low levels of ion products, ATP was still more effective than Pi in promoting calcification. Both ATP- and Pi-stimulated calcifications were found to increase with increasing levels of ion products whereas greater effectiveness of ATP over Pi remained unaltered. These observations indicate that ATP hydrolysis may initiate calcification through some mechanisms other than a simple provision of Pi in order to surpass the solubility products. Concanavalin A (Con A) was found to bind to vesicles and to enhance both ATP- and Pi-promoted calcification. Taken together, these observations suggest that ATP hydrolysis, CaxP ion products, and vesicle-associated carbohydrates are implicated in vesicle-mediated calcification.

Topics: Adenosine Triphosphate; Animals; Aorta, Thoracic; Aortic Diseases; Arteriosclerosis; Calcinosis; Calcium; Calcium Chloride; Concanavalin A; In Vitro Techniques; Phosphates; Rabbits

Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles' membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca(2+), Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on (45)Ca and (32)Pi deposition in a dose-dependent fashion with a half-maximal effect at 6-10 microg/ml. Either alpha-methylmannoside or alpha-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in (45)Ca and (32)Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N-acetylglucoside nor N-acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of le

Topics: Animals; Aorta; Aorta, Thoracic; Arteriosclerosis; Calcification, Physiologic; Cholesterol, Dietary; Collagenases; Concanavalin A; Diet, Atherogenic; Lectins; Rabbits; Spectroscopy, Fourier Transform Infrared

Effects of experimental hyperlipidemia on apoptosis and proliferation of thymocytes in response to mitogens were studied in CBA and C57Bl/6 mice. The concentrations of cholesterol in the serum and thymocyte membranes increased in both mouse strains. Spontaneous and dexamethasone-induced apoptosis in vitro and the proliferative response to phytohemagglutinin and concanavalin A were enhanced in thymocytes from C57Bl/6 mice and suppressed in cells from CBA mice. These data suggest opposite reactions of thymocyte to increased serum cholesterol concentration in these two strains, associated with stimulation and suppression of cell activity.

Topics: Animals; Apoptosis; Arteriosclerosis; Cell Membrane; Cholesterol; Concanavalin A; Dexamethasone; Diet, Atherogenic; Hyperlipidemias; In Vitro Techniques; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Phytohemagglutinins; Species Specificity; T-Lymphocytes

Immunocytochemical analyses of human plaques and experimental arterial lesions have implicated activated lymphocytes and monocytes in the pathogenesis of atherosclerosis, as demonstrated by the expression of interleukin-2 (IL-2) membrane receptors and major histocompatibility complex class II epitopes. The objective is to determine if targeting these cells with an IL-2 receptor-specific chimeric toxin, DAB486-IL-2, can inhibit experimental post-angioplasty vascular neointimal thickening. Twenty-two atherogenically modeled rabbits were treated in vivo with DAB486-IL-2 (0.1 mg/kg per day i.v.; n = 11) or placebo (n = 11) for 10 days following aortic balloon angioplasty (4 atm x 30 s each x 2 dilatations). In vitro 3H-leucine incorporation studies of mononuclear leukocyte and vascular smooth muscle cell protein synthesis inhibition by DAB486-IL-2 were also performed. Angioplasty sites were examined for evidence of hyperproliferative atherosclerotic narrowing by quantitative angiography and histomorphometry of neointimal cross-sectional area at baseline and 6 weeks after injury. In vitro Concanavalin-A stimulated rabbit mononuclear leukocyte protein synthesis was 50% inhibited by DAB486-IL-2 at a concentration (IC50) of 6 x 10(-11) M. Rabbit vascular smooth muscle cells were approximately 150-fold less sensitive to DAB486-IL-2 (IC50 = 10(-8) M). In vivo studies showed no change in angioplasty site angiographic minimum luminal diameter at 6 weeks in DAB486-IL-2 treated animals (from 2.96 +/- 0.52 to 2.96 +/- 0.48 mm; percent cross-sectional area reduction = 1 +/- 10%; P = N.S.). In control animals, luminal diameter decreased from 2.79 +/- 0.4 to 2.32 +/- 0.52 mm at 6 weeks, and percent cross-sectional area was reduced by 34 +/- 14% (P < 0.01 vs. placebo). Quantitative histomorphometric angioplasty segmental intimal cross-sectional area reduction of treated and placebo vessels also differed significantly (19 +/- 16% vs. 31 +/- 21%; P < 0.05). DAB486-IL-2 caused no adverse effects on animal survival, weight or hepatic transaminase levels. We conclude that post-angioplasty administration of the chimeric toxin DAB486-IL-2 inhibits angiographic narrowing and neointimal thickening in the atherogenic rabbit model. Although this IL-2 receptor-specific molecule was cytotoxic in vitro for activated mononuclear leukocytes and vascular smooth muscle cells, systemic toxicity did not occur in vivo at a dose comparable to that evaluated in clinical trials of this agent. Pote

Topics: Angioplasty, Balloon; Animals; Aorta, Abdominal; Arteriosclerosis; Concanavalin A; Cytotoxins; Diet, Atherogenic; Diphtheria Toxin; Female; Iliac Artery; Interleukin-2; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Muscle, Smooth, Vascular; Rabbits; Receptors, Interleukin-2; Recombinant Fusion Proteins; Recurrence

