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Chemico-Biological Interactions 188 (2010) 86–93

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

ytotoxic role of methylglyoxal in rat retinal pericytes: Involvement of a nuclearactor-kappaB and inducible nitric oxide synthase pathway

unghyun Kim, Ohn Soon Kim, Chan-Sik Kim, Nan Hee Kim, Jin Sook Kim ∗

iabetic Complications Research Center, Division of Traditional Korean Medicine (TKM) Integrated Research, Korea Institute of Oriental Medicine (KIOM), 483 Exporo, Yuseong-gu,aejeon 305-811, South Korea

r t i c l e i n f o

rticle history:eceived 4 March 2010eceived in revised form 1 July 2010ccepted 1 July 2010vailable online 6 August 2010

eywords:poptosis

nducible nitric oxide synthase

a b s t r a c t

Methylglyoxal (MGO), a cytotoxic metabolite, is produced from glycolysis. Elevated levels of MGO areobserved in a number of diabetic complications, including retinopathy, nephropathy and cardiomyopa-thy. Loss of retinal pericyte, a hallmark of early diabetic retinal changes, leads to the development offormation of microaneurysms, retinal hemorrhages and neovasculization. Herein, we evaluated the cyto-toxic role of MGO in retinal pericytes and further investigated the signaling pathway leading to cell death.Rat primary retinal pericytes were exposed to 400 �M MGO for 6 h. Retinal vessels were prepared fromintravitreally MGO-injected rat eyes. We demonstrated apoptosis, nuclear factor-kappaB (NF-�B) acti-vation and inducible nitric oxide synthase (iNOS) induction in cultured pericytes treated with MGO and

ethylglyoxaluclear factor-kappaBat retinal pericytes

MGO-injected retinal vessels. In MGO-treated pericytes, TUNEL-positive nuclei were markedly increased,and NF-�B was translocalized into the nuclei of pericytes, which paralleled the expression of iNOS. Thetreatment of pyrrolidine dithiocarbamate (an NF-�B inhibitor) or l-N6-(1-iminoethyl)-lysine (an iNOSinhibitor) prevented apoptosis of MGO-treated pericytes. In addition, in intravitreally MGO-injected rateyes, TUNEL and caspase-3-positive pericytes were significantly increased, and activated NF-�B and iNOSwere highly expressed. These results suggest that the increased expression of NF-�B and iNOS caused by

tinal

MGO is involved in rat re

. Introduction

Diabetic retinopathy is associated with progressive damagef the retinal microvasculature, including thickening of capillaryasement membranes, loss of pericytes, and acellular capillaryormation [1,2]. The loss of retinal pericytes, a hallmark of earlyiabetic retinopathy, leads to the formation of microaneurysms,etinal hemorrhages and neovasculization.

Methylglyoxal (MGO) is formed from the glycolytic interme-iate glyceraldehyde-3-phosphate [3], from the early glycationrocess by degradation of glucose or Schiff’s base, or from Amadoriroducts in the intermediate stages of glycation [4]. MGO is aeactive dicarbonyl precursor of advanced glycation end productsAGEs) [5]. It is well known that the production of AGEs is a risk fac-or for the development of diabetic complications. Coincidentally,he concentration of MGO is much higher in the plasma of diabetic

atients [6–8] and diabetic animal tissues [9]. However, currently,here is little evidence demonstrating a direct cytotoxic effect of

GO in the pathology of diabetes.

∗ Corresponding author. Tel.: +82 42 868 9465; fax: +82 42 868 9471.E-mail address: jskim@kiom.re.kr (J.S. Kim).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.07.002

pericyte apoptosis.© 2010 Elsevier Ireland Ltd. All rights reserved.

Elevated MGO levels affect cellular function by reacting withamino and sulfhydryl groups of several proteins, which may resultin diabetic complications [10]. MGO also induces apoptosis ofrat Schwann cells [11], human vascular endothelial cells [12], ratmesangial cell [13] and bovine retinal pericytes [14]. MGO-inducedreactive oxygen species (ROS) production may trigger apoptosis[15,16]. In addition, p38 MAPK activation is known to be a keysignaling intermediate of MGO-induced apoptosis in rat mesan-gial cells [17] and Schwann cells [11]. Furthermore, it was recentlyreported that both the JNK pathway [18] and alterations in growthfactor receptor signaling by MGO [19] are important for MGO-induced apoptosis. However, the underlying mechanisms of MGOcytotoxicity in retinal pericytes and whether MGO induces apop-tosis in these cells remain uncertain.

Nitric oxide (NO) is an important signaling molecule thatmediates a variety of essential physiological process, includingneurotransmission, vasodilation and host cell defense. NO is gen-erated from l-arginine by reactions catalyzed by different isoformsof nitric oxide synthase (NOS), including neuronal NOS (nNOS),

endothelial NOS (eNOS) and inducible NOS (iNOS). iNOS is usu-ally not present under normal conditions; rather, it is inducedby lipopolysaccharide or various cytokines [20]. The induction ofiNOS results in the release of excessive amounts of NO, whichis cytotoxic to neighboring cells [21,22]. Previous evidence has

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hown that an up-regulation of iNOS levels has been found inhe retinas of experimental diabetic animals and human patients23–26]. Furthermore, Zheng et al. revealed that diabetic iNOSnockout mice were protected from retinal capillary degeneration27], implicating the iNOS pathway in the degeneration of theseells.

Recent evidence suggests that enhanced apoptosis of retinalericytes is also associated with nuclear factor-kappaB (NF-�B)28,29]. It has been reported that NF-�B activation in retinal per-cytes due to hyperglycemia induces accelerated pericyte loss iniabetic retinopathy [28]. In addition, the activation of NF-�B isnown to induce the expression of iNOS, and the iNOS promoter hasonsensus NF-�B binding sites [30–34]. These findings suggest thatn NF-�B/iNOS pathway is closely associated with the degenerationf diabetic retinal capillaries. Thus, in this study, we investigatedhether MGO can induce apoptosis in rat retinal pericytes and if

o, whether MGO-induced apoptosis occurs via the induction of anF-�B/iNOS pathway.

