AGE–RAGE Stress, Stressors, and Antistressors in Health and Disease

Abstract

Adverse effects of advanced glycation end-products (AGEs) on the tissues are through nonreceptor- and receptor-mediated mechanisms. In the receptor-mediated mechanism, interaction of AGEs with its cell-bound receptor of AGE (RAGE) increases generation of oxygen radicals, activates nuclear factor-kappa B, and increases expression and release of pro-inflammatory cytokines resulting in the cellular damage. The deleterious effects of AGE and AGE–RAGE interaction are coined as “AGE-RAGE stress.” The body is equipped with defense mechanisms to counteract the adverse effects of AGE and RAGE through endogenous enzymatic (glyoxalase 1, glyoxalase 2) and AGE receptor-mediated (AGER1, AGER2) degradation of AGE, and through elevation of soluble receptor of AGE (sRAGE). Exogenous defense mechanisms include reduction in consumption of AGE, prevention of AGE formation, and downregulation of RAGE expression. We have coined AGE and RAGE as “stressors” and the defense mechanisms as “anti-stressors.” AGE–RAGE stress is defined as a shift in the balance between stressors and antistressors in the favor of stressors. Measurements of stressors or antistressors alone would not assess AGE–RAGE stress. For true assessment of AGE–RAGE stress, the equation should include all the stressors and antistressors. The equation for AGE–RAGE stress, therefore, would be the ratio of AGE + RAGE/sRAGE + glyoxalase1 + glyoxalase 2 + AGER1 +AGER2. This is, however, not practical in patients. AGE–RAGE stress may be assessed simply by the ratio of AGE/sRAGE. A high ratio of AGE/sRAGE indicates a relative shift in stressors from antistressors, suggesting the presence of AGE–RAGE stress, resulting in tissue damage, initiation, and progression of the diseases and their complications.

Advanced glycation end-products (AGEs) and its cell receptor RAGE (receptor for AGEs) have been implicated in the pathogenesis of numerous diseases (atherosclerosis, ¹ coronary artery disease, ^{2 3} hypertension, ^{4 5} cerebral vascular disease, ⁶ hyperthyroidism, ⁷ Alzheimer disease, ⁸ and end-stage renal disease, ⁹ and diabetes). ^{10 11} The adverse effects of AGEs are through nonreceptor- and receptor-mediated mechanisms. Nonreceptor-mediated mechanisms include enhanced synthesis of extracellular matrix, trapping of subendothelial low-density lipoprotein (LDL), and cross-binding with collagen. In receptor-mediated mechanism, there is an interaction of AGEs with RAGE resulting in the increased generation of oxygen radicals, activation of nuclear factor kappa B (NF-κB), and increased expressions of pro-inflammatory cytokines and cell adhesion molecules. ⁷ AGEs and AGE–RAGE interaction cause potential biological damage and hence we have coined AGE and RAGE as “stressors.” Stress is defined as a process of altered biochemical homeostasis produced by physiological or psychological or environmental stressors. ¹² The body is equipped with anti-AGE–RAGE defense mechanisms such as degradation of AGE with enzymes and AGE receptor, and circulating soluble AGE-receptor called sRAGE which competes with RAGE for AGE, to counterbalance the effects of stressors (AGE and RAGE) and we have coined this as antistressors. Excessive levels of AGE and RAGE because of increased consumption of AGE, deficiency in the AGE degradative enzymes or receptors, reduced amount of sRAGE, and increased expression of RAGE would lead to AGE-RAGE stress. A shift in the balance between stressors (AGE and RAGE) and antistressors in favor of stressors, we have coined as AGE–RAGE stress. The ratio of AGEs/sRAGE has been reported as one of the important risk marker or biomarker for disease states. ¹³ AGE/sRAGE may be one of the most important determinants of AGE–RAGE stress.

This review focuses on the AGE–RAGE stress, stressors, and antistressors. It also discusses the consequences of AGE–RAGE interaction and defense mechanisms such as degradation of AGE, downregulation of RAGE, enhancement of levels of sRAGE, and lowering of AGEs levels.

