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Biomedical subjects

H Vlassara

Publications and source records attributed to H Vlassara.

At least 55 records · Page 3Linked to original sources
PubMed1995

Antibacterial activity of lysozyme and lactoferrin is inhibited by binding of advanced glycation-modified proteins to a conserved motif.

Why diabetes is associated with abnormally high susceptibility to infection remains unknown, although two major antibacterial proteins, lysozyme and lactoferrin, have now been shown to specifically bind glucose-modified proteins bearing advanced glycation end products (AGEs). Exposure to AGE-modified proteins inhibits the enzymatic and bactericidal activity of lysozyme, and blocks the bacterial agglutination and bacterial killing activities of lactoferrin. Peptide mapping revealed a single AGE binding domain in lysozyme and two AGE binding domains in lactoferrin; each domain contains a 17- to 18- amino acid cysteine-bounded loop motif (CX15-16C) that is markedly hydrophilic. Synthetic peptides corresponding to these motifs in lysozyme and lactoferrin exhibited AGE binding activity, and similar domains are also present in other antimicrobial proteins. These results suggest that elevated levels of AGEs in tissues and serum of diabetic patients may inhibit endogenous antibacterial proteins by binding to this conserved AGE-binding cysteine-bounded domain 'ABCD' motif, thereby increasing susceptibility to bacterial infections in the diabetic population.

Amino Acid Sequence↗
PubMed1995

Advanced glycosylation endproducts in diabetic renal disease: clinical measurement, pathophysiological significance, and prospects for pharmacological inhibition.

Glucose reacts nonenzymatically with amino groups to produce a class of stable, crosslinking moieties termed advanced glycosylation endproducts (AGEs). These products act to increase vascular permeability, enhance subintimal protein and lipoprotein deposition, inactivate nitric oxide, and exert a number of toxic effects of endothelial cells. Loss of normal renal function has been found recently to cause a marked increase in the circulating levels of plasma AGEs, suggesting that these moieties may comprise one component of the so-called uremic 'middle molecules'. AGEs also form on the lipid and the protein components of LDL, forming a modified form of LDL (AGE-LDL) which is not taken up by tissue LDL receptors. Aminoguanidine offers a specific therapeutic modality for inhibiting advanced glycosylation in vivo and has been observed recently to reduce both hemoglobin-AGE and circulating LDL levels.

Animals↗
PubMed1995

Immunological evidence for the presence of advanced glycosylation end products in atherosclerotic lesions of euglycemic rabbits.

Atherosclerosis is known to be accelerated in diabetic patients, but the mechanisms of this acceleration are poorly understood. Nonenzymatic glycosylation of long-lived proteins results in the formation of advanced glycosylation end products (AGEs), which are extensively cross-linked and could contribute to atherogenesis. Oxidative modification of LDL is also an important process in atherogenesis. In vitro evidence suggests that hyperglycemia may enhance lipid peroxidation, and conversely, that increased lipid peroxidation may enhance AGE formation. If such interactions occur in vivo, we hypothesized that AGE should be found in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits in areas rich in lipids and oxidized lipoproteins. To demonstrate the presence of AGEs, we developed antisera against a specific "model" compound of AGE, 2-furoyl-4(5)-(2-furanyl)-1H-imidazole (FFI) by using FFI-hexanoic acid (FFI-HA)-protein adducts as the antigen and against AGEs in general by using AGE-albumin as the antigen. Antisera generated with FFI-HA-protein adducts recognized FFI-HA alone as well as FFI-protein adducts. Native proteins or proteins conjugated with aldehydes formed during lipid peroxidation in vitro were not recognized by these antisera. Immunocytochemistry with both FFI-specific and AGE-specific antisera revealed the presence of these epitopes in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits but not in normal aortic tissues. AGE epitopes within atherosclerotic lesions were predominantly found in similar locations as epitopes generated during modification of the lipoproteins by oxidation, consistent with the hypothesized interactions between oxidation and glycosylation. Indirect evidence in support of the in vivo presence of FFI-like structures was also obtained by the observation that both diabetic and euglycemic human subjects contained autoantibodies that recognize FFI-protein adducts. Taken together, these data provide immunological evidence for the in vivo presence of FFI-like structures and other AGE-protein adducts in atherosclerotic lesions, even in euglycemic conditions.

Adult↗
PubMed1995

Overexpression of transforming growth factor-beta 1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.

Animals↗
PubMed1995

Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes.

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (< or = 28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Administration of AGEs in vivo induces genes implicated in diabetic glomerulosclerosis.

Administration of advanced glycosylation end products (AGEs), prepared on mouse albumin, to normal young adult mice, resulted in an increase in mean glomerular volume and up-regulation of laminin B1 and alpha 1 type IV collagen mRNAs measured by competitive PCR in single microdissected glomeruli. Both glomerular hypertrophy and overexpression of genes coding for extracellular matrix were abrogated when aminoguanidine, an inhibitor of AGE cross-linking, was added to the AGEs injections, suggesting that the glomerular response to AGEs was specific. The effects of AGEs administration in vivo are comparable to those occurring in the early stage of diabetic nephropathy, suggesting a participation of AGEs in these events.