. Materials and methods

.1. Primary rat retinal pericyte cell culture

Primary retinal pericytes were isolated from Sprague–DawleySD) rat retinal microvessels using a modification of previouslyublished methods [35–38]. Briefly, eyes were enucleated fromats. Retinas were separated from eyes, then homogenized using aeflon-glass homogenizer (Wheaton, NJ, USA) and filtered though70 �m nylon mesh (BD Biosciences, CA, USA). The remain-

ng retentate was digested in 0.066% Collagenase/Dipase (Roche,annheim, Germany) in Dulbecco’s Phosphate-Buffered Saline

DPBS) for 1 h at 37 ◦C. The cellular digests were then filteredhrough a 40 �m nylon mesh. Purification of rat retinal peri-ytes was achieved with CELLection Pan Mouse IgG Kit (Invitrogenynal AS, Oslo, Norway) with mouse anti-desmin monoclonalntibody (MAB3430, Millipore, MA, USA) according to the man-facturer’s instructions. The purified cells were maintained inMEM containing 10% FBS at 37 ◦C in a humidified atmospheref a 5% CO2 incubator. Pericytes were identified by the inabilityo uptake rhodamine-conjugated, acetylated low-density lipopro-ein as described by Cacicedo et al. [39]. Our cultured cells did notontain any cells that reacted with antibodies to the endothelialell marker von Willebrand’s factor (A0082, Dako, CA, USA). Cellsetween passages 3 and 5 were used in this study. Cells were platednto appropriate culture dishes and used for experiments uponeaching 80% confluence. Standard culture medium was replacedith fresh serum-free medium 16 h before experiments. Pericytesere then incubated with the indicated concentration of MGO

n the presence or absence of the indicated reagents for 6 h andhen fixed with 4% paraformaldehyde for immunofluorescencetaining.

.2. Cell viability assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-ide (MTT) assay was performed to analyze cell survival. Briefly,

ells were plated in DMEM containing 10% FBS at a density of× 103 cells/well in a 96-well tissue culture plate, overnight. At 1ay after plating, cells were grown in serum-free DMEM for 6 h

ontaining the indicated concentration of MGO, followed by incu-ation for 1 h in the presence of 0.5 mg/mL MTT (Sigma, MO, USA).he medium was then aspirated, and the cells were dissolved insopropanol (100 �L/well). Absorbance of the converted dye was

easured at 570 nm, with a background subtraction at 630 nm inmicroplate reader (BIO-TEK, Synergy HT, USA).

teractions 188 (2010) 86–93 87

2.3. Intravitreal injection of MGO

Sixteen adult SD rats (9 weeks old) were used in this study. Eachrat was anesthetized with a 1:1 mixture xylazine hydrochloride(4 mg/kg) and ketamine hydrochloride (10 mg/kg). A single dose of8 mM MGO in a volume 3 �L was injected into the vitreous of theright eye with a microinjector (Hamilton Co., NV, USA) under a dis-secting microscope. Assuming the vitreous volume of an adult rateye to be approximately 56 �L [40], the final intravitreal concentra-tion of MGO was approximately 400 �M. For normal control, 3 �Lphysiological saline was injected into the left eye. The needle wasleft in position for 30–60 s and then slowly withdrawn to minimizefluid loss from the eye. Rats were monitored regularly for infec-tion associated with the injection site. Eye with injection-damagedlenses or retinas were excluded from the study. At 2 days after theintravitreal injection, rats were anesthetized and killed. All exper-iments were approved by the Korea Institute of Oriental MedicineInstitutional Animal Care and Use Committee.

2.4. Trypsin digested vessel preparation

The eyes were enucleated from the animals and retinas wereisolated. The retinal samples were then placed in 10% formalinfor 2 days. After fixation, the retina was incubated in trypsin (3%in sodium phosphate buffer containing 0.1 M sodium fluoride toinhibit the DNase activity) for about 60 min. The vessel structureswere isolated from the retinal cells by gentle rinsing in distilledwater. The vascular specimens were then mounted on a slide.

2.5. Assessment of apoptosis

Apoptosis was assessed by an immunofluorescence stainingfor cleaved caspase-3 and a TdT-mediated dUTP nick-end label-ing (TUNEL) kit as per the manufacturer’s instructions (Promega,WI, USA). After TUNEL staining, cells were counterstained withrhodamine labeled Wheat Germ Agglutinin (WGA) (Vector Labo-ratories, Burlingame, CA, USA) to identify the plasma membrane.The numbers of apoptotic cells and total cells per unit area (mm2)were counted by a previously described method [41], and the per-centages of apoptotic cells were calculated.

2.6. Immunofluorescence staining

The immunofluorescence staining was performed on culturedpericytes and trypsin digests. The antibodies used were mouseanti-NF-�B p65 antibody (SC-8008, Santa Cruz, CA, USA), mouseanti-NF-�B antibody (MAB3026, Chemicon International, CA, USA)and mouse anti-iNOS antibody (SC-7271, Santa Cruz). For detectionof NF-�B and iNOS, the cells were incubated with FITC-conjugatedgoat anti-mouse antibody (Santa Cruz). The cultured pericyteswere then counterstained with 4,6-diamidino-2-phenylindole. Theintensity of the fluorescence was analyzed in five randomly selectedmm2 areas using ImageJ software (NIH).

