Advanced glycation end products and diabetic retinopathy

Studies have established hyperglycemia as the most important factor in the progress of vascular complications. Formation of advanced glycation end products (AGEs) correlates with glycemic control. The AGE hypothesis proposes that hyperglycemia contributes to the pathogenesis of diabetic complications including retinopathy. However, their role in diabetic retinopathy remains largely unknown. This review discusses the chemistry of AGEs formation and their patho-biochemistry particularly in relation to diabetic retinopathy. AGEs exert deleterious effects by acting directly to induce cross-linking of long-lived proteins to promote vascular stiffness, altering vascular structure and function and interacting with receptor for AGE, to induce intracellular signaling leading to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Novel anti-AGE strategies are being developed hoping that in next few years, some of these promising therapies will be successfully evaluated in clinical context aiming to reduce the major economical and medical burden caused by diabetic retinopathy.     

Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases

The modification of free amino groups on proteins, lipids, and nucleic acids by non-enzymatic glycosylation produce a variety of complex structures named advanced glycation end products (AGEs). Glycation of these molecules participate in the development of diabetic complications and related diseases. Diabetes mellitus is characterized by short-term metabolic changes in lipid and protein metabolism, and long-term irreversible changes in vascular and connective tissue. AGEs are directly implicated in the development of chronic complications in diabetes such as nephropathy, rethinopathy, neuropathy, and other related diseases such as atherosclerosis, heart disease, stroke, and peripheral vascular disease. In this review, we aim to explain how glycation occurs in different molecules and what the pathological consequence of AGE formation in diabetes mellitus and other diseases are.     

Increased levels of vascular endothelial growth factor and advanced glycation end products in aqueous humor of patients with diabetic retinopathy.

Clinical studies have shown a relationship between diabetic retinopathy and vascular endothelial growth factor (VEGF) levels in ocular fluid. Advanced glycation end products (AGEs) have been implicated in diabetes complications, including diabetic retinopathy. Nepsilon-(carboxymethyl) lysine (CML) is a glycoxidation product that may be a marker of oxidative stress. In this study, we used enzyme-linked immunosorbent assays to determine the levels of VEGF, non-CML AGE and CML in the aqueous humor and serum of 82 Japanese patients with type 2 diabetes and 60 non-diabetic subjects. VEGF, non-CML AGE, and CML concentrations in aqueous humor and serum were then compared with the severity of diabetic retinopathy. Immunohistochemical detection analysis of non-CML AGE and CML was also performed using retinal tissues from patients with progressive diabetic retinopathy. Aqueous levels of VEGF, non-CML AGE and CML increased along with the progression of diabetic retinopathy compared to age-matched controls. After coagulation therapy, the VEGF, non-CML AGE, and CML levels were significantly reduced. Immunostaining showed diffuse co-localization of non-CML AGE and CML around microvessels and in the glial cells of proliferative membranes from patients with progressive diabetic retinopathy. These findings suggest that glycation and glycoxidation reactions (or oxidation, as revealed by CML) may contribute to both the onset and progression of diabetic retinopathy.     

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

Chelating activity of advanced glycation end-product inhibitors.

The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.     

Aldose reductase (AKR1B3) regulates the accumulation of advanced glycosylation end products (AGEs) and the expression of AGE receptor (RAGE)

Diabetes results in enhanced chemical modification of proteins by advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) precursors. These modifications have been linked to the development of several secondary diabetic complications. Our previous studies showed that aldose reductase (AR; AKR1B3) catalyzes the reduction of ALEs and AGEs precursors; however, the in vivo significance of this metabolic pathway during diabetes and obesity has not been fully assessed. Therefore we examined the role of AR in regulating ALEs and AGEs formation in murine models of diet-induced obesity and streptozotocin-induced diabetes. In comparison with wild-type (WT) and AR-null mice fed normal chow, mice fed a high-fat (HF) diet (42% kcal fat) showed increased accumulation of AGEs and protein-acrolein adducts in the plasma. AGEs and acrolein adducts were also increased in the epididymal fat of WT and AR-null mice fed a HF diet. Deletion of AR increased the accumulation of 4-hydroxy-trans-2-nonenal (HNE) protein adduct in the plasma and increased the expression of the AGE receptor (RAGE) in HF fed mice. No change in AGEs formation was observed in the kidneys of HF-fed mice. In comparison, renal tissue from AR-null mice treated with streptozotocin showed greater AGE accumulation than streptozotocin-treated WT mice. These data indicated that AR regulated the accumulation of lipid peroxidation derived aldehydes and AGEs under conditions of severe, but not mild, hyperglycemia and that deletion of AR increased RAGE-induction via mechanisms that were independent of AGEs accumulation.     

