Nerve Growth Factor Attenuates Apoptosis and Inflammation in … · length and nerve branch...

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Cornea Nerve Growth Factor Attenuates Apoptosis and Inflammation in the Diabetic Cornea Jin Hyoung Park, 1,2 Soon-Suk Kang, 2 Jae Yong Kim, 2,3 and Hungwon Tchah 2,3 1 Miso Eye Clinic, Seoul, Republic of Korea 2 Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea 3 Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea Correspondence: Hungwon Tchah, Department of Ophthalmology, Uni- versity of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Re- public of Korea; [email protected]. Submitted: April 15, 2016 Accepted: October 25, 2016 Citation: Park JH, Kang S-S, Kim JY, Tchah H. Nerve growth factor attenu- ates apoptosis and inflammation in the diabetic cornea. Invest Ophthal- mol Vis Sci. 2016;57:6767–6775. DOI: 10.1167/iovs.16-19747 PURPOSE. To examine the effects of nerve growth factor (NGF) on apoptosis and inflammation in the diabetic cornea. METHODS. To investigate the effects of NGF on glucose-induced apoptosis, we stained human corneal epithelial cells (HCECs) for annexin-V and propidium iodide (PI), and measured expression of cleaved caspase-3 and the Bcl-2–associated X protein (BAX). Moreover, to examine the effects of NGF on inflammation, we quantified the expression of interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) using multiplex cytokine analysis, and analyzed nuclear factor-jB (NF-jB) activation and NF-j-B inhibitor a (IjBa) degradation using Western blot analysis. To investigate the effects in vivo, we induced diabetes in male Sprague–Dawley rats using streptozotocin. The rats were divided into three groups: control, diabetic control, and diabetic NGF; topical NGF was applied three times daily for 3 weeks. We used the TUNEL assay to detect apoptosis in corneal tissue, and immunohistochemistry to identify cleaved caspase-3 and IL-1b. RESULTS. In HCECs, high glucose concentration (25 mM) led to reactive oxygen species (ROS) generation, apoptosis, and the release of inflammatory cytokines. Nerve growth factor markedly reduced ROS activation, annexin-PI–positive cells, and levels of cleaved caspase-3, BAX, IL-1b, and TNF-a. In the diabetic rat cornea, apoptosis and inflammation were enhanced, as were levels of cleaved caspase-3 and IL-1b. These responses were markedly reduced by NGF. CONCLUSIONS. Apoptosis and inflammation are enhanced in the diabetic cornea; NGF attenuates these responses—both in vivo and in vitro. Therefore, NGF therapy may represent a novel approach for the management of diabetic keratopathy. Keywords: apoptosis, inflammation, diabetes, cornea, nerve growth factor D iabetes is a major endocrine disease worldwide, and its prevalence is increasing. 1 The most important ocular complication associated with diabetes is diabetic retinopathy; nonetheless, diabetic keratopathy is also a common complica- tion. 2 Among the well-recognized corneal changes associated with diabetic keratopathy, 3 epithelial wound healing and nerve function have been most widely studied, mainly because they are associated with severe sight-threatening complications of diabet- ic keratopathy, such as neurotrophic ulcer, stromal opacification, surface irregularities, and microbial keratitis. 4 However, the exact pathophysiology of diabetic keratopathy remains un- known, and it is difficult to explain the various aspects of the disease by focusing solely on wound healing and nerve function. The symptoms of this condition are similar to those of dry eye syndrome, for example, foreign body sensation and changes in vision. 5,6 Diabetic keratopathy is also similar to dry eye syndrome in terms of its clinical manifestations 5,6 : superficial punctate keratitis, 7 recurrent erosions, and persistent epithelial defects. 8 In mild cases of diabetic keratopathy, the cornea may even appear disease-free on slit-lamp biomicroscopy; neverthe- less, patients complain of various symptoms that may be related to apoptosis and mild inflammation. Currently, such patients receive no targeted therapy for these symptoms. Several factors have been proposed to cause diabetic complications across many tissues: upregulation of the polyol pathway, activation of protein kinase C, hexosamine pathway flux, increased levels of advanced glycation end products (AGEs) arising from nonenzymatic reactions, and so on. 9–11 However, the recent literature suggests that all diabetic complications are the result of the following mechanism: Hyperglycemia leads to reactive oxygen species (ROS) produc- tion, and the resulting oxidative stress in turn induces apoptosis. 12 Indeed, several studies have reported that in- creased ROS production is associated with pathologic diabetic consequences in many tissues. 13,14 Our own investigations have confirmed that high levels of glucose promote apoptosis and inflammation in conjunctival epithelial cells. 15 Moreover, the ROS-induced apoptosis observed in high-glucose environments may delay epithelial wound healing in the diabetic cornea. 16,17 With specific regard to the cornea, nerve growth factor (NGF) plays an important role in pathophysiology, and exogenous NGF accelerates epithelial healing—both in rats and in humans. 18 Moreover, NGF levels in tears are elevated in patients with keratoconjunctivitis sicca, 19 and plasma NGF levels are raised in patients with vernal keratoconjunctivitis. 20 Similarly, serum NGF may be associated with altered corneal nerve fiber iovs.arvojournals.org j ISSN: 1552-5783 6767 This work is licensed under a Creative Commons Attribution 4.0 International License. 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Transcript of Nerve Growth Factor Attenuates Apoptosis and Inflammation in … · length and nerve branch...

