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Role of Aldose Reductase and Oxidative Damage inDiabetes and the Consequent Potential for

Therapeutic Options

Satish K. Srivastava, Kota V. Ramana, and Aruni Bhatnagar

Department of Human Biological Chemistry and Genetics (S.K.S., K.V.R.), University of Texas Medical Branch, Galveston,Texas 77555; and Division of Cardiology (A.B.), Department of Medicine, University of Louisville, Louisville, Kentucky40202

Aldose reductase (AR) is widely expressed aldehyde-metabo-lizing enzyme. The reduction of glucose by the AR-catalyzedpolyol pathway has been linked to the development of sec-ondary diabetic complications. Although treatment with ARinhibitors has been shown to prevent tissue injury in animal

modelsof diabetes,the clinical efficacy of these drugs remainsto be established. Recent studies suggest that glucose may bean incidental substrate of AR,whichappears to be more adeptin catalyzing the reduction of a wide range of aldehydes gen-erated from lipid peroxidation. Moreover, inhibition of theenzyme has been shown to increase inflammation-inducedvascular oxidative stress and prevent myocardial protectionassociated with the late phase of ischemic preconditioning.On the basis of these studies, several investigators have as-cribed an importantantioxidant role to theenzyme. Addition-ally, ongoing work indicates that AR is a critical componentof intracellular signaling, and inhibition of the enzyme pre-

vents high glucose-, cytokine-, or growth factor-induced ac-tivation of protein kinase C and nuclear factor--binding pro-tein. Thus, treatment with AR inhibitors prevents vascularsmooth muscle cell growth and endothelial cell apoptosis inculture and inflammation and restenosis in vivo. Additional

studies indicate that theantioxidantand signalingrolesof ARare interlinked and that AR regulates protein kinase C andnuclear factor-B via redox-sensitivemechanisms. These dataunderscore the need for reevaluating anti-AR interventionsfor the treatment of diabetic complications. Potentially, thedevelopment of newer drugs that selectively inhibit AR-mediated glucose metabolismand signaling, without affectingaldehyde detoxification, may be useful in preventing inflam-mation associated with the development of diabetic compli-cations, particularly micro- and macrovascular diseases. (En-docrine Reviews 26: 380392, 2005)

I. Introduction: Aldose Reductase and Diabetic Complications

II. AR and Antioxidant ProtectionIII. Redox Regulation of AR ActivityIV. Regulation of Intracellular Signaling by ARV. AR Inhibitors and the Treatment of Secondary Diabetic

ComplicationsVI. Conclusion

I. Introduction: Aldose Reductase and Diabetic

Complications

ALDOSE REDUCTASE (AR) is a monomeric reducednicotinamide adenine dinucleotide (NAD) phosphate(NADPH)-dependent enzyme and a member of aldo-keto

reductase superfamily. The enzyme was described in 1956 byHers (1) as a glucose-reducing activity. It was subsequentlyreported by van Heyningen (2) that high levels of AR activity

are present in the rat lens and that during diabetic and

galactosemic cataractogenesis, AR-derived polyolssorbitoland galactitolaccumulate in the ocular lens. Building onthis observation, Kinoshita et al. (3) and Varma and Kinoshita(4) demonstrated that treatment with pharmacological in-hibitors of AR ameliorated cataractogenesis in diabetic ratsand galactose-exposed rabbits. Based on these observationsit was proposed that accumulation of sorbitol in the lens, dueto AR-catalyzed reduction of glucose, causes osmotic swell-ing resulting in ionic imbalance and protein insolubilizationleading to cataractogenesis (57). A similar sequence ofevents could also account for hyperglycemic injury associ-ated with diabetic retinopathy, nephropathy, and neuropa-thy (Fig. 1).

The osmotic hypothesis that diabetic complications aredue to sorbitol accumulation in tissues has engendered ex-tensive investigations over the last three decades. Severaldrugs with varying AR-inhibiting efficacy (e.g., sorbinil, sta-til, tolrestat, and zopolrestat) have been synthesized andtested (811). Initial trials in animal models showed signif-icant protection against diabetic complications (1214). ARinhibitors, in addition to preventing diabetic and galac-tosemic cataracts (3, 4), ameliorated some of the features ofdiabetic nephropathy (15, 16) and neuropathy (1719). How-ever, clinical trials with AR inhibitors have yielded uncertainresults, in part due to the high nonspecific toxicity of thisclass of drugs.

In the mid-1980s we demonstrated that most of the inhib-

First Published Online April 6, 2005Abbreviations: AR, Aldose reductase; DAG, diaceylglycerol; DHN,

1,4-dihydroxy-2-nonene; FGF, fibroblast growth factor; GSNO, nitroso-glutathione; GSSG, oxidized glutathione; HNE, 4-hydroxynonenal;NAD, nicotinamide adenine dinucleotide; NADPH, reduced NADphosphate; NF-B, nuclear factor--binding protein; NO, nitric oxide;PKC, protein kinase C; PLC, phospholiase C; VSMC, vascular smoothmuscle cells.

Endocrine Reviews is published bimonthly by The Endocrine Society(http://www.endo-society.org), the foremost professional society serv-ing the endocrine community.

