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From: Contemporary Cardiology: Diabetes and Cardiovascular Disease, Second Edition Edited by: M. T. Johnstone and A. Veves © Humana Press Inc., Totowa, NJ
3 Diabetes and Advanced Glycoxidation End-Products
Melpomeni Peppa, MD , Jaime Uribarri, MD , and Helen Vlassara, MD
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EFERENCESINTRODUCTION
The incidence of diabetes, especially type 2 diabetes, is increasing at an alarming rate assuming epidemic proportions (1). Worldwide, 124 million people had diabetes by 1997, although an estimated 221 million people will have diabetes by the year 2010 (1).
Diabetic patients may suffer a number of debilitating complications such as retinopa- thy, nephropathy, neuropathy, and atherosclerosis resulting in cardiovascular, cere- brovascular, or peripheral vascular disease. These diabetic complications lead to huge economic and psychosocial consequences. Although the pathogenesis of type 1 diabetes is different from that of type 2 diabetes, the pathophysiology of vascular complications in the two conditions appears to be similar.
Two landmark clinical studies, the Diabetes Control and Complications Trial (DCCT)
and the United Kingdom Prospective Diabetes Study, showed that intensive control of
hyperglycemia could reduce the occurrence or progression of retinopathy, neuropathy
and nephropathy in patients with type 1 and type 2 diabetes (2,3). Although these studies
reinforce the important role of hyperglycemia in the pathogenesis of diabetic complica-
tions, the identification of the mechanisms by which hyperglycemia exerts these effects remains limited (4).
It is well known that long-term hyperglycemia leads to the formation of advanced glycation or glycoxidation end-products (AGEs), which mediate most of the deleterious effects of hyperglycemia and seem to play a significant role in the pathogenesis of diabetic complications (5,6). AGEs, together with the interrelated processes of oxidative stress and inflammation, may account for many of the complications of diabetes (5,6).
Evidence for this emerges not only from an increased number of in vitro and in vivo studies exploring the role of AGEs in different pathologies, but also from studies dem- onstrating significant improvement of features of diabetic complications by inhibitors of the glycoxidation process (7–13).
In the following review we will provide a general overview of the nature, formation, and action of AGEs and recent evidence on their pathogenic potential in the initiation and progression of diabetic complications. We will conclude delineating possible therapeutic interventions based on this new knowledge.
ADVANCED GLYCOXIDATION END-PRODUCTS Endogenous Advanced Glycoxidation End-Products Formation It is now appreciated that normal living is associated with spontaneous chemical transformation of amine-containing molecules by reducing sugars in a process described since 1912 as the Maillard reaction. This process occurs constantly within the body and at an accelerated rate in diabetes (5,6). Reducing sugars react in a nonenzymatic way with free amino groups of proteins, lipids, and guanyl nucleotides in DNA and form Schiff base adducts. These further rearrange to form Amadori products, which undergo rear- rangement, dehydration, and condensation reactions leading to the formation of irrevers- ible moieties called AGEs. Among all naturally occurring sugars, glucose exhibits the slowest glycation rate, although intracellular sugars such as fructose, threose, glucose- 6-phosphate, and glyceraldehyde-3-phosphate form AGEs at a much faster rate (5,6,14).
Faster and more efficient than the modification of proteins is the glycoxidation of lipids that contain free amines producing advanced lipoxidation end-products (5,6,15) (Fig. 1). AGEs such as JN-carboxymethyl-lisine (CML) can also form through autoxida- tion of glucose or ascorbate (16,17). Metal-catalyzed autoxidation of glucose is accom- panied by the generation of reactive oxygen species as superoxide radicals, which can undergo dismutation to hydrogen peroxides (18). Physiological glycation processes also involve the modification of proteins by reactive oxoaldehydes that come from the deg- radation of glucose, Schiff base adducts, Amadori products, glycolytic intermediates, and lipid peroxidation. Among them, glyoxal, methylglyoxal (MG), and 3-deoxyglu- cosone have been more extensively studied. Under normal conditions, in vivo produced oxoaldehydes are metabolized and inactivated by enzymatic conversion to the corre- sponding aldonic acids and only a small portion proceeds to form AGEs (5,6,19).
Despite the identification of numerous AGE compounds that exist in nature the elu-
cidation of the structure of pathogenic AGEs remains elusive. Pentosidine, CML, and
MG derivatives are among the well-characterized compounds (5,6) that are commonly
used as AGE markers in many studies. AGEs are immunologically distinct, but can co-exist
on the same carrier proteins such as albumin, hemoglobin, collagen, or lipoproteins at
different stages of the glycation process, some more unstable than others. This adds to the
challenges presented to chemists and biologists interested in their characterization.
Exogenous Sources of Advanced Glycoxidation End-Products
AGEs can also be introduced in biological systems from exogenous sources. Methods of food processing (heating in particular) have a significant accelerating effect in the generation of diverse highly reactive F-G-dicarbonyl derivatives of glyco- and lipoxidation reactions that occur in complex mixtures of nutrients (20–23).
About 10% of a single AGE-rich meal is absorbed into the body (24,25). Food-derived AGEs, rich in MG, CML, and other derivatives, are potent inducers of oxidative stress and inflammatory processes. As with endogenous AGEs these processes can be blocked by antioxidants and anti-AGE agents (26), pointing to many similarities (structural and biological) between exogenous and endogenous AGEs.