Cornea

Nerve Growth Factor Attenuates Apoptosis andInflammation in the Diabetic Cornea

Jin Hyoung Park,1,2 Soon-Suk Kang,2 Jae Yong Kim,2,3 and Hungwon Tchah2,3

1Miso Eye Clinic, Seoul, Republic of Korea2Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea3Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea

Correspondence: Hungwon Tchah,Department of Ophthalmology, Uni-versity of Ulsan College of Medicine,Asan Medical Center, 88 Olympic-ro43-gil, Songpa-gu, Seoul 05505, Re-public of Korea;[email protected].

Submitted: April 15, 2016Accepted: October 25, 2016

Citation: Park JH, Kang S-S, Kim JY,Tchah H. Nerve growth factor attenu-ates apoptosis and inflammation inthe diabetic cornea. Invest Ophthal-

mol Vis Sci. 2016;57:6767–6775. DOI:10.1167/iovs.16-19747

PURPOSE. To examine the effects of nerve growth factor (NGF) on apoptosis and inflammationin the diabetic cornea.

METHODS. To investigate the effects of NGF on glucose-induced apoptosis, we stained humancorneal epithelial cells (HCECs) for annexin-V and propidium iodide (PI), and measuredexpression of cleaved caspase-3 and the Bcl-2–associated X protein (BAX). Moreover, toexamine the effects of NGF on inflammation, we quantified the expression of interleukin-1b(IL-1b) and tumor necrosis factor-a (TNF-a) using multiplex cytokine analysis, and analyzednuclear factor-jB (NF-jB) activation and NF-j-B inhibitor a (IjBa) degradation using Westernblot analysis. To investigate the effects in vivo, we induced diabetes in male Sprague–Dawleyrats using streptozotocin. The rats were divided into three groups: control, diabetic control,and diabetic NGF; topical NGF was applied three times daily for 3 weeks. We used the TUNELassay to detect apoptosis in corneal tissue, and immunohistochemistry to identify cleavedcaspase-3 and IL-1b.

RESULTS. In HCECs, high glucose concentration (‡25 mM) led to reactive oxygen species(ROS) generation, apoptosis, and the release of inflammatory cytokines. Nerve growth factormarkedly reduced ROS activation, annexin-PI–positive cells, and levels of cleaved caspase-3,BAX, IL-1b, and TNF-a. In the diabetic rat cornea, apoptosis and inflammation were enhanced,as were levels of cleaved caspase-3 and IL-1b. These responses were markedly reduced byNGF.

CONCLUSIONS. Apoptosis and inflammation are enhanced in the diabetic cornea; NGFattenuates these responses—both in vivo and in vitro. Therefore, NGF therapy may representa novel approach for the management of diabetic keratopathy.

Keywords: apoptosis, inflammation, diabetes, cornea, nerve growth factor

Diabetes is a major endocrine disease worldwide, and itsprevalence is increasing.1 The most important ocular

complication associated with diabetes is diabetic retinopathy;nonetheless, diabetic keratopathy is also a common complica-tion.2 Among the well-recognized corneal changes associatedwith diabetic keratopathy,3 epithelial wound healing and nervefunction have been most widely studied, mainly because they areassociated with severe sight-threatening complications of diabet-ic keratopathy, such as neurotrophic ulcer, stromal opacification,surface irregularities, and microbial keratitis.4 However, theexact pathophysiology of diabetic keratopathy remains un-known, and it is difficult to explain the various aspects of thedisease by focusing solely on wound healing and nerve function.

The symptoms of this condition are similar to those of dryeye syndrome, for example, foreign body sensation and changesin vision.5,6 Diabetic keratopathy is also similar to dry eyesyndrome in terms of its clinical manifestations5,6: superficialpunctate keratitis,7 recurrent erosions, and persistent epithelialdefects.8 In mild cases of diabetic keratopathy, the cornea mayeven appear disease-free on slit-lamp biomicroscopy; neverthe-less, patients complain of various symptoms that may be relatedto apoptosis and mild inflammation. Currently, such patientsreceive no targeted therapy for these symptoms.