2.7. Evaluation of NF-�B DNA binding activity

Nuclear proteins were isolated from retinal pericytes usingnuclear and cytoplasmic extraction kits (NE-PERTM Nuclear andCytoplasmic Extraction Reagents, Pierce, IL, USA). NF-�B DNA bind-ing activity was evaluated using an ELISA-based EZ-detectedTM

Transcription Factor Kit for NF-�B p65 (Pierce) according to the

manufacturer’s instructions. Briefly, 10 �L of nuclear extracts con-taining 40 �g proteins was added to the wells covered witholigonucleotide containing the NF-�B consensus binding site. Theactive transcription factor bound to the consensus sequence wasincubated with a specific primary antibody (NF-�B p65) and then

88 J. Kim et al. / Chemico-Biological Int

Fig. 1. Dose-dependent cytotoxic effects of MGO in rat retinal pericytes. Cytotox-icity was measured by the MTT assay (A) and TUNEL staining (B). (C) Quantitativeanalysis of TUNEL-positive cells. Pericytes were incubated for 6 h with the indicatedconcentration of MGO. Values in the bar graphs represent means ± SE, n = 4. *p < 0.05vs. control group; **p < 0.01 vs. control group.

Fig. 2. Effects of pyrrolidine dithiocarbamate (PDTC) and l-N6-(1-iminoethyl)-lysine (lquantitative analysis of TUNEL-positive cells and (C) MTT assay results. Pericytes were incof PDTC and l-NIL. Values in the bar graphs represent means ± SE, n = 4. *p < 0.01 vs. contr

eractions 188 (2010) 86–93

with a secondary HRP conjugated antibody. Chemiluminescencewas measured with a luminometer. All samples were run intriplicates. A wild-type consensus oligonucleotide, provided as acompetitor for NF-�B binding, and a mutated consensus oligonu-cleotide with no effect on NF-�B binding was used in order tomonitor the specificity of the assay.

2.8. Western blotting analysis

Proteins were extracted from retinal vessels, and thenapproximately 20 �g of protein lysates was separated bySDS–polyacrylamide gel electrophoresis and transferred to nitro-cellulose membranes (Biorad, CA, USA). Membrane was probedwith mouse anti-iNOS antibody (Santa Cruz), and then the immunecomplexes were visualized with an enhanced chemiluminescencedetection system (Amersham Bioscience, NJ, USA).

2.9. Statistical analysis

The data from cultured cells were analyzed with the paired t-testand a one-way analysis of variance (ANOVA) followed by Tukey’smultiple comparison test. Statistical analyses were performed byGraphPad Prism 4.0 (GraphPad, CA, USA).

3. Results

3.1. MGO-induced apoptosis of cultured rat retinal pericytes

Fig. 1A shows the absorbance of MTT formazan after incubationwith MTT for 1 h, which is believed to be proportional to the numberof living cells. MGO treatment elicited a dose-dependent cytotox-icity on rat retinal pericytes. MGO slightly reduced cell survival,as measured by the MTT assay, at a concentration of 200 �M. The

viability of cells incubated with 400 �M MGO alone was approxi-mately 60% compared to that of control cells (Fig. 1A). To investigatewhether this MGO-induced cell death was due to apoptosis, apop-totic cell death was identified by the TUNEL assay (Fig. 1B). In theTUNEL assay, the ratio of the apoptotic cell number to the total cell

-NIL) on apoptosis of MGO-treated rat retinal pericytes. (A) TUNEL staining, (B)ubated with 400 �M MGO in the absence or presence of increasing concentrationsol group; #p < 0.01 vs. MGO-treated group.

J. Kim et al. / Chemico-Biological In

Fig. 3. MGO-induced NF-�B activation in rat retinal pericytes. (A) Subcellular local-icV#

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zation of NF-�B p65 subunits. The middle panel depicts DAPI nuclear staining as aounterstain. (B) Analysis of NF-�B DNA binding activity by an ELISA-based assay.alues in the bar graphs represent means ± SE, n = 4. *p < 0.01 vs. control group;p < 0.01 vs. MGO-treated group.

umber was increased to 42.3 ± 6.71% when treated with 400 �MGO, with an apparent dose-dependency (Fig. 1C).

.2. Effect of PDTC on MGO-induced NF-�B activation andpoptosis in rat retinal pericytes

We examined the cytoprotective effects of pyrrolidine dithio-arbamate (PDTC, a selective inhibitor of NF-�B) on MGO-treatedericytes. Pericytes were incubated with PDTC and 400 �M MGOnd were examined for cell viability and apoptosis. As shown inig. 2A and B, the enhanced apoptotic cell death by MGO wasignificantly suppressed, by 54%, with 10 �M PDTC. The viabilityf cells incubated with MGO alone was approximately 60% com-ared to that of controls, whereas PDTC suppressed MGO-inducedell death in a dose-dependent manner (Fig. 2C). Next, we fur-her investigated whether MGO induced a nuclear translocationf NF-�B p65 in rat retinal pericytes with an immunofluorescencetaining method. In untreated cells, positive staining for NF-�B p65as hardly detectable in nuclei. As expected, there was increasedositive staining for NF-�B p65 in the nuclei of cells treated with

00 �M MGO (Fig. 3A). The MGO-induced increases in nuclear NF-B p65 staining were significantly attenuated by the treatmentith 10 �M PDTC, as shown in Fig. 3A. To confirm the results of

mmunofluorescence staining and evaluate NF-�B activation quan-itatively, we performed an ELISA-based NF-�B assay, measuring

teractions 188 (2010) 86–93 89

NF-�B binding. Similarly, MGO-treated rat retinal pericytes exhib-ited a significantly higher activity of NF-�B than control cells,whereas the level of activated NF-�B in MGO/PDTC-co-treated ratretinal pericytes was significantly lower, by 60%, than those ofMGO-treated cells (Fig. 3B).