Animal studies have demonstrated the close relationship between increased dietary AGE intake and development and/or progression of many diabetes-related complica- tions. Nephropathy, postinjury restenosis, accelerated atherosclerosis, and delayed wound healing were significantly inhibited by lowering dietary AGE intake (27–30). Sebekova and associates demonstrated in the remnant-kidney rat model that feeding an AGE-rich diet for 6 weeks increases kidney weight and causes proteinuria, independent of changes in glomerular filtration rate, pointing to the detrimental effect of such diet on the kidney (31). Of particular interest are studies showing that a low-glycotoxin environment can prevent or delay significantly autoimmune diabetes in successive generations of nonobese diabetic (NOD) mice (32) and to improve the insulin-resistant state in db/db (+/+) mice (33). Reduction in exposure to exogenous AGEs of db/db (+/+) mice, lacking in leptin receptor and thus prone to insulin resistance and type 2 diabetes, led to amelioration of the insulin resistance and marked preservation of islet structure and function (33).
Clinical studies have further confirmed the above laboratory data. Studies in diabetic
patients with normal renal function and nondiabetic patients with chronic renal insuffi-
ciency, another condition with elevated serum AGE levels, demonstrated that lowering
dietary AGE intake can significantly decrease circulating AGE levels followed by par-
Fig. 1. Glycation and oxidation of lipids. Glycation of phospholipids containing a free amino group enhances the oxidation of the fatty acid chain (ref. 141).allel changes in circulating inflammatory markers such as C-reactive protein (34–37).
These preliminary but striking findings added further credence to the hypothesis that exogenous AGEs, in addition to being major determinants of the total AGE pool (Figs.
2 and 3), may be powerful modulators of the inflammatory state that is common in conditions such as chronic renal insufficiency (Fig. 4). This is highly relevant to human aging as it is associated with loss of renal function, often significant (38).
Tobacco smoke is another exogenous source of AGE. Tobacco curing is essentially a Maillard “browning” reaction, as tobacco is processed in the presence of reducing sugars.
Combustion of these adducts during smoking gives rise to reactive, toxic AGE formation
Fig. 2. Multifactorial influences determining circulating AGE levels.Fig. 3. Serum AGEs correlate with dietary AGE intake in humans. Association between daily dietary AGE content (assessed by dietary history) and serum AGE levels, measured as CML, in a large cross-section of chronic renal failure patients on dialysis (ref. 35).
(39). Total serum AGE, or AGE-apolipoprotein (apo)-B levels have been found to be significantly higher in cigarette smokers than in nonsmokers. Smokers and especially diabetic smokers have high AGE levels in their arteries and ocular lenses (40).
ADVANCED GLYCOXIDATION END-PRODUCTS METABOLISM Steady-state serum AGE levels reflect the balance of oral intake, endogenous forma- tion, and catabolism of AGEs. AGE catabolism is dependent on both tissue degradation and renal elimination.
Cells such as tissue macrophages can ingest and degrade AGEs via specific or nonspe-
cific receptors (5,6,41). Mesenchymal cells such as endothelial and mesangial cells seem
to participate also in AGE elimination (42). It has been postulated that insulin may
accelerate macrophage scavenger receptor-mediated endocytic uptake of AGE proteins
through the IRS/PI3 pathway (43). Cellular removal of AGEs is processed largely
through endocytosis and further intracellular degradation resulting in the formation of
low-molecular-weight AGE peptides, which are released to the extracellular space and
circulation (5,41,44). These peptides undergo a variable degree of reabsorption and
further catabolism in the proximal nephron and the rest is excreted in the urine. Therefore,
effective AGE elimination is dependent on normal renal function (5,41,44,45). We have
recently found that diabetic patients with normal renal function have a significantly lower
urinary AGE excretion than healthy controls. This impaired renal AGE clearance second-
Fig. 4. Changes of circulating AGEs and markers of inflammation during dietary AGE modula- tion. Percent change of serum AGEs (CML, MG, and LDL-CML), C-reactive protein (CRP), tumor necrosis factor (TNF)-F and vascular adhesion molecule (VCAM)-1 in a group of stable diabetic patients fed either AGE-restricted or regular diet for up to 6 weeks (ref. 34).ary to increased tubular reabsorption of AGE peptides may be a factor in the high-serum AGE levels obeserved in these patients (46).
Other intracellular protective systems also help to limit the accumulation of reactive AGE intermediates. Methylglyoxal is first converted by glyoxalase-I to S-
D-lacto- ylglutathione in the presence of reduced glutathione as an essential cofactor, and then converted to
D-lactate by glyoxalase-II. The significance of such systems is supported by studies in which overexpression of glyoxalase-I prevented hyperglycemia-induced AGE formation and increased macromolecular endocytosis (47). These systems, however, could still be overwhelmed by high AGE conditions such as diabetes, renal failure, or sustained excess dietary AGE intake.
ADVANCED GLYCOXIDATION END-PRODUCTS INTERACTIONS AGEs can cause pathological changes in tissues by multiple receptor-dependent and receptor-independent processes. A characteristic of AGEs is their ability for covalent crosslink formation that leads to alterations of the structure and function of proteins (5,6,41). It is now clear that short- and long-lived molecules alike including circulating proteins, lipids, or intracellular proteins and nucleic acids can be modified (5,6,41,48).
