BASIC SCIENCE

Involvement of advanced glycation end products,oxidative stress and nuclear factor-kappaBin the development of diabetic keratopathy

Junghyun Kim & Chan-Sik Kim & Eunjin Sohn &

Il-Ha Jeong & Hyojun Kim & Jin Sook Kim

Received: 31 August 2010 /Revised: 4 November 2010 /Accepted: 4 November 2010 /Published online: 23 November 2010# Springer-Verlag 2010

AbstractBackground The purpose of the experiment reported herewas to assess the involvement of advanced glycation endproducts (AGEs), oxidative stress, and nuclear factorkappa-B (NF-κB) activation in the development of diabetickeratopathy.Methods Diabetes was induced by intraperitoneal strepto-zotocin injection in male Sprague–Dawley rats. Thethickness of the cornea was measured. Apoptosis wasdetected by TUNEL assay and western blot for caspase-3.The expression of AGEs and 8-hydroxydeoxyguanosine(8-OHdG) were studied by immunohistochemistry incorneal tissues of normoglycaemic and diabetic rats. NF-κBactivation was evaluated by electrophoretic mobility shiftassay and southwestern histochemistry.Results Corneal edema was observed in diabetic rats. Thethickness of cornea was higher in diabetic than in controlrats. AGEs were accumulated in corneal tissues. 8-OHdGand NF-κB were identified in corneal epithelium, stromaand endothelium, and its expressions were greater indiabetic than in those of control rats. Diabetes inducessignificant alterations in rat corneal tissue structure.Conclusions The higher expression of AGE, 8-OHdG andNF-κB in corneal tissues of diabetic rats suggests that thesefactors are involved in apoptosis and in subsequent cornealalterations related to diabetic keratopathy.

Keywords Apoptosis . Advanced glycation end products .

Diabetic keratopathy

Introduction

Diabetes mellitus, a chronic disease characterized byhyperglycemia, is caused by diminished insulin secretionor resistance to insulin. Chronically high glucose is themajor cause of diabetic retinopathy, nephropathy andneuropathy [1, 2]. Diabetic retinopathy, cataract, refractiveerror and keratopathy are the most common ocularcomplications of diabetes. Corneal disorders associatedwith diabetic keratopathy is histologically characterized bysubepithelial deposits, thickening of the subepithelialbasement membrane and altered morphological appearancein the corneal epithelium and endothelium [3–5]. Althoughit is not known whether these anatomical changes directlyaffect corneal function, many clinical evidences haveshown that patients with diabetes have functional abnor-malities, such as abnormal wound repair, recurrent cornealerosion, persistent epithelial defects, persistent cornealedema and increased endothelial permeability to fluoresceinafter intraocular surgery [6–9]. The pathogenic mechanismunderlying these corneal abnormalities is not entirely clear.

Advanced glycation end-products (AGEs) have beenproposed for the potential causative factor of diabeticcorneal complications [10, 11]. AGEs are sugar-derivedirreversible protein modifications that have been implicatedin the pathogenesis of diabetic complications, such asretinopathy, nephropathy and neuropathy [12]. In patientswith diabetes, AGEs can increase abnormally and accumu-late on tissue and organs that develop chronic complica-tions of diabetes [13]. AGEs has induced structural and

J. Kim : C.-S. Kim : E. Sohn : I.-H. Jeong :H. Kim :J. S. Kim (*)Diabetic Complications Research Center, Division of TraditionalKorean Medicine (TKM) Integrated Research,Korea Institute of Oriental Medicine (KIOM),483 Exporo, Yuseong-gu,Daejeon 305-811, South Koreae-mail: jskim@kiom.re.kr

Graefes Arch Clin Exp Ophthalmol (2011) 249:529–536DOI 10.1007/s00417-010-1573-9

functional alterations of the plasma and the extracellularmatrix proteins. The interaction between AGEs and theirreceptors (RAGE) causes the formation of oxygen radicalsand the release of pro-inflammatory cytokines [14–17]. Inthe human eye, AGEs have been detected in the cornealstroma, lens, Descemet’s membrane, basement membraneof the corneal epithelium and the lamina cribrosa [10, 18–20].The level of AGEs fluorescence in diabetic cornea is higherthan that in normal cornea. Moreover, treatment withaminoguanidine, AGEs inhibitor, prevented diabetic cornealstructural abnormalities, such as the degeneration of intra-cellular organelles, cytoplasmic vacuole formation andedema in the corneal stroma [11, 21]. Based on these results,it is likely that the accumulation of AGEs is also related tothe development of diabetic keratopathy. However, the exactrole of AGEs in diabetic keratopathy remains to beelucidated. We therefore use an in vivo model to furtherinvestigate the role of AGEs accumulation in the diabeticcornea.