Stressors (AGE and RAGE)

1. AGEs

   AGEs comprise of chemical structures such as N-ε-carboxymethyl-lysine (CML), N-ε-carboxyethyl-lysine (CEL), pyrraline, pentosidine, and argpyrimidine. ¹⁴ CML modifications of proteins are predominant AGEs. ¹⁵ AGEs are heterogeneous groups of irreversible adducts formed by nonenzymatic glycation and glyoxidation of proteins, lipids, and nucleic acid with reducing sugars. ^{16 17} There are two sources of AGEs, in vivo, endogenous, and exogenous. Endogenous AGE formation in normal individual occurs slowly. Hyperglycemia accelerates formation of AGEs. ¹⁸ Endogenous sources comprise of glycation, polyol pathway, and glyoxidation. Glycation is nonenzymatic reaction of proteins, lipids, and nucleic acids with reducing sugars. ^{16 17} In the polyol pathway, aldolase reductase or sorbitol dehydrogenase acts on glucose to form intermediary products which bind to proteins to form AGEs. ^{19 20} Formation of AGE through glyoxidation pathway involves reactive oxygen species (ROS). Generation of superoxide anions in the mitochondria or redox-sensitive mechanism that generates hydroxyl radicals forms glyoxal and methylglyoxal (MGO). These agents react with different biomolecules to produce AGEs. ^{20 21} Exogenous sources of AGEs include foods high in AGE content (red meat, cheese, crispy brown crackers, fatty cookies sweetened with sugars, cream, and animal fat), ²² cooking at high temperature in dry heat (frying, broiling, grilling, roasting, and baking), ²³ and cigarette smoking. ²⁴

   In humans, there is a significant increase in plasma levels of AGEs within 2 hours following oral intake of single AGE-rich diet. ²⁵ There is a positive correlation between dietary AGE content and serum/tissue levels of AGEs. ²⁶ About 10% to 30% of AGE (CML) is absorbed in the gastrointestinal tract and is delivered to the liver and other organs. ²⁷ Thirty-three percent of the absorbed AGEs is excreted in the urine and the rest accumulates in the body. ²⁸

2. Receptor for AGE

   There are mainly three receptors for AGEs: (a) full-length RAGE, (b) cleaved RAGE (cRAGE), and (c) endogenous secretory RAGE (esRAGE). Full-length RAGE ( Fig. 1 ) is a multiligand receptor and a member of the immunoglobulin superfamily of cell surface molecule. ²⁹ It has three extracellular domains including v-type that possesses ligand binding properties, and two c-type immunoglobulin domains C1 and C2, a transmembrane helix, and a short cytosolic tail. ³⁰ The fourth transmembrane domain anchors RAGE in the membrane and is connected to the highly charged fifth intracellular domain that interacts with cytosolic transduction molecule. RAGE is expressed in a wide range of cells including monocytes, macrophages, endothelial cells, adipocytes, and podocytes. Besides AGEs, S100, calgranulin, ³¹ amphoterin ³² amyloid-B, and other fibrillar proteins ³³ can bind with RAGE.

Fig. 1.

Diagrammatic representation of full-length RAGE and sRAGE. Full-length RAGE consists of intracellular tail, transmembrane domain, and extracellular domain, comprising of C ₁ , C ₂ , and V domain. V domain binds with AGE. sRAGE is comprised of cRAGE and esRAGE. Both cRAGE and esRAGE lack transmembrane domain and intracellular tail. AGE, advance glycation end-product; RAGE, cell bound receptor for AGE; sRAGE, soluble receptor for AGE; cRAGE, cleaved RAGE; esRAGE, endogenous secretory RAGE; C, constant; V, variable.

Adverse Effects of AGE and RAGE

AGE induces adverse effects in the body by two separate mechanisms: (a) nonreceptor and (b) receptor-mediated mechanisms.

1. Nonreceptor-mediated mechanism

   Functional properties of extracellular matrix are affected by AGEs. Accumulation of AGEs on protein of extracellular matrix leads to the formation of cross-links, which traps other local macromolecules. ³⁴ The properties of collagen are altered through AGE–RAGE intermolecular covalent bond or cross-linking. ³⁵ Cross-linking of AGEs on collagen and elastin increases the extracellular matrix area which increases the stiffness of the artery. ³⁶ Glycation increases the synthesis of collagen. ³⁷ Cross-linking makes the collagen insoluble to the hydrolytic enzymes. ³⁸ AGE-linked collagen is less susceptible to hydrolytic turnover and becomes stiff. Cross-linking of AGE with elastin reduces the elasticity of arterioles. Cross-linking increases the synthesis of collagen and reduces the quantity of elastin. AGE cross-linking with protein depends upon both sugar concentration and turnover rate of body proteins. LDL is sensitive to AGE cross-linking resulting in decreased uptake by LDL receptors. ³⁹ AGE cross-linking of proteins of lens induces functional changes in lens ¹⁷ and AGE cross-linking of the proteins of renal tissue induces thickness of basement membrane of glomerulus, ⁴⁰ and many other organs (retina, kidney nerves, hypertension, and atherosclerosis. ^{4 41}