Several factors have been proposed to cause diabeticcomplications across many tissues: upregulation of the polyolpathway, activation of protein kinase C, hexosamine pathwayflux, increased levels of advanced glycation end products(AGEs) arising from nonenzymatic reactions, and so on.9–11

However, the recent literature suggests that all diabeticcomplications are the result of the following mechanism:Hyperglycemia leads to reactive oxygen species (ROS) produc-tion, and the resulting oxidative stress in turn inducesapoptosis.12 Indeed, several studies have reported that in-creased ROS production is associated with pathologic diabeticconsequences in many tissues.13,14 Our own investigations haveconfirmed that high levels of glucose promote apoptosis andinflammation in conjunctival epithelial cells.15 Moreover, theROS-induced apoptosis observed in high-glucose environmentsmay delay epithelial wound healing in the diabetic cornea.16,17

With specific regard to the cornea, nerve growth factor(NGF) plays an important role in pathophysiology, andexogenous NGF accelerates epithelial healing—both in rats andin humans.18 Moreover, NGF levels in tears are elevated inpatients with keratoconjunctivitis sicca,19 and plasma NGF levelsare raised in patients with vernal keratoconjunctivitis.20 Similarly,serum NGF may be associated with altered corneal nerve fiber

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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length and nerve branch density in patients with diabeticperipheral neuropathy.21 We recently performed an in vitrostudy in which NGF may have attenuated apoptosis in culturedprimary human conjunctival epithelial cells (HCECs) exposed tohyperosmolar stress.22 These findings suggested that NGF playsan important additional role in several corneal diseases.

However, the effect of NGF on the enhanced apoptosis andinflammation of the diabetic cornea has not yet beenelucidated. Hence, we hypothesized that the high-glucoseenvironment in diabetes enhances apoptosis and inflammationby promoting ROS production in the cornea, and that thiseffect can be reversed by NGF. Therefore, the aim of thecurrent study was to examine the antiapoptotic and anti-inflammatory effects of NGF in the diabetic cornea, usinghuman corneal epithelial cells (HCECs) and the streptozotocin(STZ)-induced diabetic rat cornea.

MATERIALS AND METHODS

Corneal Epithelial Cell Line

Telomerase-immortalized HCECs were kindly donated by JamesV. Jester (Gavin Herbert Eye Institute, University of California,Irvine, CA, USA).23 The cells were cultured in bronchialepithelium growth medium (Lonza, Walkersville, MD, USA)that was supplemented with 5 mg/mL insulin (Clonetics, SanDiego, CA, USA), 0.5 mg/mL hydrocortisone (Clonetics), 6.5ng/mL triiodothyronine (Clonetics), 10 ng/mL transferrin(Clonetics), 10 ng/mL retinoic acid (Clonetics), 0.13 mg/mLbovine pituitary extract (Clonetics), a mixture of 50 mg/mLgentamicin:50 ng/mL amphotericin (Clonetics), 5 ng/mLhuman epidermal growth factor (EGF) (Sigma-Aldrich Corp.,St. Louis, MO, USA), and 0.15 mg/mL bovine serum albumin(BSA; Sigma-Aldrich Corp.) at 378C in an atmosphere of 5%CO2. Cells were subcultured with 0.25% trypsin-EDTA every 3or 4 days and then used in this study.

NGF and NGF mRNA Levels in HCECs

Human CECs were cultured in glucose-added medium (0, 5, 25mM) for 24 hours; total RNA was then isolated using theRNeasy mini kit in accordance with the manufacturer’sprotocol (Qiagen, Inc., Hilden, Germany). First-strand cDNAwas synthesized from 0.5 lg total RNA using murine leukemiavirus reverse transcriptase (Fermentas, Inc., Foster, CA, USA).The primer sequences specific for the NGF transcripts(GenBank accession no. NM_002506) were 50-CAC ACT GAGGTG CA-30 (forward) and 50-TGA TGA CCG CTT GCT CCT GT-30 (reverse); for the tropomyosin receptor kinase A (TrkA)transcripts, the primers were 50- GTC AAG GCA CTG AAG GAGGC-30 (forward) and 50-TGG CAT CAG GTC CAT GGG AT-30

(reverse; GenBank accession no. NM_ 001012331); for the b-actin transcripts, the primers were 50-GCC AAG GTC ATC CATGAC AAC-30 (forward) and 50-GTC CAC CAC CCT GTT GCTGTA-30 (reverse; GenBank accession no. NM_002046.3).

The concentrations of b-NGF in the supernatant weredetermined using a human ELISA kit (Quantikine; R&DSystems, Inc., Minneapolis, MN, USA) in accordance with themanufacturer’s protocol.

