1162 Diabetes Volume 67, June 2018

Inhibition of Soluble Epoxide 2 Ameliorates Diabetic Keratopathy and Impaired Wound Healing in Mouse Corneas

Haijing Sun,1 Patrick Lee,1 Chenxi Yan,1,2 Nan Gao,1 Jiemei Wang,3 Xianqun Fan,2 and Fu-Shin Yu1

Diabetes 2018;67:1162–1172 | https://doi.org/10.2337/db17-1336

EPHX2 (encoding soluble [sEH]) con- diabetes a leading cause of blindness throughout the world verts biologically active epoxyeicosatrienoic acids (EETs), (1). In addition to the aforementioned complications, var- anti-inflammatory and profibrinolytic effectors, into the ious types of corneal disorders are also relatively common in less biologically active metabolites, dihydroxyeicostrienoic patients with diabetes (2,3). Abnormalities of the cornea, acids. We sought to characterize the expression and the termed diabetic keratopathy (DK), are resistant to conven- function of EPHX2 in diabetic corneas and during wound tional treatment regimens (for a comprehensive review, see healing. The expression of EPHX2 at both mRNA and pro- Ljubimov [3]). Unlike diabetic retinopathy or cataracts, DK tein levels, as well as sEH enzymatic activity, was markedly patients usually do not have detectable symptoms; however, upregulated in the tissues/cells, including corneal epithelial once the cornea is injured, delayed epithelial wound healing cells as well as the retina of human type 2 and mouse type 1 is often observed (4) and may be associated with sight- (streptozotocin [STZ] induced) and/or type 2 diabetes. threatening complications such as stromal opacification, Ephx2 depletion had no detectable effects on STZ-induced surface irregularity, and microbial keratitis (5). Chronic hyperglycemia but prevented the development of tear fl deficiency. Ephx22/2 mice showed an acceleration of low-grade in ammation and persistent oxidative stress are thought to be two major contributing pathogenic factors for

COMPLICATIONS hyperglycemia-delayed epithelium wound healing. More- over, inhibition of sEH increased the rate of epithelium wound the development of diabetic complications (3). However, the closure and restored hyperglycemia-suppressed STAT3 molecules and signaling pathways leading to these patho- activation and heme oxygenase-1 (HO-1) expression in genic events remain incompletely understood. the diabetic corneas. Treatment of diabetic corneas with Corneal epithelium debridement is an ideal model to cobalt protoporphyrin, a well-known HO-1 inducer, re- study re-epithelialization, delayed wound healing, and ul- stored wound-induced HO-1 upregulation and accelerated ceration in the cornea (6). Using this model, we performed delayed wound healing. Finally, Ephx2 depletion enhanced a genome-wide cDNA array analysis and observed that sensory innervation and regeneration in diabetic corneas diabetes caused a general decline, at the transcriptional level, at 1 month after epithelial debridement. Our data suggest in both uninjured and healing corneal epithelial cells (CECs). that increased sEH activity may be a contributing factor In unwounded cells, 31 (loci) were upregulated and for diabetic corneal complications; targeting sEH pharma- 72 genes were downregulated (7). Among these genes, cologically or supplementing EETs may represent a new, EPHX2, encoding soluble epoxide hydrolase (sEH), was adjunctive therapy for treating diabetic keratopathy. unique; its expression was not significantly altered in re- sponse to wounding and yet increased .14.8- and 11.9-fold in diabetic unwounded and healing epithelia when compared With the recent rapid increase in the prevalence of diabetes, with that of normal corneas, respectively. the associated ocular complications such as retinopathy, is a polyunsaturated omega-6 fatty acid cataract, uveitis, and neurophthalmic disorders have made and is the precursor that is metabolized to a wide range of