3.3. Effect of l-NIL on MGO-induced iNOS expression andapoptosis in rat retinal pericytes

As shown in Fig. 2A and B, the pretreatment of cells with 50 �Ml-N6-(1-iminoethyl)-lysine (l-NIL, a selective inhibitor of iNOS)significantly suppressed MGO-induced pericyte apoptosis. In theMTT assays, the decreased viability of cells incubated with 400 �MMGO was restored by l-NIL in a dose-dependent manner (Fig. 2C).Moreover, a significant increase in iNOS-positive immunofluores-cence was detected in the cytoplasm of rat retinal pericytes afterthe pericytes were treated with 400 �M MGO for 6 h. As shownin Fig. 4, an increase of 86 ± 4.8% of iNOS-positive immunofluo-rescence was observed after the cells were treated with 400 �MMGO, in comparison to that of untreated cells (p < 0.01). Further-more, the increased iNOS-positive immunofluorescence inducedby 400 �M MGO was significantly decreased after the pretreat-ment with 50 �M l-NIL (Fig. 4). Interestingly, iNOS cytoplasmicimmunofluorescence, induced by MGO, was also inhibited bytreatment with 10 �M PDTC. The level of activated NF-�B in MGO/l-NIL-co-treated rat retinal pericytes was not significantly inhibitedby treatment of 50 �M l-NIL in MGO-treated rat retinal pericytes(Fig. 3B).

3.4. Apoptosis in retinal microvascular cells from intravitreallyMGO-injected eyes

We examined the cytotoxic effect of MGO on retinal peri-cytes in vivo. Eyes were intravitreally injected with 400 �M MGOand were examined for apoptosis. To characterize the apopto-sis of retinal vascular cell, we applied the TUNEL assay andcleaved caspase-3 immunostaining. In the retinal trypsin digestsof normal eye, a TUNEL-positive nucleus was barely detected. Incontrast, in retinal vessels from MGO-injected eyes, many TUNEL-positive microvascular cells and numerous fragmented nuclei wereobserved (Fig. 5A). Similarly, enhanced cleaved caspase-3-positiveendothelial cells and pericytes were observed in retinal capillar-ies from MGO-injected eyes (Fig. 5B). Pericytes were identified bytheir morphology. In whole mount retinal digests, the endothe-lial cell nuclei occurring within the vessel wall, are large, oval andprotrude luminally. Pericyte nuclei are small, round, and protrudelaterally from the vessel wall. The number of TUNEL-positive retinalvascular cells was significantly increased 4.5-fold in MGO-injectedeyes compared to normal eyes (Fig. 5C, p < 0.01).

3.5. NF-�B activation and iNOS expression in retinal pericytes ofMGO-injected eyes

NF-�B in retinal pericytes was evaluated by immunostainingusing anti-NF-�B antibody MAB3026. This antibody recognizes anepitope overlapping the nuclear localization signal of the p65 sub-unit of the NF-�B heterodimer [42]. Thus, this antibody selectivelybinds the I�B-free, activated form of NF-�B. Using this antibody, weobserved that the activated NF-�B was mainly found in the roundor oval nuclei of retinal pericytes in retinal capillaries from MGO-injected eyes (Fig. 6A). The NF-�B-positive nuclei of pericytes were

scattered throughout the vascular network and were more frequentin MGO-injected eyes than in normal eyes. To confirm the results ofimmunostaining and evaluate NF-�B activity in a quantitative way,we also performed an ELISA-based NF-�B assay. Similarly, the reti-nal vessels of MGO-injected eyes presented a significantly higher

90 J. Kim et al. / Chemico-Biological Interactions 188 (2010) 86–93

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ig. 4. MGO-induced iNOS expression in rat retinal pericytes. (A) Immunofluoresceericytes. Values in the bar graphs represent means ± SE, n = 4. *p < 0.01 vs. control

ctivity of NF-�B than their normal counterparts (Fig. 6C, p < 0.01).ikewise, a remarkable increase in the iNOS-positive pericytes wasbserved in retinal vessels of MGO-injected eyes by immunostain-ng (Fig. 6B). In Western blot analysis, the iNOS protein was greatlyncreased in retinal vessels of MGO-injected eyes compared to nor-

al eyes (Fig. 6D).

. Discussion

MGO is a major precursor of AGEs and is increased in diabeticissues [9]. The formation of AGEs in retinal tissue closely correlatesith the development of diabetic retinopathy [43,44]. Recently, itas reported that MGO induced the apoptosis of bovine retinal per-

cytes by oxidative stress and that MGO also led to the activation ofF-�B in bovine retinal pericytes [14]. Similarly, NF-�B activation,nder diabetic conditions, induced accelerated pericyte loss [28]nd retinal capillary cell death [29]. In addition, human retinal per-cytes underwent apoptosis along with a dramatic increase in MGO

hen incubated with an NO donor [45] Moreover, the iNOS genes known to have a consensus NF-�B binding site in its promoteregion and to be regulated by NF-�B [30–34]. Thus, we hypothe-ized that NF-�B and iNOS are key players in the mechanism byhich MGO induces apoptosis of retinal pericytes. To elucidate

he mechanisms of MGO-induced apoptosis in retinal pericytes, weocused on identifying a putative NF-�B/iNOS signaling pathway.

The plasma methylglyoxal levels are estimated to be about.5 �M in healthy individuals and can increase twofold in diabetes8], while others demonstrated that plasma methylglyoxal con-entration in poorly controlled human diabetic patients is about

00 �M [46]. In some previous studies, methylglyoxal in a rangef 10–1000 �M caused the impairment of vasoreactivity in ratesenteric arteries in a concentration-dependent manner [47],

nd the treatment of methylglyoxal (200–800 �M) for 6 h inducedpoptosis in bovine retinal pericytes in a concentration-dependent

ining for iNOS (green). (B) The immunoreactivity of iNOS in l-NIL- or PDTC-treated; #p < 0.01 vs. MGO-treated group.

manner [14]. Moreover, cells produce large amounts of methylgly-oxal [48]. Therefore intracellular levels are probably much higherthan plasma levels [9]. Based on previous experiments, we choosethe dose of 400 �M methylglyoxal and exposure time to mimicthe exposure to elevated levels of methylglyoxal under diabeticconditions.

