Effect of chronic hypoxia on RAGE and its soluble forms in lungs and plasma of mice.

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor. Alternative splicing and enzymatic shedding produce soluble forms that protect against damage by ligands including Advanced Glycation End products (AGEs). A link between RAGE and oxygen levels is evident from studies showing RAGE-mediated injury following hyperoxia. The effect of hypoxia on pulmonary RAGE expression and circulating sRAGE levels is however unknown. Therefore mice were exposed to chronic hypoxia for 21 d and expression of RAGE, sheddases in lungs and circulating sRAGE were determined. In addition, accumulation of AGEs in lungs and expression of the AGE detoxifying enzyme GLO1 and receptors were evaluated. In lung tissue gene expression of total RAGE, variants 1 and 3 were elevated in mice exposed to hypoxia, whereas mRAGE and sRAGE protein levels were decreased. In the hypoxic group plasma sRAGE levels were enhanced. Although the levels of pro-ADAM10 were elevated in lungs of hypoxia exposed mice, the relative amount of the active form was decreased and gelatinase activity unaffected. In the lungs, the RAGE ligand HMGB1 was decreased and of the AGEs, only LW-1 was increased by chronic hypoxia. Gene expression of AGE receptors 2 and 3 was significantly upregulated. Chronic hypoxia is associated with downregulation of pulmonary RAGE protein levels, but a relative increase in sRAGE. These alterations might be part of the adaptive and protective response mechanism to chronic hypoxia and are not associated with AGE formation except for the fluorophore LW-1 which emerges as a novel marker of tissue hypoxia.

Dual second harmonic generation and up-conversion photoluminescence emission in highly-optimized LiNbO3 nanocrystals doped and co-doped with Er3+ and Yb3.

Preparation from the aqueous alkoxide route of doped and co-doped lithium niobate nanocrystals with Er3+ and Yb3+ ions, and detailed investigations of their optical properties are presented in this comprehensive work. Simultaneous emission under femtosecond laser excitation of second harmonic generation (SHG) and up-conversion photoluminescence (UC-PL) is studied from colloidal suspensions according to the lanthanide ion contents. Special attention has been paid to produce phase pure nanocrystals of constant size (∼20 nm) thus allowing a straightforward comparison and optimization of the Er content for increasing the green UC-PL signals under 800 nm excitation. An optimal molar concentration at about 4 molar% in erbium ions is demonstrated, that is well above the concentration usually achieved in bulk crystals. Similarly, for co-doped LiNbO3 nanocrystals, different lanthanide concentrations and Yb/Er content ratios are tested allowing optimization of the green and red up-conversion excited at 980 nm, and analysis of the underlying mechanisms from excitation spectra. All together, these findings provide valuable insights into the wet-chemical synthesis and potential of doped and co-doped LiNbO3 nanocrystals for advanced applications, combining both SHG and UC-PL emissions from the particle core.

Nonenzymatic glycosylation, sulfhydryl oxidation, and aggregation of lens proteins in experimental sugar cataracts.

The formation of sugar-cataracts has been hypothesized to involve the nonenzymatic glycosylation, sulfhydryl oxidation, and aggregation of lens proteins. Cataractous lenses of diabetic and galactosemic rats were analyzed for glycosylated lysine residues in crystallins. A five- and a ten-fold increase in glycosylated lysine residues was measured in galactose and diabetic cataracts, respectively. The modification was predominant in the insoluble fraction of the lens homogenate. The proteins were further examined for the presence of disulfide bonds and high molecular weight aggregates. After careful disruption of the lens in a nitrogen environment, a cloudy solution was obtained from cataractous lenses whereas a clear solution was obtained from normal lenses. The absorbance at 550 nm of the solution of both the galactosemic and the diabetic cataracts could be decreased by approximately 50% with the addition of dithioerythritol (50 mM). The presence of high molecular weight aggregates was ascertained by sucrose gradient centrifugation and gel filtration chromatography. The proteins were heterogenous in size and showed a mol wt range of 36 to greater than 176 million daltons. Treatment with dithioerythritol induced a marked decrease in the amount of high molecular weight proteins. These data suggest that sugar cataracts of experimental animals have, in common with human cataracts, the presence of high molecular weight aggregates which are in part linked by disulfide bonds.