0163-769X/05/$20.00/0 Endocrine Reviews 26(3):380392Printed in U.S.A. Copyright 2005 by The Endocrine Society

doi: 10.1210/er.2004-0028

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itors synthesized by pharmaceutical companies were notselective for AR but also inhibited other members of the

aldo-keto reductase superfamily such as aldehyde reductase(20). In addition, we also demonstrated that increased os-motic pressure due to accumulation of polyols in galac-tosemic and diabetic cataractogenesis could not be the maincause of cataractogenesis (2123). Antioxidants such as bu-tylated hydroxytoluene and 6-hydroxy-2,5,7,8-tetramethe-nyl-chroman-2-carboxylic acid (Trolox) prevented diabeticcataractogenesis in rats and significantly attenuated lensopacity in galactose-fed rats even though the polyol levels inthe lens were extremely high (8090 mm), indicating thatpolyol accumulation per se is not sufficient for cataractogen-esis and that other metabolic changes accompanying ARactivation may be critical and important modulators of the

cataractous effects of hyperglycemia and diabetes (Fig. 2).Because AR utilizes NADPH to catalyze glucose reduc-tion, we, as well as other investigators, suggested that tissueinjury associated with high glucose may be due, in part, toincreased NADPH utilization by the reduction of glucose byAR (24, 25). In the presence of normal glucose (5.5 mm),AR-catalyzed reduction represents less than 3% of total glu-cose utilization, whereas in the presence of high glucose (20mm), more than 30% of the glucose is used by AR (26),suggesting that the profound increase in the AR-catalyzedreductive pathway may impose a significant strain onNADPH supply. Because NADPH is used for several criticalreductive metabolic steps, such as the detoxification of re-active oxygen species and hydroperoxides (e.g., by the glu-

tathione reductase/glutathione peroxidase system), a largedrain on the NADPH pool could compromise the ability of

the cell to protect itself from oxidative stress.Another reason for the inconsistent effects of AR inhibitors

may relate to posttranslation modification of the enzyme. Asdocumented extensively in our previous publications (2628), the oxidation state of a single cysteine residue located atthe active site of the enzyme regulates both substrate andinhibitor binding and thus, given the extensive oxidativechanges in diabetes, it is likely that in diabetic tissues AR is

FIG. 2. Oxidative stress is the main contributor to diabetic catarac-togenesis. Digital image analysis was performed on lensescultured innormal glucose (5.5 mM; NG), high glucose (50 mM; HG) without orwith antioxidants on d 8. Sorbitol content was measured by using gaschromatography. The data represent mean SD (n 4). **,P 0.001;#, Pnonsignificant in HG-treated group compared withNG-treatedgroup. BHT, Butylated hydroxytoluene.

FIG. 1. Involvement of polyol pathwayin diabetic complications. During hy-perglycemia, reduction of glucose tosorbitol by AR constitutes the first andtherate-limitingstep of thepolyolpath-way that converts glucose to fructose

via sorbitol dehydrogenase (SDH). Inthis pathway both NADPH and NAD

are consumed as cofactors for the en-zymes AR andSDH. Osmotic stressdueto accumulation of sorbitol and oxida-tive stress due to changes in the ratio ofNADPH/NADP and reduced NAD(NADH)/NAD are the major cause of

various complications of secondary di-abetes. ROS, Reactive oxygen species.

Srivastava et al. Aldose Reductase and Diabetic Complications Endocrine Reviews, May 2005, 26(3):380392 381


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insensitive to inhibitors due to oxidative modification. Insupport of this view, we have demonstrated that AR inhuman erythrocytes exists in two forms, activated and un-activated,and in hyperglycemiatotalactivity of AR increases(29). Multiple forms of AR have also been reported by otherinvestigators (30, 31), and our in vitro data suggest that ox-idants can induce a variety of changes in the catalytic activityof the enzyme, although which of these forms occur in vivoand how they are interconverted remains unclear. Never-theless, the view that the hyperglycemia changes the sensi-tivity of AR to inhibition is further supported by the obser-vation that prolonged culture of endothelial cells in highglucose progressively decreases the efficacy of sorbinil inpreventing sorbitol accumulation (32).

In contrast to inhibitor data, newer molecular approacheshave provided more unambiguous evidence for the involve-ment of AR in diabetic complications. In these studies trans-genic overexpression of AR gene selectively in the lens wasfound to accelerate diabetic and galactosemic cataract for-mation in mice (33). Additionally, ubiquitous overexpression

of the AR gene increased the rate of neuropathic changes indiabetic animals (3436). Collectively, these data arguestrongly that AR is an important component of high glucose-induced metabolic changes that underlie the development ofsecondary diabetic complications. Nevertheless, despite thisevidence, the exact mechanism by which AR contributes tothe development of diabetic complications remains unclear.Persistent increase in extracellular glucose levels inducespleiotrophic changes in metabolism that elicit polygenic re-sponses (Fig. 3). Extensive changes in the activation of pro-tein kinases and the accumulation of advanced glycation endproducts have been reported (3739), although their rela-tionships with modulatory influences on AR remain unclear.

In principle, AR could contribute to both protein kinaseactivation and advanced glycation end product accumula-tion. In addition, AR could catalyze the formation of potentprotein glycating agents and induce oxidative stress.

