Low-dose radiation exposure and protection against atherosclerosis in ApoE(-/-) mice: the influence of P53 heterozygosity.

We recently described the effects of low-dose γ-radiation exposures on atherosclerosis in genetically susceptible (ApoE(-/-)) mice with normal p53 function. Doses as low as 25 mGy, given at either early or late stage disease, generally protected against atherosclerosis in a manner distinctly nonlinear with dose. We now report the influence of low doses (25-500 mGy) on atherosclerosis in ApoE(-/-) mice with reduced p53 function (Trp53(+/-)). Single exposures were given at either low or high dose rate (1 or 150 mGy/min) to female C57BL/6J ApoE(-/-) Trp53(+/-) mice. Mice were exposed at either early stage disease (2 months of age) and examined 3 or 6 months later, or at late stage disease (7 months of age) and examined 2 or 4 months later. In unirradiated mice, reduced p53 functionality elevated serum cholesterol and accelerated both aortic root lesion growth and severity in young mice. Radiation exposure to doses as low as 25 mGy at early stage disease, at either the high or the low dose rate, inhibited lesion growth, decreased lesion frequency and slowed the progression of lesion severity in the aortic root. In contrast, exposure at late stage disease produced generally detrimental effects. Both low-and high-dose-rate exposures accelerated lesion growth and high dose rate exposures also increased serum cholesterol levels. These results show that at early stage disease, reduced p53 function does not influence the protective effects against atherosclerosis of low doses given at low dose rate. In contrast, when exposed to the same doses at late stage disease, reduced p53 function produced detrimental effects, rather than the protective effects seen in Trp53 normal mice. As in the Trp53 normal mice, all effects were highly nonlinear with dose. These results indicate that variations in p53 functionality can dramatically alter the outcome of a low-dose exposure, and that the assumption of a linear response with dose for human populations is probably unwarranted.

Low-dose radiation exposure and atherosclerosis in ApoE⁻/⁻ mice.

The hypothesis that single low-dose exposures (0.025-0.5 Gy) to low-LET radiation given at either high (about 150 mGy/min) or low (1 mGy/min) dose rate would promote aortic atherosclerosis was tested in female C57BL/6J mice genetically predisposed to this disease (ApoE⁻/⁻). Mice were exposed either at an early stage of disease (2 months of age) and examined 3 or 6 months later or at a late stage of disease (8 months of age) and examined 2 or 4 months later. Changes in aortic lesion frequency, size and severity as well as total serum cholesterol levels and the uptake of lesion lipids by lesion-associated macrophages were assessed. Statistically significant changes in each of these measures were observed, depending on dose, dose rate and disease stage. In all cases, the results were distinctly non-linear with dose, with maximum effects tending to occur at 25 or 50 mGy. In general, low doses given at low dose rate during either early- or late-stage disease were protective, slowing the progression of the disease by one or more of these measures. Most effects appeared and persisted for months after the single exposures, but some were ultimately transitory. In contrast to exposure at low dose rate, high-dose-rate exposure during early-stage disease produced both protective and detrimental effects, suggesting that low doses may influence this disease by more than one mechanism and that dose rate is an important parameter. These results contrast with the known, generally detrimental effects of high doses on the progression of this disease in the same mice and in humans, suggesting that a linear extrapolation of the known increased risk from high doses to low doses is not appropriate.

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.

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.

Receptor for advanced glycation end products (AGEs) has a central role in vessel wall interactions and gene activation in response to circulating AGE proteins.