1Departments of Ophthalmology and Anatomy and Cell Biology, Wayne State Received 3 November 2017 and accepted 17 March 2018. University School of Medicine, Detroit, MI H.S., P.L., and C.Y. contributed equally to this work. 2Department of Ophthalmology, Shanghai Ninth Peoples’ Hospital, Shanghai Jiao © 2018 by the American Diabetes Association. Readers may use this article as Tong University School of Medicine, Shanghai, People’s Republic of China long as the work is properly cited, the use is educational and not for profit, and the 3Department of Pharmaceutical Sciences, Eugene Applebaum College of Phar- work is not altered. More information is available at http://www.diabetesjournals macy and Health Sciences, Wayne State University, Detroit, MI .org/content/license. Corresponding author: Fu-Shin Yu, [email protected]. diabetes.diabetesjournals.org Sun and Associates 1163 biologically and clinically important eicosanoid molecules by blood glucose levels .300 mg/dL within 4 weeks postinjec- 2/2 various , including enzymes of the COX, lipoxygenase, tion and thereafter (24). Ephx2 mice on a B6 background and cytochrome P450 (CYP) monooxygenase pathways (8). were a gift from Joan Graves (National Institutes of Health The CYP enzymes generate four bioactive [NIH]/National Institute of Environmental Health Sciences epoxyeicosatrienoic acids (EETs) by metabolizing arachidonic [NIEHS]) and were induced to develop diabetes in the same acid: 5,6-, 8,9-, 11,12-, and 14,15-EET. EETs contribute to manner as diabetic mice. the regulation of vascular tone, cardiovascular homeostasis, nociception, inflammatory response, angiogenesis, and cell Evaluation of Tear Secretion – proliferation (9–11). All EETs are then further metabolized Tear secretion was determined with phenol red impregnated by sEH (10,12), which is encoded by the EPHX2, and cotton threads (Zone-Quick, Tokyo, Japan). The threads are converted into inactive or less active 1,2-diols, dihydroxy- were placed in the medial canthus for 1 min and the length eicosatrienoic acids (DHETs) (13). A decrease in EET avail- of the wetted part, turning red on soaking tears, was photo- ability, due to an increased degradation by sEH, has been graphed and measured. found to be a deleterious mechanism associated with Corneal Epithelial Debridement Wound various disease states such as cardiac hypertrophy, ath- Diabetic and age-matched normal mice were anesthetized by erosclerosis, , pain, and diabetes (14–17). an intraperitoneal injection of xylazine (7 mg/kg) and ke- Accordingly, inhibition of sEH exerts beneficial actions in tamine (70 mg/kg) plus topical proparacaine, and a 1.5-mm controlling or ameliorating these human diseases and pa- circular wound was first demarcated with a trephine in the thologies (13,18,19). The role of EPHX2 has also been central cornea, followed by the removal of epithelial cells explored in the pathogenesis of diabetic complications, within the circle with a blunt scalpel blade under a dissecting particularly in nephropathy; inhibition of sEH activity by microscope (Zeiss). Two corneas were pooled in one tube, gene deletion and by pharmacological inhibitor of EPHX2 stored at 280°C. The collected cells were marked as un- reduced renal inflammation and injury in diabetic mice in an wounded (0 h). The progress of wound healing was mon- NF-kB–related manner (20). Moreover, the gain-of-function itored by fluorescence staining for epithelial defects and 55Arg polymorphism variant is found to be associated with photographed with a slit lamp microscope. At the end of acute kidney injury after cardiac surgery in patients without healing, the corneas were either snap frozen in optimal preexisting chronic kidney disease (21). Our cDNA array cutting temperature compound for cryostat sectioning or data, showing that hyperglycemia caused marked upregula- markedwiththesamesizetrephineforCECcollection, tion of EPHX2 in both unwounded and healing CECs (7), marked as healing CECs. suggest that EPHX2 may contribute to the pathogenesis of DK (22). Western Blotting fi fi In this study, we rst con rmed the diabetes-associated Western blotting of CECs was performed as follows. Cell expression of EPHX2 in CECs. We assessed its role during lysates with an equal amount of proteins (20 mg) were diabetes-impaired epithelium wound healing and sensory separated with 5–15% gradient SDS-PAGE and transferred nerve innervation and regeneration. Moreover, we evaluated to pore size 0.2 mmol/L nitrocellulose membrane. The the therapeutic potential of the local application of sEH membranes were stained with EPHX2 (ab133173; Abcam), inhibitors and its downstream gene, heme oxygenase-1 HO-1 (ab13243; Abcam), pSTAT3 (T705; Cell Signaling HO-1 ( ), to treat delayed diabetic wound healing. Technology), pSTAT3 (S727; Cell Signaling Technology), STAT3 (Cell Signaling Technology), or nonmuscle b-actin RESEARCH DESIGN AND METHODS (A1978; Sigma-Aldrich), followed by incubation with horse- Ethics Statement radish peroxidase–conjugated donkey secondary antibodies All investigations using animals conformed to the regula- (1:5,000 dilution; Jackson ImmunoResearch Laboratories). tions of the Association for Research in Vision and The bands were visualized with enhanced chemiluminescence Ophthalmology (ARVO) Statement for the Use of Animals in (SuperSignal), and the images were acquired using Kodak Ophthalmic and Vision Research, the National Institutes Image Station 4000R Pro. Band intensity was analyzed using of Health, and the guidelines of the Animal Investigation Carestream Molecular Imaging Software. Actin was used as Committee of Wayne State University (WSU). Human au- loading control. topsy corneas with or without diabetes were obtained from Michigan Eye Bank, without any personal information ex- Immunohistochemistry of Mouse Corneas cept age, sex, and cause of death. Mouse eyes were enucleated, embedded in Tissue-Tek optimal cutting temperature compound, and frozen in liquid nitrogen. Animals and Induction of Diabetes Human 6-mm-thick sections were cut and mounted to poly- Six-week-old C57BL/6 mice, both males and females pur- L-–coated glass slides, fixed in 4% paraformaldehyde, chased from The Jackson Laboratory, were induced to blocked with 10 mmol/L PBS containing 2% BSA for 1 h at develop diabetes with streptozotocin (STZ) as described room temperature, and incubated with rabbit primary EPHX2 previously (23,24). Mice were considered as diabetic with or HO-1 antibodies. This was followed by a secondary 1164 Ephx2 in Diabetic Corneas Diabetes Volume 67, June 2018 antibody, fluorescein isothiocyanate anti-rabbit (1:100; observed that EPHX2 immunoreactivity was primarily found Jackson ImmunoResearch Laboratories). Slides were mounted in the epithelium layer; its staining intensity increased with with Vectashield mounting medium containing DAPI mount- time post-STZ treatment (Fig. 1A). The presence of recombi- ing media. Controls were similarly treated with rat or rabbit nant mouse EPHX2 in the first antibody incubation abol- IgG, as well as using the depletion of primary antibodies with ished immunoreactivity (Fig. 1A,insets,8weeks,).Since mouse recombinant EPHX2 (10:1). The sections were exam- EPHX2 was mostly expressed in the epithelium, we per- ined under a Nikon ECLIPSE 90i microscope. The center of formed Western blotting of CECs isolated at different times unwounded or the leading edge of healing corneas was photo- after STZ injection. Figure 1B revealed that EPHX2 was graphed. expressed in normal CECs; elevated expressions were detected at different time points post-STZ; relative intensities, nor- Whole-Mount Immunostaining and Quantitation of malized to actin, were 24.2 pre-STZ treatment, and increas- Innervation of B6 Mouse Corneas ing to 34.8, 49.1, and 42.8 at 2, 4, and 8 weeks, respectively Whole-mount immunostaining and quantitation of inner- (Fig. 1B). EPHX2 encodes a phosphatase at the N terminus vation of B6 mouse corneas were performed as described in and an epoxide hydrolase at the COOH terminus (9). sEH our previous study (25). In brief, the enucleated eyes were activity was assessed in diabetic CECs (Fig. 1C). Basal activity fixed and the corneas were isolated and further fixed for of sEH was detected in nondiabetic (NL) CECs, and STZ an additional 10 min. The corneas were cut radially into treatment resulted in 2.95-, 4.68-, and 5.21-fold increases at six standardized sections and were incubated at 37°C in 2, 4, and 8 weeks, respectively (Fig. 1C). Figure 1D shows the 20 mmol/L EDTA for 30 min, followed by 2-day incuba- immunostaining of human corneas, one from a 66-year-old tion in 0.025% hyaluronidase and 0.1% EDTA in PBS. male (NL) and the other from a 56-year-old female patient The tissues were blocked at room temperature for 2 h in with severe diabetic retinopathy; stronger EPHX2 staining PBS–Triton X-100 containing 2% BSA, followed by incuba- was seen in diabetic human corneal epithelium compared with tion overnight at 4°C with antibody against b-tubulin III. that of a patient without diabetes. Taken together, these results After secondary antibody, the tissues were mounted and indicate that sEH activity increases in diabetic mouse CECs. examined under a confocal microscope (TCS SP2; Leica, To determine the amounts of EETs and their metab- Heidelberg, Germany). Innervation in a region was calculated olite DHETs, we pooled epithelial cells from eight corneas as the percent area positive for b-tubulin III staining by from the control and STZ diabetic mice, two each, and ImageJ. assessed the levels of EETs. Among four EETs, 8.9-EET sEH Activity Assay and Epoxy/Dihydroxy Fatty Acid was detected with 0.30 and 0.43 ng per sample for NL and Measurement 0.17 and 0.24 ng per sample for diabetic CECs, respec- sEH enzymatic activity was measured using Cayman Chem- tively. 11,12-EET was barely detectable only in normal ical Cell-Based Assay Kit (600090) with 50 mg of cell lysates. CECs (0.01). Tissue DHET levels were below the limit of For EET/DHET measurement, collected CECs from normal quantification. and diabetic mice were processed and epoxy/dihydroxy Ephx22/2 fatty acids were analyzed by the Lipidomics Core Facility Diabetes Induction in Mice Ephx22/2 at WSU using standardized liquid chromatography–mass mice have been used as models for hypertension Ephx2 spectrometry methods as described previously (26). and cardiovascular diseases (8,27,28). We obtained - knockout (Ephx2-KO)miceonB6backgroundfromDr. Statistical Analysis Darryl C. Zeldin (NIH/NIEHS). An early study using in- The statistical analyses were performed using the software traperitoneal injection of 50 mg/kg/day STZ for 3 days GraphPad Prism 6. Data were presented as means 6 SD. reported that Ephx2 deletion prevented the development Experiments with two treatments and/or conditions were of diabetes (29). We used 50 mg/kg/day STZ injected in- 2 2 analyzed for statistical significance using two-tailed unpaired traperitoneally for 5 days. As shown in Fig. 2, Ephx2 / mice Student t test. Experiments with two groups were analyzed had similar average random blood glucose levels and became using one-way ANOVA (Fig. 1C), and more than two groups hyperglycemic in a similar time fashion with the control were analyzed with two-way ANOVA to determine overall B6 mice (Fig. 2A); no differences in their body weight were differences. A Bonferroni posttest was performed to de- observed as well (Fig. 2B). Hence, Ephx2 depletion had termine statistically significant differences. Significance was minimal effects on the development of diabetes induced accepted at P , 0.05. Experiments were repeated at least by STZ. Our results were similar to those reported by twice to ensure reproducibility. Elmarakby et al. (20). Using Western blotting, we showed that diabetes induced EPHX2 expression in wild-type (WT), 2/2 RESULTS but not in Ephx2 ,mice(Fig.2C). Elevated Expression of EPHX2 in CECs of STZ-Treated One of the characteristics of DK in patients is decreased Mice tear secretion. Whereas no difference was observed in NL 2/2 To determine whether EPHX2 was also expressed at the WT and Ephx2 mice, Schirmer test revealed that STZ protein level, we first performed immunohistochemistry and treatment significantly decreased tear secretion in WT (65% diabetes.diabetesjournals.org Sun and Associates 1165