Glycation of one such molecule, low-density lipoprotein (LDL), leads to its delayed receptor-mediated clearance and subsequent deposition in the vessel wall, contributing to atherosclerosis and macrovascular disease (5,6,41,48).
Intracellular AGEs are reported to form at a rate up to 14-fold faster under high- glucose conditions, although the impact of intracellular glycation can be partially coun- tered by the high turnover and short half-life of many cellular proteins (49).
Experimental work conducted over the last 15 years has led to the recognition of an AGE-receptor system and soluble AGE-binding proteins. The interaction of AGEs with these proteins leads either to endocytosis and degradation or to cellular activation (5,6,41).
In addition to these receptor pathways, AGEs can also induce cellular activation via intracellularly generated glycoxidant derivatives or via free radical generation (5,6,41).
The first cell surface AGE-binding protein receptor identified was AGE-R1, with characteristic membrane-spanning and signal domains homologous to a 48kD component of the oligosaccharyltransferase complex-48 (5,41,50–52).This component has recently been shown to be linked to AGE removal and supression of undue oxidative stress and cell-activation events (53). An 80kD protein or AGE-R2, identical to a tyrosine-phospho- rylated protein located largely within the plasma membrane was found involved in bind- ing and forming complexes with adaptor molecules such as Shc and GRB-2. AGEs are highly efficient stimuli for AGE-R2 phosphorylation indicating its possible involvement in AGE-signaling (5,41,54–57). AGE-receptor-3 or Galectin-3, known as Mac-2 or car- bohydrate-binding protein-35, is also known to interact with the G-galactoside residue of several cell surface and extracellular matrix (ECM) glycoproteins, via the carbohydrate recognition domain (5,41,54–58). AGE-R3 or Galectin-3 is only weakly detectable on cell surfaces under basal conditions, but becomes highly expressed with age and diabetes (55). AGE-R3 exhibits high-binding affinity for AGEs and appears to enhance AGE internalization and degradation in macrophages, astrocytes, and endothelial cells (5,41,54–57).
The expression of AGE receptors in mesangial cells and monocytes/macrophages in
NOD mice and in diabetic patients was found to correlate with the severity of diabetic
complications (5,41,52). AGE-receptor-3 knockout mice exhibited accelerated diabetic
glomerulopathy, associated with marked renal/glomerular AGE accumulation implying a beneficial role for AGE-R3 in AGE clearance (58). Recent in vitro studies imply a possible direct role of Galectin-3 in the pathogenesis of atherosclerosis and diabetic glomerulopathy (5,41,57,58).
The receptor for advanced glycation end-products (RAGE), a well-characterized multiligand member of the immunoglobulin superfamily, is viewed as an AGE-binding intracellular signal-transducing peptide, which mediates diverse cellular responses rather than as a receptor involved in AGE endocytosis and turnover. Several other distinct ligands have been described for RAGE including amyloid, amphoterin, and S100/
calgranulins (5,41,59–62). RAGE is present at low levels in adult animals and humans, but is later upregulated regardless of diabetic vascular disease (62). RAGE expression is increased in sites of increased AGE accumulation such as vasculature, neurons, lympho- cytes, and tissue-invading mononuclear phagocytes. In the kidney, RAGE is expressed in glomerular visceral epithelial cells (podocytes) but not in mesangium or glomerular endothelium (59). Diabetic RAGE-transgenic mice exhibit renal vascular changes char- acteristic of diabetic nephropathy (60). In contrast, brief infusion of a soluble truncated RAGE is reported to intercept diverse processes such as endothelial leakage, atheroscle- rosis, and inflammatory bowel disease (59).
Other well-studied AGE-binding molecules are the macrophage scavenger receptors, class A (MSR-A) and class B (MSR-B). MSR-A, better known as receptors for oxidized LDL may also play a role in endocytic uptake and degradation of AGE-proteins in vivo (5,41,63,64). CD36, a member of MSR-B receptors, is a highly glycosylated 88-kD protein expressed on macrophages which, although not restricted to AGE uptake, may contribute to AGE-mediated foam cell formation (65). The class B type I scavenger receptor (SR-BI) is also considered as an AGE-interactive molecule; it is suggested that it contributes to reverse cholesterol transport by suppressing selective uptake of high- density lipoprotein cholesterol efflux (HDL-CE) by liver and cholesterol efflux from peripheral cells to HDL (66). Additionally, recently cloned LOX-1 and SREC, novel scavenger receptors expressed in vascular endothelial cells are awaiting studies to deter- mine their affinity for AGE-proteins (67).
Another molecule with significant AGE-binding affinity and intriguing anti-AGE properties is lysozyme. Lysozyme is a well-characterized natural host-defense protein thought to exert antibacterial effects through the catalytic degradation of the peptidogly- can component of the bacterial wall (68). Lysozyme is found in saliva, nasal secretions, milk, mucus, serum, and in lysosomes of neutrophils and macrophages. Against all predictions, however, a novel AGE-binding site was mapped to a 17–18 amino acid hydrophilic domain (ABCD motif or AGE-binding cysteine-bounded domain), bounded on both sides by cysteines and located within one of the two lysozyme catalytic regions (68). Lysozyme enhances the uptake and degradation of AGE proteins by macrophages, apparently via an AGE-specific receptor pathway not well defined thus far (68). Lysozyme administration to diabetic mice, however, increases AGE clearance, suppresses mac- rophage and mesangial cell-specific gene activation in vitro, and improves albuminuria, thus presenting an unusual combination of advantages, which have stimulated interest in this native substance as a potential therapeutic target (69). A novel receptor that mediates AGE-induced chemotaxis in rabbit smooth muscle cells has also been identified (70).