Cytotoxicity induced by MGO has been previously reported invarious cells (rat Schwann cells, human vascular endothelial cells,rat mesangial cell and bovine retinal pericytes) [11–14]. In thisstudy, we demonstrated that MGO exposure results in toxicity tocultured rat retinal pericytes and rat retinal capillary pericytes,specifically resulting in an induction of apoptosis in these cells(Figs. 1 and 5). These data appear to suggest a probable involve-ment of MGO in the pathogenesis of diabetic retinal pericyte loss.In our recent study, it was reported that oxidative DNA damagecaused by MGO is involved in apoptosis of rat retinal pericytes [41].Present study successfully showed that retinal pericytes showedNF-�B activation in the presence of MGO in vitro and in vivo. NF-�B activation is responsive to reactive oxygen species (ROS) [49].These data indicate that the production of ROS by MGO on pericytescould be associated with increases in NF-�B activation.

Previous in vitro work demonstrated that the activation ofNF-�B by high glucose in bovine retinal pericytes induced theexpression of Bax and pro-apoptotic cytokines, such as tumornecrosis factor-alpha [28]. In addition, we showed that PDTC, anNF-�B inhibitor, suppressed apoptosis of rat retinal pericytes likelythrough the inhibition of nuclear translocation of NF-�B (Fig. 3A).These results suggest that the MGO-induced NF-�B nuclear translo-cation is involved in the cytotoxic mechanism of MGO on rat retinalpericytes. PDTC has been extensively used as a NF-�B inhibitor.

However, the molecular basis for the action of PDTC has beenclaimed to be due to its antioxidant or metal chelator activity[50]. Although, the dithiocarboxyl group on PDTC is expected toexert antioxidant effect [51], PDTC is a far more potent NF-�B

J. Kim et al. / Chemico-Biological Interactions 188 (2010) 86–93 91

Fig. 5. Retinal capillary cell apoptosis. The trypsin-digested retinal vessels froma normal control eye (CON) and MGO-injected eye (MGO) were stained with (A)TUNEL (green) and (B) cleaved caspase-3 (red). Arrow indicates fragmented nucleiof apoptotic cells. Arrowhead and asterisk indicate caspase-3-positive pericyteaTmi

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Fig. 6. NF-�B activation and iNOS expression in retinal vessels. (A) Representativephotomicrographs of retinal vessels from a normal control eye (CON) and MGO-injected eye (MGO). Positive immunoreactivity for activated NF-�B (arrowhead) inMGO-injected eye (arrowhead). (B) Immunofluorescence localization of iNOS pro-

nd endothelial cell, respectively. 400× magnification. (C) Quantitative analysis ofUNEL-positive nuclei. All data are expressed as means ± SE, n = 8. *p < 0.01 vs. nor-al rats. (For interpretation of the references to color in this figure legend, the reader

s referred to the web version of the article.)

nhibitor as compared to most antioxidants [52]. This suggests thathe inhibitory effect of PDTC on apoptosis of rat retinal pericytes isttributable, at least in part, to its antioxidative as well as NF-�Bnhibitory effect.

In several ocular diseases, the alteration of iNOS expression haseen reported. The induction of iNOS and excessive production ofO were observed in uveitis, retinitis, glaucoma and cataract for-ation [53–56]. The concentration of NO in the vitreous body was

levated in diabetes patients with diabetic retinopathy [57]. In fact,iabetes-induced alterations of retinal structure and microvesselsere reduced in diabetic iNOS knockout mice [27]. l-NIL, an iNOS

elective inhibitor, prevented apoptosis of retinal pericytes (Fig. 2And B). These findings provide evidence that iNOS expression byGO is, at least partially, responsible for the apoptosis of retinal

ericytes. Furthermore, NO decreased the activity and expressionf glyoxalase I, a critical detoxifying enzyme of MGO in retinal per-cytes [45]. NO is extremely reactive with superoxide anion radicalO2

−), generating peroxynitrite (ONOO−) [58], which functions asn oxidant to proteins, vitamins, and DNA [59]. MGO causes theeneration of NO and superoxide anion radical, leading to peroxyni-rite formation and triggers apoptosis of cultured rat aortic smooth

uscle cells [60]. These previous findings demonstrate a possibleytotoxic role of NO and peroxynitrite in retinal pericytes.

Finally, we examined the possible link between NF-�B activa-

ion and iNOS expression in MGO-treated rat retinal pericytes. BothF-�B activation and iNOS expression (Figs. 4 and 6) were demon-

trated in retinal pericytes treated with MGO. PDTC treatment notnly decreased MGO-induced apoptosis, but also prevented NF-�Buclear translocation (Fig. 3), as well as, iNOS expression (Fig. 4) in

tein (arrow) in a MGO-injected eye. 400× magnification. (C) Analysis of NF-�B DNAbinding activity by ELISA-based assay. (D) Western blot analysis of iNOS in retinalvessels. Values in the bar graphs represent means ± SE, n = 8. *p < 0.01 vs. normalrats.

MGO-treated pericytes. Importantly, the iNOS promoter is knownto have a consensus binding site for NF-�B [30–34]. Thus, theseresults provide strong support for the hypothesis that NF-�B/iNOSsignaling pathway contributes to retinal pericyte death caused byMGO.