   Glycation affects apoprotein B and phospholipid component of LDL resulting in functional alteration in LDL clearance and increased susceptibility to oxidative modification. ^{42 43} Glycation of LDL decreases its recognition by LDL receptors. ⁴⁴ Glycated LDL has the capacity to stimulate the mitogen-activated protein kinase (MAPK) signaling pathways in vascular smooth muscle cells that increase the cell proliferation or differentiation. ^{40 45} AGEs interfere with the reverse cholesterol transport through suppression of scavenger receptor B1 (sR-B1)-mediated uptake of cholesterol ester from high-density lipoprotein (HDL) by liver and sR-B1-mediated cholesterol efflux from peripheral cells. ⁴⁶ AGE induces accumulation of cholesterol and its ester in macrophages in vitro. ⁴⁷

   Glycated albumin alters the binding of drugs in plasma in diabetes. ⁴⁸ It plays role in the platelet activation and aggregation. ⁴⁹ Glycated fibrinogen impairs fibrinolysis ⁵⁰ and increases fibrin gel permeability resulting in the formation of less thrombogenic fibrin network. ⁵¹ Glycation of immunoglobulin (IgG) is associated with inflammation and is target for auto-antibodies in rheumatoid arthritis. ⁵² Among all the AGEs, MGO is the major immune-suppressant in patients with diabetes. ⁵²

2. Receptor-mediated mechanism

   The effects of interaction of AGEs with RAGE are summarized in Fig. 2. Interaction of AGEs with RAGE results in the generation of ROS and activation of NF-κB. AGE–RAGE interaction directly increases the generation of ROS through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ⁵³ which in turn activates NF-κB. Binding of AGEs with RAGE stimulates various signaling pathways including MAPKs, extracellular regulated kinases (Erk) 1 and 2, phosphatidyl-inositol 3 kinase/c-Jun-N-terminal kinase, p21 Ras, and the Janus kinases. ^{54 55} The net result of these signaling mechanisms is the activation of NF-κB and subsequent transcription of numerous proinflammatory genes shown in Table 1 . ^{31 35 56} Interaction of AGEs with RAGE in monocytes induces chemotaxis which accelerates the migration of monocytes into subendothelial space. ^{57 58} Binding of AGEs with RAGE in monocyte-macrophage increases the expression and generation of interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), platelet-derived growth factor (PGDF), and insulin-like growth factor 1 (IGF-1) ^{59 60 61} and increases uptake of glycated LDL. ⁶² Interaction of AGEs with RAGE decreases endothelial barrier function and hence increases permeability of endothelial cell layer ^{63 64} and vascular smooth muscle cells proliferation and production of fibronectin. ^{65 66}

Fig. 2.

The effects of interaction AGE with RAGE or sRAGE. Interaction of AGE with RAGE increases ROS, NF-κB, VCAM-1, growth factor, and cytokines. Interaction of AGE with sRAGE counteracts the effect of AGE–RAGE interaction. ↑, increase; (-), decrease; AGE, advance glycation end-product; RAGE, cell bound receptor for AGE; sRAGE, soluble receptor for AGE; ROS, reactive oxygen species; NF-κB, nuclear factor kappa B; VCAM-1, vascular cell adhesion molecule 1; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; PDGF, platelet-derived growth factor; IGF-1, insulin-like growth factor-1.

Table 1. Increased gene expressions of some molecules by activated NF-κB ¹⁶ .

Cell adhesion molecules	ELAM-1
	ICAM-1
	VCAM-1
Cytokines and chemokines	IL-1, IL-1β, IL-2, IL-6, IL-8, TNF-α, G-CSF, M-CSF, MCP-1
Acute phase proteins	Serum amyloid A precursor
	Angiotensinogen
	Complement factor C4
	Compliment factor B
Others	Nitric oxide synthase
	Hemeoxygenase-1
	Growth factors

Abbreviations: ELAM-1, endothelial leukocyte adhesion molecule 1; G-CSF, granulocyte colony stimulating factor; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MCP-1, monocyte chemotactic protein; M-CSF, monocyte colony stimulating factor; NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.