Figure 1—EPHX2 expression was increased in diabetic mouse cornea. Eight-week-old C57BL/6 (WT) mice were intraperitoneally injected with five doses of 50 mg/kg STZ diluted in citrate buffer, injected intraperitoneally (diabetes [DM]). A: Immunohistochemistry showing EPHX2 expression (green) in NL and diabetic mice weeks after STZ injection. The cryostat sections were also counterstained with DAPI showing nuclei (blue). Insets in STZ 8 weeks show primary antibody absorbed with mouse recombinant EPHX2. E, epithelium; S, stroma. B: Western blot analysis of EPHX2 expression in mouse CECs 2, 4, and 8 weeks post–STZ treatment. For each sample, 20 mg total protein from collected CECs was analyzed by WesternblotwithEPHX2antibody.ThenumbersaboveEPHX2bands(63kDa) are the pixels analyzed with ImageJ, and below (bold font) are the intensities normalized with corresponding actin band densities. C: sEH activity was measured by using an sEH Cell-Based Assay Kit. CECs were collected and 50 mg of total protein lysis was loaded for the assay, according to the protocol of the kit. Asterisks on top of columns are P value results compared with the control. *P , 0.05; **P , 0.01 (one-way ANOVA). Data are representative of independent experiments (mean + SD, n = 3). D: Immunohistochemistry of human corneas stained with EPHX2 antibody. NL, a donor cornea from 66-year-old male patient without diabetes; DM, a donor cornea from 53-year-old female patient without severe diabetic retinopathy. The figure, except D, is the representative of three independent experiments.