Phosphatidylinositol 3'-kinase-dependent activation of renal mesangial cell Ki-Ras and ERK by advanced glycation end products

Advanced glycation end products (AGEs) are produced by the non-enzymatic glycation of proteins and lipids. AGE levels are pathologically elevated in a number of inflammatory diseases and in diabetes mellitus. There is evidence that AGEs, acting through the receptor for AGEs, contribute to diabetic complications. Nephropathy is a major complication of diabetes mellitus. However, the initiating molecular events that trigger diabetic renal disease are unknown. Renal mesangial cells produce excess extracellular matrix in response to treatment with transforming growth factor-beta, and excess mesangial cell matrix production, by impairing glomerular filtration, contributes to diabetic nephropathy. AGEs are known to trigger the autocrine production and release of transforming growth factor-beta. However, it is unclear how AGEs signal in mesangial cells. Here we show that treatment of mesangial cells with AGEs and with the receptor for AGEs agonist S100 triggers activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) pathways. AGEs trigger the GTP loading of mesangial cell Ras, and AGE activation of ERK requires Ras. We observe that Ki-Ras, but not Ha-Ras, is the target of AGE action. Surprisingly, inhibition of PI3K blocks both ERK and Ki-Ras activation. We also observe that activation of ERK and the PI3K target kinase protein kinase-B is blocked with free radical scavengers, indicating a role for reactive oxygen species in AGE recruitment of PI3K. Thus, AGEs signal to Ki-Ras and ERK through reactive oxygen species-dependent activation of PI3K.     

Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.     

The combination of high glucose and advanced glycation end-products (AGEs) inhibits the mineralization of osteoblastic MC3T3-E1 cells through glucose-induced increase in the receptor for AGEs.

Type 1 diabetes mellitus is known to be associated with reduced bone mass and increased bone fractures. This is thought to be due to a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption, but the precise mechanism is unknown. In this study, we examined whether or not high glucose or advanced glycation end-products (AGEs), which play key roles in the pathogenesis and complications of diabetes, affect the differentiation of osteoblastic MC3T3-E1 cells. First, MC3T3-E1 cells were incubated in media containing either 22 mM glucose, 22 mM mannitol, 300 microg/ml AGE2, or 300 microg/ml AGE3. Each of these agents alone did not affect the mineralization of the cells by von Kossa staining and Alizarin red staining. However, high glucose but not mannitol or AGEs markedly increased mRNA expression of AGE receptor (RAGE) by real-time PCR. Next, we examined the combined effects of high glucose and AGEs on the differentiation of MC3T3-E1 cells. The combination of 22 mM glucose and 300 microg/ml AGE2 significantly inhibited the mineralization of MC3T3-E1 cells, and 22 mM glucose in combination with either 300 microg/ml AGE2 or AGE3 apparently decreased osteocalcin mRNA expression. These results suggest that high glucose or AGEs alone might have no effect on osteoblastic differentiation, but their combination could additionally or synergistically inhibit osteoblastic mineralization through glucose-induced increase in RAGE expression.     

Pyridoxamine traps intermediates in lipid peroxidation reactions in vivo: evidence on the role of lipids in chemical modification of protein and development of diabetic complications

Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5-10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of alpha-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases.     

AGE/RAGE signalling regulation by miRNAs: Associations with diabetic complications and therapeutic potential

Excessive formation of advanced glycation end-products (AGEs) presents the most important mechanism of metabolic memory that underlies the pathophysiology of chronic diabetic complications. Independent of the level of hyperglycaemia, AGEs mediate intracellular glycation of the mitochondrial respiratory chain proteins leading to excessive production of reactive oxygen species (ROS) and amplification of their formation. Additionally, AGEs trigger intracellular damage via activation of the receptor for AGEs (RAGE) signalling axis that leads to elevation of cytosolic ROS, nuclear factor kappaB (NF-κB) activation, increased expression of adhesion molecules and cytokines, induction of oxidative and endoplasmic reticulum stress. Recent studies have identified novel microRNAs (miRNAs) involved in the regulation of AGE/RAGE signalling in the context of diabetic micro- and macrovascular complications. The aim of this review is to discuss the emerging role of miRNAs on AGE/RAGE pathway and the potential use of several miRNAs as novel therapeutic targets.     

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.     