Antistressors (AGE–RAGE Defense Mechanism)

The body is equipped with defense mechanisms to counteract the deleterious effects of AGE–RAGE stressors. These antistressors can be classified into two types: endogenous and exogenous.

1. Endogenous antistressors

   Endogenous antistressors include enzymatic degradation of AGEs, AGE receptor-mediated degradation of AGE, and sRAGE.

   1. Enzymatic degradation of AGEs

      Glyoxalase-1 (GLO1) and glyoxalase-2 (GLO2) degrade reactive dicarbonyls prior to the formation of AGEs. Reactive dicarbonyls, MGO, react with reduced glutathione to form hemithioacetal. ⁶⁷ The hemithioacetal is converted to s-2-hydroacetalglutathione by GLO1. GLO2 converts s-2-hydroacetalglutathione to α-hydroxyl and releases reduced glutathione. ⁶⁸ Overexpression of GLO1 in endothelial cells in vitro under hyperglycemic conditions reduced the levels of dicarbonyls ⁶⁹ and this effect was associated with correction of the defects in angiogenesis ⁷⁰ and vascular relaxation. ⁷¹ Similarly, overexpression of GLO1 in lens and retinal capillary pericytes respectively protected against hyperglycemia-induced protein modifications ⁷² and apoptosis. ⁷³ GLO1 is the key enzyme in antiglycation defense system because this is a rate-limiting step in the glyoxalase pathway and it prevents the storage of reactive dicarbonyls. ⁷⁴

   2. AGE-receptor-mediated degradation of AGE

      Besides RAGE, AGE can bind to other cell surface receptors such as advanced glycation end-products receptors (AGER1, AGER2, AGER3). The first cell receptor discovered in connection with AGE endocytosis was AGER1. ⁷⁵ This protein has significant AGE-specific binding capacity and hence was named AGER1.Although AGER2 does not directly binds with AGE, it is effectively phosphorylated by AGE and has been suggested to play a role in early stages of AGE signaling. ⁷⁵ AGER3 has a high affinity to bind with AGE. ⁷⁶ Its exact role is not known. However, it has been suggested that AGER3 may regulate the turnover of AGE and maintain the integrity of tissue. ⁷⁷ It is upregulated in hyperglycemia and after exposure to AGEs. ⁷⁸ AGER1 has been studied in detail. AGER1 protein is present in most of the cells and tissues including macrophages, ⁷⁹ mesangial cells, ⁸⁰ and mononuclear cells ⁸¹ mediate the uptake and degradation of AGE by kidney. ⁸² AGER1 accelerates the uptake and removal of AGE and blocks cellular AGE–RAGE-mediated generation of ROS and proinflammatory cytokines. ^{80 83} AGER1 also counteracts AGE-induced oxidative stress through inhibition of RAGE signaling. ^{80 83} Degradation of AGE by AGER1 produces AGE peptides which normally filter through glomerular membrane. The filtrate undergoes variable degree of tubular reabsorption or further catabolism in the proximal tubules and is excreted in the urine. ⁸⁴ There is an inverse correlation between serum levels of AGE and renal function. ⁸⁵ Renal disease is associated with reduced excretion of AGEs. ⁸⁶ Since AGER1 and RAGE compete for AGE, low levels of AGER1 would increase the binding of AGE with RAGE and hence increase in the oxidative stress and inflammation.

      There is a downregulation of AGER1 by AGE-rich diet ⁸³ and diabetes. ⁸⁷ There is a reduction in both RAGE and AGER1 with consumption of low AGE-rich diet. Also, there is an inverse correlation between AGER1 and intracellular levels of AGEs and positive correlation between AGER1 and urinary AGE levels in nondiabetic individuals. ⁸⁸ However, the levels of RAGE are high while that of AGER1 reduced in diabetic patients in spite of full antidiabetic therapy. ⁸⁹ With the consumption of low AGE-rich diet for 4 months, the levels of AGER1 were restored, while those of RAGE were suppressed. ⁸⁹ These data suggest that the reduction in AGER1 gene contributes to the complications in diabetes due to elevation of AGE and consequences of AGE–RAGE interaction. It has been reported that interruption of AGER1-dependent uptake of AGEs and subsequent degradation accelerates glomerular renal pathologies in spontaneous nonobese diabetic mice. ⁸⁷ It has also been shown that the patients with severe diabetic complications have reduced expression of AGER1 in circulating mononuclear cells and elevated serum levels of AGEs. ⁸¹ AGER1/RAGE ratio may serve as a biomarker for pathogenesis of AGE–RAGE-mediated disease conditions. High ratio of AGER1/RAGE would protect from the deleterious effects of interaction of AGE with RAGE.