2/2 of NL mice), but not Ephx2 mice (Fig. 2D and E), before, diabetes significantly delayed wound healing in WT suggesting an overall protective effect of Ephx2 depletion. B6 mouse corneas (45.15 6 6.75% RWA; P , 0.01); this 2/2 delay, however, was markedly attenuated in Ephx2 mice Depletion of Ephx2 Attenuates Pathogenesis of DK (13.5 6 5.95% RWA). Having shown that EPHX1 expression and sEH activities Immunohistochemistry analysis revealed that there was were elevated in diabetic corneas and that diabetes can be little EPHX2 staining in normoglycemic corneas, but intense 2/2 induced in Ephx2 mice, we next investigated the effects staining of EPHX2 in the entire epithelia of both unwounded of Ephx2 depletion on epithelial wound closure in the and wounded diabetic corneas was observed (Fig. 3C). corneas (Fig. 3). Ten weeks after induction of diabetes, 2/2 the Ephx2 diabetic and age-matched WT diabetic mice Ephx2 Deficiency Restores the Wound-Induced STAT3 were wounded with a 1.5-mm diameter epithelium debride- Signaling in the Healing Diabetic Corneas ment procedure. The wounds were allowed to heal for 24 h, Epithelial wounding is known to activate the JAK/STAT3 and the progress of wound healing was monitored by corneal pathway, leading to wound closure in vitro and in vivo (30). fluorescence staining for epithelial defects and photo- EETs have been shown to mediate STAT3 signaling graphed with a slit lamp microscope (Fig. 3A). The remaining in cardiomyocytes (31). Using STAT3 phosphorylation as wound area (RWA) at 24 h postwounding was calculated a marker of activation, we assessed the effects of hypergly- (Fig. 3B). For NL corneas, Ephx2 depletion exhibited no cemia and EPHX2 expression on wound-induced STAT3 detectable effects on the rate of epithelium wound signaling (Fig. 4). Phosphorylation at both T705 and S727 closure, 15.5 6 6.5% vs. 17.25 6 6.25% RWA. As we showed can be detected at basal levels in unwounded (0 h) WT 1166 Ephx2 in Diabetic Corneas Diabetes Volume 67, June 2018