Materials and methods

Animal experimental design

Diabetes was induced by a single injection of streptozotocin(STZ, 60 mg/kg body weight, i.p.) in Sprague–Dawley(SD) rats. Age-matched control rats (aged 7 weeks) wereinjected with the vehicle only. After 1 week of induction ofdiabetes, the blood glucose level was measured from thetail vein. The glucose assay employed was an enzymaticassay based on glucose oxidase and peroxidase (glucose B-Test Wako, Osaka, Japan). Rats with a plasma glucose levelgreater than 300 mg/dl were considered as diabetes-inducedrats. The animals were divided into two groups: (1) normalSD rats (n=8), and (2) STZ-induced diabetic rats (n=8). Allprocedures involving rats were performed in accordancewith the ARVO Statement for the Use of Animals inOphthalmic and Vision Research, and approved by theKorea Institute of Oriental Medicine Institutional AnimalCare and Use Committee.

Histopathological analysis

Thirteen weeks after diabetes induction, the eyes wereenucleated from the animals and fixed in 10% phosphate-buffered formalin for 24 hours and embedded in paraffinwax. The eyes were then cut into 5-μm-thick sections andstained with hematoxylin and eosin (H&E). Using eightcorneal sections per experimental group, the thicknesses ofthe cornea, corneal epithelium and stroma in the centralregion of cornea were measured in five random fields persection.

Corneal autofluorescence measurement

Corneal autofluorescence was measured as described byYucel et al. [11]. Briefly, corneas were homogenized inphosphate-buffered saline (PBS). Samples were brought to0.5 mg protein/ml in PBS and autofluorescence wasdetermined at 340 nm excitation and 460 nm emissionwavelength using a spectrofluorometer (Synergy HT,BioTek, USA). Fluorescence intensity was expressed permilligram of protein. Protein concentrations in all sampleswere measured spectrophotometrically using a Lowry assaywith bovine serum albumin as a standard. AGE-modifiedbovine serum albumin (AGE-BSA, MBL international,Woburn, MA, USA) and a buffer blank were used for thepositive and negative controls respectively.

Apoptosis assay

To determine whether diabetes-induced accumulation ofAGEs is able to induce apoptosis in corneal cells, theTUNEL (terminal deoxynucleotidyl transferase dUTP nickend labeling) assay was performed with a kit (DeadEndapoptosis detection system, Promega, Madison, WI, USA)according to the manufacturer’s instructions. Apoptoticcells were detected using fluorescein-conjugated streptavidin.As negative controls, sections were incubated by omittingeither terminal deoxynucleotidyl transferase enzyme orstreptavidin. For quantitative analysis, TUNEL-positivenuclei were then counted per unit area (0.32 mm2).

Western blot analysis

Activation of cleavage of caspase-3 was also determined bywestern blot analysis. The corneas were excised from theeyeball under an optical microscope. Proteins wereextracted from the corneas, and then separated usingSDS–polyacrylamide gel electrophoresis and transferred tonitrocellulose membranes (Biorad, Hercules, CA, USA).Membranes were probed with rabbit anti-cleaved caspase-3antibody (Cell Signaling, Beverly, MA, USA). The immunecomplexes were then visualized with an enhanced chemi-luminescence detection system (ECL; Amersham Bioscience,Piscataway, NJ, USA). Protein expression levels weredetermined by analyzing the signals captured on the nitrocel-lulose membranes, using an image analyzer (Las-3000, FujiPhoto, Tokyo, Japan).

Immunohistochemical staining

Immunohistochemistry was performed as previously described[22]. Antibodies were mouse anti-AGEs (1:200, Cosmo bio,Tokyo, Japan) and rabbit anti-8-hydroxydeoxyguanosine(1:250, 8-OHdG, Abcam, Cambridge, MA, USA). For

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detection of AGEs and 8-OHdG, the sections were incubatedwith the LSAB kit (DAKO, CA, USA) and visualized using3,3’-diaminobenzidine tetrahydrochloride. Negative controlsfor immunohistochemistry were run by incubating thesections with nonimmune serum instead of the primaryantibody.