Reduction of glucose by AR leads to the formation ofsorbitol, which, in some tissues, is further oxidized to fruc-tose upon sorbitol dehydrogenase-catalyzed oxidation. Theconversion of glucose to fructose (the polyol pathway)results not only in the utilization of NADPH, but also NAD.As a result, increased activity of the polyol pathway duringhyperglycemia could lead to a depletion of NADPH andaccumulation of reduced NAD. This shift in the redox stateof pyridine coenzymes recapitulates the metabolic pheno-type of hypoxia and has been proposed to induce a state ofpseudohypoxia resulting in hypoxia-like responses (4042).Polyol pathway-mediated alterations in pyridine nucleotideshave been linked to diverse metabolic changes such as thesynthesis of nitric oxide (NO) and activation of protein ki-nases (39). Specifically, it has been proposed that the increasein NADH due to elevated polyol pathway activity couldincrease the synthesis of diacylglycerol (DAG) from dihy-droxyacetone phosphate (43). DAG levels could also increase

by stress that activates phospholipase C (PLC in Fig. 4). DAGis an essential activator of the classical and novel members

of the protein kinase C family, and DAG-dependent activa-tion ofthese kinases is thought to play a key rolein mesangialexpansion and smooth muscle cells proliferation induced byhigh glucose (4446). Inhibitors of specific protein kinase C(PKC) isoforms are currently under clinical trial for the treat-ment of diabetic complications (47, 48).

II. AR and Antioxidant Protection

Due to its ability to reduce glucose, AR is involved inseveral tissue-specific metabolic pathways. For instance, inthe kidney, AR participates in osmoregulation (49, 50) and,in seminal vesicles, in the generation of fructose (1). The

FIG. 3. Pathways of hyperglycemia-induced oxidative stress. In-creased superoxide production in the mitochondria during hypergly-cemia causes increased oxidative stress. During hyperglycemia theratio NADPH/NAD decreases due to excessive use of NADPH for thereduction of glucose to sorbitol. Consequently, the NADPH availabil-ity to maintain glutathione in the reduced form catalyzed by gluta-thione reductase decreases,which causes oxidative stress. Increase inadvanceglycationend products (AGEs)formationand bindingof AGEto its cognate receptor (RAGE) also increases oxidative stress. In-creased DAG formation in hyperglycemia activates PKC and subse-quently induces oxidative stress via a PKC-dependent activation ofNAD(P)H oxidase.

FIG. 4. Pathways of increased DAG in hyperglycemia. Increasedstress conditions because of autooxidation of glucose, osmotic stress,oxidative stress, and hypoxia during polyol pathway hyperactivitycan contribute to increase in DAG levels through a cascade of signalsthat activate phospholipase C (PLC) or directly through glycolysis.

AGE, Advance glycation end product; ROS, reactive oxygen species;SDH, sorbitol dehydrogenase.

382 Endocrine Reviews, May 2005, 26(3):380 392 Srivastava et al. Aldose Reductase and Diabetic Complications


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observation that AR and related proteins are up-regulated byfibroblast growth factor (FGF) and other mitogens suggeststhat AR may be involved in cell growth or growth factor-induced changes in cellular metabolism (5161). Our studiesshow that in vitro homogenous AR catalyzes the reduction ofa large series of saturated and unsaturated aldehydes with103- to 104-fold higher efficiency than glucose (62). The en-zyme is particularly efficient in reducing medium- to long-chain (C-6 to C-18) aldehydes such as those generated in highabundance during lipid peroxidation (63, 64). The enzymealso catalyzes the reduction of the glutathione conjugates ofunsaturated aldehydes, in many cases with efficiency higherthan that of the parent free aldehyde (6567). The reductionof glutathione conjugates by AR may be a protective mech-anism that may be useful in minimizing the reactivity of thealdehyde function unquenched by glutathiolation. In addi-tion, our recent studies show that AR is also an efficientcatalyst for the reduction of core aldehydesor aldehydesgenerated in oxidized phospholipids (Refs. 6570 and Fig. 5).

The role of AR in detoxification is supported by structural

studies showing that the active site of AR lacks ionic residuecharacteristic of carbohydrate-binding proteins (7176). In-stead, x-ray analyses of AR crystals reveal a highly plasticand hydrophobic active site (73, 74). This site could poten-tially accommodate a wide range of structures if the alde-hyde function were to orient itself appropriately between thetwo ionic active site residues, His-110 and Tyr-48, whichparticipate in acid-base catalysis. The structure of the binarycomplex of the enzyme with NADP also indicates a pro-found conformational change upon NADPH binding (72). Ithas been suggested that the energy released as a result of thisinteraction is used for stabilization of the transition state withlittle demand from energy stabilization due to substrate

binding (77). Hence, as a result of high-affinity interactionwith NADPH, AR functions as an unusually promiscuousaldehyde removase, features that seem to be critically re-quired of a detoxification enzyme involved in the removal ofa wide range of aldehydes and glutathione aldehyde adductsgenerated during lipid peroxidation.