The extended interaction of aldoses with proteins or lipids results in nonenzymatic glycation and oxidation, ultimately forming AGEs, the presence of which in the plasma and vessel wall is associated with diabetic vascular complications. We show here that AGE albumin in the intravascular space interacts with the vessel wall via binding to an integral membrane protein, receptor for AGE (RAGE), a member of the immunoglobulin superfamily, resulting in clearance from the plasma and induction of interleukin 6 mRNA. Intravenously infused 125I-AGE albumin showed a rapid phase of plasma clearance with deposition in several organs. Rapid removal of 125I-AGE albumin from the plasma was prevented by administration of a soluble, truncated form of RAGE, which blocked binding of 125I-labeled AGE albumin to cultured endothelial cells and mononuclear phagocytes, as well as by pretreatment with anti-RAGE IgG. Ultrastructural studies with AGE albumin-colloidal gold conjugates perfused in situ showed that in murine coronary vasculature this probe was taken up by endothelial plasmalemmal vesicles followed by transport either to the abluminal surface or by accumulation in intracellular vesicular structures reminiscent of endosomes and lysosomes. Consequences of AGE-RAGE interaction included induction of interleukin 6 mRNA expression in mice. These data indicate that RAGE mediates the interaction of AGEs with the vessel wall, both for removal of these glycated proteins from the plasma and for changes in gene expression.

Cardiomyocytes express albumin binding proteins.

We investigated whether cardiomyocytes express specific albumin binding proteins (ABP) which may function in the dissociation of fatty acids from their non-covalent complexes with albumin. The experiments were performed on rat neonatal cardiomyocytes (freshly isolated and up to 3 days in culture) and on an enriched sarcolemmal fraction isolated from adult rabbit ventricular myocardium. Three types of experiments were conducted: (a) identification of ABP on electroblots of cardiomyocytes and sarcolemmal extracts reacted with [125I]-bovine serum albumin ([125I]Alb); (b) kinetic assays of [125I]Alb interaction with cardiomyocytes (at 37 degrees C), and with a sarcolemmal fraction (at 4 degrees C); (c) affinity isolation of ABP from solubilized radioiodinated sarcolemmal proteins interacted with an albumin-agarose matrix. The investigation showed that: first, two pairs of polypeptides (ABP of M(r) 18 and 31 kDa) in either cardiomyocytes or sarcolemmal fraction reacted on electroblots with [125I]Alb; second, the binding of the latter to cardiomyocytes was saturable and competed by unlabeled albumin: 50 microM albumin reduced by approximately 90% the binding of radiolabeled albumin. The sarcolemmal fraction bound [125I]Alb with a Kd of 3.66 x 10(-7) M. Thirdly, among the sarcolemmal proteins retained by the albumin-agarose matrix (18 and 31 kDa), the most prominent was the lower band (approximately 16 kDa) of the 18 kDa pair of ABP. The observations revealed that albumin interacts with relatively high affinity with specific binding sites on cardiomyocyte sarcolemma. This interaction may be a recognition step for subsequent fatty acid dissociation and translocation.

Selective radioiodination of the apical and luminal cell surfaces: in vitro and in situ experiments on vascular endothelial cells with Iodogen-coated Sephadex.

A gentle and nonexpensive agent for selective radioiodination of the cell surface proteins was obtained by plating aliquots of Iodogen on dried Sephadex beads 50-60 microns in diameter. Iodogen-coated Sephadex inherits Iodogen properties: it is stable and virtually insoluble in water, allowing rapid iodination of the cell surface proteins in the solid phase with 125I-. Iodination is terminated by simply removing the beads. The agent was tested on bovine aortic endothelial cells in culture and on rabbit aortic endothelial cells in situ. Light and electron microscopic studies revealed that during radioiodination, apparently no ultrastructural modifications occurred in the endothelial cells. In addition, experiments with 51Cr (used as an indicator of endothelial cell injury) demonstrated that during iodination the cell integrity was preserved. The technique reported here may be generally applied for selective radioiodination of the apical surface proteins of various cultured cells and of the luminal endothelial surface of large blood vessels.

Evaluation of a new method for the prevention of neonatal anemia.