      Metabolic states including diabetes, hyperlipidemia, uremia, and aging are associated with upregulation of RAGE, AGER2, and AGER3, most probably because of elevation of AGE levels in these conditions. However, AGER1 expression is dependent on AGE levels. ⁸¹ Reduction in the expression of AGER1 in human monocytes is associated with elevated levels of sRAGE in patients with severe diabetic complications. ⁸¹ There was a positive correlation between expression of AGER1 and serum levels of sRAGE in complication-free diabetic patients suggesting that sRAGE modulates macrophage/monocyte AGER1. ⁸¹

   3. Soluble receptor for AGE

      There are three well-described receptors for AGE: full-length RAGE, cRAGE, and esRAGE. Full-length RAGE has been described in the detail in the receptor for AGE (RAGE) section of this review. sRAGE, cRAGE, and esRAGE are diagrammatically presented in Fig. 1 . cRAGE is proteolytically cleaved from full-length RAGE. ⁹⁰ esRAGE is formed from alternative splicing of full-length RAGE mRNA. ⁹¹ Measurement of total soluble RAGE (sRAGE) includes cRAGE and esRAGE (sRAGE ELISA kit), while measurement of esRAGE measures only esRAGE (esRAGE ELISA kit). Since total sRAGE includes both cRAGE and esRAGE, cRAGE is determined by subtracting esRAGE from sRAGE. Serum sRAGE is approximately five times higher than esRAGE in healthy subjects. ^{9 92 93}

      cRAGE and esRAGE lack cytosolic tail and transmembrane domain and circulate in the blood. They bind with AGE but does not activate intracellular singling. ⁹³ Soluble receptors are competitive inhibitor of AGE–RAGE interaction and may also serve as scavenger receptor for circulating AGEs. ⁹⁴ sRAGE competes with full-length RAGE for binding with AGEs and functions as decoy, and hence has a cytoprotective effects against adverse effects of AGE–RAGE interaction. AGEs interact with sRAGE before they interact with full-length RAGE. ⁹⁵ Low levels of serum sRAGE will allow high levels of AGEs to interact with RAGE and hence deleterious effect on the cells. Low serum levels of sRAGE have been implicated in the pathophysiology of numerous diseases. ^{1 2 3 4 5 6 7 8 9 10 11} However, diabetes and chronic renal disease and their complications are associated with high levels of serum sRAGE. ^{9 13} One would have expected that high levels of sRAGE would have protected the development of diabetes, chronic kidney disease. The reason for this discrepancy may be due to the elevation of levels of AGEs greater than the elevation of serum levels of sRAGE. Prasad et al ^{9 13} have reported that in the end-stage renal disease there is an increase in levels of both AGEs and sRAGE more so in AGEs than sRAGE. ⁹

      It has been reported by Zhou et al ⁹⁶ that the levels of AGEs and RAGE in carotid arterial wall are elevated in Zucker diabetic rats. They also showed that the balloon injury in carotid artery of these rats further increased the levels of AGE and RAGE and produced neointimal hyperplasia. Therapy with sRAGE reduced neointimal growth significantly. Treatment of diabetic apoE-deficient mice with sRAGE completely suppressed atherosclerosis. ⁹⁷ McNair et al ⁹⁸ have reported that low serum levels of sRAGE are a predictor of restenosis following percutaneous coronary intervention (PCI). These data suggest that sRAGE exerts an antagonist effects by binding RAGE ligands and preventing their signaling through membrane bound RAGE.

2. Exogenous antistressors

   Exogenous antistressors can be categorized as follows: (I) reduction in AGE consumption, (II) cessation of smoking, (III) prevention of AGE formation, (IV) AGE breakers, (V) downregulation of RAGE expression, (VI) elevation of sRAGE, (VII) administration of recombinant sRAGE.

