Page 1 of 34 Diabetes

Sun et al 1

Inhibition of Soluble Epoxide 2 Ameliorates Diabetic Keratopathy and Impaired

Wound Healing in Mouse Corneas

Haijing Sun1*, Patrick Lee1*, Chenxi Yan1,2*, Nan Gao, Jiemei Wang3, Xianqun Fan2, and Fu

Shin Yu1

Departments of Ophthalmology and Anatomy/Cell Biology, Wayne State University School of Medicine, 4717 St. Antoine Blvd., Detroit, MI, 48201, USA; Department of Ophthalmology, Shanghai Ninth Peoples’ Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Rd, Shanghai 200011, People’s Republic of China. 3Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA

*These authors contributed equally to this work.

Running title: Ephx2 in diabetic corneas

Key words: diabetic neurotrophic keratopathy, soluble 2, wound healing,

epoxyeicosatrienoic acids

Number of words in text: 3922

Number of figures: 8

Number of Tables: 0

Correspondence to: FuShin X. Yu, Ph.D., Kresge Eye Institute, Wayne State University

School of Medicine, 4717 St. Antoine Blvd, Detroit, MI, 48201

Tel: (313) 5771657; Email: [email protected]

Diabetes Publish Ahead of Print, published online April 3, 2018 Diabetes Page 2 of 34

Sun et al 2

ABSTRACT

EPHX2 (soluble , sEH) converts biologically active epoxyeicosatrienoic

acids (EETs), antiinflammatory and profibrinolytic effectors into the less biologically active

metabolites, dihydroxyeicostrienoic acids. We sought to characterize the expression and the

function of EPHX2 in diabetic corneas and during wound healing. The expression of EPHX2 at

both mRNA and protein levels, as well as sEH enzymatic activity, were markedly upregulated in

the tissues/cells, including corneal epithelial cells as well as the retina of human type2, mouse

type1 (STZinduced) and/or type2 diabetes. Ephx2depletion had no detectable effects on

STZinduced hyperglycemia but prevented the development of tear deficiency. Ephx2-/- mice

showed an acceleration of hyperglycemiadelayed epitheliumwound healing. Moreover,

inhibition of sEH increased the rate of epitheliumwound closure and restored hyperglycemia

suppressed STAT3 activation and heme oxygenase-1 (HO1) expression in the diabetic

corneas. Treatment of diabetic corneas with Cobalt protoporphyrin, a wellknown HO1 inducer,

restored woundinduced HO1 upregulation and accelerated delayed wound healing. Finally,

Ephx2depletion enhanced sensory innervation and regeneration in diabetic corneas at 1

month after epithelial debridement. Our data suggests that increased sEH activity may be a

contributing factor for diabetic corneal complications; targeting sEH pharmacologically or

supplementing EETs may represent a new, adjunctive therapy for treating diabetic keratopathy.

Key words: diabetic keratopathy, Soluble Epoxide Hydrolase 2, corneal epithelial wound

healing, Heme Oxygenase 1. Page 3 of 34 Diabetes

Sun et al 3

Introduction

With the recent rapid increase in the prevalence of diabetes mellitus (DM), the

associated ocular complications such as retinopathy, cataract, uveitis, and neurophthalmic

disorders have made diabetes a leading cause of blindness throughout the world (1). In addition

to the aforementioned complications, various types of corneal disorders are also relatively

common in DM patients (2; 3). Abnormalities of the cornea, termed diabetic keratopathy (DK),

are resistant to conventional treatment regimens [for a comprehensive review, please see (3)].

Unlike diabetic retinopathy or cataracts, DK patients usually do not have detectable symptoms;

however, once the cornea is injured, delayed epithelial wound healing is often observed (4) and

may be associated with sightthreatening complications such as stromal opacification, surface

irregularity, and microbial keratitis (5). Chronic lowgrade inflammation and persistent oxidative

stress are thought to be two major contributing pathogenic factors for the development of

diabetic complications (3). However, the molecules and signaling pathways leading to these

pathogenic events remain incompletely understood.