In conclusion, our study shows that MGO may enhance theexpression of iNOS by activating NF-�B in rat retinal pericytes,which leads to apoptosis of these cells. PDTC or l-NIL has the abilityto attenuate the increase in iNOS expression directly or indirectly,resulting in the prevention of apoptosis. Taking together, it seemslikely that NF-�B/iNOS pathway plays a critical role in the regula-tion of retinal pericyte apoptosis caused by MGO.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

This research was supported by a grant [K09030 and K10040]from the Korea Institute of Oriental Medicine (KIOM).

References

[1] J. Cunha-Vaz, R. Bernardes, Nonproliferative retinopathy in diabetes type 2.Initial stages and characterization of phenotypes, Prog. Retin. Eye Res. 24 (2005)355–377.

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[2] S. Roy, T. Sato, G. Paryani, R. Kao, Downregulation of fibronectin overexpres-sion reduces basement membrane thickening and vascular lesions in retinas ofgalactose-fed rats, Diabetes 52 (2003) 1229–1234.

[3] S.A. Phillips, P.J. Thornalley, The formation of methylglyoxal from triose phos-phates. Investigation using a specific assay for methylglyoxal, Eur. J. Biochem.212 (1993) 101–105.

[4] P.J. Thornalley, A. Langborg, H.S. Minhas, Formation of glyoxal, methylglyoxaland 3-deoxyglucosone in the glycation of proteins by glucose, Biochem. J. 344(Pt 1) (1999) 109–116.

[5] M. Brownlee, Lilly Lecture 1993. Glycation and diabetic complications, Diabetes43 (1994) 836–841.

[6] A.C. McLellan, P.J. Thornalley, J. Benn, P.H. Sonksen, Glyoxalase system in clin-ical diabetes mellitus and correlation with diabetic complications, Clin. Sci. 87(1994) 21–29.

[7] P.J. Beisswenger, S.K. Howell, R.M. O’Dell, M.E. Wood, A.D. Touchette, B.S. Szw-ergold, Alpha-Dicarbonyls increase in the postprandial period and reflect thedegree of hyperglycemia, Diabetes Care 24 (2001) 726–732.

[8] A. Lapolla, R. Flamini, A. Dalla Vedova, A. Senesi, R. Reitano, D. Fedele, E. Basso, R.Seraglia, P. Traldi, Glyoxal and methylglyoxal levels in diabetic patients: quan-titative determination by a new GC/MS method, Clin. Chem. Lab. Med. 41 (2003)1166–1173.

[9] E.W. Randell, S. Vasdev, V. Gill, Measurement of methylglyoxal in rat tissuesby electrospray ionization mass spectrometry and liquid chromatography, J.Pharmacol. Toxicol. Methods 51 (2005) 153–157.

10] J. Zeng, M.J. Davies, Evidence for the formation of adducts and S-(carboxymethyl)cysteine on reaction of alpha-dicarbonyl compounds withthiol groups on amino acids, peptides, and proteins, Chem. Res. Toxicol. 18(2005) 1232–1241.

11] M. Fukunaga, S. Miyata, B.F. Liu, H. Miyazaki, Y. Hirota, S. Higo, Y. Hamada,S. Ueyama, M. Kasuga, Methylglyoxal induces apoptosis through activation ofp38 MAPK in rat Schwann cells, Biochem. Biophys. Res. Commun. 320 (2004)689–695.

12] A.A. Akhand, K. Hossain, H. Mitsui, M. Kato, T. Miyata, R. Inagi, J. Du, K. Takeda,Y. Kawamoto, H. Suzuki, K. Kurokawa, I. Nakashima, Glyoxal and methylglyoxaltrigger distinct signals for map family kinases and caspase activation in humanendothelial cells, Free Radic. Biol. Med. 31 (2001) 20–30.

13] W.J. Huang, C.W. Tung, C. Ho, J.T. Yang, M.L. Chen, P.J. Chang, P.H. Lee, C.L.Lin, J.Y. Wang, Ras activation modulates methylglyoxal-induced mesangial cellapoptosis through superoxide production, Ren. Fail. 29 (2007) 911–921.

14] J. Kim, J.W. Son, J.A. Lee, Y.S. Oh, S.H. Shinn, Methylglyoxal induces apoptosismediated by reactive oxygen species in bovine retinal pericytes, J. Korean Med.Sci. 19 (2004) 95–100.

15] J. Du, H. Suzuki, F. Nagase, A.A. Akhand, X.Y. Ma, T. Yokoyama, T. Miyata, I.Nakashima, Superoxide-mediated early oxidation and activation of ASK1 areimportant for initiating methylglyoxal-induced apoptosis process, Free Radic.Biol. Med. 31 (2001) 469–478.

16] F. Amicarelli, S. Colafarina, F. Cattani, A. Cimini, C. Di Ilio, M.P. Ceru, M. Miranda,Scavenging system efficiency is crucial for cell resistance to ROS-mediatedmethylglyoxal injury, Free Radic. Biol. Med. 35 (2003) 856–871.

17] B.F. Liu, S. Miyata, Y. Hirota, S. Higo, H. Miyazaki, M. Fukunaga, Y. Hamada, S.Ueyama, O. Muramoto, A. Uriuhara, M. Kasuga, Methylglyoxal induces apopto-sis through activation of p38 mitogen-activated protein kinase in rat mesangialcells, Kidney Int. 63 (2003) 947–957.

18] W.H. Chan, H.J. Wu, N.H. Shiao, Apoptotic signaling in methylglyoxal-treatedhuman osteoblasts involves oxidative stress, c-Jun N-terminal kinase, caspase-3, and p21-activated kinase 2, J. Cell. Biochem. 100 (2007) 1056–1069.