1. Reduction in AGE consumption

   Consumption of AGE-rich diet such as read meat, cheese, cream, butter, animal fat, and sugars should be reduced. They increase the levels of AGEs in the body. ²³

2. Cessation of smoking

   Cigarette smoking should be stopped because it increases the serum levels of AGEs. ²⁴

3. Prevention of AGE formation

   The measures for the prevention of formation of AGE formation are summarized in Table 2 .

   1. Cooking

      Avoid cooking at high temperature with dry heat (frying, broiling, grilling, roasting, and baking). Cook at low temperature in moist heat. ²³

   2. Agents that reduce formation of AGEs

   1. Acidic ingredients (lemon juice, vinegar). ⁹⁹

   2. Phytochemicals from pomegranates, ¹⁰⁰ berries and grapes, ¹⁰¹ inhibit the formation of AGEs.

   3. Drugs:

      Aminoguanidine, a hydralazine compound, inhibits formation of AGEs. ¹⁰² It has been reported that aminoguanidine prevents the diabetic vascular complications in diabetic animals. ¹⁰³ In a placebo controlled clinical trial, aminoguanidine reduced glomerular filtration rate and proteinuria and prevented deterioration of retinopathy in diabetic patients. ¹⁰⁴ Action II trial reported that aminoguanidine produces side effects such as flu-like symptoms, hepatic abnormalities, gastrointestinal disorders, and anemia. ¹⁰⁵ Further clinical trials were terminated because of concern over the side effects. ¹⁰⁵

      Angiotensin-converting enzyme (ACE) inhibitor ramipril reduces fluorescent AGE in diabetic patients. ¹⁰⁶ Angiotensin II receptor blockers (telmisartan and losartan) reduced the formation of AGEs in cell culture. ¹⁰⁷ Valsartan ¹⁰⁸ and candesartan ¹⁰⁹ lowered the serum levels of AGE in hypertensive patients with diabetes. Atorvastatin ¹¹⁰ and cerivastatin ¹¹¹ reduced the serum levels of AGE in diabetic or prediabetic or diabetic kidney disease.

      Biguanide derivative (metformin), an antidiabetic drug, reduces the serum levels of AGE in women with polycystic ovary syndrome. ¹¹² Thiazolidine derivative (pioglitazone), an antidiabetic drug, inhibits AGE formation by trapping dicarbonyl compounds. ¹¹³ There are other drugs including α-lipoic acid, ¹¹⁴ aspirin, ¹¹⁵ taurine, ¹¹⁶ pentoxifylline, ¹¹³ resveratrol, ¹¹⁷ and curcumin ¹¹⁸ that are potential inhibitors of AGE formation.

   4. Vitamins

      Certain vitamins reduce the formation of AGEs. Benfotiamine (vitamin B1) ¹¹⁹ and pyridoxamine, a natural form of vitamin B6, ¹²⁰ vitamin C, ¹²¹ vitamin D, ¹²² and vitamin E, ¹²³ reduce the formation of AGEs.

   5. AGE cross-link breaker

      AGE cross-link breaker fragments α-carbonyl compounds by cleaving the carbon–carbon bond between carbonyls. Alagebrium (ALT-711) nonenzymatically breaks the established cross-linking AGE with adjacent long-lived collagen and elastin ¹²⁴ and reduces the levels of AGE. It has also been shown to reduce arterial stiffness. ¹²⁵

4. Downregulation of RAGE expression

   Downregulation of RAGE expression would reduce the availability of RAGE to interact with AGE resulting in reduction of adverse effects of AGE–RAGE interaction. Table 3 shows the agents that reduce the expression of RAGE. In statin groups, simvastatin inhibits the expression of RAGE via decreases in the myeloperoxidase-dependent formation of AGEs. ¹²⁶ RAGE expression is downregulated in vitro by atorvastatin. ¹²⁷ Angiotensin II receptor blockers, telmisartan ¹²⁸ and candesartan, ¹²⁹ downregulate the expression of RAGE. Metformin, a biguanide derivative used in the treatment of diabetes, downregulates the expression of RAGE in the vascular endothelium. ¹³⁰ Thiazolidinediones (pioglitazone, rosiglitazone) downregulate the expression of RAGE in human endothelial cells. ¹³¹ Nifedipine, a calcium channel blocker, reduces the RAGE expression in vascular endothelium exposed to AGE. ¹³² Curcumin, a condiment used in cooking, downregulates the RAGE expression in cultured hepatic cells. ¹³³ Resveratrol downregulates the expression of RAGE in the vascular smooth muscle cells. ^{134 135}