Measuring of NF-κB acitivty

For electrophoretic mobility shift assay (EMSA), nuclearextracts were prepared with a kit according to themanufacturer’s instructions (Pierce Biotechnology, Rock-ford, IL, USA). EMSA assay was performed by incubating10 μg of nuclear protein extract with IRDye 700-labeledNF-κB oligonucleotide (LI-COR, Lincoln, NE, USA) orunlabelled probe for cold competition. EMSA gels wereanalysed and images were captured and quantified using theLI-COR Odyssey infrared laser imaging system. To localizethe NF-κB activity, southwestern histochemistry was alsoperformed as described by Kim et al. [23].

Statistical analysis

Statistical evaluation of the results was performed usingtwo-tailed Student’s t-test using GraphPad Prism 4.0software (GraphPad Software, San Diego , CA, USA).

Results

Body weight and blood glucose

Body weights and fasting blood glucose levels at thebeginning and at the end of the experiment are summarizedin Table 1. At the end of the study, all diabetic rats hadmarked hyperglycemia and lower body weight compared tothe normal rats (p<0.01).

Histopathological analysis

In the cornea of STZ-induced diabetic rats, perinuclear clearareas were observed, and the stroma was edematous. Thebasement membrane was thickened and multilaminated.The total thicknesses of the central cornea of STZ-induceddiabetic rats were significantly higher than those of normal

rats. Specifically, the thicknesses of the stroma weresignificantly increased in STZ-induced diabetic rats(Table 2).

AGEs accumulation in diabetic cornea

Sato et al. [24] reported that corneal autofluorescencevalues were significantly correlated with AGEs levels inthe corneal tissue from diabetic patients. Thus, wemeasured corneal AGEs autofluorescence. Autofluores-cence detected in the diabetic group (10.13±2.31) wasfound to be significantly greater than that in the control(4.25±1.36) (Fig. 1). In addition, the immunostaining ofAGEs was performed in the corneal sections. In normalrats, there was a weak staining for AGEs within the cornea(Fig. 2a), whereas a striking increase in immunoreactivestraining for AGEs was observed in diabetic rats (Fig. 2b).This increased immunoreactivity of AGEs was mainlyobserved at the site of the corneal epithelium, stroma andendothelium.

Apoptosis of corneal cells

To characterize the apoptotic damage of corneal cells indiabetes, we applied two assays, the TUNEL assay anddetection of active caspase-3. In the cornea of normal rats, aTUNEL-positive nucleus was barely detected (Fig. 3a). Indiabetic rats, many TUNEL-positive cells and somefragmented nuclei were observed in the corneal epithelial,stromal and endothelial cells (Fig. 3b). In quantitativeanalysis, the number of TUNEL-positive cells was in-creased five-fold in the cornea of diabetic rats compared tothe normal rats (Fig. 3c). In western blot analysis, theexpression of cleaved caspase-3 protein was greatlyincreased in the diabetic corneas compared to the normalcontrol (Fig. 3d). This result indicated that several cornealcells are undergoing apoptosis under diabetic conditions.

Oxidative DNA damage in diabetic cornea

To confirm the oxidative stress status in corneal cells underdiabetic conditions, we assessed the formation of oxidativestress specific markers. Representative patterns of theimmunohistochemical localization of 8-OHdG in thecorneal are shown in Fig. 4a and b. 8-OHdG marker shows

Group Mean body weight (g) Mean plasma glucose (mg/dl)

Beginning Final Beginning Final

NOR 189.46±8.15 494.46±8.73 111.25±7.71 124.93±10.8

DM 177.42±11.10 229.25±10.66* 399.58±45.51* 409.78±68.10*

Table 1 Body weight and plas-ma glucose levels

*Statistically significant differ-ence compared with normalcontrol group (p<0.01).

NOR, normal control rats; DM,STZ-induced diabetic rats.

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nuclear and/or perinuclear localization in epithelial, stromaland endothelial cells. In STZ-induced diabetic rats, anincreased immunoreactivity of 8-OHdG was observed. Theexpression of 8-OHdG followed a similar pattern of AGEsaccumulation.