19] A.V. Cantero, M. Portero-Otin, V. Ayala, N. Auge, M. Sanson, M. Elbaz, J.C. Thiers,R. Pamplona, R. Salvayre, A. Negre-Salvayre, Methylglyoxal induces advancedglycation end product (AGEs) formation and dysfunction of PDGF receptor-beta: implications for diabetic atherosclerosis, FASEB J. 21 (2007) 3096–3106.

20] S. Moncada, R.M. Palmer, E.A. Higgs, Nitric oxide: physiology, pathophysiology,and pharmacology, Pharmacol. Rev. 43 (1991) 109–142.

21] H.Y. Yun, V.L. Dawson, T.M. Dawson, Nitric oxide in health and disease of thenervous system, Mol. Psychiatry 2 (1997) 300–310.

22] C. Nathan, Q.W. Xie, Nitric oxide synthases: roles, tolls, and controls, Cell 78(1994) 915–918.

23] A.M Abu El-Asrar, S. Desmet, A. Meersschaert, L. Dralands, L. Missotten, K.Geboes, Expression of the inducible isoform of nitric oxide synthase in the reti-nas of human subjects with diabetes mellitus, Am. J. Ophthalmol. 132 (2001)551–556.

24] A. Carmo, J.G. Cunha-Vaz, A.P. Carvalho, M.C. Lopes, Nitric oxide synthaseactivity in retinas from non-insulin-dependent diabetic Goto-Kakizaki rats:correlation with blood–retinal barrier permeability, Nitric Oxide 4 (2000)590–596.

25] Y. Du, M.A. Smith, C.M. Miller, T.S. Kern, Diabetes-induced nitrative stress in theretina, and correction by aminoguanidine, J. Neurochem. 80 (2002) 771–779.

26] E.A. Ellis, D.L. Guberski, B. Hutson, M.B. Grant, Time course of NADH oxidase,inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in theBBZ/WOR rat, Nitric Oxide 6 (2002) 295–304.

27] L Zheng, Y. Du, C. Miller, R.A. Gubitosi-Klug, S. Ball, B.A. Berkowitz, T.S. Kern,

Critical role of inducible nitric oxide synthase in degeneration of retinal cap-illaries in mice with streptozotocin-induced diabetes, Diabetologia 50 (2007)1987–1996.

28] G. Romeo, W.H. Liu, V. Asnaghi, T.S. Kern, M. Lorenzi, Activation of nuclearfactor-kappaB induced by diabetes and high glucose regulates a proapoptoticprogram in retinal pericytes, Diabetes 51 (2002) 2241–2248.

[

[

eractions 188 (2010) 86–93

29] R.A. Kowluru, P. Koppolu, S. Chakrabarti, S. Chen, Diabetes-induced activationof nuclear transcriptional factor in the retina, and its inhibition by antioxidants,Free Radic. Res. 37 (2003) 1169–1180.

30] H. Zhang, X. Chen, X. Teng, C. Snead, J.D. Catravas, Molecular cloning and anal-ysis of the rat inducible nitric oxide synthase gene promoter in aortic smoothmuscle cells, Biochem. Pharmacol. 55 (1998) 1873–1880.

31] C.J. Lowenstein, E.W. Alley, P. Raval, A.M. Snowman, S.H. Snyder, S.W. Russell,W.J. Murphy, Macrophage nitric oxide synthase gene: two upstream regionsmediate induction by interferon gamma and lipopolysaccharide, Proc. Natl.Acad. Sci. U.S.A. 90 (1993) 9730–9734.

32] Q.W. Xie, Y. Kashiwabara, C. Nathan, Role of transcription factor NF-kappa B/Relin induction of nitric oxide synthase, J. Biol. Chem. 269 (1994) 4705–4708.

33] K.F. Beck, R.B. Sterzel, Cloning and sequencing of the proximal promoter of therat iNOS gene: activation of NFkappaB is not sufficient for transcription of theiNOS gene in rat mesangial cells, FEBS Lett. 394 (1996) 263–267.

34] M.E. de Vera, R.A. Shapiro, A.K. Nussler, J.S. Mudgett, R.L. Simmons, S.M. MorrisJr., T.R. Billiar, D.A. Geller, Transcriptional regulation of human inducible nitricoxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2promoter, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 1054–1059.

35] J. Cai, O. Kehoe, G.M. Smith, P. Hykin, M.E. Boulton, The angiopoietin/Tie-2 sys-tem regulates pericyte survival and recruitment in diabetic retinopathy, Invest.Ophthalmol. Vis. Sci. 49 (2008) 2163–2171.

36] A. Capetandes, M.E. Gerritsen, Simplified methods for consistent and selectiveculture of bovine retinal endothelial cells and pericytes, Invest. Ophthalmol.Vis. Sci. 31 (1990) 1738–1744.

37] T. Kondo, K. Hosoya, S. Hori, M. Tomi, S. Ohtsuki, H. Takanaga, E. Nakashima,H. Iizasa, T. Asashima, M. Ueda, M. Obinata, T. Terasaki, Establishment ofconditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and theirapplication in a co-culture system using retinal capillary endothelial cell line(TR-iBRB2), Cell Struct. Funct. 28 (2003) 145–153.

38] Y.L. Wang, Y.N. Hui, B. Guo, J.X. Ma, Strengthening tight junctions of reti-nal microvascular endothelial cells by pericytes under normoxia and hypoxiainvolving angiopoietin-1 signal way, Eye 21 (2007) 1501–1510.

39] J.M. Cacicedo, S. Benjachareowong, E. Chou, N.B. Ruderman, Y. Ido, Palmitate-induced apoptosis in cultured bovine retinal pericytes: roles of NAD(P)Hoxidase, oxidant stress, and ceramide, Diabetes 54 (2005) 1838–1845.