Nonenzymatic browning in vivo: possible process for aging of long-lived proteins.

The incubation of lens proteins with reducing sugars leads to the formation of fluorescent yellow pigments and cross-like similar to those reported in aging and cataractous human lenses. Called nonenzymatic browning or the Maillard reaction, this aging process also occurs in stored foods. Reducing sugars condense with the free amino group of proteins, then rearrange and dehydrate to form unsaturated pigments and cross-linked products. Although most proteins in living systems turn over with sufficient rapidity to avoid nonenzymatic browning, some, such as lens crystallins and skin collagen, are exceptionally long-lived and may be vulnerable.

Brilliant molecular nanocrystals emerging from sol-gel thin films: towards a new generation of fluorescent biochips.

To develop highly sensitive biosensors, we made directly available to biological aqueous solutions organic nanocrystals previously grown in the pores of sol-gel films. Through the controlled dissolution of the sol-gel surface, we obtained emerging nanocrystals that remained strongly anchored to the sol-gel coating for good mechanical stability of the final sensing device. We demonstrated that in the presence of a solution of DNA functionalized with a molecular probe, the nanocrystal fluorescence is strongly quenched by Förster resonance energy transfer thus opening the way towards very sensitive fluorescent biosensors through biomolecules grafted onto fluorescent nanocrystals. Finally, this controlled dissolution, involving weak concentrated NaOH solution, is a generic process that can be used for the thinning of any kind of sol-gel layer.

Suppressor of fused links fused and Cubitus interruptus on the hedgehog signalling pathway.

The Hedgehog (Hh) family of signalling proteins [1] mediate inductive interactions either directly or by controlling the transcription of other secreted proteins through the action of Gli transcription factors, such as Cubitus interruptus (Ci) [2]. In Drosophila, the transcription of Hh targets requires the activation of the protein kinase Fused (Fu) and the inactivation of both Suppressor of fused (Su(fu)) and Costal-2 (Cos-2) [3]. Fu is required for Hh signalling in the embryo and in the wing imaginal disc and acts also as an antitumorigen in ovaries [4]. All fu- phenotypes are suppressed by the loss of function of Su(fu) [5]. Fu, Cos-2 and Ci are co-associated in vivo in large complexes that are bound to microtubules in a Hh-dependent manner [6,7]. Here we investigate the role of Su(fu) in the intracellular part of the Hh signalling pathway. Using the yeast two-hybrid method and an in vitro binding assay, we show that Su(fu), Ci and Fu can interact directly to form a trimolecular complex, with Su(fu) binding to both its partners simultaneously. Su(fu) and Ci also co-immunoprecipitate from embryo extracts. We propose that, in the absence of Hh signalling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci.

Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline.

Pyrraline is one of the major Maillard compounds resulting from the reaction of glucose with amino compounds at slightly acidic pH. For in vivo studies, monoclonal pyrraline antibodies were raised after immunization of Balb/c mice with keyhole limpet hemocyamin-caproyl pyrraline conjugate. Of 660 hybridoma clones from one donor, 260 produced an antibody to the free hapten, two of which named Pyr-A and Pyr-B also cross-reacted with L-lysyl pyrraline. Using Pyr-B antibody and an ELISA, a gradual increase in pyrraline immunoreactivity was observed in serum albumin incubated with glucose or 3-deoxyglucosone. Plasma pyrraline levels increased fourfold (P less than 0.001) in Sprague-Dawley rats upon induction of diabetes with streptozotocin and were twofold increased in randomly selected plasmas from diabetic humans. Highly specific pyrraline immunoreactivity was detected in sclerosed glomeruli from diabetic and old normal kidneys as well as in renal arteries with arteriolosclerosis and in perivascular and peritubular sclerosed extracellular matrix and basement membranes. The preferential localization of pyrraline immunoreactivity in the extracellular matrix strengthens the notion that the advanced glycosylation reaction may contribute to decreased turnover and thickening of the extracellular matrix in diabetes and aging.