Corneal epitheliumdebridement is an ideal model to study reepithelialization, delayed

wound healing, and ulceration in the cornea (6). Using this model, we performed a genome

wide cDNA array analysis and observed that diabetes caused a general decline, at the

transcriptional level, in both uninjured and healing corneal epithelial cells (CECs). In unwounded

cells, 31 (loci) were upregulated and 72 genes were downregulated (7). Among these

genes, EPHX2, encoding soluble epoxide hydrolase (sEH), was unique; its expression was not

significantly altered in response to wounding and yet increased over 14.8 and 11.9 fold in

diabetic unwounded and healing epithelia when compared to that of normal corneas,

respectively. Diabetes Page 4 of 34

Sun et al 4

Arachidonic acid is a polyunsaturated omega6 fatty acid and is the precursor that is metabolized to a wide range of biologically and clinically important eicosanoid molecules by various , including enzymes of the COX, lipoxygenase and cytochrome P450 (CYP) monooxygenase pathways (8). The CYP enzymes generate four bioactive regioisomeric 1 acids (EETs) by metabolizing : EETs: 5,6, 8,9, 11,12 and

14,15EET. EETs contribute to the regulation of vascular tone, cardiovascular homeostasis, nociception, inflammatory response, angiogenesis, and cell proliferation (911). All EETs are then further metabolized by sEH, (10; 12) which is encoded by the EPHX2 and are converted into inactive or less active 1,2diols, dihydroxyeicosatrienoic acids (DHETs) (13). A decrease in EET availability, due to an increased degradation by sEH, has been found to be a deleterious mechanism associated with various disease states such as cardiac hypertrophy, atherosclerosis, , pain and diabetes (1417). Accordingly, inhibition of sEH exerts beneficial actions in controlling or ameliorating these human diseases and pathologies (13; 18;

19). The role of EPHX2 has also been explored in pathogenesis of diabetic complications, particularly in nephropathy: inhibition of sEH activity by gene deletion and by pharmacological inhibitor of EPHX2 reduced renal inflammation and injury in diabetic mice in NFκB related manner (20). Moreover, the gainoffunction 55Arg polymorphism variant is found to be associated with acute kidney injury following cardiac surgery in patients without preexisting chronic kidney disease (21). Our cDNA array data, showing that hyperglycemia caused marked upregulation of EPHX2 in both unwounded and healing CECs (7), , suggests that EPHX2 may contribute to the pathogenesis of DK (22).

In this study, we first confirmed the diabetesassociated expression of EPXH2 in CECs.

We assessed its role during diabetesimpaired epitheliumwound healing and sensory nerve innervation and regeneration. Moreover, we evaluated the therapeutic potential of the local Page 5 of 34 Diabetes

Sun et al 5

application of sEH inhibitors and its downstream gene, heme oxygenase1 (HO-1), to treat

delayed diabetic wound healing.

Diabetes Page 6 of 34

Sun et al 6

RESEARCH DESIGN AND METHODS

Ethics statement

All investigations using animals conformed to the regulations of the ARVO Statement for the

Use of Animals in Ophthalmic and Vision Research, the National Institutes of Health, and the guidelines of the Animal Investigation Committee of Wayne State University. Human autopsy corneas with or without diabetes were obtained from Michigan Eye Bank, without any personal information except age, gender, the cause of death.

Animals and Induction of Diabetes

Sixweekold C57BL/6 mice, both males and females purchased from the Jackson

Laboratory, were induced to develop diabetes with Streptozotocin (STZ) as described previously

(23; 24). Mice were considered as diabetic with bloodglucose levels >300 mg/dl within four weeks postinjection and thereafter (24). Ephx2-/- mice on a B6 background were a gift from

Joan Graves (NIH/NIEHS) and were induced to develop DM in the same manner as DM mice.