Animals↗
PubMed1995

Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits.

BACKGROUND: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are shown to colocalize with atheromatous lesions and to trigger complex chemical and biological responses through interaction with vessel wall elements. In diabetes and renal insufficiency, atherosclerosis is common, as are elevated levels of serum and vascular tissue AGEs. In the present study, AGEs supplied exogenously to normal animals elicited vascular and renal pathology. MATERIALS AND METHODS: Nondiabetic rabbits were injected intravenously with low doses of AGE-modified rabbit serum albumin (AGE-RSA, 16 mg/kg/day) for 4 months alone, or combined with a brief terminal period (2 weeks) of a cholesterol-rich diet (CRD) (2% cholesterol, 10% corn oil). AGE-RSA associated expression of vascular cell adhesion molecules and the development of atheromatous changes within the aorta were determined by immunohistology. RESULTS: The AGE content of aortic tissue increased by 2.2-fold in AGE-treated and by 3.2-fold in AGE + CRD-treated rabbits compared with normal saline-treated control rabbits (p < 0.025 and 0.001, respectively). Serum AGE levels in AGE groups rose up to 3-fold above the controls (p < 0.025 and p < 0.01). Ascending aortic sections from AGE-treated rabbits showed significant focal intimal proliferation, enhanced endothelial cell adhesion with infrequent intimal macrophages. oil-red-O staining lipid deposits and positive focal expression of vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), a pattern not observed in controls. These AGE-induced changes were markedly enhanced in animals cotreated with AGEs and a brief period of CRD. Lesions consisted of multifocal atheromas, containing foam cells, massive lipid droplets, and strong endothelial expression of VCAM-1 and ICAM-1 restricted to the affected areas. CONCLUSIONS: This study provides in vivo evidence for a causal relationship between chronic AGE accumulation and atherosclerosis independent of diabetic hyperglycemia, and suggests the utility of this animal model for the study of diabetic vascular disease in relation to glycation.

Albumins↗
PubMed1995

Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor complex.

BACKGROUND: Advanced glycation end products (AGE), the reactive derivatives of nonenzymatic glucose-protein condensation reactions, are implicated in the multiorgan complications of diabetes and aging. An AGE-specific cellular receptor complex (AGE-R) mediating AGE removal as well as multiple biological responses has been identified. By screening an expression library using antibody against a previously identified component of the AGE-R complex p90, a known partial cDNA clone was isolated with homology to galectin-3, a protein of diverse identity, and member of the galectin family. MATERIALS AND METHODS: To explore this unexpected finding, the nature of the interactions between galectin-3 and AGE was studied using intact macrophage-like RAW 264.7 cells, membrane-associated and recombinant galectin-1 through -4, and model AGE-ligands (AGE-BSA, FFI-BSA). RESULTS: Among the members of this family (galectin-1 through 4), recombinant rat galectin-3 was found to exhibit high-affinity 125I-AGE-BSA binding with saturable kinetics (kD 3.5 x 10(7) M-1) that was fully blocked by excess unlabeled naturally formed AGE-BSA or synthetic FFI-BSA, but only weakly inhibited by several known galectin-3 ligands, such as lactose. In addition to the p90, immunoprecipitation with anti-galectin-3, followed by 125I-AGE-BSA ligand blot analysis of RAW 264.7 cell extracts, revealed galectin-3 (28 and 32 kD), as well as galectin-3-associated proteins (40 and 50 kD) with AGE-binding activity. Interaction of galectin-3 with AGE-BSA or FFI-BSA resulted in formation of SDS-, and beta-mercaptoethanol-insoluble, but hydroxylamine-sensitive high-molecular weight complexes between AGE-ligand, galectin-3, and other membrane components. CONCLUSIONS: The findings point toward a mechanism by which galectin-3 may serve in the assembly of AGE-R components and in the efficient cell surface attachment and endocytosis by macrophages of a heterogenous pool of AGE moieties with diverse affinities, thus contributing to the elimination of these pathogenic substances.

Animals↗
PubMed1994

Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats.

High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur in diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosis of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were administered daily to healthy rats alone or in combination with the AGE inhibitor aminoguanidine. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), significant periodic acid/Schiff reagent-positive deposits, basement membrane widening, and mesangial extracellular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with significant loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and functional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. The effects are AGE-specific, as they are ameliorated by a pharmacological AGE inhibitor, aminoguanidine.

Aging↗
PubMed1994

Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGE-modified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extracellular matrix alpha 1(IV) collagen, laminin B1, and transforming growth factor beta 1 mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.

Actins↗
PubMed1994

Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency.