Measuring Intracellular ROS in HCECs

The HCECs were seeded onto six-well plates. After cellattachment and growth for 1 day, the cells were treated withthe respective concentrations of glucose (0, 5, or 25 mM—with or without 20 ng/mL human recombinant NGF; catalogno. 256-GF/CF; R&D Systems, Inc.) for 24 hours at 378C. Thecells were then incubated to allow treatment-induced ROS

production. We measured intracellular ROS levels in HCECs byincubating the cells with the redox-sensitive fluorescent dye2 0,7 0-dichlorofluorescein diacetate (DCFH-DA; MolecularProbes, Life Technologies, Grand Island, NY; 10 lM) at 378Cfor 30 minutes in the dark; the cells were then detached fromthe culture wells using 0.25% trypsin-EDTA and washed twiceusing ice-cold phosphate-buffered saline (PBS). Flow cytomet-ric measurements (BD Biosciences, Inc., San Diego, CA, USA)were performed six times for each treatment. The meanfluorescence intensity was quantified using CELLQuest soft-ware (BD Biosciences, Inc.). Cells that had been incubatedwithout DCFH-DA were used as the negative control. Thedistribution of fluorescent DCF on the cell monolayer wasvisualized and photographed under a fluorescence microscope(Olympus, Tokyo, Japan).

Assessing Apoptosis in HCECs

The HCECs were cultured in media with the respective glucoseconcentrations, with or without human NGF, for 24 hours.Cells were then harvested and washed using ice-cold PBS. Cellpellets were collected by centrifugation, resuspended inbinding buffer including 0.01 M HEPES/NaOH (pH 7.4), 0.14M NaCl, 2.5 mM CaCl2, and incubated with fluoresceinisothiocyanate (FITC)-labeled annexin-V (BD Biosciences,Inc.) and propidium iodide (PI; BD Biosciences, Inc.) at roomtemperature in the dark for 15 minutes. Cells were thenanalyzed using fluorescence-activated cell sorting Calibur flowcytometry (BD Biosciences, Inc.).

Western Blot Analysis in HCECs

The HCECs were cultured in glucose media with or withoutNGF for 24 hours; they were then collected and lysed using alysis solution (10 mM Tris, 10 mM NaCl, 2 mM EDTA, 25 mMNaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride(PMSF), proteinase and phosphatase inhibitor cocktail, 0.5%Triton X-100, pH 7) to detect the levels of cleaved caspase-3,Bcl-2–associated X protein (BAX), NF-j-B inhibitor a (IjBa) andnuclear factor-jB (NF-jB)-p65. In each case, the total proteinconcentration in the supernatant was determined using theBradford method. Aliquots of protein (30 lg) were boiled inequal volumes of Laemmli sample and were loaded into a 12%or 15% acrylamide gel and SDS–PAGE and were electrophoret-ically transferred to nitrocellulose filters (Amersham, LittleChalfont, UK). The blots were treated with antibodies againstcleaved caspase-3 (1:1,000; catalog no. 9661; Cell SignalingTechnology, Danvers, MA, USA), BAX (1:1,000; catalog no.2772; Cell Signaling Technology), IjBa (1:1,000; catalog no.9247; Cell Signaling Technology), NF-jB-p65 (1:1,000; catalogno. sc-109; Santa Cruz Biotechnology, Santa Cruz, CA, USA),and with an antibody for b-actin (1:10,000; Sigma-AldrichCorp.) used as a loading control for overnight at 48C. Afterthree 10-minute washes using Tris-buffered saline (TBS)containing 0.1% Tween-20, the membranes were incubatedwith horseradish peroxidase–conjugated anti-IgGs (1:10,000)(Thermo Fisher Scientific, Inc., Waltham, MA, USA). Therespective target proteins of the antibodies were visualizedusing enhanced chemiluminescence reagents (Santa CruzBiotechnology).

Multiplex Cytokine Analysis in HCECs

Concentrations of the proinflammatory cytokines interleukin-1b(IL-1b) and tumor necrosis factor-a (TNF-a) in the supernatantsof HCECs cultured for 24 hours in normal or high-glucosemedium, with or without NGF, were determined usinginflammatory cytokine human magnetic 5-plex panel (catalog

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no. LHC0003M; Thermo Fisher Scientific, Inc.) that usesmultiplex bead technology. The human inflammatory magnetic5-plex panel contains all the reagents that are intended for usewith the Luminex 200 dual laser detection system withxPONENT software (Luminex, Inc., Austin, TX, USA).

Animal Model

Six-week-old male Sprague-Dawley rats with an initial weight of150 to 160 g were acclimated for 1 week. The rats wererandomly divided into three groups: diabetic (n ¼ 6), diabeticNGF (n¼ 6), and age-matched normal control (n¼ 6) groups.Diabetes was induced by intraperitoneal injection of 65 mg/kgSTZ in ice-cold 0.5 M citrate buffer (pH 4.5). The control groupreceived the citrate buffer only. After 24 hours, the bloodglucose levels were determined in the rats that had receivedthe injection. A glucose concentration > 300 mg/dL inheparinized tail vein blood (measured using a glucometer)was considered as indicating successful induction. All exper-iments conformed to the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research and werereviewed and approved by the Institutional Animal Care andUse Committee of the Asan Institute for Life Sciences at theAsan Medical Center. The committee abides by the Institute ofLaboratory Animal Resources guide.