Based on our substrate specificity studies showing high-

affinity reduction of glutathione conjugates by AR (66, 67),we reasoned that the active site of the enzyme must conformto a specificglutathione-binding domain. Consistent with thepresence of specific interactions between the amino acid res-idues of glutathione and the AR active site, we found thatalterations in the structure of glutathione diminished thecatalytic efficiency for the reduction of glutathione-aldehydeconjugates and that nonaldehydic conjugates of glutathioneor glutathione analogs displayed active site inhibition (67).Molecular dynamics calculations suggest that the conjugatesadopt a specific low-energy configuration at the active site(Fig. 6). Mutations of the active site residues identified bythese calculations selectively decreased catalysis of the glu-tathione-aldehyde conjugates, without affecting reduction ofthe free aldehyde. The high specificity and selectivity withwhich the enzyme catalyzes the reduction of glutathioneconjugates suggest that such conjugates may be in vivo sub-strates of the enzyme. Indeed, our metabolic studies with4-hydroxynonenal (HNE) show AR-dependent reduction ofits glutathione adduct in heart, smooth muscle, and eryth-

rocytes (7881).Reduction of aldehyde-glutathione conjugates by AR may

be of physiological significance, particularly under condi-tions of oxidative stress when other antioxidant mechanismsare overwhelmed. Under these conditions, AR, by reducing

both free aldehydes and their glutathione conjugates, couldpromote efficient removal and detoxification of unsaturatedaldehydes, which are the major electrophilic end products oflipid peroxidation. Thus, investigations into the structureand kinetics of AR point toward a general detoxification roleof the enzyme, indicating that lipid aldehydes and theirglutathione conjugates are physiological substrates of theenzyme and that glucose is perhaps an incidental substrate,

which is used only when it is high in concentration or oc-casionally for tissue-specific metabolism.

Consistent with its role as a detoxification enzyme, AR hasbeen found to be regulated by aldehydes generated fromlipid peroxidation (64, 70, 82), thiol-reducing agents (83 88),metal ions (8991), and NO (9297). Tissue levels of AR arealso increased under conditions of high oxidative stress such

FIG. 5. Reduction of lipid aldehydes byaldose reductase. The bold numbers in

parentheses represent reference num-bers.



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as iron overload (98), alcoholic liver disease (99), heart failure(100), and vascular inflammation (53, 101). Moreover, AR isalso specifically up-regulated during vasculitis, specificallyin the areas of high HNE formation, and inhibition of ARincreases the concentration of free HNE and protein-HNEadducts, accompanied by a 3-fold increase in the number ofapoptotic cells (102). Our studies show that AR is also up-regulated in the heart by brief episodes of ischemia (103).This increase in AR was prevented by inhibitors of PKC andNO synthase, indicating that the up-regulation of AR is reg-ulated by cardioprotective signaling associated with the latephaseof ischemicpreconditioning. Furthermore, a posttrans-lational activation of AR during myocardial ischemia has

been reported (104). The functional significance of the in-crease in AR in the ischemic heart is underscored by theobservation that inhibition of AR abolishes the cardiopro-tective effects of late preconditioning (105107). Collectively,these data provide support to the view that AR is a compo-nent of an antioxidant defense mechanism, which protectstissues from the harmful effect of lipid peroxidation productssuch as HNE that accumulate during conditions of oxidativestress such as ischemia and inflammation.

Although HNE and related aldehydes are metabolized byseveral different pathways, metabolism via AR could rep-resent a true detoxification mechanism. In most cells, HNEand related aldehydes readily form glutathione conjugates;

however, the formation of glutathione-HNE itself may not besufficient for detoxification. The glutathione-aldehyde con-jugates are toxic. They induce DNA damage and stimulateradical formation (108110). Therefore, reduction of the glu-tathione-aldehyde conjugates by AR may be necessary tosubstantially annul the reactivity of the conjugate and todecrease free radical generation. Nonetheless, the mecha-nisms by which AR-dependent aldehyde metabolism regu-lates the development of diabetic complications remain un-clear, and the contribution of AR to cell growth andantioxidant defense requires further elucidation. In particu-lar, it remains to be clarified how AR expression and activityare regulated and which components of intracellular signal-ing are regulated by AR.

III. Redox Regulation of AR Activity

As discussed above, a component of the hyperglycemicinjury is due to an increased flux of glucose through thepolyol pathway because the inhibition of AR prevents someof the diabetic complications. Although inefficacy of AR in-hibitors could be due to their nonspecificity and hypersen-sitivity of selected individuals, the limited long-term efficacyof the AR inhibitors could be attributed to the posttransla-tional modification in AR that alters ligand binding andcatalysis. In our earlier studies we found that AR isolatedfrom individuals with different levels of hyperglycemia dis-played altered kinetic properties and was less sensitive tohydantoin inhibitors such as sorbinil compared with theenzyme isolated from the tissues of normoglycemic subjects(29). Similar changes were observed at various stages ofpurification of AR in the absence of a reducing reagent suchas dithiothreitol or -mercaptoethanol from the tissues ofnormal subjects or from recombinant bacteria overexpress-ing AR (30, 31). Both theMichaelis-Menten constant (Km)andinhibition constant (Ki) values of the enzyme significantlyincreased upon its in vitro thiol modification by HNE (64, 82),nitrosoglutathione (GSNO) (92), and oxidized glutathione(GSSG) (83).

The high sensitivity of AR to oxidants such as H2O2 andNO was attributed to a highly reactive cysteine (Cys-298)residue present at the active site of the enzyme (88). OxidantssuchasH2O2 cause enzyme inactivation. Also glutathiolationof the Cys-298 results in a significant inactivation of theenzyme (83, 88, 92). However, depending on the conditionsof the reaction and the nature of the NO donor used, AR iseither S-thiolated (inactivated enzyme) or S-nitrosated (ac-tivated enzyme). On the basis of these observations, we hy-pothesized that NO regulates the intracellular activity of ARand consequently the flux of glucose via the polyol pathway(9298). Because, in hyperglycemia, NO synthesis is signif-icantly less compared with normoglycemic subjects, it has

been reasoned that the AR activity may be up-regulated indiabetic tissues (111, 112).