Atherosclerosis develops rapidly in patients with diabetes or renal insufficiency. Plasma lipoprotein profiles are frequently abnormal in these conditions and reflect an elevation in the level of the apoprotein B (ApoB)-containing components very low density lipoprotein (VLDL) and low density lipoprotein (LDL). High levels of circulating advanced glycation end products (AGEs) also occur in diabetes and end-stage renal disease (ESRD). These products arise from glucose-derived Amadori products and include AGE-modified peptides (AGE-peptides) which result from the catabolism of AGE-modified tissue proteins. AGE-peptides have been shown to crosslink protein amino groups and to accumulate in plasma as a consequence of renal insufficiency. To address potential mechanisms for the dyslipidemia of diabetes and ESRD, we investigated the possibility that circulating AGEs react directly with plasma lipoproteins to prevent their recognition by tissue LDL receptors. AGE-specific ELISA showed a significantly increased level of AGE-modified LDL in the plasma of diabetic or ESRD patients compared with normal controls. AGE-LDL formed readily in vitro when native LDL was incubated with either synthetic AGE-peptides or AGE-peptides isolated directly from patient plasma. LDL which had been modified by AGE-peptides in vitro to the same level of modification as that present in the plasma of diabetics with renal insufficiency exhibited markedly impaired clearance kinetics when injected into transgenic mice expressing the human LDL receptor. These data indicate that AGE modification significantly impairs LDL-receptor-mediated clearance mechanisms and may contribute to elevated LDL levels in patients with diabetes or renal insufficiency. This hypothesis was further supported by the observation that the administration of the advanced glycation inhibitor aminoguanidine to diabetic patients decreased circulating LDL levels by 28%.

Animals↗
PubMed1994

Advanced glycation end products contribute to amyloidosis in Alzheimer disease.

Alzheimer disease (AD) is characterized by deposits of an aggregated 42-amino-acid beta-amyloid peptide (beta AP) in the brain and cerebrovasculature. After a concentration-dependent lag period during in vitro incubations, soluble preparations of synthetic beta AP slowly form fibrillar aggregates that resemble natural amyloid and are measurable by sedimentation and thioflavin T-based fluorescence. Aggregation of soluble beta AP in these in vitro assays is enhanced by addition of small amounts of pre-aggregated beta-amyloid "seed" material. We also have prepared these seeds by using a naturally occurring reaction between glucose and protein amino groups resulting in the formation of advanced "glycosylation" end products (AGEs) which chemically crosslink proteins. AGE-modified beta AP-nucleation seeds further accelerated aggregation of soluble beta AP compared to non-modified "seed" material. Over time, nonenzymatic advanced glycation also results in the gradual accumulation of a set of posttranslational covalent adducts on long-lived proteins in vivo. In a standardized competitive ELISA, plaque fractions of AD brains were found to contain about 3-fold more AGE adducts per mg of protein than preparations from healthy, age-matched controls. These results suggest that the in vivo half-life of beta-amyloid is prolonged in AD, resulting in greater accumulation of AGE modifications which in turn may act to promote accumulation of additional amyloid.

Alzheimer Disease↗
PubMed1994

Serum advanced glycosylation end products: a new class of uremic toxins?

Irreversible advanced glycosylation end products (AGE) are shown to induce tissue damage by a variety of chemical and cellular mechanisms. When administered to normal animals, AGE-modified albumin reacts covalently with tissue proteins to produce complex vascular alterations closely resembling diabetic vasculopathy. In humans, the clearance of serum with renal insufficiency and is severely impaired in diabetic patients with end-stage renal disease (ESRD). The renal clearance of AGE-peptides was estimated at 0.72 +/- 0.23 ml/min for normal subjects, and 0.61 +/- 0.2 ml/min for diabetics with normal glomerular filtration rate (GFR) (p value NS). After comparing different modes of treatment of patients with ESRD, we noted that, although conventional hemodialysis is ineffective, high-flux dialysis (HF) markedly reduces AGE levels only to return to near-pretreatment levels within 3 h. In chronic ambulatory peritoneal dialysis (CAPD)-treated patients, serum AGE are similar to those achieved by hemodialysis. In contrast, AGE levels in diabetics with renal transplantation decreased 8 h after surgery and remained within the normal range over the long term. Human serum AGE peptides (M.W. between 2,000 and 6,000) are shown to retain strong cross-linking activity with collagen in vitro. AGE peptides in human circulation may represent a thus far unrecognized class of reactive and potentially toxic substances which can exacerbate extrarenal vascular pathology, through their covalent attachment onto matrix proteins.

Animals↗
PubMed1993

Advanced glycosylation endproduct-specific receptors on human and rat T-lymphocytes mediate synthesis of interferon gamma: role in tissue remodeling.

During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.

Animals↗
PubMed1993

Lipid advanced glycosylation: pathway for lipid oxidation in vivo.

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.

Adult↗