Activation of NF-κB in diabetic cornea

We investigated whether the downstream effect of AGEsassociated mechanistically with the NF-κB pathway. Nuclearextracts from corneal cells were subjected to analysis forNF-κB DNA-binding activity as measured by EMSA. Theresults showed that NF-κB DNA-binding activity wassignificantly increased in the diabetic rats compared to thenormal rats (Fig. 5a). In situ NF-κB activity was alsodetected using southwestern histochemistry. Normal corneasgenerally lacked activated NF-κB staining in any of thecorneal layers (Fig. 5b), whereas in all the examined diabeticcorneas, a marked NF-κB activity was mainly found to benuclear in the corneal epithelial and endothelial cells, butdetected weakly in stromal cells (Fig. 5c). These observa-tions indicate that diabetic states strongly induced NF-κBDNA-binding activity in corneas.

Discussion

Ocular complications associated with diabetes includediabetic keratopathy [3–5]. However, the mechanism thatleads to alteration of corneal cell function in diabetes is notcompletely understood. It was recently reported thatincreased AGEs accumulation and NF-κB activation byAGE/RAGE interaction contribute to the development of

diabetic ocular complications, such as diabetic retinopathyand lacrimal gland dysfunction [14, 25–27]. In the presentstudy, we investigated whether AGEs accumulations con-tributes to corneal disease in STZ-induced diabetic rats. TheSTZ rat model of diabetes could serve as a useful model forconsidering the nature of diabetic keratopathy [21].

In STZ-induced diabetic rats, we demonstrated thealteration of cornea, such as an increased corneal thicknessand apoptotic damage in corneal cells compared to normalrats. These changes are analogous to those described insimilar animal studies and in humans with diabetes [4, 21,28]. In addition, the enhanced AGEs accumulation and theoxidative DNA damage in diabetic corneas were observed.In addition, in both type 1 and type 2 diabetes, theendothelial cell density was reduced 11% in type 1 diabetesand 5% in type 2 diabetes compared to age-matchedhomogeneous normal control groups [28]. Ultrastructuralalterations of corneal cells and stromal edema wereobserved in diabetic rats [21, 29]. Corneal autofluorescencedue to AGEs increased in patients with proliferativediabetic retinopathy [24]. Pentosidine level was higher inhuman diabetic corneas than in control corneas [18]. N"-(carboxymethyl)lysine immunoreactivity was observed inthe epithelial basement membrane in the corneas of patientswith diabetes [10]. Moreover, the accumulation of AGEs inbovine corneal endothelial cell induced apoptosis via thegeneration of reactive oxygen [15]. Based on these results,it is likely that the apoptosis of corneal cells is related to theaccumulation of AGEs and the oxidative stress.

Under diabetic conditions, AGEs accumulate in varioustissues, and lead to a gradual decline in tissue function andto the pathogenesis of diabetic complication, includingnephropathy and retinopathy. AGEs enhanced apoptosis inretinal pericytes, corneal endothelial cells, neuronal cells,and renal mesangial cells [15, 16, 30]. A major way inwhich AGEs exert their cellular effects is generallymediated by interaction with the receptor for AGEs [31].The interaction of AGEs and RAGE activates its down-stream signaling and overproduction of cytokines andreactive oxygen species (ROS) [32]. In the current study,results of AGEs and 8-OHdG immunohistochemistryclearly demonstrated the accumulation of AGEs and thepresence of oxidative DNA damage in the diabetic cornealcells respectively. The oxidation of guanine to form8-hydroxy-2’-deoxyguanosine (8-OHdG) is a marker ofoxidative DNA damage [33]. Results of the present studyrevealed a correlation between the apoptotic damage in thediabetic cornea and the intense nuclear localization of8-OHdG observed in this area. These findings also correlatewith previous data demonstrating an enhanced AGEsaccumulation in diabetic cornea, and provide strongevidence that nuclear oxidative DNA damage by AGEsaccumulation is responsible, at least in part, for the

Table 2 Corneal thickness

Total cornea (μm) Epithelium (μm) Stroma (μm)

NOR 135.6±8.5 33.4±3.3 101.8±7.3

DM 214.4±11.6* 42.5±4.9* 171.2±10.7*

*Statistically significant difference compared with normal controlgroup (p<0.01).

NOR, normal control rats; DM, STZ-induced diabetic rats.