40] B.A. Berkowitz, R.A. Lukaszew, C.M. Mullins, J.S. Penn, Impaired hyaloidalcirculation function and uncoordinated ocular growth patterns in experimen-tal retinopathy of prematurity, Invest. Ophthalmol. Vis. Sci. 39 (1998) 391–396.

41] O.S. Kim, J. Kim, C.S. Kim, N.H. Kim, J.S. Kim, KIOM-79 prevents methyglyoxal-induced retinal pericyte apoptosis in vitro and in vivo, J. Ethnopharmacol. 129(2010) 285–292.

42] C. Kaltschmidt, B. Kaltschmidt, T. Henkel, H. Stockinger, P.A. Baeuerle, Selec-tive recognition of the activated form of transcription factor NF-kappa B by amonoclonal antibody, Biol. Chem., Hoppe-Seyler 376 (1995) 9–16.

43] K. Koga, S. Yamagishi, T. Okamoto, Y. Inagaki, S. Amano, M. Takeuchi, Z. Makita,Serum levels of glucose-derived advanced glycation end products are asso-ciated with the severity of diabetic retinopathy in type 2 diabetic patientswithout renal dysfunction, Int. J. Clin. Pharmacol. Res. 22 (2002) 13–17.

44] J. Miura, S. Yamagishi, Y. Uchigata, M. Takeuchi, H. Yamamoto, Z. Makita, Y.Iwamoto, Serum levels of non-carboxymethyllysine advanced glycation end-products are correlated to severity of microvascular complications in patientswith Type 1 diabetes, J. Diabetes Complications 17 (2003) 16–21.

45] A.G. Miller, D.G. Smith, M. Bhat, R.H. Nagaraj, Glyoxalase I is critical for humanretinal capillary pericyte survival under hyperglycemic conditions, J. Biol.Chem. 281 (2006) 11864–11871.

46] A. Lapolla, R. Reitano, R. Seraglia, G. Sartore, E. Ragazzi, P. Traldi, Evaluationof advanced glycation end products and carbonyl compounds in patients withdifferent conditions of oxidative stress, Mol. Nutr. Food Res. 49 (2005) 685–690.

47] O. Brouwers, P.M. Niessen, G. Haenen, T. Miyata, M. Brownlee, C.D. Ste-houwer, J.G. De Mey, C.G. Schalkwijk, Hyperglycaemia-induced impairmentof endothelium-dependent vasorelaxation in rat mesenteric arteries is medi-ated by intracellular methylglyoxal levels in a pathway dependent on oxidativestress, Diabetologia 53 (2010) 989–1000.

48] F.W. Chaplen, W.E. Fahl, D.C. Cameron, Evidence of high levels of methylglyoxalin cultured Chinese hamster ovary cells, Proc. Natl. Acad. Sci. U.S.A. 95 (1998)5533–5538.

49] S. Ghosh, M.J. May, E.B. Kopp, NF-kappa B and Rel proteins: evolutionarilyconserved mediators of immune responses, Annu. Rev. Immunol. 16 (1998)225–260.

50] R. Schreck, B. Meier, D.N. Mannel, W. Droge, P.A. Baeuerle, Dithiocarbamatesas potent inhibitors of nuclear factor kappa B activation in intact cells, J. Exp.Med. 175 (1992) 1181–1194.

51] R. Bessho, K. Matsubara, M. Kubota, K. Kuwakado, H. Hirota, Y. Wakazono, Y.W.Lin, A. Okuda, M. Kawai, R. Nishikomori, et al., Pyrrolidine dithiocarbamate,a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation, pre-vents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes,Biochem. Pharmacol. 48 (1994) 1883–1889.

52] C.H Kim, J.H. Kim, C.Y. Hsu, Y.S. Ahn, Zinc is required in pyrrolidine dithiocar-

bamate inhibition of NF-kappaB activation, FEBS Lett. 449 (1999) 28–32.

53] F. Becquet, Y. Courtois, O. Goureau, Nitric oxide in the eye: multifaceted rolesand diverse outcomes, Surv. Ophthalmol. 42 (1997) 71–82.

54] M. Inomata, M. Hayashi, S. Shumiya, S. Kawashima, Y. Ito, Involvement ofinducible nitric oxide synthase in cataract formation in Shumiya cataract rat(SCR), Curr. Eye Res. 23 (2001) 307–311.

ical In

[

[

[

[

J. Kim et al. / Chemico-Biolog

55] Y. Ito, T. Nabekura, M. Takeda, M. Nakao, M. Terao, R. Hori, M. Tomohiro, Nitricoxide participates in cataract development in selenite-treated rats, Curr. EyeRes. 22 (2001) 215–220.

56] G.C. Chiou, Review: effects of nitric oxide on eye diseases and their treatment,J. Ocul. Pharmacol. Ther. 17 (2001) 189–198.

57] D.C. Tsai, S.H. Chiou, F.L. Lee, C.K. Chou, S.J. Chen, C.H. Peng, Y.H. Kuo,C.F. Chen, L.L. Ho, W.M. Hsu, Possible involvement of nitric oxide inthe progression of diabetic retinopathy, Ophthalmologica 217 (2003) 342–346.

[

[

teractions 188 (2010) 86–93 93

58] J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman, Apparenthydroxyl radical production by peroxynitrite: implications for endothelialinjury from nitric oxide and superoxide, Proc. Natl. Acad. Sci. U.S.A. 87 (1990)

1620–1624.

59] H. Ischiropoulos, A.B. Al-Mehdi, Peroxynitrite-mediated oxidative proteinmodifications, FEBS Lett. 364 (1995) 279–282.

60] J. Li, J. Su, W. Li, W. Liu, B.T. Altura, B.M. Altura, Peroxynitrite induces apoptosisin canine cerebral vascular muscle cells: possible relation to neurodegenerativediseases and strokes, Neurosci. Lett. 350 (2003) 173–177.