Cardiovascular complications are one of the major causes

FIG. 6. Molecular modeling showing two possible orien-

tations of glutathione-propanal bound to AR. Orienta-tions 1 and 2 are shown in panels A and B, respectively.Carbon, nitrogen, sulfur, and oxygen atoms are colored

gray, blue,yellow, and red, respectively. Potential hydro-gen bonds are indicated with dashed lines. [Reproducedwith permission from B. L. Dixit et al.: J Biol Chem275:2158721595, 2000 (67).]



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of mortality in patients with prolonged diabetes (113). Therole of AR in cardiovascular complications, especially inatherosclerosis (100), restenosis subsequent to balloon injury(53, 57), and cardiac preconditioning (103106), has beenextensively investigated. Vascular endothelial cells are themain source of NO for vascular smooth muscle cells becausein the vasculature, NO synthase is present mainly in theendothelium (114). The NO secreted by endothelial cellscould readily form GSNO with glutathione that is abundantin vascular smooth muscle cells (VSMC). The GSNO formedcould, in turn, readily S-glutathiolate AR at Cys-298 (92).Although GSSG (50100 m) also S-glutathiolates AR, it isunlikely that the physiological source of S-glutathiolation ofAR (or other proteins) could be GSSG because the concen-tration of GSSG in cells rarely exceeds 2025 m. BecauseS-glutathiolation inactivates AR, under normoglycemic con-ditions, it appears likely that a significant fraction of AR invascular tissues is present in an inactive form, whereas inhyperglycemia a decrease in NADPH/NADP ratio andother factors would decrease NO and the AR would be in the

active form. Indeed, we have demonstrated that, in diabeticrat aorta, both AR activity and sorbitol levels are more than20-fold higher than nondiabetic aorta (95). Daily ip injectionsof NO synthase inhibitor, NG-nitro-l-argininemethyl ester orapplication of nitroglycerin patch significantly increased theactivity of aorta AR as well as sorbitol formation in bothnondiabetic and streptozotocin-diabetic rats. On the otherhand, daily injections of NO synthase substrate, l-arginine,significantly decreased AR activity and sorbitol content inaortas of both diabetic and nondiabetic rats. Similar resultswere obtained when aortic rings from normal and diabeticmice were incubated with l-arginine and NG-nitro-l-argin-inemethyl ester, whereas, both NO synthase substrate and

inhibitor had no effect on AR activity or sorbitol content ofaorta from endothelial NO synthase-null mice (97). Interest-ingly, it was observed that S-glutathiolation of AR in VSMC

by NO donors such as GSNO, which inactivates the enzyme,is reversible. Thus, the changes in glucose levels that alter thelevels of NO would change the AR activity, sorbitol levels,and related effects in diabetic subjects. Hence, in addition toglycemic control, NO donors or drugs that increase NO lev-els could represent one treatment modality for the preven-tion or treatment of diabetic complications. Because hyper-glycemia causes increased generation of reactive oxygenspecies by autooxidation of glucose and other metabolicpathways, antioxidants would also have beneficial effects.

Diabetic complications such as cataractogenesis, retinopa-thy, and cardiovascular effects have been shown to be ame-liorated by antioxidants such as butylated hydroxytoluene,

butylated hydroxyanisole, Trolox, ascorbate, vitamin E, N-acetyl cysteine, pyruvate, etc. (115120).

IV. Regulation of Intracellular Signaling by AR

Diabetes is a major risk factor for the development ofcardiovascular disease. It is associated with a 2- to 4-foldhigher risk of cardiovascular disease, and it accelerates theprogression and increases the severity of atherosclerotic le-sion formation in peripheral, coronary, and cerebral arteries

(121125). Moreover, diabetics have a higher propensity forrestenosis after percutaneous transluminal coronary angio-plasty (126). Even though coronary stenting significantlyreduces restenosis, diabetes remains a powerful predictor ofin-stent restenosis (127). Processes that lead to an increase insmooth muscle cell growth in diabetic as well as nondiabeticrestenotic vessels have not been identified but, given theobservation that high levels of protein-HNE adducts areassociated with proliferative vascular lesion, it appears thatproducts of lipid peroxidation, such as HNE, play a signif-icant role in modulating the growth of vascular lesion. Thisis supported by our observation that treatment with lowconcentrations (2 m) of HNE increases VSMC growth inculture, although at higher concentrations HNE induced ap-optotic cell death (53). To test the role of HNE in vascularproliferation, we determined how changes in its metabolismvia AR would affect its mitogenic activity. Surprisingly, wefound that inhibition of AR prevented VSMC growth inculture (53). Serum-starved VSMC cultured in 1 m HNEshowed increased proliferation compared with cells cultured

without HNE, as determined by MTT (3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and cellcount, whereas under similar conditions, HNE caused apo-ptosis in vascular endothelial cells and lens epithelial cells(Fig. 7). Both proliferation and apoptosis were attenuated byinhibiting AR using two structurally different AR inhibitors,sorbinil andtolrestat, andalso by using antisenseAR or smallinterfering RNA. In addition to HNE-stimulated growth,smooth muscle cells growing in response to serum FGF,TNF, or high glucose were also sensitive to AR inhibitors(128). Moreover, inhibition of AR also prevented smoothmuscle cell growth in vivo: we balloon-injured normal anddiabetic rat carotid artery and followed restenosis by quan-

tifying the neointima formation (101). In both normal anddiabetic rats, AR inhibitor significantly (45%) prevented neo-intimal hyperplasia (Fig 8). Interestingly, the neointimal hy-