Evaluation of Tear Secretion

Tear secretion was determined with phenol red–impregnated cotton threads (Zone

Quick, Tokyo, Japan). The threads were placed in the medial canthus for 1 minute and the

length of the wetted part, turning red on soaking tears, was photographed and measured.

Corneal Epithelial Debridement Wound

DM and agematched normal mice were anesthetized by an intraperitoneal injection of

xylazine (7 mg/kg) and ketamine (70 mg/kg) plus topical proparacaine and an 1.5 mm circular

wound was first demarcated with a trephine in the central cornea, followed by the removal of

epithelial cells within the circle with a blunt scalpel blade under a dissecting microscope (Zeiss).

Two corneas were pooled in 1 tube, stored at −80 ◦C. The collected cells are marked as

unwounded (0h). The progress of wound healing was monitored by fluorescence staining for

epithelial defects and photographed with a slit lamp microscope. At the end of healing, the Page 7 of 34 Diabetes

Sun et al 7

corneas were either snapfrozen in OCT for cryostat sectioning or marked with the same size

trephine for CEC collection, marked as healing CECs.

Western Blotting

Western blotting of CECs was performed as described, cell lysates with equal amount of

proteins (20 ug) were separated were separated with 5% to 15% gradient SDSPAGE,

transferred to pore size 0.2 uM nitrocellulose membrane. The membranes were stained with

EPHX2 (Abcam, ab133173), HO1 (Abcam, ab13243), pSTAT3 (T705), pSTAT3 (S727), (Cell

signal0), STAT3 (Cell signal), or nonmuscle βactin (Sigma, A1978), followed by incubation

with HRPconjugated donkey secondary antibodies (1:5000 dilution; Jackson ImmunoResearch

Laboratories), and the bands were visualized with ECL (SuperSignal) and the images were

acquired using Kodak Image Station 4000R Pro. Band intensity was analyzed using Carestream

Molecular Imaging Software. Actin was used as loading control.

Immunohistochemistry of mouse corneas.

Mouse eyes were enucleated and embedded in TissueTek OCT compound, and frozen

in liquid nitrogen. Human Six micrometerthick sections were cut and mounted to poly

coated glass slides, fixed in 4% paraformaldehyde, blocked with 10 mM BPS containing 2%

BSA for 1 hour at RT, and incubated with rabbit primary EPHX2, or HO1 antibodies. This was

followed by a secondary antibody, FITC antirabbit (Jackson ImmunoResearch Laboratories

1:100). Slides were mounted with Vectashield mounting medium containing DAPI mounting

media. Controls were similarly treated with rat or rabbit IgG, as well as using the depletion of

primary antibodies with mouse recombinant EPHX2 (10:1). The sections were examined under

a Nikon ECLIPSE 90i microscope. The center of unwounded, or the leading edge of healing

corneas were photographed.

Whole mount Immunostaining and quantitation of innervation of B6 mouse Corneas Diabetes Page 8 of 34

Sun et al 8

Whole mount Immunostaining and quantitation of innervation of B6 mouse Corneas were performed as described in our previous study (25). Briefly, the enucleated eyes were fixed and the corneas were isolated and further fixed for an additional 10 minutes. The corneas were cut radially into 6 standardized sections and were incubated at 37°C in 20 mmol/L EDTA for 30 minutes, followed by 2day incubation in 0.025% hyaluronidase and 0.1% EDTA in PBS. The tissues were blocked at RT for 2 hours in PBS–Triton X100 containing 2% BSA, followed by incubation overnight at 4°C with antibody against βtubulin III. After secondary antibody, the tissues were mounted and examined under a confocal microscope (TCS SP2; Leica,

Heidelberg, Germany). Innervation in a region was calculated as the percent area positive for β tubulin III staining by Image J. sEH activity assay and epoxy/dihydroxy fatty acids measurement

sEH enzymatic activity was measured using Cayman’s CellBased Assay Kit (600090)

with 50 g of cell lysates. For EET/DHETs measurement, collected ECEs from normal and

diabetic mice were processed and epoxy/dihydroxy fatty acids were analyzed by the Lipidomics

Core Facility at WSU using standardized LCMS methods as described earlier (26).