All animals received their respective eye drops three times aday during the experimental period, beginning 1 day after theintraperitoneal injection and ending 21 days after the injection.The control and diabetic control groups received one drop (40lL) of PBS three times a day. The diabetic NGF group receivedone drop of rat NGF-b (200 lg/mL; catalog no. N2513; Sigma-Aldrich Corp.) three times a day.

Histopathology and Immunohistochemistry in theRat Cornea

In all three groups, 3 weeks after intraperitoneal injection, theeyes were enucleated; all the corneas were buttoned and fixed

in 10% phosphate-buffered formalin for 24 hours before beingembedded in paraffin wax. The eyes were then cut into 4-lm-thick sections and stained using hematoxylin and eosin.Cleaved caspase-3 and IL-1b expression was analyzed byimmunohistochemistry (IHC; Envision-HRP detection system;Dako, Carpinteria, CA, USA). After deparaffinization andantigen retrieval using 0.01 M sodium citrate buffer (pH 6.0)in a pressure cooker at full power for 10 minutes, tissuesections were treated with 3% H2O2 for 5 minutes. Theanticleaved caspase-3 (1:50; Cell Signaling Technology) anti-body and anti-IL-1b antibody (1:50; Santa Cruz Biotechnology)were added to the slides, which were then incubated at 48Covernight. The slides were then incubated with the Envisionreagent for 30 minutes and with 3,30-diaminobenzidine (DAB)chromogen for 10 minutes; they were counterstained withMeyer’s hematoxylin and mounted. Careful rinses using severalchanges of TBS-0.1% Tween buffer were performed at eachstep. As a negative control, rat IgG1 isotype control was usedto exclude the primary antibody. The slides were evaluated andphotographed using a Leica Microphoto FXA microscope(Leica, Wetzlar, Germany).

Apoptosis Assay in the Rat Cornea

Apoptosis was detected under different conditions using theTdT-mediated dUTP nick-end labeling (TUNEL) technique (CellDeath Detection Kit; Roche Diagnostics, Seoul, South Korea),which labels the cut ends of DNA fragments in the nuclei ofapoptotic cells. The nuclei of all cells were stained byincubation with 40-6-diamidino-2-phenylindole (DAPI; VectorLaboratories, Burlingame, CA, USA).

Statistical Analysis

All quantitative experiments were performed at least six times,and data are shown as the mean 6 standard deviation.Statistical significance was determined using Student’s t-test,and P values < 0.05 were considered statistically significant.

FIGURE 1. Reduction in transcribed nerve growth factor (NGF) mRNA, secreted NGF, and transcribed tropomyosin receptor kinase A (TrkA) mRNAin human corneal epithelial cells cultured with high concentrations of glucose. (A) Expression level of NGF mRNA determined usingsemiquantitative RT-PCR, with b-actin as a control. Data for one of six experiments and quantitative mean densitometry results are shown. (B)Production of NGF protein, determined using ELISA, in HCECs cultured for 24 hours in normal medium (untreated control), or in mediumcontaining normal glucose (NG; 5 mM), high glucose (HG; 25 mM), or high glucose with NGF (20 ng/mL). (C) Expression level of TrkA mRNAdetermined using semiquantitative RT-PCR, with b-actin as a control. The data of one representative experiment and quantitative densitometry areshown. *P < 0.05 compared with normal glucose.

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RESULTS

Human NGF mRNA in HCECs Exposed to High

Glucose Levels

Levels of NGF transcripts and secreted NGF were deter-

mined in HCECs exposed to normal-glucose (5 mM) or high-

glucose medium (25 mM) for 24 hours. Levels both of NGFmRNA and of NGF secretion were significantly lower afterexposure to high glucose levels (P < 0.05; Figs. 1A, 1B).Tropomyosin receptor kinase A expression was checked byRT-PCR, which mediates NGF activity as receptor, and themRNA was significantly lower in high glucose (P < 0.05; Fig.1C).

FIGURE 2. Fluorescence of 20,70-dichlorofluorescein diacetate (DCFH-DA) oxidized by intracellular ROS was detected by flow cytometry andquantitatively measured. The mean fluorescence intensity of the flow cytometry was quantified as mean 6 SD. *P < 0.05 compared with normalglucose. **P < 0.05 compared with high glucose. High glucose generates dichlorofluorescein (DCF)-sensitive reactive oxygen species (ROS) inhuman corneal epithelial cells (HCECs), and nerve growth factor (NGF) reduces high glucose–induced ROS. HCECs were cultured for 24 hours inmedium containing normal glucose (NG; 5 mM), high glucose (HG; 25 mM), or high glucose with NGF (20 ng/mL); they were then incubated withcell-permeable DCFH-DA (10 lM) for 45 minutes. Cells were detached using trypsin digestion and washed with PBS. (A) Distribution of fluorescentDCF on the cell monolayer, photographed under a fluorescence microscope. Representative data of one of six experiments are shown. (B)Quantitative flow cytometric measurements.