End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen.

Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.

Early and advanced glycosylation end products. Kinetics of formation and clearance in peritoneal dialysis.

The chronic contact of glucose-containing dialysate and proteins results in the deposition of advanced glycation end products (AGEs) on peritoneal tissues in patients treated by peritoneal dialysis (PD), yet plasma levels of the AGE pentosidine are significantly lower in PD than in hemodialysis (HD). We measured glycation of peritoneal proteins in patients on PD over the time course of intraperitoneal equilibration of fresh peritoneal dialysate. The glycated content of peritoneal proteins (furosine method) was initially identical to plasma but increased 200% within 4 h due to in situ glycation as also demonstrated in vitro. In contrast, peritoneal proteins contained a 2-4 x greater content of the AGE pentosidine at all equilibrium time points. Plasma protein furosine content was identical in patients on PD and on HD. Fractionation by gel filtration of serum from patients on PD and HD revealed that > 95% of the pentosidine was linked to proteins > 10,000 mol wt; < 1% to proteins < 10,000 mol wt; and < 1%, free. Neither HD nor PD affected protein-bound pentosidine. The HD treatment decreased free and < 10,000 mol wt bound pentosidine. However clearance of protein-associated pentosidine by the peritoneal membrane may explain lower steady state levels in patients treated by PD.

Age-related normalization of the browning rate of collagen in diabetic subjects without retinopathy.

The age-related changes in collagen-linked fluorescence (browning) were investigated in skin from subjects with long-standing type I diabetes. Overall browning rates were 2.4 times higher in diabetics than in controls (P less than 0.02) and slope intercept accurately reflected the mean age of onset of diabetes (11.6 vs. 11.2 yr), suggesting that the browning process has the attributes of a biological clock. Browning rates were not different in controls and diabetics without retinopathy (P greater than 0.05) but were 2.4 (P less than 0.05) and 2.7 (P less than 0.01) times increased in the presence of background and proliferative retinopathy, respectively. Compared with subjects with retinopathy, individual browning rates since onset of diabetes decreased with advancing age in subjects free of retinopathy (P less than 0.001). Extrapolation revealed that they would become identical to that of nondiabetic subjects by the age of 66.4 yr. These results suggest the presence of a mechanism that controls the browning rate of collagen in diabetics who do not develop retinopathy.

Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy.

Advanced glycation end products (AGEs) include a variety of protein adducts whose accumulation alters the structure and function of tissue proteins and stimulates cellular responses. They have been implicated in tissue damage associated with diabetic complications. To assess the possible link between AGE accumulation and the development of diabetic nephropathy (DN), we have examined the immunohistochemical localization of various AGE structures postulated to date, i.e., pentosidine, Nepsilon-(carboxymethyl)lysine (CML), and pyrraline, in diabetic and control kidneys. CML and pentosidine accumulate in the expanded mesangial matrix and thickened glomerular capillary walls of early DN and in nodular lesions and arterial walls of advanced DN, but were absent in control kidneys. By contrast, pyrraline was not found within diabetic glomeruli but was detected in the interstitial connective tissue of both normal and diabetic kidneys. Although the distribution of pyrraline was topographically identical to type III collagen, distribution of pentosidine and CML was not specific for collagen type, suggesting that difference in matrix protein composition per se could not explain heterogeneous AGE localization. Since oxidation is linked closely to the formation of pentosidine and CML, we also immunostained malondialdehyde (MDA), a lipid peroxidation product whose formation is accelerated by oxidative stress, assuming that local oxidative stress may serve as a mechanism of pentosidine and CML accumulation. Consistent with our assumption, diabetic nodular lesions were stained positive for MDA. These findings show that AGE localization in DN varies according to AGE structure, and suggest that the colocalization of markers of glycoxidation (pentosidine and CML) with a marker of lipid peroxidation reflects a local oxidative stress in association with the pathogenesis of diabetic glomerular lesions. Thus, glycoxidation markers may serve as useful biomarkers of oxidative damage in DN.