Diabetic kidney disease: a role for advanced glycation end-product receptor 1 (AGE-R1)?

Diabetic patients are postulated to be in a perpetual state of oxidative stress and inflammation at sites where chronic complications occur. The accumulation of AGEs derived from both endogenous and exogenous sources (such as the diet) have been implicated in the development and progression of diabetic complications, particularly nephropathy. There has been some interest in investigating the potential for reducing the AGE burden in chronic disease, through the action of AGE “clearance” receptors, such as the advanced glycation end-product receptor 1 (AGE-R1). Reducing the burden of AGEs has been linked to attenuation of inflammation, slower progression of diabetic complications (in particular vascular and renal complications) and has been shown to extend lifespan. To date, however, there have been no direct investigations into whether AGE-R1 has any role in modulating normal kidney function, or specifically during the development and progression of diabetes. This mini-review will focus on the recent advances in knowledge around the mechanistic function of AGE-R1 and the implications of this for the pathogenesis of diabetic kidney disease.     

A review of the anti-inflammatory properties of antidiabetic agents providing protective effects against vascular complications in diabetes

The global prevalence of Type 2 diabetes mellitus and its associated complications are growing rapidly. Although the role of hyperglycemia is well recognized in the pathophysiology of diabetic complications, its exact underlying mechanisms are not fully understood. In this regard, accumulating evidence suggests that the role of inflammation appears pivotal, with studies showing that most diabetic complications are associated with an inflammatory response. Several classes of antidiabetic agents have been introduced for controlling glycemia, with evidence that these pharmacological agents may have modulatory effects on inflammation beyond their glucose-lowering activity. Here we review the latest evidence on the anti-inflammatory effects of commonly used antidiabetic medications and discuss the relevance of these effects on preventing diabetic complications.     

Pyridoxamine, an inhibitor of advanced glycation and lipoxidation reactions: a novel therapy for treatment of diabetic complications

Pyridoxamine (PM), originally described as a post-Amadori inhibitor of formation of advanced glycation end-products (AGEs), also inhibits the formation of advanced lipoxidation end-products (ALEs) on protein during lipid peroxidation reactions. In addition to inhibition of AGE/ALE formation, PM has a strong lipid-lowering effect in streptozotocin (STZ)-induced diabetic and Zucker obese rats, and protects against the development of nephropathy in both animal models. PM also inhibits the development of retinopathy and neuropathy in the STZ-diabetic rat. Several products of reaction of PM with intermediates in lipid autoxidation have been identified in model reactions in vitro and in the urine of diabetic and obese rats, confirming the action of PM as an AGE/ALE inhibitor. PM appears to act by a mechanism analogous to that of AGE-breakers, by reaction with dicarbonyl intermediates in AGE/ALE formation. This review summarizes current knowledge on the mechanism of formation of AGE/ALEs, proposes a mechanism of action of PM, and summarizes the results of animal model studies on the use of PM for inhibiting AGE/ALE formation and development of complications of diabetes and hyperlipidemia.     

Immunohistochemical study of N-epsilon-carboxymethyl lysine (CML) in human brain: relation to vascular dementia

Background  

Advanced glycation end-products (AGEs) and their receptor (RAGE) occur in dementia of the Alzheimer's type and diabetic microvascular disease. Accumulation of AGEs relates to risk factors for vascular dementia with ageing, including hypertension and diabetes. Cognitive dysfunction in vascular dementia may relate to microvascular disease resembling that in diabetes. We tested if, among people with cerebrovascular disease, (1) those with dementia have higher levels of neuronal and vascular AGEs and (2) if cognitive dysfunction depends on neuronal and/or vascular AGE levels.     

The in vitro glycation of human serum albumin in the presence of Zn(II)

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.     

Advanced glycation and endothelial functions: a link towards vascular complications in diabetes

The formation of advanced glycation end-products (AGEs), also called the Maillard reaction, occurs ubiquitously and irreversibly in patients with diabetes mellitus, and its consequences are especially relevant to vascular dysfunctions. The interaction of AGEs with their receptors (RAGE) has been implicated in the development of vascular complications. This interaction elicits remarkable vascular cell changes analogous to those observed in diabetes mellitus, including angiogenic and thrombogenic responses of endothelial cells, increased oxidative stress, and functional alterations in vascular tone control. This review focuses on AGEs formation, the interaction with their specific receptors and how the triggered intracellular events determine functional alterations of vascular endothelium. Finally, some potential pharmacological approaches undertaken to circumvent the deleterious effects of AGEs are also discussed.     

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.