Figure 2—Hyperglycemia, growth, and tear secretion in WT and Ephx22/2 micetreatedwithSTZ.Eight-week-oldWTB6andEphx22/2 mice were intraperitoneally injected with five doses of 70 mg/kg STZ diluted in citrate buffer (diabetes [DM]). Blood glucose (A) and body weight (B)were measured at 11:00 A.M. at 5-day intervals starting from the next day of last STZ injection. C: Western blotting showing EPHX2 expression in cornea epithelial cells extracted from WT and Ephx22/2 mice at 8 weeks post–STZ injection, with b-actin staining as internal control for protein loading. The number on the left side indicates protein molecular weights (the predicted molecular weight: 63 kDa for EPHX2). D: Schirmer test measurements of tear secretion in NL and diabetic mice using phenol red–impregnated cotton threads. Red color is indicative of wet threads. E: Tear secretion was quantitated and represented as the length (mm) of red threads (mean + SD; unpaired Student t test; n =5;*P , 0.05). Three independent experiments were performed, and one representative image for each condition is presented.

2/2 2/2 and Ephx2 mouse corneas with or without diabetes. Ephx2 mice with or without diabetes. Wounding induced 2/2 Wounding-induced phosphorylation (24 h postwounding) HO-1 expression in normal WT and Ephx2 (;2.7-fold) was observed at the T705 site in nondiabetic WT (6.26-fold but not diabetic mice, whereas Ephx2 depletion reversed the over the control, 0-h WT-N, value set as 1 vs. 24-h WT-N) diabetes-induced suppression of HO-1 expression at the 2/2 and Ephx2 (13.1-fold, 24-h KO-N) mice, but not diabetic mRNA (a 2.53-fold increase) (Fig. 5A) and protein level mouse corneas (1.05-fold, 24-h WT-D). Ephx2 depletion (Fig. 5B and C). prevented hyperglycemia-suppressed STAT3 phosphoryla- tion (13.4-fold, 24-h KO-D). Similar patterns were observed sEH Inhibition Accelerates Epithelial Wound Healing and for site S727 as well. Promotes HO-1 Expression in Diabetic Mouse Corneas There were contradicting reports regarding the effects of Ephx2 Deficiency Enhances Hyperglycemia-Suppressed Ephx2 depletion on kidney function (20,27), presumably Expression of HO-1 in Healing Diabetic Corneas because of the phosphatase activity of the EPHX2 gene. Since HO-1 induction was found to be Src/STAT3 dependent To test the effects of sEH inhibition on corneal wound in breast cancer cells (32), we therefore assessed HO-1 healing, we used two structurally different sEH-specific expression in response to wounding and/or hyperglycemia. inhibitors, t-AUCB (33) and GSK2256294A (34) (Fig. 6). Without wounding, HO-1 levels were low in WT and These two inhibitors had no noticeable effects on wound diabetes.diabetesjournals.org Sun and Associates 1167