The genomic organization, chromosomal location, and several prevalent gene poly-
morphisms of some of the AGE-R-related molecules have come to light in the past few
years. For instance, a recent screening using single-strand conformational polymorphism
analysis and direct sequencing of allelic polymerase chain reaction fragments in 48 type 1 diabetics with or without nephropathy showed variants of AGE receptors, mutations, and polymorphisms that correlated with the presence and the severity of complications, albeit only weakly (71).
ADVANCED GLYCOXIDATION END-PRODUCTS AND DIABETIC MICROANGIOPATHY
Diabetic microangiopathy is a broad term that describes changes in microvascular beds in which endothelium and associated mural cells function are progressively dis- rupted, resulting in occlusion, ischemia, and organ damage. Although kidney and retina are most commonly affected, diabetic microangiopathy can occur in a wide range of tissues such as peripheral nerves and skin (4). A large number of studies have supported the pathogenic role of AGE in diabetic microangiopathy (4–6,41), even as their exact role is still under investigation.
Diabetic Nephropathy
The prevalence of diabetic nephropathy has increased dramatically and is now the first cause of end-stage renal disease requiring renal replacement therapy worldwide (72).
Although the genetic background is important in determining susceptibility to diabetic nephropathy, exposure to chronic hyperglycemia leading to the subsequent activation of multiple pathogenic pathways appears to be the main initiating factor (2,3,4–6,41).
Diabetic nephropathy occurs in up to 30%–40% of diabetic patients. The initial abnor- malities include glomerular hyperfiltration and hyperperfusion resulting in microalbu- minuria, increased glomerular basement membrane thickening, and mesangial ECM deposition. These processes are followed by mesangial hypertrophy, diffuse and nodu- lar glomerulosclerosis, tubulointerstitial fibrosis, and eventually progressive renal failure (73).
IN VITRO DATA
The ability to culture cells that are affected by AGEs has provided an important insight into the mechanisms of action of these adducts, their receptors and the way they may contribute to tissue dysfunction in diabetes. In vitro, AGEs bind to renal mesangial cells through AGE receptors, which initiate overproduction of matrix proteins and changes in the expression of matrix metalloproteinases and proteinase inhibitors (74,75). Exposure of rat mesangial cells to AGE-rich proteins results in mesangial oxidative stress and activation of RAGE or other processes, e.g., protein kinase C-G (76) or angiotensin II causing, for instance, in vitro inhibition of nephrin gene expression (77) or induction of apoptosis and secretion of vascular endothelin growth factor (VEGF) and monocyte chemotactic peptide-1 proteins, events that were prevented by N-acetylcysteine (78).
AGEs also stimulate production of collagen IV and fibronectin in glomerular endothelial cells (79).
ANIMAL STUDIES
Immunohistochemical studies of kidneys from normal and diabetic rats show that
glomerular basement membrane, mesangium, podocytes, and renal tubular cells accumu-
late high levels of AGEs with AGE concentrations rising with age and more rapidly with
diabetes (80,81). Moreover, the intensity of CML immunostaining is greatest in the areas
of extensive glomerular sclerosis characteristic of advanced diabetic nephropathy (82).
Short-term exogenous AGE administration to normal, nondiabetic animals has repro- duced some of the vascular defects associated with clinical diabetic nephropathy includ- ing induction of basement membrane components (e.g., F1-collagen IV) or transforming growth factor (TGF)-G (82,83). Furthermore, chronic treatment of animals with AGE albumin can reproduce glomerular hypertrophy, basement membrane thickening, extra- cellular mesangial matrix expansion and albuminuria, all consistent with findings of diabetic nephropathy (82,83).
The role of AGEs in the pathogenesis of diabetic nephropathy has been supported by studies in transgenic animals. RAGE overexpression in diabetic mice resulted in increased albuminuria, elevated serum creatinine, renal hypertrophy, mesangial expan- sion, and glomerulosclerosis (60), although blockade of RAGE by soluble truncated RAGE suppressed structural and functional components associated with nephropathy in db/db mice (58).
AGE inhibitors have been shown to prevent AGE accumulation in renal structures and diabetic nephropathy in diabetic animal models (84–87). Aminoguanidine ameliorated overexpression of F1-type IV collagen, laminin B1, TGF-G, and platelet-derived growth factor, all associated with glomerular hypertrophy (87). ORB-9195 administration to diabetic rats resulted in a reduction in the progression of diabetic nephropathy by block- ing type IV collagen and overproduction of TGF-G and VEGF (88). In the same context, the AGE-breaker, ALT-711, has also been shown to afford renoprotection to diabetic animals (89).