FIG. 7. Inhibition of AR attenuates proliferation of VSMC and apo-ptosisof human umbilical vein endothelial cells (HUVEC)and humanlens epithelial cells (HLEC). Growth-arrested cells were pretreatedwith or without AR inhibitors, sorbinil and tolrestat (10 M) for 24 hfollowed by stimulation with TNF (2 nM) for an additional 24 h. Cell

viability was determined by measuring the incorporation of [3H]thy-midine (10 Ci/ml), added 6 h before the end of the experiment. Theextent of cell proliferation or apoptosis is expressed as percentagechange in cell viability. **, P 0.001; *, P 0.01; and #, P 0.001in highglucose (HG)AR inhibitor group compared with HG-treatedgroup alone.



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perplasia was associated with increased nuclear factor--bind-ing protein (NF-B) activation (128). This was significantlyprevented by sorbinil, suggesting that AR is required for sig-naling pathways responsible for NF-B activation and for theproliferation of smooth muscle cells in vascular lesions. To-gether, these observations suggest that AR is essential forsmooth muscle cell growth induced by several mitogenic path-ways and that inhibition of AR interrupts their growth in cul-ture and in vivo (53, 57, 128).

Although the mechanism by which AR regulates growthremains unclear, several of the key signaling pathways re-lated to cell growth are sensitive to AR inhibition. Signifi-cantly, our studies reveal that inhibition of AR preventsactivation of the transcription factor NF-B stimulated witheither TNF (Fig. 9),FGF, angiotensinII, or high glucose (101,128). Inhibition of AR, however, does not seem to directlyinterfere with the DNA binding of NF-B, but preventsNF-B activation by extinguishing signaling events up-stream to dissociation of NF-B from IB and the nucleartranslocation of NF-B. This appears to be due to the inhi-

bition of phosphorylation and degradation of IB in cellstreated with AR inhibitors. Significantly, inhibition of ARdoes not prevent NF-B when the cells are stimulated byphorbol esters, indicating that inhibition of AR does notinterfere with the IB-NF-B signaling pathway but preventssignaling events upstream to PKC (128).

Similar to the effects observed with TNF, inhibition orablation of AR also interrupts the activation of the PKC-NF-B by high glucose (101). The sensitivity of both highglucose- and TNF-mediated cell growth to AR inhibitorssuggests that both stimuli might activate overlapping path-ways. Indeed, previous studies have shown that treatmentwith anti-TNF antibodies prevents accelerated restenosis indiabetic vessels (129), and in our laboratory treatment withanti-TNF antibodies was effective in preventing high glu-cose-induced smooth muscle cell growth in culture (K. V.Ramana, R. Tammali, A. Bhatnagar, and S. K. Srivastava,unpublished observations). Hence, it appears that the mito-genic effects of high glucose may be mediated, in part, bystimulation of cytokine release and autocrine activation of

FIG. 8. AR inhibitor attenuates NF-B activation in bal-loon-injured normal and diabetic rat arteries. Sections ofballoon-injured arteries were obtained from nondiabeticand diabetic rats 10 and 21 d after balloon injury and werestained with anti-p65 antibodies. Immunoreactivity is ev-ident asa dark brown stain, whereas the nonreactive areasdisplay only the background color. The bar graph showsmean SEM values of the percent of neointima stained bythe anti-p65 antibody. *,P 0.05 in tolrestat vs. control;,

P 0.05 in diabetic vs. control; and #, P 0.05 diabetic tolrestat vs. diabetic without tolrestat. [Reprinted with per-mission from K. V. Ramana et al.: Diabetes 53:29102920,2004 (101). 2004 American Diabetes Association.]

FIG. 9. Inhibition of AR attenuatesTNF-induced activation of NF-B inVSMC and human umbilical vein en-dothelial cells (HUVEC). Quiescent

VSMC (A) and HUVEC (B) were pre-incubated without or withthe indicatedconcentrations of sorbinil for 24 h andthen stimulated with 0.1 nM TNF for1 h. The nuclear extracts were pre-pared, and NF-B activity was mea-sured by EMSA. [A, Reproduced withpermission from K. V. Ramana et al.:

J Biol Chem 277:3206332070, 2002(128); and B, reproduced with permis-sion from K. V. Ramana et al.: FASEB

J 18:12091218, 2004 (134).]



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the cell growth pathways. How glucose could induce therelease of cytokines is unclear. However, the recent obser-vationthat thereleaseof TNF is regulated by PKC (130 132)suggests that activation of PKC by high glucose could triggerTNF release and stimulate growth. Because inhibition/ablation of AR prevents both PKC activation (Fig. 10) andTNF signaling, it could interrupt the initial trigger events aswell as subsequent autocrine stimulation of mitogenic sig-naling in cells exposed to high glucose (101, 133).