Statistical analysis

The statistical analyses were performed using the software GraphPad Prism 6. Data was

presented as means ± SD. Experiments with two treatments and/or conditions were analyzed

for statistical significance using 2tailed unpaired Student's t test. Experiments with two groups

were analyzed using oneway ANOVA (Fig. 1C), and more than two groups were analyzed with

twoway ANOVA to determine overall differences and a Bonferroni posttest was performed to

determine statistically significant differences. Significance was accepted at p<0.05. Experiments

were repeated at least twice to ensure reproducibility. Page 9 of 34 Diabetes

Sun et al 9

RESULTS

Elevated expression of EPHX2 in corneal epithelial cells of STZ-treated mice.

To determine whether EPHX2 was also expressed at the protein level, we first

performed immunohistochemistry and observed that EPHX2 immunoreactivity was primarily

found in the epithelium layer; its staining intensity increased with time postSTZ treatment (Fig.

1A). The presence of recombinant mouse EPHX2 in the first antibody incubation abolished

immunoreactivity (inserts, 8 weeks, Fig.1A). Since EPHX2 was mostly expressed in the

epithelium, we performed Western blotting of CECs isolated at different times after STZ

injection. Figure 1B revealed that EPHX2 was expressed in normal CECs; elevated

expressions were detected at different time points postSTZ; relative intensities, normalized to

actin, were 24.2 preSTZ treatment, and increasing to 34.8, 49.1 and 42.8 at 2, 4 and 8 weeks,

respectively (Fig. 1B). EPHX2 encodes a phosphatase at the Nterminal and an epoxide

hydrolase at the Cterminal (9). sEH activity was assessed in diabetic CECs (Fig. 1C). Basal

activity of sEH was detected in NL CECs, and STZ treatment resulted in 2.95, 4.68, and 5.21

fold increases at 2, 4 and 8 weeks, respectively (Fig. 1C). Figure 1D is the immunostaining of

human corneas, one from a 66 year old male (NL) and the other from a 56 year old female

patient with severe diabetic retinopathy; stronger EPHX2 staining was seen in diabetic human

corneal epithelium compared that of to a nondiabetic patient. Taken together, these results

indicate that sEH activity increases in diabetic mouse CECs.

To determine the amounts of EETs and their metabolites DHETs, we pooled epithelial

cells from 8 corneas, of the control and STZ diabetic mice, two each, and assessed the levels of

EETs. Among 4 EETs, 8.9EET was detected with 0.30 and 0.43 ng per sample for nondiabetic

and 0.17 and 0.24 ng per sample for diabetic CECs, respectively. 11,12EET was barely Diabetes Page 10 of 34

Sun et al 10

detectable only in normal ECEs (0.01). Tissue DHET levels were below the limit of

quantification.

Diabetes induction in Ephx2-/- mice.

Ephx2-/- mice have been used as models for hypertension and cardiovascular diseases

(8; 27; 28). We obtained Ephx2knockout mice on B6 background from Dr. Darryl C. Zeldin

(NIH/NIEHS). An early study using intraperitoneal injection of 50 mg/kg/day STZ for 3 days

reported that Ephx2deletion prevented the development of diabetes (29). We used 50 mg/kg

1day-1 STZ, injected intraperitoneally for 5 days. As shown in Figure 2, Ephx2/ mice had similar average random blood glucose levels and became hyperglycemic in a similar time fashion with the control B6 mice (Fig. 2A); no differences in their body weight were observed as well (Fig.