HUMAN STUDIES
Biopsy samples from kidneys from diabetic subjects have demonstrated increased AGE deposition at AGE-specific binding sites throughout the renal cortex (90,91). Spe- cific AGE compounds (e.g., CML, pyralline, and pentosidine) have been identified in renal tissue of diabetics with or without end-stage renal disease; AGE accumulation appeared to parallel the severity of diabetic nephropathy (92). Also, whereas low-level RAGE expression in normal control human subjects was restricted to podocytes, glom- eruli of patients with diabetic nephropathy demonstrated diffuse upregulation of RAGE expression in podocytes, colocalizing with synaptopodin expression (93). A recent study in kidney biopsies from patients with diabetic nephropathy showed significant reduction of nephrin, an important regulator of the glomerular filter integrity. In the same study, cultured podocytes showed significant downregulation in nephrin expression when glycated albumin was added (77).
In a clinical study of type 1 diabetic patients serum levels of AGEs increased signifi- cantly as patients progressed from normal to microalbuminuria, clinical nephropathy and hemodialysis and correlated positively with urinary albumin excretion (94).
Diabetic Retinopathy
Diabetic retinopathy occurs in three-fourths of all persons with diabetes for more than 15 years (95) and is the most common cause of blindness in the industrialized world (96).
It is primarily a disease of the intraretinal blood vessels, which become dysfunctional in response to hyperglycemia with progressive loss of retinal pericytes and eventually endothelial cells leading to capillary closure and widespread retinal ischemia (97).
It has been shown that AGEs disturb retinal microvascular homeostasis by inducing
pericyte apoptosis and VEGF overproduction (98). In vitro work in bovine retinal endot-
helial cells showed that AGEs induced VEGF overproduction through generation of
oxidative stress and downstream activation of the protein kinase C pathway (99). In vitro studies in retinal organ cultures showed increased glyoxal-induced CML formation, a dose-dependent induction of apoptotic molecules and increased cell death, events that were prevented by anti-AGE agents and antioxidants (100).
AGEs were found to retard the growth of pericytes and exert an acute toxicity to these cells (98). In vitro, rat retinal vascular cells exposed to AGE show abnormal endothelial nitric oxide synthase expression, which may account for some of the vasoregulatory abnormalities observed in the diabetic vasculature (101). In vitro studies in human donor eyes showed that vitreous collagen undergoes glycation as well as copper and iron glycoxidation, leading to structural and functional impairment and possibly retinopathy (102).
Within a few months of diabetes, AGEs are already found to accumulate in vascular basement membrane and retinal pericytes of rats (103).
When nondiabetic animals were infused with AGEs for several weeks, significant amounts of these adducts distributed around and within the pericytes, colocalized with AGE receptors and induced basement membrane thickening (104) leading to loss of retinal pericytes (105). In contrast, the inhibition of AGE formation by aminoguanidine, a well known AGE inhibitor, prevented microaneurysm formation, endothelial prolifera- tion, and pericyte loss (97). A combination of antioxidants and AGE inhibitors has been shown to prevent AGE-induced apoptosis in primary (rat) retinal organ cultures (100), although the administration of monoclonal antibodies, which recognize Amadori-modi- fied glycated albumin, reduced the thickening of the retinal basement membrane in db/
db mice, implying that even early glycated adducts may play a role in diabetic retinopathy (104). More studies are needed to confirm these data, however.
A study comparing postmortem human retinas between diabetic subjects with dia- betic retinopathy and nondiabetic subjects, found that CML and VEGF immunoreactivi- ties, which were not evident in the control subjects, were distributed around blood vessels of diabetic retinas. Both VEGF and CML expression was greater in subjects with prolif- erative diabetic retinopathy (106). These data suggest that CML could have a role in VEGF expression in diabetic retinopathy.
In a clinical study of type 1 diabetics (38 males and 47 females) a significant elevation of serum AGE levels was found associated with severe diabetic retinopathy. CML-AGE levels were also increased at the stage of simple diabetic retinopathy suggesting a pos- sible role of CML in the early phases of this condition (92).
Similarly, increased pentosidine levels were found in the majority of vitreous samples from diabetic patients with diabetic retinopathy compared to controls indicating that glycation occurs and is accelerated in human diabetic vitreous (107). This was further confirmed in another clinical study which involved 72 type 2 diabetics, in which sugar- induced AGEs correlated with the severity of retinopathy (108).
Diabetic Neuropathy
About half of all people with diabetes experience some degree of diabetic neuropathy, which can present either as polyneuropathy or mononeuropathy (109). Diabetic neuropa- thy can also affect the central and the autonomic nervous systems. Level of hyperglyce- mia seems to determine the onset and progression of diabetic neuropathy (110,111).
In vitro studies have shown that glycation of cytoskeletal proteins such as tubulin,
actin, and neurofilament results in slow axonal transport, atrophy, and degeneration
(110). Additionally, glycation of laminin, an important constituent of Schwann cell basal
laminae, impairs its ability to promote nerve fiber regeneration (111). The process of glycation increases the permeability of proteins, albumin, nerve growth factor, and immunoglobulin G across the blood–nerve barrier (112) leading to protein accumula- tion in the central nervous system (113).
Diabetic rats show reduction in sensory motor conduction velocities and nerve action potentials and reduction in peripheral nerve blood flow and all these abnormalities can be prevented by pretreatment with anti-AGE agents such as aminoguanidine (114,115).