Blood sampling for diagnostic purposes is a major cause of neonatal anemia. We propose a new method of blood sampling which preserves the infant's erythrocytes. Upon drawing of 1.5-ml blood samples, the syringe is centrifuged and erythrocytes are injected back into the circulation. Using rabbits as an experimental model, we documented the efficiency of this method in decreasing the need for foreign blood transfusion. Results of a variety of laboratory tests performed with plasma obtained with the new method were identical to those achieved with whole blood. Subsequent testing did not show increased risk of contamination or bleeding phenomena secondary to heparin load. Red blood cells obtained by centrifugation were morphologically normal. This new method may be effective in reducing the need for foreign blood transfusions in neonates.

Identification of albumin-binding proteins in capillary endothelial cells.

Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit.

Chemical mapping of the luminal surface of normal rabbit aortic and coronary endothelium was investigated cytochemically to establish a baseline for further comparison with the biochemical changes possibly induced by the experimental hypercholesterolemia. Morphometric analysis showed that in the aortic endothelium the plasma membrane exposes a large number of uniformly-distributed positively-charged groups of high pKa, and a heterogeneous pattern of dense anionic groups of low pKa. Among the latter, only a third was represented by neuraminidase-cleavable sialic acids. These are constituted by various classes of N-, and O-substituted sialyl residues in glycoconjugates, most frequent being those non-O-acetylated at C8 or C9. Among the oligosaccharides detected with lectins, very abundant were the glycoconjugates containing mannosyl and subterminal galactosyl, whereas N-acetyl-glucosamine, terminal galactosyl and N-acetyl-galactosaminyl moieties were rather poorly represented. The density of the latter two markedly increased after its unmasking by neuraminidase treatment. Coated pits contained both anionic and cationic sites, but only few sialic acids and saccharide residues in significantly lower amounts than plasma membrane. The membrane of plasmalemmal vesicles displayed a high number of cationic sites and mannosyl residues, but very few anionic groups, sialyl residues, and galactosyl and N-acetyl-galactosaminyl moieties. Coronary endothelium displayed a chemical pattern similar to aorta, with some differences, especially in the frequency of some oligosaccharides. Vena cava was low in acidic groups but rather rich in galactose. Plasmalemmal vesicles were only occasionally labeled by the probes used. Monocyte surface exhibited a high density of anionic sites, and binding sites for wheat germ agglutinin and Ricinus communis agglutinin. No mononuclear cells were observed adhering to endothelial surface.

Prelesional events in atherogenesis. Changes induced by hypercholesterolemia in the cell surface chemistry of arterial endothelium and blood monocytes, in rabbit.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.

Charge effect on binding, uptake and transport of ferritin through fenestrated endothelium.

The binding, uptake and transport of hydrosoluble macromolecules as a function of their electric charge have been investigated in mouse pancreatic exocrine fenestrated endothelium. After intravenous injection of ferritin derivatives with different isoelectric points (3.7, 4.5, 7.0 and 8.4) specimens of pancreas were collected at 5, 25, 45 and 60 min and processed for electron microscopy. Although the concentrations (10 mg/ml) and injected volumes (1 ml/100 g body weight) were identical, the tracers with similar molecular weight (Mr approximately 960,000), but with different net electric charge, followed different routes in endocytotic and transcytotic processes. The endothelial cell internalizes ferritins into multivesicular bodies and lysosomes. While anionic ferritins are endocytosed via plasmalemmal vesicles (fluid phase endocytosis), the cationic derivatives are taken up exclusively by coated pits (adsorptive endocytosis). The anionic and neutral macromolecules seem to be transcytosed via plasmalemmal vesicles. The circulating polycations are aggregated by plasma proteins and transcytosed as such either in fluid phase by large vacuoles, or by adsorption on the coated structures. The first mechanism is physiological, the latter probably appears in unusual conditions by which the endothelium participates in the plasma clearance. Our findings indicate that, for the transport of macromolecules, the capillary endothelium can be accounted not only as a size barrier but also as a selective-differentiated charge barrier with complex and multiple mechanisms for the modulation of its transport systems. The endocytotic and transcytotic processes undergo the effect of net electric charge of the plasma components.