2B). Hence, Ephx2depletion had minimal effects on the development of diabetes induced by

STZ. Our results were similar to those reported by Elmarakby et al (20). Using Western

blotting, we showed that diabetes induced EPHX2 expression in WT but not in Ephx2-/- mice

(Fig. 2C).

One of the characteristics of DK in patients is decreased tear secretion. While no

difference was observed in NL WT and Ephx2-/- mice, Schirmer’s test revealed that STZ treatment significantly decreased tear secretion in WT (65% of NL mice), but not Ephx2-/- mice

(Fig. 2D&E)., suggesting an overall protective effect of Ephx2depletion.

Depletion of Ephx2 attenuates pathogenesis of diabetic keratopathy

Have shown that EPHX1 expression and sEH activities were elevated in diabetic

corneas and that diabetes can be induced in Ephx2-/- mice, we next investigated the effects of

Ephx2depletion on epithelial wound closure in the corneas (Fig. 3). Ten weeks after induction of the diabetes, the Ephx2-/- DM and agematched WT DM were wounded with a 1.5mm

diameter epithelium debridement procedure. The wounds were allowed to heal for 24 h and the Page 11 of 34 Diabetes

Sun et al 11

progress of wound healing was monitored by corneal fluorescence staining for epithelial defects

and photographed with a slit lamp microscope (Fig. 3A). The remaining wound area at 24 h

post wounding was calculated (Fig. 3B). For nondiabetic corneas, Ephx2depletion exhibited no

detectable effects on the rate of epitheliumwound closure, 15.5±6.5% versus 17.25±6.25%

remaining wound area (RWA). As we showed before, diabetes significantly delayed wound

healing in WT B6 mouse corneas (45.15±6.75% RWA, p<0.01); this delay, however, was

markedly attenuated in Ephx2/ mice (13.5±5.95% RWA).

Immunohistochemistry analysis revealed that there was little EPHX2 staining in

normoglycemia corneas, intense staining of EPHX2 in the entire epithelia of both unwounded

and wounded DM corneas was observed (Fig. 3C).

Ephx2 deficiency restores the wound-induced STAT3 signaling in the healing diabetic

corneas

Epithelial wounding is known to activate the JAK/STAT3 pathway, leading to wound

closure in vitro and in vivo (30). EETs have been shown to mediate STAT3 signaling in

cardiomyocytes (31). Using STAT3 phosphorylation as a marker of activation, we assessed the

effects of hyperglycemia and EPHX2 expression on woundinduced STAT3 signaling (Fig. 4).

Both phosphorylation at T705 and S727 can be detected at basal levels in unwounded (0h) WT

and Ephx2/ mouse corneas with (D) or without (N) diabetes. Wounding (24 hpw)induced

phosphorylation was observed at the T705 site in nondiabetic WT (6.26 fold over the control,

0h WTN, value set as 1 vs 24h WTN) and Ephx2-/- (13.1 fold, 24h KON) mice, but not

diabetic mouse corneas (1.05 fold, 24h WTD). Ephx2depletion prevented hyperglycemia

suppressed STAT3 phosphorylation (13.4 fold, 24 KOD). Similar patterns were observed for

site S727 as well. Diabetes Page 12 of 34

Sun et al 12

Ephx2-deficiency enhances hyperglycemia-suppressed expression of heme oxygenase 1

in healing diabetic corneas.

Since HO1 induction was found to be Src/STAT3dependent in breast cancer cells (32),

we therefore assessed HO1 expression in response to wounding and/or hyperglycemia.

Without wounding, HO1 levels were low in WT and Ephx2-/- mice with or without diabetes.

Wounding induced HO1 expression in normal WT and Ephx2/ (~2.7 fold) but not diabetic mice

whereas Ephx2depletion reversed the diabetesinduced suppression of HO1 expression at the

mRNA (a 2.53 fold increase, Fig 5A) and protein level (Fig. 5B & 5C).

sEH inhibition accelerates epithelial wound healing and promotes HO-1 expression in

diabetic mouse corneas.