FIGURE 3. Hematoxylin and eosin staining of the corneas of the three experimental groups, control (Con), diabetic (DM), and diabetic NGF(DMþNGF), and quantitative analysis of total corneal thickness in the three groups. (A) Histopathologic analysis in the cornea of diabetic ratshowing perinuclear clear areas and thickened basement membranes in the corneal epithelium, as well as stromal edema. These changes in thediabetic cornea were partially reversed by NGF treatment. Representative data of one of six experiments are shown. Magnification: 3100. (B) Meandifferences in total corneal thickness in the control, diabetic, and diabetic with NGF treatment groups.

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ROS in HCECs Exposed to High Glucose Levels

When HCECs were exposed to high glucose levels (25 mM),more cells had high-intensity fluorescence than when the cellswere cultured in normal glucose (5 mM; Figs. 2A, 2B).Furthermore, fluorescence intensity was greater in cellsexposed to high glucose levels than in those incubated innormal glucose, a conclusion that could be confirmed usingfluorescent microscopy (Figs. 2A, 2B). The increased fluores-cence intensity by high glucose levels was attenuated by NGFtreatment (Figs. 2A, 2B).

Histopathologic Analysis in the Rat Cornea

In the corneal epithelium of the STZ-induced diabetic rat,perinuclear clear areas and thickened basement membraneswere observed (Fig. 3A). Furthermore, the corneal stroma wasthicker due to edema (Fig. 3A). The total thickness of the

diabetic cornea was significantly higher than that of the normalcornea (Fig. 3B). These changes in the diabetic cornea wereconsistently reversed by NGF treatment (Figs. 3A, 3B).

Apoptosis in HCECs and Rat Cornea

The percentage of FITC-positive cells, which was 12.1% innormal-glucose medium, increased to 25.2% in high-glucosemedium (Fig. 4A). In high-glucose medium with NGFtreatment, the percentage of FITC-positive cells was 12.4%,implying that the apoptosis induced by high glucose had beenpartially suppressed by treatment with NGF (Fig. 4A). Thepercentage of PI-positive cells, which was 4.7% in normal-glucose medium, increased to 14.2% in high-glucose medium.The necrosis induced by high glucose had been partiallysuppressed (7.9%) by NGF treatment (Fig. 4A). Western blotanalysis revealed that the levels of cleaved caspase-3 and BAXwere significantly increased in a high-glucose environment

FIGURE 4. Fluorescence-activated cell sorting Calibur flow cytometry and Western blot analysis for cleaved caspase-3 and Bcl-2–associated Xprotein (BAX) in human corneal epithelial cells (HCECs) treated with the indicated concentrations of glucose. Nerve growth factor (NGF) preventshigh glucose–induced apoptosis in HCECs. Cells were treated for 24 hours with normal glucose (NG; 5 mM), high glucose (HG; 25 mM), or highglucose with NGF (20 ng/mL). (A) Cellular apoptosis was determined using flow cytometry. The percentage of the total cells in the quadrantscorresponded to the number of early apoptotic cells (annexin-V–positive cells; Q4-1, lower right), late apoptotic cells (annexin-V–positive andpropidium iodide (PI)-positive cells; Q2-1, upper right), and necrotic cells (annexin-V–negative and PI-positive cells; Q1-1, upper left). Fluoresceinisothiocyanate–positive rates were calculated as the sum of the values in quadrants Q2-1 and Q4-1. The PI-positive rates were calculated as the sumof the values in quadrants Q1-1 and Q2-1. (B) Expression of cleaved caspase-3 and BAX was analyzed and quantified by Western blotting aftertreatment for 24 hours with normal glucose (NG; 5 mM), high glucose (HG; 25 mM), or high glucose with NGF; b-actin was used as a control.Representative data of one of six experiments and quantitative mean densitometry results are shown. *P < 0.05 compared with normal glucose. **P< 0.05 compared with high glucose.

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(Fig. 4B). However, NGF markedly suppressed the highglucose–induced increase in levels of cleaved caspase-3 andBAX (Fig. 4B).

In the corneas of the control group, few TUNEL-positivenuclei were detected (Fig. 5). In the diabetic corneas, manyTUNEL-positive cells, as well as some fragmented nuclei, wereobserved across the entire cornea; TUNEL-positive cells wereseen in many corneal epithelial cells in particular (Fig. 5).Conversely, barely any TUNEL-positive nuclei were observed inthe diabetic NGF group (Fig. 5). That is, the NGF treatmentmarkedly reversed the apoptotic response in the diabeticcornea.

Immunohistochemically, the epithelium, stroma, and endo-thelium of the diabetic cornea showed intense staining forcleaved caspase-3 (Figs. 6A, 6C). However, no immunohisto-chemical staining for cleaved caspase-3 was observed in thediabetic cornea with NGF treatment (Figs. 6B, 6D).