Fig. 1 Corneal AGE fluores-cence values in the normal rat(□) and diabetic rat (■). Valuesin the bar graphs representmeans ± SE, n=8. *p<0.01 vsnormal rats

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apoptotic damage of diabetic corneal cells. However, it waspreviously reported that the number of apoptotic or necroticcells in diabetic rat cornea was not increased compared tonormal corneas [34]. In contrast, ultrastructural apoptoticchanges were detected in keratocyte of diabetic rat cornea[29]. There are several possible reasons for this discrepancybetween animal studies. This discrepancy might be ascribedto the severity of diabetes and different diabetes durations.

Next, we investigated NF-κB activity in diabeticcorneas. NF-κB is a common downstream signal pathwayby AGEs [35]. NF-κB is also involved in the production ofROS and the releases of pro-apoptotic cytokines [15–17].Therefore, the activation of NF-κB and its higher presence

in diabetic corneas suggest that AGEs/RAGE interactionleads to NF-κB activation, sustained oxidative stress, andpro-apoptotic cytokine transcription, thus mediating tissuedamage of cornea, as previously attributed to NF-κB indiabetic retinopathy [14].

Although corneal stroma has an inherent tendency toimbibe water, the normal cornea maintains a constantthickness via the barrier and pump functions of cornealendothelium [36, 37]. Tight junctions in endothelial cellswork as physical barriers, and ion pumps help outwardmovement of the water from the corneal stroma to theanterior chamber [36, 38, 39]. In the present study, severecorneal edema and the apoptotic change of the endothelial

Fig. 2 AGEs accumulation. Immunohistochemical localization of AGEs in the normal rat (a), diabetic rat (b) and negative control (c). A strongimmunoreaction of AGEs was observed in the corneal epithelial, stromal and endothelial cells (magnified inset) of diabetic rats. Scale bar=50 μm

Fig. 3 Apoptosis of cornealcell. The lens sections from thenormal rat (a) and diabetic rat(b) were stained with TUNEL(green). Apoptotic corneal cellswere observed in diabetic rats.Scale bar=50 μm. c Quantita-tive analysis of TUNEL-positivecells in the normal rat (□) anddiabetic rat (■). All data areexpressed as mean ± SE, n=8.*p<0.01 vs normal rats. dWestern blot analysis of cleavedcaspase-3. NOR, normal rat;DM, diabetic rat

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cells occurred in STZ-induced diabetic rats. We consideredthat this corneal edema can be induced as a result of adamage of either the anatomical barrier or the pumpfunctions of corneal endothelial cells.

Previous studies with animal models have indicated aprotective effect of aminoguanidine, AGEs inhibitor,against diabetic nephropathy [40–42], retinopathy [43]and keratopathy [11, 21]. In addition, a clinical studyindicated that antioxidant therapy can improve the ocularsurface complications of diabetic patients [44]. Taken

together, our results for the first time showed that thealterations of corneal cells observed in an animal model ofdiabetes allow us to postulate that the pathogenesis ofdiabetic keratopathy may be associated in part with theaccumulation of AGE, oxidative stress, and activation ofNF-κB. Moreover, these similar mechanisms by AGEs/RAGE axis have been previously demonstrated in otherocular tissues [14, 45–47]. Our in vivo experiment providedthe supporting evidence for the role of AGEs and NF-kB inthe development of diabetic keratopathy.

Fig. 4 Immunofluorescence staining of 8-OHdG. Representativephotomicrographs of corneas from the normal rat (a), diabetic rat (b)and negative control (c). Diabetic corneal epithelial, stromal and

endothelial cells (magnified inset) showed strong immunoreactivityfor 8-OHdG. Scale bar=50 μm

Fig. 5 NF-κB activation in dia-betic cornea. a NF-κB DNA-binding activity measured byEMSA. All data are expressedas mean ± SE, n=8. *p<0.01 vsnormal rats. b,c Southwesternhistochemistry. Representativephotomicrographs of corneasfrom the normal rat (b) anddiabetic rat (c). Positive signalsfor activated NF-κB mainlydetected in nucleus of diabeticcorneal epithelial and endotheli-al cells. Scale bar=50 μm

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Acknowledgments This research was supported by a grant[L08010, K09030] from the Korea Institute of Oriental Medicine(KIOM).

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