There were contradicting reports regarding the effects of Ephx2depletion on kidney

function (20; 27), presumably because of the phosphatase activity of EPHX2 gene. To test the

effects of sEH inhibition on corneal wound healing, we used two structurally different sEH

specific inhibitors, tAUCB (33) and GSK2256294 (34) (Fig. 6). These two inhibitors had no

noticeable effects on wound healing in normal corneas (panels 1, 2, 1’ and 2’, Fig. 6AD). The

delayed wound healing in diabetic corneas was significantly accelerated by tAUCB (from

36.82±13.03% to 15.57±6.78% RWA) and GSK (from 33.27±11.46% to 16.51±4.22 RWA) (Fig.

6AD). Similar to Ephx2depletion, inhibition of sEH also promoted HO1 expression in diabetic while it exhibited no effects on normal corneas in response to wounding (Fig. 6E and F).

Induction of HO-1 expression accelerates delayed epithelium wound healing

Having shown that inhibition of sEH increased woundinduced HO1 expression in

diabetic corneas, we next investigated its role in accelerating wound healing in diabetic corneas

using Cobalt protoporphyrin (CoPP), a wellknown HO1 inducer (35). As expected, CoPP

applied through subconjunctival injection stimulated HO1 expression at the protein levels in Page 13 of 34 Diabetes

Sun et al 13

healing diabetic corneas, while this expression was undetectable in the control, vehicletreated

eyes (Fig. 7A). These HOI expressing epithelia had a significantly higher healing rate

compared to vehicletreated diabetic corneas, 28.78±4.18% vs 41.4±6.36% RWA (Fig. 7B and

C).

Ephx2-depletion enhances sensory innervation and regeneration in diabetic corneas.

We previously showed in rodent models of diabetes, there were decreases in the density

of sensory nerve fibers/endings in the corneas (7; 24; 25). Using whole mount confocal

microscopy, we first examined sensory nerves in unwounded corneas. There was a significant

decrease in n the density of nerves at the center of the cornea of diabetic WT mice (Fig. 8A1

and A3), 71.9±2.9% of the control, nondiabetic WT mice (set as value 1 or 100%, Fig. 8B).

While Ephx2depletion had no significant effects on nondiabetic corneas (Fig. 8A2 and A4), it

increased the density of nerve fibers/endings of diabetic mouse corneas (105% of the control

with no statistical significance, Fig. 8B).

The regeneration of the basal nerve plexus was also examined at 1 month post

epithelium wounding, at which time the wound center remained uncovered by the nerve plexus

(Fig. 8A5A8). While in the WT vs DM cornea, the region not covered by nerves was larger with

a reduced nerve density of (comparing Fig. 8A5 and A7), 52.9±2.2% vs 38.9±6.9%, p<0.05), in

Ephx2-/- corneas, the densities of sensory nerves in normal and diabetic corneas (Fig. 8A6 and

A8) were similar, 51.1 ±2.2% vs 59.5±10.% (Fig. 8B).

Diabetes Page 14 of 34

Sun et al 14

DISCUSSION

In this study, we evaluated EPHX2 expression and role in the pathogenesis of DK in a

B6 mouse model of human diabetes. We showed for the first time that EPHX2 expression was elevated in diabetic cornea as well as retina with or without wounding. Unlike previous reports showing the failure of STZ to induce DM in Ephx2-/- mice, we observed no significant differences in the course of STZinduced DM between WT B6 and Ephx2-/- mice. However, unlike WT mice, Ephx2-/- mice did not develop diabetesassociated dry eye symptoms. Moreover, Ephx2 depletion increased the rate of epithelial wound closure, ameliorated sensory nerve degeneration in unwounded corneas, and enhanced their regeneration in wounded diabetic corneas while exhibiting no detectable effects on the nondiabetic corneas. Importantly, two structurally different sEH inhibitors accelerated delayed epithelial wound healing in the diabetic corneas to a level similar to that of NL mice. Inhibition of sEH also restored hyperglycemia suppressed expression of HO1, a factor necessary for proper wound healing in the cornea.