Pentosidine content was increased in cytoskeletal proteins of the sciatic nerve of streptozotocin induced diabetic rats and decreased after islet transplantation (111).
Pentosidine content was found elevated in cytoskeletal and myelin protein extracts of sural nerve from human subjects (116). The sural and peroneal nerves of human diabetic subjects contain AGEs in the perineurium, endothelial cells, pericytes of endoneural microvessels, and in myelinated and unmyelinated fibers; a significant correlation has been observed between the intensity of CML accumulation and myelinated fiber loss (117). At the submicroscopic level, AGE deposition appeared focally, as irregular aggre- gates in the cytoplasm of endothelial cells, pericytes, axoplasm and Schwan cells of both myelinated and unmyelinated fibers. Interstitial collagen and basement membrane of the perineurium also exhibited similar deposits. The excessive accumulation of intra and extracellular AGEs in human diabetic peripheral nerve supports the view of a causative role for these substances in the development of diabetic neuropathy (117). Furthermore, AGE accumulation in the vasa nervorum could worsen wall thickening with occlusion and ischemia and secondarily segmental demyelination (118).
Diabetic Dermopathy
Skin, like other tissues, accumulates glycoxidation products in diabetes (119–121), which can account for alterations in physicochemical properties leading to the diabetic skin-related disorders (61,122). AGE-related changes on diabetic skin are similar to those observed in aged skin and include altered tissue oxygen delivery (123), growth factor activity (124–127), vascular, skin, fibroblast, and inflammatory cell dysfunction (128–131), increased metalloproteinase production (132), and defective collagen remod- eling (122–130). In skin, inhibitors of AGE formation, such as aminoguanidine were shown to prevent AGE accumulation and subsequent collagen crosslinking, to improve angiogenesis and to restore the activity of various growth factors (133–137).
From a pathogenic point of view, through the above described effects, AGEs could partially explain the delayed wound healing observed in diabetes. Recently, it has been showed that dietary AGEs can modify wound healing rate in db/db (+/+) mice by altering the total body AGE burden (32). Another study has shown acceleration of wound healing in db/db mice by RAGE-receptor blockade further supporting a role for AGEs in the pathogenesis of delayed diabetic wound healing (61).
ADVANCED GLYCOXIDATION END-PRODUCTS AND MACROANGIOPATHY
The two most frequent patterns of macrovascular disease are atherosclerosis, which
leads to thickening of the intima, plaque formation, and eventual occlusion of the vascular
lumen and stiffness of the arterial wall, which leads to ventricular hypertrophy. Based on
the existing literature, AGEs play a significant role in the pathogenesis of both manifes-
tations of cardiovascular disease (CVD).
In Vitro Studies
In vitro studies have shown that AGEs have complex properties that promote vascular disease including an ability to form chemically irreversible intra- and intermolecular crosslinks with matrix proteins in the vascular wall increasing arterial rigidity (5,138).
Also, the interaction of AGEs with endothelial cell receptors induces increased vascular permeability, increases procoagulant activity, migration of macrophages and T-lympho- cytes into the intima (initiating a subtle inflammatory response), and impairment of endothelium-dependent relaxation (139). Impaired endothelial relaxation and endothe- lial migration of monocytes are generally considered to be among the first steps in atherogenesis. At the same time, AGE-induced activation of monocyte/macrophages leads to the release of a variety of inflammatory cytokines and growth factors, which induce over production of extracellular vascular wall matrix (5,140).
Glycoxidation of LDL cholesterol makes this molecule less recognizable by the native LDL receptor, which results in delayed clearance, increased plasma levels and eventually enhanced uptake of cholesterol by the scavenger receptors on macrophages and vascular smooth muscle cells. This process, finally, results in lipid-laden foam cells formation in the arterial intima and atherosclerosis (5,48,141). Glycated LDL has also been shown to stimulate production of plasminogen activator inhibitor-1 (PAI-1) and to reduce genera- tion of tissue plasminogen activator (tPA) in cultured human vascular endothelial cells (142). These effects that could potentially increase thrombotic vascular complications in vivo were prevented by treatment with aminoguanidine.
Regarding HDL the net effect of glycation on this molecule is altered lipoprotein function and decreased ability to prevent monocyte adhesion to aortic endothelial cells, an important initial event in the development of atherosclerosis (143). The physiological significance of this finding, however, remains to be further substantiated. Additionally, in vitro glycation of lipoprotein(a), an independent risk factor for CVD, has been shown to amplify lipoprotein(a)-induced production of PAI-1 and further reduced tPA genera- tion from vascular endothelial cells (144,145).
More recently it has become apparent that the vascular wall also produces superoxide, mostly via enzymes similar to the neutrophil oxidase. All cell types in the vascular wall produce reactive oxygen species (ROS) derived from superoxide-generating protein complexes similar to the leukocyte nicotinamide adenine dinucleotide phosphate oxi- dase. AGE have been shown to enhance vascular oxidase activity (146) and increased vascular oxidase activity has been associated with diabetes (147).