Inflammation in HCECs and Rat Cornea

Levels of NF-jB-p65 were much higher in HCECs that had beenincubated in high glucose concentrations; however, NGFconsiderably reduced expression levels of NF-jB-p65 in HCECs(Fig. 7A). In high-glucose medium–incubated cells, IjBa levelswere markedly reduced, while NGF treatment prevented thedegradation of IjBa (Fig. 7A). The promoter regions of IL-1band TNF-a both contain a binding site or sites for NF-jB;therefore, we next determined whether NGF could reduce thelevels of secreted IL-1b and TNF-a in the supernatants ofHCECs cultured in either normal or high glucose. The HCECsexposed to high glucose levels secreted significantly more TNF-a and IL-1b than those incubated in normal glucose levels (Fig.7B); NGF treatment significantly reduced the levels of secretedTNF-a and IL-1b in high-glucose medium (Fig. 7B).

Immunohistochemically, the epithelium, stroma, and endo-thelium of the diabetic cornea showed intense staining for IL-1b (Fig. 8B). However, weak immunohistochemical staining forIL-1b was observed in the diabetic cornea after NGF treatment(Fig. 8C).

DISCUSSION

Previous studies have confirmed that dry eye symptoms indiabetes are correlated with the severity of the diabeticretinopathy.5,24 In relation to this, several investigators have

suggested that dry eye symptoms in diabetes are associatedwith peripheral polyneuropathy caused by reduced cornealsensitivity,6,25 altered homeostasis of the cornea,26 or ocularsurface dysfunction caused by tear film instability and reducedtear secretion.27 Nevertheless, the exact pathophysiologicalchanges that occur in the diabetic cornea remain controversial.

Recently, several studies have suggested that the initial stepin the immunopathogenic mechanism responsible for dry eyesyndrome is desiccating stress on ocular surface epithelialcells.28,29 The glucose concentration in tears is tightly linked toblood glucose,30 as in dry eye syndrome. Therefore, wehypothesized that high glucose–induced biochemical andultrastructural changes in the cornea, especially in the cornealepithelium, lead to desiccating stress, and that such changesare the most important factor leading to dry eye in diabeticpatients whose corneas look disease-free—more importantthan structural changes in the nerves or basement membrane.

It has been suggested that NGF plays a role in ocular surfacehealing and sensitivity in corneal and conjunctival cells.18,31

Moreover, Kang et al.22 showed that NGF has an antiapoptoticeffect in hyperosmotic stress–induced apoptosis of conjuncti-val epithelial cells. Therefore, to determine the suitability ofNGF as a target-specific growth factor, we assessed NGF mRNAand NGF production in HCECs cultured in high glucose. Next,to assess whether high glucose levels enhance ROS, apoptosis,and inflammation in corneal epithelial cells, as well as whetherNGF can reverse these pathologic changes, we evaluated ROS,apoptosis, and inflammation in HCECs cultured in highglucose, with or without NGF. With a view to obtaining invivo evidence, we then investigated histopathologic, apoptot-ic, and inflammatory changes in the corneas of diabetic rats,with or without NGF treatment.

The results show that NGF mRNA and NGF production aresignificantly reduced in HCECs cultured in high glucose, andthat high glucose levels affect mitochondria-dependent caspaseactivity, enhancing ROS production and increasing the numberof apoptotic cells. In addition, high glucose levels significantlyenhance expression of NF-jB-p65, as well as of the proin-flammatory cytokines IL-1b and TNF-a in HCECs. This suggeststhat ROS is associated with NF-jB activation, and that it mayenhance the inflammatory response in HCECs, as well as in thetissues or cells of diabetic patients. That is, we havedemonstrated that high glucose levels enhance ROS, apoptosis,and inflammation in HCECs. Furthermore, we have shown—byimaging mitochondria-dependent caspase activity—enhancedapoptosis in the corneas of STZ-induced diabetic rats. Notably,

FIGURE 5. TdT-mediated dUTP nick-end labeling (TUNEL) assay of the three experimental groups: control (Con), diabetic (DM), and diabetic NGF(DMþNGF). Many apoptotic cells (green, i.e., TUNEL-positive, nuclei) were detected in the corneal epithelium, stroma, and endothelium of thediabetic rat, whereas only a few TUNEL-positive apoptotic cells were seen in the corneal epithelial layer of the diabetic rat after NGF treatment.Representative data of one of six experiments are shown. Magnification: 3100.

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although apoptotic cells were detected across the entirecornea, the strongest apoptotic responses were detected inbasal corneal epithelial cells, perhaps because apoptosis is anintracellular reaction, and the basal corneal epithelial celllayers are the greatest in number and have the strongestcellular activity. Our results also show that enhancedinflammation in the cornea of STZ-induced diabetic rats isassociated with IL-1b. Unlike apoptosis, immunoreactivity forIL-1b was detected evenly across the entire cornea, suggestingthat secreted inflammatory cytokines from damaged cells aredistributed across the cornea. Histopathology demonstratedcorneal stromal edema and active apoptotic changes in cornealepithelium. That is, enhanced apoptosis and inflammation inthe diabetic cornea can be demonstrated both histopatholog-ically and immunohistochemically.