Studies delineating the role of AR in intracellular signalingsuggest an alternative paradigm for understanding the con-tribution of this enzyme to the development of diabetic com-plications and the efficacy of AR inhibitors against secondarydiabetic complications, despite the detoxification role of theenzyme. Hence, inhibition of inflammation could representone mechanism by which beneficial effects could be derivedfrom inhibiting AR. Our studies show that key steps in theinflammatory process, such as NF-B activation and the in-crease in the expression of adhesion molecules ICAM-1 andVCAM-1 and monocyte adhesion, could be prevented by

inhibiting AR (Fig. 11) (134). Moreover, inhibition of AR alsoprevents the cytotoxic effects of TNF on endothelial cells.

Our studies have also shown that inhibition of AR bysorbinil or tolrestat prevents TNF-induced increase in Baxand Bad and the down-regulation of Bcl-2 (135, 136). Inhi-

bition of AR also abrogates activator protein 1 DNA bindingactivity and prevents the activation of caspase-3, c-Jun N-terminal kinase, and p38 MAPK in cells stimulated by TNF,suggesting that AR could be a critical regulator of TNF-induced apoptotic signaling in endothelial cells (136). Giventhe key role of the endothelium in regulating atherogenesisand restenosis, the salutary effects of AR inhibitors on thesecells could further contribute to the ability of these drugs to

limit inflammation and vascular adhesion. Although theoverall effects of inhibiting AR during atherosclerotic lesionformation remain to be studied, they arelikely to be complex.Given that inhibition of AR could prevent inflammatorychanges as well as increase the accumulation of lipid per-oxidation products, inhibition of AR could yield highly con-text-dependent results, and the benefits of inhibiting AR may

be specific to the extent of lesion progression and hypergly-cemia and/or may require concurrent administration of an-tioxidants. Such duality and complexity of effects is, how-

ever, expected. AR regulates oxidative and inflammatoryresponses, both of which could be beneficial or harmful de-pending upon the context and the extent to which they arestimulated and the rate at which they are resolved. Regard-less, much work remains to be done to further our under-standing of the enigmatic role of AR and its contribution ofantioxidant defenses as well as the development of second-ary diabetic complications.

V. AR Inhibitors and the Treatment of Secondary

Diabetic Complications

Despite the initial promise, outcomes of clinical trials withAR inhibitors have been disappointing. Most studies revealonly modest improvement with multiple side effects. How-ever, despite such negative data, it would be unfortunate ifanti-AR therapy were to be relegated to a historical footnote.Clinical trials with AR inhibitors were designed and con-ducted with only limited information about the enzyme and

with little or no understanding of its physiological role inprocesses other than the reduction of glucose. Moreover,most clinical trials were designed to examine only a limitedset of physiological and pathological end points and weremostly focused on diabetic neuropathy. However, subse-quent to initial clinical trials, much has been learned aboutthe enzyme and its role in glucose metabolism, detoxifica-tion, inflammation, and growth that will be critical in rede-signing clinical trials with a larger number of pathologicalindices and end points.

Given the extensive data showing that by catalyzing thereduction of lipid-derived aldehydes and their glutathioneconjugates (62, 66, 67), AR protects against oxidative stress

(101, 128), it may be beneficial to try a combination therapywith AR inhibitor and antioxidants. Because long-term di-abetes is associated with increased oxidative stress, the ben-eficial effects of AR inhibitors may be diminished by a con-current accumulation of lipid peroxidation products.Therefore, to prevent this, it may be necessary to concur-rently administer antioxidants that prevent lipid peroxida-tion or enhance the expression of antioxidant enzymes thatcan compensate for the lack of AR. Treatment with antioxi-dants may also keep the enzyme in the reduced form and

FIG. 10. Ablation of AR attenuates cytokines,growthfactors, high glucose, HNE butnot phor-bol 12-myristate 13-acetate (PMA)-induced ac-tivation of PKC in VSMC. The VSMC weretransiently transfected with AR antisense orscrambled control oligonucleotides. Subse-quently, the cells were stimulated with TNF(0.1nM), basic FGF (bFGF) (5 ng/ml),platelet-derived growth factor (PDGF)-AB (5 ng/ml),angiotensin II (Ang-II; 2 M),HNE(1 M), highglucose (HG, 50 mM),orPMA(10nM)for4h.Themembrane-bound PKC activity was determinedby using the Promega Signa TECT PKC assaysystem (Promega Corp., Madison, WI). Barsrepresent mean SE (n 4). *,P 0.001in ARantisense group compared with control group.[A portion of the data was reproduced with per-mission from K. V. Ramana et al.: J Biol Chem277:3206332070, 2002 (128).]



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thus prevent drug resistance that develops due to AR mod-ification under conditions of chronic hyperglycemia. Addi-tionally, treatment with AR inhibitors could be restricted toa specific stage of disease development. Thus, for instance,inhibition of AR may be more beneficial during early stagesof diabetes, when oxidative stress is low, than during laterstages of the disease when, due to increased oxidative stressand oxidative modification of the enzyme, it may be difficultto inhibit AR or to accrue significant benefits from anti-ARinterventions without further increasing oxidative stress.