5. Elevation of soluble receptors (sRAGE, esRAGE)

   The drugs that affect the expression and levels of soluble receptors (sRAGE, esRAGE) are shown in Table 4 . ACE inhibitors, ramipril, upregulated the expression of sRAGE in the aorta of streptozotocin-induced diabetic rats and increased the serum levels of sRAGE. ¹³⁶ Perindopril increased the serum levels of sRAGE in patients with type 1 diabetes. ¹¹⁰

   Among the statins, pitavastatin and pravastatin elevated the serum levels of sRAGE in angina patients with coronary atherosclerosis. ¹¹⁰ Atorvastatin increased the serum levels of sRAGE and esRAGE in hypercholesterolemic patients with type 2 diabetes and upregulated the expression of sRAGE and esRAGE in THP1 cells in vitro. ¹³⁷ Lovastatin increases the sRAGE levels by inducing RAGE shedding. ¹³⁸ Fluvastatin stimulates the production of sRAGE and esRAGE in vitro. ¹³⁸ Antidiabetic drug, metformin, increased the serum levels of sRAGE in patients with metabolic syndrome. ¹³⁹ Insulin increased the serum levels of sRAGE and esRAGE in Chinese patients with type 1 diabetes. ¹⁴⁰ Insulin also stimulated the shedding of sRAGE from membrane-bound receptor for AGE in cell culture. ¹⁴⁰ Rosiglitazone, a thiazolidine derivative, increased the serum levels of sRAGE and esRAGE. ¹⁴¹

6. Exogenous administration of sRAGE

   Exogenous administration of sRAGE suppressed development of atherosclerosis and restenosis, prevented destabilization of vulnerable plaques, and reduced ischemia reperfusion-induced myocardial injuries. ¹⁶ Administration of recombinant sRAGE protected ischemic stroke in animal model, ¹⁴² reduced carotid artery stenosis in mice, ⁶⁶ and completely suppressed atherosclerosis in apoE-deficient mice. ⁹⁷ Possibility exists that administration of exogenous sRAGE would raise the serum levels of sRAGE which will combine with AGE, resulting in the reduced interaction of AGE with RAGE and hence reduction in pathophysiology of the disease.

Table 2. Agents that reduce the formation of AGEs.

Cooking		Cooking food in moist heat 23
Acidic ingredients		Lemon juice and vinegar 99
Phytochemicals		Berries, grapes, 101 pomegranate 100
Drugs	Aminoguanidine	Pimagedine 102
	ACE-inhibitor	Ramipril 106
	Ang II-receptor blockers	Telmisartan, 107 losartan, 107 valsartan, 108 candesartan 109
	Statins	Atorvastatin, 110 cerivastatin 111
	Antidiabetic drugs	Metformin, 112 pioglitazone 113
	Other drugs	Aspirin, 115 pentoxifylline 113
Vitamins		Benfotiamine (B1), 119 pyridoxamine (B6), 120 vitamin C, 121 D, 122 E 123
Other		α-lipoic acid, 114 resveratrol, 117 curcumin 118

Abbreviations: AGE, advance glycation end-product; ACE, angiotensin-converting enzyme; Ang II, angiotensin II.

Table 3. Agents that downregulate the expression of receptor for advanced glycation end-products.

Statins	Atorvastatin, 127 Simvastatin 126
Angiotensin II-receptor blockers	Telmisartan, 128 Candesartan 129
Biguanidine derivatives	Metformin 130
Thiazolidinediones	Rosiglitazone, pioglitazone, 131
Calcium channel blocker	Nifedipine 132
Others	Curcumin 133

Table 4. Agents that elevate sRAGE expression.

Angiotensin-converting enzyme inhibitors	Ramipril, 136 Perindopril 136
Statins	Atorvastatin, 137 pitavastatin, 110 pravastatin, 110 fluvastatin, 138 lovastatin 138
Antidiabetic drugs	Rosiglitazone, 141 metformin, 139 insulin 140

Abbreviation: sRAGE, soluble receptor for advance glycation end-product.