Animal Studies
Indirect evidence for AGE involvement in CVD emerges from histological studies that show increased AGE deposits in aortic atherosclerotic lesions, even in the absence of diabetes (140,148). These AGE deposits correlate with the degree of atheroma (146). In more direct experiments, prolonged intravenous infusion of glycated rabbit serum albu- min into nondiabetic rabbits promoted intimal thickening of the aorta (149). Addition- ally, exogenous administration of AGE-modified albumin in healthy nondiabetic rats and rabbits was correlated with significantly increased AGE levels (approximately sixfold) in aortic tissue samples and increased vascular permeability together with markedly defective vasodilatory responses to acetylcholine and nitroglycerin. These abnormalities were prevented or reduced by the combined administration of aminoguanidine (150).
Infusion of diabetic red blood cells (carrying AGEs) into normal rats increased vascular
permeability in these animals and this effect was prevented by blockade of RAGE (151).
Local application of AGEs to the vessel wall-enhanced intimal hyperplasia, indepen- dently of diabetes or hypercholesterolemia, in a model of atherosclerosis in the rabbit (152).
In recent studies, dietary AGE restriction was associated with significant reduction in circulating AGE levels and a significant reduction in neointimal formation, 4 weeks after arterial injury in apo-E knockout mice (Fig. 5). This study also showed markedly decreased macrophage infiltration in the lesioned areas of the vessel wall (28). Addition- ally, after 8 weeks on the low AGE diet, diabetic apo-E knockout mice showed significant suppression of atherosclerotic lesions accompanied by reduced circulating AGE levels and decreased expression of inflammatory molecules (29) (Fig. 6). These studies support the view that exogenous AGEs have a significant vasculotoxic effect.
Fig. 5. Dietary AGE restriction and neointimal proliferation in apolipoprotein E-deficient non-db mice. A, low AGE diet. B, regular AGE diet (ref. 28).
Fig. 6. Dietary AGE restriction prevents atheroma formation in apoE knock out mice. A, low AGE diet. B, high AGE diet (ref. 29).
Inhibition of AGE formation by aminoguanidine has been shown to lead to reduced atherosclerotic plaque formation in cholesterol-fed rabbits (140,153). Aminoguanidine administration to rats has been shown to prevent diabetes-induced formation of fluores- cent AGE and crosslinking of arterial wall connective tissue proteins (153). Oral admin- istration of 2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), an inhibitor of both glycoxidation and lipoxidation reactions in rats, following balloon injury of the carotid artery, effectively prevented the intimal thickening that typically accompanies this injury (7). These studies support a causative role for AGEs in athero- sclerosis.
Treatment of apo-E deficient diabetic mice with the soluble extracellular domain of RAGE also suppressed diabetic atherosclerosis in a glycemia- and lipid-independent manner (154).
Treatment of rats with streptozotocin-induced diabetes with the AGE-breaker ALT- 711 for 1–3 weeks reversed the diabetes-induced increase of large artery stiffness as measured by systemic arterial compliance, aortic impedance, and carotid artery compli- ance and distensibility (8). Administration of the same compound produced significant improvement of diabetes-induced myocardial structural changes in rats with streptozotocin- induced diabetes (155). N-Phenylthiazolium bromide (PTB), another AGE breaker, has led to marked reduction in AGE-collagen from tail tendons in rats (13). These findings support the role of AGE accumulation in causing arterial stiffness.
Treatment of diabetic rats with hydralazine and olmesartan showed equal renoprotective effect despite differential effect on the renin–angiotensin system. The fact that both drugs effectively suppress AGE formation suggests a critical role for AGEs in this nephropathy model (156).
Human Studies
Currently, human studies provide only indirect support for a role of AGEs in initiating CVD. Long-term interventional trials will be able to prove this link. Highly suggestive of such role are the findings of AGE deposits in the atherosclerotic plaque of arteries from diabetic patients (157,158), and chronic renal failure patients with or without diabetes (159). An autopsy study showed increased colocalized deposition of AGE and apo-B in aortic atherosclerotic lesions in end-stage renal disease patients with or without diabetes compared to controls. These deposits correlated with the duration of hemodialysis, but not with the duration of diabetes (160). A significant correlation between serum AGE- apo-B levels, tissue accumulation of AGE-collagen and severity of atherosclerotic le- sions has been described in a group of nondiabetic patients with coronary artery occlusive disease requiring bypass surgery (159). Histological sections of human aortas obtained from postportem examination of diabetic subjects showed a correlation between AGE tissue accumulation and aortic stiffness (161). A significant CML deposition in athero- sclerotic plaques was also observed that correlated with the extent of the atherosclerotic changes. Moreover, AGE receptors were identified in the cellular components of the lesions with the same distribution pattern as AGE (162).
A cross-sectional study showed that type 2 diabetic patients had increased serum AGE
levels and impaired endothelium-dependent and endothelium-independent vasodilata-
tion compared to healthy control subjects. Data analysis showed a significant inverse
correlation between serum AGE levels and endothelium-dependent vasodilatation of the
brachial artery, a well-established marker of early atherosclerosis. In multiple regression
analysis, serum AGE levels were the only factors, which correlated independently with the endothelium-dependent vasodilatation (163).
Administration of an AGE-restricted diet for several weeks in a group of diabetic patients was associated with significant reduction of serum levels of AGEs and vascular cell adhesion molecule-1, an indicator of endothelial dysfunction (34). A recent abstract reported acute endothelial dysfunction in response to the ingestion of an AGE-rich bev- erage in diabetic subjects (164). An obvious implication of these striking results is that a low AGE diet would reverse endothelial dysfunction among diabetes patients, but this remains to be further proven.