Although changes in cellular metabolism and tissue injury

have been monitored in both animal studies and clinicaltrials, changes in inflammation and inflammatory markershave not been carefully examined. In view of recent studiesshowing that inhibition of AR prevents multiple inflamma-

tory pathways (Refs. 101, 128, and 134; Fig. 12), it may benecessary to examine how AR inhibitors affect systemic andlocal inflammation during diabetes. This appears to be par-ticularly critical because chronic inflammation has emergedto be one of the critical features of diabetic complications,particularly cardiovascular disease. Moreover, based on datashowing that inhibition of AR prevents restenosis, vascularsmooth muscle growth, and endothelial cell apoptosis (101,134136), it may be possible to design animal and clinicalstudies to test the efficacy of anti-AR therapy against micro-and macrovascular complications of diabetes, which appear

to be the most serious and prevalent outcomes of prolongeddiabetes. Because protection against vascular changes wasaccompanied by an inhibition of cell signaling involved ininflammation, it appears likely that AR inhibitors may be

FIG. 11. Inhibition of AR attenuates TNF-induced monocyte adhesion and cell surface expression of intercellular adhesion molecule (ICAM)-1and vascular cell adhesion molecule (VCAM)-1 in human umbilical vein endothelial cells (HUVEC). Quiescent HUVEC were preincubatedwithout or with 10 M sorbinil/tolrestat for 24 h, and then stimulated with 2 nM TNF for 24 h. After 24 h of incubation, THP-1 (monocytes)cells were added, and the incubation continued for another 12 h. Cell adhesion assays were performed using light microscope. To determinecellsurface expressionof adhesion moleculesafter stimulation with TNF, thecellswere fixed in 4% formaldehyde,and theexpression of ICAM-1and VCAM-1 was measured by ELISA using anti-VCAM-1 and anti-ICAM-1 antibodies, respectively. Bars represent mean SE (n 4). ***,

P 0.001 in TNF- AR inhibitors-treated group compared with TNF-treated group. [Reproduced with permission from K. V. Ramana et

al.: FASEB J 18:12091218, 2004 (134).]

FIG. 12. Schematic representation ofAR involvement in cell signaling. Cyto-kines, growthfactors,and hyperglycemiagenerate ROS and cause peroxidation oflipids resulting in the generation of toxiclipid aldehydes such as HNE. AR effi-ciently reduces HNE and its conjugatewith glutathione (GSH) to 14-dihy-droxy-2-nonene (DHN) and GS-DHN, re-spectively. The reduced products of alde-hydes may be involved in the cytotoxicsignaling leading to cell death or growth

via activation of phospholipase C (PLC)/PKC/NF-B pathway. Paracrine effectsof cytokines such as TNF amplify hy-perglycemia and other signals as shownin the pathway.



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effective against diseases other than diabetes, particularlythose that are associated with high levels of cytokine gen-eration and inflammation such as rheumatoid arthritis andsepsis. Acute intervention with AR inhibitors during sepsisappears to be particularly attractive because short-term treat-ment will promote recovery by inhibiting both cytokine sig-naling and production, without the risk of long-term treat-ment with AR inhibitors that may chronically increaseoxidative stress.

Finally, to derive maximal benefits from AR inhibition, itis imperative to have specific and selective inhibitors. Al-though the currently available AR inhibitors bind to theenzyme with high affinity, they also display high levels ofnonspecific toxicity. Moreover, in the absence of detailedpharmacokinetic studies, it is unclear whether the dose of ARinhibitors used for clinical studies was effective in inhibitingAR. Hence, further studies are warranted to carefully deter-mine the extent of enzyme inhibition in human tissues for agiven dose of AR inhibitor and whether their efficacy persistsin diabetic tissues. More importantly, however, it appears

that it may be necessary to design more specific inhibitors ofAR that could selectively inhibit the ability of the enzyme tocatalyze the reduction of glutathione conjugates and glucose,without inhibiting aldehyde detoxification. Our structure-activity studies (66, 67) with free and glutathione-conjugatedaldehydes suggest that there are distinct glutathione- andaldehyde-binding domains on the enzyme, and selectivemodification of the enzyme active site could prevent recog-nition and reduction of glutathione conjugates without af-fecting aldehyde reduction. These results suggest the inter-esting possibility that the signaling and detoxification rolesof AR could be regulated independently of each other andthat more selective inhibitors could be designed to selectively

prevent cell injury without compromising antioxidant defense.

VI. Conclusion

Due to its ability to reduce a wide variety of aldehydes,ranging from membrane phospholipids to glucose, AR playsa complex role in cellular metabolism and signaling. Al-though identified initially as a glucose-reducing enzyme, theenzyme is now believed to be an important component ofantioxidant defense involved in the removal and detoxifi-cation of reactive aldehydes generated by lipid peroxidation.Inhibition of AR has been shown to prevent the developmentof diabetic complications in animal models; however, a crit-

ical evaluation of the clinical efficacy of AR inhibitors awaitsa clearer understanding of the role of AR in regulating in-flammation and cell growth. More selective and effectiveinhibitors are needed to specifically inhibit the cytotoxic roleof AR in cell signaling without affecting its detoxificationrole. Such inhibitors are likely to be more effective in treatingsecondary diabetic complications by preventing inflamma-tion due to chronic hyperglycemia.

Acknowledgments

Address all correspondence and requests for reprints to: Satish K.Srivastava, Department of Human Biological Chemistry and Genetics,

University of Texas Medical Branch, Galveston, Texas 77555. E-mail:[email protected]

This work was supported, in part, by NIH Grants DK36118, EY01677,HL55477, and HL59378.

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