Radical AGEing in Alzheimer's disease.

The pathological presentation of Alzheimer's disease, the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intracellular and extracellular filamentous protein aggregates that form lesions termed neurofibrillary tangles and senile plaques, respectively. Several independent parameters have been suggested as the primary factor that is responsible for this pathogenesis, including apolipoprotein epsilon genotype, hyperphosphorylation of cytoskeletal proteins, or metabolism of amyloid beta. However, at present, no one theory explains adequately the host of complex biochemical and pathological facets of the disease. Recent findings suggest that age-related increases in oxidative stress and protein glycation either individually, or more probably in a synergistic manner, could, exclusive of the other theories or in concert with them, account for all aspects of Alzheimer's disease.

Isolation and characterization of a blue fluorophore from human eye lens crystallins: in vitro formation from Maillard reaction with ascorbate and ribose.

A blue fluorophore, named LM-1 was isolated from human eye lens crystallins. The fluorescence property of LM-1 (excitation/emission, 366/440 nm) is similar to the fluorescence originating during non-enzymatic glycation (Maillard reaction) of proteins with the reducing sugars. LM-1 accumulates linearly with age in highly cross-linked water insoluble crystallins and is present at higher levels in cataractous lenses. The fluorophore could be synthesized by incubation of bovine serum albumin (BSA) with ribose, but not with glucose or fructose. Incubation of bovine lens crystallins with ascorbic acid (ASA) and its oxidative products, dehydroascorbic acid (DHA) and 2,3-diketogulonic acid (DKG) in presence of oxygen resulted in LM-1 formation. When oxygen was removed from the system, only DHA and DKG could synthesize LM-1, but not ASA, suggesting that ASA oxidation is obligatory for LM-1 synthesis. Modification of lysine residues on BSA prior to incubation with ribose resulted in corresponding decrease in LM-1 formation. Since ASA concentration is unusually high in lens and has been found to be a powerful glycating agent of crystallins and since LM-1 does not form with hexoses, it is likely that ASA is the major precursor of LM-1.

Detection of nonenzymatic browning products in the human lens.

The nonenzymatic browning or Maillard reaction is an aging process in stored foods. The initial stage of this reaction, nonenzymatic glycosylation, has been shown to occur in the human lens. The possible occurrence of further steps of the Maillard reaction involving lysine residues and glucose has been investigated. A lipid-free protein extract from a pool of human cataractous lenses was reduced, alkylated, and digested with pronase. The digest was reduced with [3H]borohydride, acid hydrolyzed and fractionated by Sephadex G-15 chromatography. The fractions eluting ahead of epsilon-1-deoxyglucitolyllysine were pooled and separated with an amino acid analyzer. Four fluorescent, yellow, and radioactive peaks were obtained. One of these, which co-eluted with tyrosine, was isolated, acetylated, and further analyzed by reverse phase chromatography using HPLC. Two new peaks were separated which co-chromatographed with lysine derivatives isolated from the nonenzymatic browning reaction of alpha-tert-butyloxycarbonyllysine with glucose. Control experiments showed that they were not artifacts due to acid hydrolysis of epsilon-glucitolyllysine. These results suggest that dehydration and rearrangement of the Amadori product, epsilon-fructosyllysine, has occurred in vivo, thus leading to the formation of at least two nonenzymatic browning products.

Protein modification by the degradation products of ascorbate: formation of a novel pyrrole from the Maillard reaction of L-threose with proteins.