Figure 3—EPHX2 deficiency changed corneal epithelial wound healing rate and gene expression in CEC. The corneas of WT and Ephx22/2 mice with (diabetes [DM]) or without (NL) STZ treatment were wounded by epithelium debridement (1.5 mm diameter). A: The remaining wounds at 24 h postwounding were visualized by fluorescein staining under slit lamp. B:Analysisoffluorescent-stained areas using Adobe Photoshop software. The denuded area was measured as the numbers of pixels; the results are presented as percentage of healed (pixels of remaining wound/pixels of original wound) and are representative of two independent experiments (n = 5 each). **P , 0.01 (one-way ANOVA with Bonferroni posttest). C: Immunohistochemistry showing EPHX2 expression in NL and diabetic corneas before (0) and 24 h after wounding (24). The cryostat sections were also counterstained with DAPI showing nuclei. Insets in DM (24) panels are the control staining of the cornea with primary antibodies in the presence of 10-fold recombinant mouse EPHX2. The figure is representative of three corneas per condition from two independent experiments. healing in normal corneas (Fig. 6A–D,panels1,2,19,and29). 16.51 6 4.22 RWA) (Fig. 6A–D). Similar to Ephx2 depletion, The delayed wound healing in diabetic corneas was signif- inhibition of sEH also promoted HO-1 expression in diabetic icantly accelerated by t-AUCB (from 36.82 6 13.03% to corneas, whereas it exhibited no effects on normal corneas in 15.57 6 6.78% RWA) and GSK (from 33.27 6 11.46% to response to wounding (Fig. 6E and F).

Figure 4—Impaired STAT3 signaling in WT but not Ephx22/2 diabetic corneas during epithelial wound healing. CECs were harvested from one cornea of WT or Ephx22/2 (KO) mice with (D) or without diabetes (N) during epithelium debridement (0) or 24 h postwounding (24 h) and subjected to Western botting with STAT3 phosphorylation site-specific antibodies (T705 or S727) or pan-STAT3 antibody to normalize the loading. Two independent experiments were performed (n = 3), and one representative image for each condition is presented. The number under each lane is the number of pixels determined using ImageJ and numbers in parentheses are those normalized with total STAT3 reading; the numbers after parentheses are relative increases/decreases, with WT unwounded NL corneas as 1. HPW, hours postwounding. 1168 Ephx2 in Diabetic Corneas Diabetes Volume 67, June 2018

Figure 5—Expression and distribution of HO-1 in unwounded and healing corneas of WT and Ephx22/2 mice with or without diabetes. CECs were harvested as described in Fig. 4 and subjected to real-time PCR (A)andWesternblotting(B)analysesfortheexpressionofHO-1inunwounded(0) and healing (24 h) corneas of WT (W or WT) or Ephx22/2 (K or KO) mice with (D) or without diabetes (N). The results in panel A were first normalized with the levels of b-actin and then compared with the levels of WT NL unwounded (value 1), presented as fold changes. n =5;**P , 0.01 (two-way ANOVA with Bonferroni posttest among the healing CECs). C: Immunohistochemistry showing HO-1 expression and distribution in unwounded (0 h) and healing (24 h) corneas of WT and Ephx22/2 mice with or without diabetes. The cryostat sections were also counterstained with DAPI showing nuclei. The figure is representative of three corneas per condition from two independent experiments. DM, diabetes.