Assessment of AGE–RAGE Stress

As mentioned earlier, AGE–RAGE stress is defined as a shift in balance between stressors and antistressors in favor of stressors ( Fig. 3 ). Measurements of stressors (AGE, RAGE) or antistressors (degradation of AGE by GLO1, GLO2, AGER1, and AGER 2), and sRAGE would not measure AGE–RAGE stress. A formula using all stressors and antistressors would provide a true AGE–RAGE stress. A ratio of AGE + RAGE/GLO1 + GLO2 + AGER1 + AGER2 + sRAGE would provide a true index of AGE–RAGE stress. This ratio of AGE–RAGE stress can be determined in animal studies. It would be cumbersome to use this ratio in human beings. AGE and sRAGE can be measured in the blood samples from human; however, human tissues are required to measure the receptors such as AGER1, AGER2, and RAGE and that is not easy. We have, therefore, suggested that the ratio of AGE/sRAGE would be a simple and feasible measure of AGE–RAGE stress. An increase in the ratio of AGE/sRAGE would indicate a relative shift in stressors from antistressors, suggesting the presence of AGE–RAGE stress at cellular and organ levels. AGE/RAGE ratio has been suggested as a risk marker of disease process. ¹³ Increased ratio of AGE/sRAGE has been implicated in pathogenesis of restenosis following PCI ⁹⁸ hyperthyroidism, ⁷ and end-stage renal disease. ⁹ The sensitivity, specificity, positive and negative predictive value, and accuracy of AGE/sRAGE should be determined with large number of control and a particular group of patients. Using receiver operating characteristics (ROC) curve analysis, we have reported that the sensitivity and specificity of AGE/sRAGE ratio were 73% and 70%, respectively, in identifying patients with hyperthyroidism. ⁷ The sensitivity, specificity, positive predictive value, and negative predictive value of AGE/sRAGE ratio with optimal cut value of 2.75 were 84.88%, 80.95%, 94.81%, and 56.67%, respectively, in identifying patients with end-stage renal disease. ⁹ The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of AGE/sRAGE ratio were 100%,83%, 85%, 100%, and 91%, respectively, in predicting restenosis following PCI. ⁹⁸ These values were obtained using methods described by Glas et.al. ¹⁴³ The data suggest that the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the ratio of AGE/sRAGE in identifying the risk factor or predicting the disease condition appear to be excellent. The AGE/sRAGE ratio is, therefore, an appropriate measure of AGE–sRAGE stress.

Fig. 3.

Schematic representation of AGE–RAGE stress, stressors, and antistressors. AGE, advance glycation end-product; RAGE, cell bound receptor for AGE; sRAGE, soluble receptor for AGE; AGER1, advance glycation end-product receptor 1; AGER2, advance glycation end-product receptor 2; GLO1, glyoxalase 1; GLO2, glyoxalase 2.

Conclusions

Stress is defined as a process of altered biochemical homeostasis produced by physiological, psychological or environmental stressors. In the present context, AGE and RAGE are stressors, and GLO1, GLO2, AGER1, and AGER 2 which degrade AGE and sRAGE are antistressors. Low levels of AGEs are due to reduction in the formation and consumption of AGE and degradation of AGE enzymatically (GLO1, GLO2) and through receptors (AGER1, AGER2). AGE–RAGE stress occurs when excess AGE and RAG are produced that could overwhelm the normal antistressors. In other word, AGE–RAGE stress is defined as a shift in the balance between stressors and antistressors in favor of stressors. Measurements of only stressors or antistressors would not provide an index of AGE–RAGE stress. The ratio of AGE + RAGE/sRAGE + GLO1 + GLO2 + AGER1 + AGER2 would be a true measure of AGE–RAGE stress. The measurement of this ratio is very feasible in animal studies. However, the measurement of this ratio of AGE–RAGE stress is not feasible in humans because one has to take tissue from human to measure RAGE and AGER1 and AGER2. We have, therefore, suggested that AGE/sRAGE ratio would be simple and feasible index of AGE–RAGE stress in clinical practice and for experimental studies. It is concluded that AGE/sRAGE ratio is one of the important determinants of AGE–RAGE stress. A high ratio would indicate a relative shift in stressors from antistressors suggesting the presence of AGE–RAGE stress that may partly be involved in the pathogenesis of numerous diseases and their complications.