Nevertheless, the most important finding in our currentstudy is that NGF can reverse all these phenomena—both invitro and in vivo. Taken together, our results indicate thatdiabetic hyperglycemia enhances apoptosis and inflammationin the cornea—especially in the corneal epithelium—and thatthis occurs without any severe neurotrophic ulcerativechanges. This may explain the unusually severe symptoms ofdry eye that occur in patients with diabetes whose corneasappear disease-free. In addition, our results confirmed thatNGF can successfully suppress this enhanced apoptosis andinflammation in the diabetic cornea.

There is evidence that high glucose can enhance apoptosis,ROS production, and inflammation. And a number of studieshave been done in both humans and animal models toinvestigate the ability of topical NGF to accelerate the healing

FIGURE 6. Immunofluorescence staining for cleaved caspase-3 in the cornea of the experimental groups: the diabetic (DM) and diabetic NGF(DMþNGF) groups. (A) Representative photomicrographs of the corneas of diabetic rats (magnification: 3100). (B) Representativephotomicrographs of the corneas of diabetic rats after nerve growth factor (NGF) treatment (magnification: 3100). (C) Representativephotomicrographs of the corneal epithelium, Bowman’s layer, and anterior stroma in diabetic rats (magnification: 3400). (D) Representativephotomicrographs of the corneal epithelium, Bowman’s layer, and anterior stroma of diabetic rats after NGF treatment (magnification: 3400).Diabetic corneal epithelial, stromal, and endothelial cells showed immunoreactivity for cleaved caspase-3. Diabetic corneal epithelial cells showedespecially strong immunoreactivity. Only weak or no immunoreactivity for cleaved caspase-3 was seen in the diabetic cornea after NGF treatment.

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process in cornea ulcers. However, to our knowledge, thecurrent study is the first to identify that NGF can act as anantiapoptotic and anti-inflammatory agent in the diabeticcornea, which has been demonstrated by both in vitro and invivo experiments.

In summary, high glucose in diabetes induces apoptosis andinflammation in the cornea by promoting ROS formation; thesechanges are most apparent in the corneal epithelial cells.Furthermore, the biochemical changes associated with apo-ptosis and inflammation occur without severe structural

change, and this may explain the dry eye symptoms seen in

diabetic patients. We have provided evidence that these

pathologic changes can be reversed by NGF treatment. Hence,

NGF could be used to treat mild to severe diabetic keratopathy,

and targeted prevention of apoptosis may represent a novel

therapeutic approach to managing the diabetic cornea. The

main limitation of our current study was its in vitro and animal

model design; in future, nonrandomized and randomized

clinical trials will be needed to confirm our findings.

FIGURE 7. Western blot analysis for nuclear factor (NF)-jB-p65 and IjBa, and multiplex cytokine analysis for interleukin (IL)-1b and tumor necrosisfactor (TNF)-a, in human corneal endothelial cells (HCECs) treated with the indicated concentrations of glucose. Nerve growth factor (NGF) inhibitshigh glucose–activated NF-jB and inflammatory cytokines in HCECs. (A) The expression of NF-jB-p65 (nuclear fraction) and IjBa was analyzed andquantified by Western blotting, using b-actin as a control, after treatment for 24 hours with normal glucose (NG; 5 mM), high glucose (HG; 25 mM),or high glucose with NGF (20 ng/mL). Representative data of one of six experiments and quantitative mean densitometry results are shown. (B)After identical treatment, IL-1b and TNF-a expression were quantified using multiplex cytokine analysis. The data are presented as the mean 6 SD ofat least six independent experiments. *P < 0.05 compared with normal glucose. **P < 0.05 compared with high glucose.

FIGURE 8. Immunofluorescence staining for interleukin (IL)-1b in the cornea of the three experimental groups: control (Con), diabetic (DM), anddiabetic NGF (DMþNGF). (A) Representative photomicrographs of the corneas of control rats (magnification: 3100). (B) Representativephotomicrographs of the corneas of diabetic rats (magnification: 3100). (C) Representative photomicrographs of the corneas of diabetic rats afterNGF treatment (magnification: 3100). Diabetic corneal epithelial, stromal, and endothelial cells showed strong immunoreactivity for IL-1b. Onlyweak or no immunoreactivity for IL-1b was seen in the diabetic cornea after NGF treatment.

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Acknowledgments

The authors thank Eun Young Lee for figure file preparation.

Supported by a grant (NRF-2016R1A2B1011341) from the NationalResearch Foundation of Korea, Seoul, Republic of Korea.

Disclosure: J.H. Park, None; S.-S. Kang, None; J.Y. Kim, None;H. Tchah, None

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