Induction of HO-1 Expression Accelerates Delayed (Fig. 8A, 2 and 4), it increased the density of nerve fibers/ Epithelium Wound Healing endings of diabetic mouse corneas (105% of the control Having shown that inhibition of sEH increased wound- with no statistical significance) (Fig. 8B). induced HO-1 expression in diabetic corneas, we next in- The regeneration of the basal nerve plexus was also vestigated its role in accelerating wound healing in diabetic examined at 1 month postepithelium wounding, at which corneas using cobalt protoporphyrin (CoPP), a well-known time the wound center remained uncovered by the nerve HO-1 inducer (35). As expected, CoPP applied through plexus (Fig. 8A,5–8). Whereas in the WT versus diabetic subconjunctival injection stimulated HO-1 expression at cornea, the region not covered by nerves was larger with the protein levels in healing diabetic corneas, whereas this a reduced nerve density (52.9 6 2.2% vs. 38.9 6 6.9%; P , 2/2 expression was undetectable in the control, vehicle-treated 0.05) (Fig. 8A, compare 5 and 7), in Ephx2 corneas, the A – eyes (Fig. 7 ). These HO-1 expressing epithelia had a sig- densities of sensory nerves in normal and diabetic corneas fi ni cantly higher healing rate compared with vehicle-treated (Fig. 8A, 6 and 8) were similar (51.1 6 2.2% vs. 59.5 6 10.%) 6 6 diabetic corneas (28.78 4.18% vs. 41.4 6.36% RWA) (Fig. 8B). (Fig. 7B and C). DISCUSSION Ephx2 Depletion Enhances Sensory Innervation and Regeneration in Diabetic Corneas In this study, we evaluated EPHX2 expression and role in the We previously showed in rodent models of diabetes that pathogenesis of DK in a B6 mouse model of human diabetes. there were decreases in the density of sensory nerve fibers/ We showed for the first time that EPHX2 expression was endings in the corneas (7,24,25). Using whole-mount elevated in diabetic corneas as well as retinas with or without confocal microscopy, we first examined sensory nerves in wounding. Unlike previous reports showing the failure of 2/2 unwounded corneas. There was a significant decrease in the STZ to induce diabetes in Ephx2 mice, we observed no density of nerves at the center of the cornea of diabetic WT significant differences in the course of STZ-induced diabetes 2/2 mice (Fig. 8A, 1 and 3), 71.9 6 2.9% of the control, non- between WT B6 and Ephx2 mice. However, unlike WT 2/2 diabetic WT mice (set as value 1 or 100%) (Fig. 8B). Whereas mice, Ephx2 mice did not develop diabetes-associated dry Ephx2 depletion had no significant effects on NL corneas eye symptoms. Moreover, Ephx2 depletion increased the rate diabetes.diabetesjournals.org Sun and Associates 1169

Figure 6—Effects of sEH inhibitors on diabetic epithelial wound healing and HO-1 expression. NL and diabetic mice were subconjunctivally injected with 5 mL control (PBS) or EPHX2 inhibitor t-AUCB (10 nmol/L; Cayman) or GSK2256294A (10 nmol/L; MedchemExpress) 4 h before epithelium debridement. Corneas were photographed at 24 h postwounding (A and D), and the wound sizes were fluorescence stained and calculated. Results are presented as the mean of the RWA (B and E). C and F: Real-time PCR analysis of HO-1 expression in CECs. The results were first normalized with the levels of b-actin and then compared with the levels of WT NL unwounded (value 1, C1, and C19), presented as fold changes. n =5;*P , 0.05; **P , 0.01 (two-way ANOVA with Bonferroni posttest among the healing CECs). DM, diabetes.

of epithelial wound closure, ameliorated sensory nerve de- diabetic, compared with normal, mice (20). The elevated generation in unwounded corneas, and enhanced their re- EPHX2 expression in diabetic tissues is consistent with its generation in wounded diabetic corneas while exhibiting no role as a stress response gene (39,40). detectable effects on the NL corneas. Importantly, two EETs have been shown to promote organ and tissue structurally different sEH inhibitors accelerated delayed regeneration, including liver regeneration, kidney and epithelial wound healing in the diabetic corneas to a level lung compensatory growth, corneal neovascularization, similar to that of NL mice. Inhibition of sEH also restored and retinal vascularization (41). Our study showed a trend hyperglycemia-suppressed expression of HO-1, a factor nec- of decreases in the levels of 8,9-EET as well as 11,12-EET essary for proper wound healing in the cornea. Our study in diabetic, compared with normal, CECs. Local administra- revealed that elevated EPHX2 in diabetic corneas is a path- tion of EETs accelerates wound epithelialization and neo- ogenic factor for DK and suggested that supplementing vascularization in a mouse ear wound model (42). Our study, EETs and/or inhibiting sEH may prevent or treat DK. however, suggested that in the normoglycemic mice, low- EPHX2 has been found to be expressed ubiquitously in ering sEH had no effect on epithelial wound closure, many tissues. It has, however, also been detected as a stress suggesting that supplementing extra EETs may not have response gene associated with many human diseases beneficial effects on wound healing in normoglycemic (14–17). In the literature, results of EPHX2 expression in corneas. Given the fact that EETs exhibit potent protective the diabetic mouse kidney were mixed, from a decrease in effects, including anti-inflammation, we speculate that over- the cytosol (36), no significant changes (20), or an increase expression of EPHX2 may contribute to low-grade inflam- (37,38) in whole kidney extracts. We showed that the mation in some tissues, such as the kidney and the cornea, enzymatic activity of sEH was also elevated during the course but not in others, such as the liver; although it has been of STZ-induced diabetes in CECs. This is consistent with the linked to the hepatic inflammatory response in fatty liver report showing a great decrease in the EETs/DHETs in disease (37). 1170 Ephx2 in Diabetic Corneas Diabetes Volume 67, June 2018