Ascorbate (vitamin C) degradation products can undergo non-enzymatic glycation (Maillard reaction) with proteins to form highly crosslinked structures with brown pigmentation and characteristic fluorescence. Proteins in the body, especially the long-lived proteins develop similar changes during aging and diabetes. Several studies have shown excessive degradation of ascorbate in plasma in diabetes, and in ocular lens during aging and cataract formation. Recent studies have suggested that ascorbate degradation products-mediated glycation plays a role in lens pigmentation and cataract formation. However, the precise chemical nature of ascorbate-specific advanced glycation end-products are not known. Here, we report the purification and characterization of a glycation end-product derived from one of the major degradation products of ascorbate, L-threose. This compound was characterized to be 2-acetamido-6-(3-(1,2-dihydroxyethyl)-2-formyl-4-hydroxymethyl-1- pyrrolyl)hexanoic acid (formyl threosyl pyrrole or FTP) formed by the condensation of epsilon-amino group of lysine with two molecules of threose. Formation of FTP occurred rapidly in the incubation of threose and lysine and reached plateau level within a day. We have developed a sensitive assay for its quantification in proteins based on enzyme digestion followed by HPLC. Ribonuclease A and human lens crystallins incubated with L-threose showed time- and sugar concentration-dependent increases in FTP, reaching 8.2 and 2.48 nmol per mg protein, respectively after one week of incubation. Human plasma proteins showed a peak with identical retention time as that of purified FTP under two different HPLC conditions. FTP may be used as a sensitive marker to assess ascorbate-mediated protein glycation and modifications in aging and diabetes.

Chromatographic evidence for pyrraline formation during protein glycation in vitro and in vivo.

Pyrraline (epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an advanced Maillard reaction product formed from 3-deoxyglucosone in the non-enzymatic reaction between glucose and the epsilon-amino group of lysine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by immunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodology. To clarify this issue, we investigated pyrraline formation in proteins following alkaline hydrolysis and quantitation by high-performance liquid chromatography on a C18 reverse-phase column. Time- and sugar concentration-dependent increase in pyrraline formation was noted in serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyglucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at slightly acidic pH, confirming its synthetic pathway through this Maillard reaction intermediate. Low levels of pyrraline (< 10 pmol/mg protein) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, pyrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatography. Plasma from diabetic patients showed a significant increase in pyrraline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg protein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabetic plasma.

Collagen aging in vitro by nonenzymatic glycosylation and browning.

Aging and diabetes mellitus are associated with cross-linking and nonenzymatic glycosylation of collagen. Incubation of tendon fibers with reducing sugars results in increased breaking time in urea similar to that seen in aging, and in nonenzymatic glycosylation and browning. Effect of a sugar is proportional to the amount of sugar available in the open chain form. The increase in breaking time correlates with the appearance of chromophores characteristic of crosslinked browning products. Collagen altered by nonenzymatic browning may play a role in some age-like major complications of diabetes.

The mechanism of collagen cross-linking in diabetes: a puzzle nearing resolution.

Considerable interest has been focused in recent years on the mechanism of collagen cross-linking by high glucose in vitro and in vivo. Experiments in both diabetic humans and in animals have shown that over time collagen becomes less soluble, less digestible by collagenase, more stable to heat-induced denaturation, and more glycated. In addition, collagen becomes more modified by advanced products of the Maillard reaction, i.e., immunoreactive advanced glycation end products and the glycoxidation markers carboxymethyllysine and pentosidine. Mechanistic studies have shown that collagen cross-linking in vitro can be uncoupled from glycation by the use of antioxidants and chelating agents. Experiments in the authors' laboratory revealed that approximately 50% of carboxymethyllysine formed in vitro originates from pathways other than oxidation of Amadori products, i.e., most likely the oxidation of Schiff base-linked glucose. In addition, the increase in thermal stability of rat tail tendons exposed to high glucose in vitro or in vivo was found to strongly depend on H2O2 formation. The final missing piece of the puzzle is that of the structure of the major cross-link. We speculate that it is a nonfluorescent nonultraviolet active cross-link between two lysine residues, which includes a fragmentation product of glucose linked in a nonreducible bond labile to both strong acids and bases.

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

Chromatographic quantitation of plasma and erythrocyte pentosidine in diabetic and uremic subjects.

Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P less than 0.001) and a 23-fold (P less than 0.001) elevation of mean +/- SD plasma protein pentosidine in diabetic (2.4 +/- 1.2 pmol/mg) and uremic (21.5 +/- 10.8 pmol/mg) subjects compared with healthy (0.95 +/- 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 +/- 0.4 pmol/mg hemoglobin, P less than 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.