P388D1 macrophage-like cells have previously been shown to produce both mitogenic and inhibitory regulators of porcine smooth muscle cell (pSMC) growth. The mitogenic activity was shown to have a molecular mass of > 10 kDa while the inhibitory activity was in the range of 2-6 kDa. In the present study, we present a novel dialysis culture system where P388D1 cells were grown in dialysis membranes with a 12 kDa cut-off which allowed continuous production of fractions of the culture medium. Using pSMC as target cells, mitogenic activity was found to be retained by the dialysis membrane while the low molecular mass inhibitory activity passed freely through the membrane. The effect of the macrophage-activators phorbol myristate acetate (PMA), concanavalin A (ConA) and interferon-gamma in combination with lipopolysaccharide (IFN gamma/LPS) were investigated in the dialysis culture system. PMA, ConA and IFN gamma/LPS were found to enhance the production of mitogenic activity by P388D1 cells. PMA also increased the production of growth-inhibitory activity, while ConA abolished inhibitor production and IFN gamma/LPS had no effect on the amount of inhibitory activity produced by P388D1 cells. The experiments show that the balance of production of mitogenic and inhibitory activities by macrophages can be modulated by agents that alter the state of activation of the cells. This could be of profound significance in the influence of macrophages on smooth muscle cell growth during the development of atherosclerosis.

Topics: Animals; Arteriosclerosis; Cell Division; Cell Line; Concanavalin A; Culture Media, Serum-Free; Culture Techniques; Dialysis; Interferon-gamma; Kinetics; Lipopolysaccharides; Macrophage Activation; Macrophages; Models, Biological; Rabbits; Swine; Tetradecanoylphorbol Acetate; Thymidine

In order to assess the possible utility of lectin binding to identify the cellular components of fixed arterial lesions we studied lectin binding in experimental rabbit and monkey vessels, as well as in human atherosclerotic arteries obtained at surgery. The avidin-biotin-peroxidase technique was used to localize the binding of the following biotinylated lectins: Concanavalin A (Con A), Dolicho biflorus agglutinin (DBA), soybean agglutinin (SBA), peanut agglutinin (PNA), Phaseolus vulgaris agglutinin (PHA), Ricinus communis agglutinin (RCA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin (UEA). PHA demonstrated specific cytoplasmic staining of macrophages in rabbit, monkey, and human tissues and differentiated macrophages from other cell types in atherosclerotic lesions. When morphometric comparisons were made between lesion PHA staining and another macrophage marker, acid lipase, very similar results were obtained. Con A, RCA, and WGA stained macrophages intensely and differentiated them from other cell types in normal reticuloendothelial tissues and lesions, but also stained smooth muscle cells and endothelial cells when these cells developed lipid vacuoles. UEA stained the endothelium of vasa vasorum consistently in human arteries, but staining of artery lumen endothelium was variable. Endothelial cells of rabbit or monkey vessels did not stain with UEA. DBA, PNA, and SBA did not consistently stain any cellular structures in arteries. PHA was found to be an excellent marker to differentiate and quantify macrophages in glutaraldehyde or formalin-fixed, paraffin-embedded experimental and human atherosclerotic lesions. Con A, RCA and WGA merit further detailed study in conjunction with other histochemical tests as possible markers of functional changes in arterial cells during lesion development.

Topics: Animals; Aorta; Arteries; Arteriosclerosis; Concanavalin A; Endothelium; Histocytochemistry; Humans; Immunoenzyme Techniques; Lectins; Macaca fascicularis; Macrophages; Peanut Agglutinin; Phytohemagglutinins; Plant Lectins; Rabbits; Soybean Proteins; Wheat Germ Agglutinins

Delayed appearance of atherosclerotic lesions in cerebral arteries has been observed not only in man but also in monkeys and rabbits submitted to atherogenic diets. Previous observations of ours had shown a Con A positive reaction ("glycocalyx" or "surface coat") at the luminal surface and in the plasmalemmal vesicles of aortic endothelial cells of rabbits an other laboratory animals. The "surface coat" is now reputed the site of lipoproteinlipase activity whose importance in atherogenesis has recently been stressed. In our present observations, the endothelial cells Con A reactivity after Bernhard and Avrameas which was not previously studied in the cerebral arteries of rabbits and monkeys has resulted always lacking in this arterial district. Those observations may help explaining delayed appearance of atherosclerotic lesions in cerebral arteries.

Topics: Animals; Aorta; Arteriosclerosis; Cerebral Arteries; Cholesterol, Dietary; Concanavalin A; Coronary Disease; Coronary Vessels; Endothelium; Intracranial Arteriosclerosis; Macaca fascicularis; Rabbits

Topics: Adult; Arteriosclerosis; Cell Division; Concanavalin A; Humans; Immunity, Innate; Leukocyte Count; Middle Aged; T-Lymphocytes, Regulatory

Modifications of the aortic endothelial surface coat have been visualized at SEM with the use of the Con A-haemocyanin method. After fifteen days of an atherogenic diet, a strong increase of the reactive coat was evident in areas near the orifice of the collateral branches. In other areas, the reaction appeared to be intensely diminished.

Topics: Animals; Aorta; Arteriosclerosis; Cholesterol, Dietary; Concanavalin A; Diet, Atherogenic; Hemocyanins; Microscopy, Electron, Scanning; Rabbits; Receptors, Concanavalin A; Time Factors

Methylprednisolone injections inhibit the thickness increase of the Concanavalin A reactive coat at the endothelial cell surface both in rabbits on a hypercholesterolic diet or submitted to pig aortic homogenate injections.

Topics: Animals; Aorta; Arteriosclerosis; Carbohydrate Metabolism; Cell Membrane; Cholesterol, Dietary; Concanavalin A; Diet, Atherogenic; Freund's Adjuvant; Methylprednisolone; Microscopy, Electron, Scanning; Rabbits; Swine; Tissue Extracts

Topics: Animals; Aorta; Arteriosclerosis; Cholesterol, Dietary; Concanavalin A; Diet, Atherogenic; Endothelium; Guinea Pigs; Histocytochemistry; Lectins; Microscopy, Electron; Rabbits; Rats