Figure 7—Effects of HO-1 induction on diabetic epithelial wound healing. Diabetic mice were subconjunctivally injected with 5 mL PBS (control [Cont]) or HO-1 inducer CoPP (1 mg/mL; Sigma-Aldrich) 4 h before epithelium debridement. A: CECs were collected and subjected to Western blotting with HO-1 antibody, with actin as the loading control. B:At24hpostwounding,theremainingwoundswerefluorescent stained and photographed. C: The wound sizes were calculated and results were presented as the mean of the RWA. **P , 0.01 (unpaired Student t test; n =5).

In addition to being potentially involved in tissue in- clear whether the defects were due to the general health flammation, our study also suggests that the elevated affected by a decrease in EETs, the increase in the ratio of EPHX2 expression may contribute to the delayed epithelium EETs to DHETs in epithelial cells, or the defects in the wound wound closure and sensory nerve regeneration in diabetic response. The fact that sEH inhibitors accelerated epithelial corneas. Most studies of EPHX2 focused on its effects on wound closure in diabetic corneas suggests that the cellular injuries associated with endothelia, including ischemic car- levels of EETs play a beneficial role for wound closure. diomyopathy (43), vascular remodeling (44), and renal injury Although most studies show similar beneficial effects during diabetes (20), and associated with hypertension (28). of Ephx2 deficiency and pharmacological inhibition of sEH, Thecorneaisanavasculartissue,andassuch,theeffectsof Ephx2 depletion has been shown to worsen angiotensin II– diabetes-induced upregulation on impaired wound healing induced cardiac dysfunction since it aggravated myocardial are likely due to the direct effects on CECs. However, it is not fibrosis and increased cardiac inflammation (45). The

Figure 8—Effects of EPHX2 deficiency on sensory nerve innervation and regeneration in B6 mouse corneas. A 1.5-mm epithelium debridement wound was created in the corneas of WT and Ephx2 mice with (DM) or without diabetes (NL). One month postwounding, unwounded (UW) and 30 days postwound (30 DPW) corneas of WT and Ephx22/2 mice were excised, stained with tubulin III for sensory nerves, and visualized with whole-mount confocal microscopy. The center of the cornea was photographed (A) and the innervation was calculated as the percent area positive for b-tubulin III by ImageJ (B). The results are representative of two independent experiments (n = 3 each), and indicated P values were generated using two-way ANOVA with Bonferroni posttest. **P , 0.01. diabetes.diabetesjournals.org Sun and Associates 1171 adverse effects of Ephx2 depletion observed on cardiac Research to Prevent Blindness (to Kresge Eye Institute). The WSU Lipidomics Core fibrosis may be related to the depleted lipid phosphatase Facility is supported by a grant from the NIH National Center for Research Resources and sEH activities (45). In our model, both Ephx2 deletion (S10RR027926). fl and sEH inhibition exhibited similar effects on epithelial Duality of Interest. No potential con icts of interest relevant to this article were reported. wound closure, indicating the importance of sEH activity or Author Contributions. H.S., P.L., and C.Y. performed laboratory testing and the cellular concentrations of EETs in mediating the epithe- edited and checked accuracy of the manuscript. N.G. performed laboratory testing. lial wound response, which was impaired in diabetic tissues. J.W. and X.F. contributed to the discussion and reviewed and revised the manuscript. Our study also revealed a correlation between sEH ac- F.-S.Y. was responsible for study design and recruitment, contributed to sample tivity and the expression of HO-1, a stress-inducible protein collection and data analysis, and reviewed and edited the manuscript. F.-S.Y. is the with a potential anti-inflammatory effect. HO-1 has been guarantor of this work and, as such, had full access to all the data in the study and shown to play an important role in skin injury and wound takes responsibility for the integrity of the data and the accuracy of the data analysis. healing (46). The induction of HO-1, the rate-limiting Prior Presentation. This study was presented at the Annual Meeting of in heme degradation, represents a key event in ARVO, Seattle, WA, 1–5 May 2016. cellular responses to pro-oxidative and proinflammatory References insults(47).Inthecornea,theincreasedexpressionof 1. Threatt J, Williamson JF, Huynh K, Davis RM. 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