Arterial stiffness increases with duration of diabetes largely as the result of the effect of AGE on connective tissue and matrix components (165). Oral administration of ALT- 711, a novel nonenzymatic breaker of AGE crosslinks, significantly improved arterial compliance and decreased pulse pressure in older individuals with vascular stiffening compared to placebo (9). These results strongly suggest that AGE have a pathogenic role in arterial stiffness.
ANTIADVANCED GLYCOXIDATION END-PRODUCT STRATEGIES As the understanding of the biology of AGE has evolved, new strategies to forestall their adverse effects have developed. Several approaches seeking to decrease exogenous AGE intake, decrease or inhibit endogenous AGE formation, reduce AGE effects on cells, and break pre-existing AGE crosslinks have been explored.
Diet
As diet provides a significant source of exogenous AGE, recent work has focused on determining whether it represents a modifiable risk factor for the development of AGE- induced pathology. In vivo studies have demonstrated that the typical diabetes-related structural changes seen in experimental animals fed with standard AGE-enriched diets could be prevented by dietary AGE restriction (27–30). A recent 6-week study in diabetic patients compared the effects of two nutritionally equivalent diets differing only by their AGE content and demonstrated 30%–50% reduction of serum AGE levels and a signifi- cant reduction in the levels of inflammatory factors in the subjects receiving the diet with low AGE content (34). A significant reduction of circulating AGE levels was also observed in a study including nondiabetic renal failure patients undergoing peritoneal dialysis treatment (35–37). These studies support the notion of a significant contribution of dietary AGE intake to the body pool of AGE and make evident that dietary AGE restriction is a feasible and safe strategy to decrease the body AGE burden.
Metabolic Factors
As hyperglycemia enhances AGE formation it is obvious that intensive treatment of hyperglycemia can modify the body AGE pool. Indeed, diabetic rats with good metabolic control exhibited lower levels of pentosidine, and lower intensity of collagen-linked fluorescence glycation and oxidation compared to rats with bad metabolic control (166).
Skin collagen glycation, glycoxidation, and crosslinking were lower in a large group of
type 1 diabetic patients under long-term intensive vs conventional treatment, as was
shown in a cohort of patients studied in the DCCT (119).
Antioxidants
Several studies have proposed various antioxidants as anti-AGE agents, including vitamin E (167), N-acetylcysteine (168), taurine (169), F-lipoic acid (170), penicillamine (171), and nicarnitine (172). Also, pyruvate is a potent scavenger of ROS such as H2O2 and O2- that also minimizes the production of OH by the Haber-Weiss reaction. Addi- tionally, it inhibits the initial reaction of glucose with free amino groups that results in Schiff base formation, as documented by in vitro data (173,174). Despite the existing data, however, further studies are needed to establish the effectiveness of treatment with antioxidants as a strategy in reducing AGE levels.
Agents That Prevent Advanced Glycoxidation End-Products Formation A large number of in vitro and in vivo studies have been conducted using agents that prevent AGE formation to modify diabetic complications. These agents act by inhibiting postAmadori advanced glycation reactions or by trapping carbonyl intermediates (gly- oxal, methylglyoxal, 3-deoxyglucosone) and thus inhibiting both advanced glycation and lipoxidation reactions. Aminoguanidine (11,13), ALT-946 (11,175,176), 2-3- Diaminophenazine (11,177), thiamine pyrophosphate (178), benfotiamine (179,180), and pyridoxamine (181–183) constitute known representatives of the first group of agents, although ORB-9195 is a derivative- representative of the second group of agents (7,184–186).
Advanced Glycoxidation End-Product Crosslink Breakers
Recently, a promising therapeutic strategy has been to attack the irreversible intermo- lecular AGE crosslinks formed in biological systems providing prevention or reversal of various diabetes- and aging-related complications. This approach aims to “break”
preaccumulated AGE and help renal elimination of resulting smaller peptides. PTB was originally studied (187) and more recently ALT-711 (8,13,188). Long-term studies are in progress to establish the safety of this new category of anti-AGE agents
Advanced Glycoxidation End-Products Antibody (A717)
A monoclonal antibody that neutralizes the effects of excess glycated albumin has been studied and shown to offer significant primary or secondary prevention of diabetic nephropathy (189,190).
Antihypertensive Agents
Recently, losartan and olmesartan, antihypertensive drugs known to act through angio- tensin receptor inhibition, have been shown to decrease AGE formation (191). Hydralazine, another antihypertensive agent whose effect does not involve the renin–angiotensin sys- tem, has AGE-inhibitory effects similar to those of low-dose olmesartan (192). The renoprotective effects shown by these drugs suggest that they derive not only from the drugs effect on lowering blood pressure and blocking angiotensin but also from reduced AGE formation (193).
CONCLUSIONS
An increasing body of evidence indicates that AGEs play a significant role in the
pathogenesis of diabetic complications. Further studies, however, are still needed to
elucidate the exact role of AGEs in this area. More importantly, as these studies progress,
new approaches to therapy to reduce the life-threatening impact of these complications would develop. Dietary restriction of AGE intake appears as a novel and important intervention tool that deserves further development.
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