Our study revealed that elevated EPHX2 in diabetic corneas is a pathogenic factor for DK and suggested that supplementing EETs and/or inhibiting sEH may prevent or treat DK.

EPHX2 has been found to be expressed ubiquitously in many tissues. It has, however, also been detected as a stress response gene associated with many human diseases (1417).

In the literature, results of EPHX2 expression in the diabetic mouse kidney was mixed, from a decrease in the cytosol (36), no significant changes (20), or an increase (37; 38) in whole kidney extracts. We showed that the enzymatic activity of sEH was also elevated during the course of

STZinduced diabetes in CECs. This is consistent with the report showing a great decrease in the EETs/DHEETs in diabetic, compared to normal, mice (20). The elevated EPHX2 expression in diabetic tissues is consistent with its role as a stress response gene (39; 40). Page 15 of 34 Diabetes

Sun et al 15

EETs have been shown to promote organ and tissue regeneration, including liver

regeneration, kidney and lung compensatory growth, corneal neovascularization, and retinal

vascularization (41). Our study showed a trend of decreases in the levels of 8,9EET as well

11,12EET in diabetic, compared to normal nondiabetic CECs. Local administration of EETs

accelerates wound epithelialization and neovascularization in a mouse earwound model (42).

Our study however suggested that in the normoglycemic mice, lowering sEH had no effect on

epithelial wound closure, suggesting that supplementing extra EETs may not have beneficial

effects on wound healing in normoglycemic corneas. Given the fact that EETs exhibit potent

protective effects, including antiinflammation, we speculate that overexpression of EPHX2 may

contribute to lowgrade inflammation in some tissues such as the kidney and the cornea, but not

in others such as the liver, although it has been linked to the hepatic inflammatory response in

fatty liver disease (37).

In addition to being potentially involved in tissue inflammation, our study also suggests

that the elevated EPHX2 expression may contribute to the delayed epitheliumwound closure

and sensory nerve regeneration in diabetic corneas. Most studies of EPHX2 focused on its

effects on injuries associated with endothelia including ischemic cardiomyopathy (43), vascular

remodeling (44), and renal injury during diabetes (20), and associated with hypertension (28).

The cornea is an avascular tissue, and as such, the effects of diabetesinduced upregulation on

impaired wound healing is likely due to the direct effects on CECs. However, it is not clear

whether the defects were due to the general health affected by a decrease in EETs, the

increase in the ratio EETs/ DHETs in epithelial cells, or due to the defects in the wound

response. The fact that sEH inhibitors accelerated epithelial wound closure in diabetic corneas

suggests that the cellular levels of EETs play a beneficial role for wound closure.

While most studies show similar beneficial effects of Ephx2 deficiency and

pharmacological inhibition of sEH, Ephx2depletion has been shown to worsen angiotensin II Diabetes Page 16 of 34

Sun et al 16 induced cardiac dysfunction since it aggravated myocardial fibrosis and increased cardiac inflammation (45). The adverse effects of Ephx2depletion observed on cardiac fibrosis may be related to the depleted lipid phosphatase and sEH activities (45). In our model, both Ephx2 deletion and sEH inhibition exhibited similar effects on epithelial wound closure, indicating the importance of sEH activity or the cellular concentrations of EETs in mediating the epithelial wound response, which was impaired in diabetic tissues.

Our study also revealed a correlation between sEH activity and the expression of Heme oxygenase1 (HO1), a stressinducible protein with a potential antiinflammatory effect. HO1

has been shown to play an important role in skin injury and wound healing (46). The induction of

HO1, the ratelimiting in heme degradation, represents a key event in cellular

responses to prooxidative and proinflammatory insults (47). In the cornea, the increased

expression of HO1 modulates inflammation and promotes wound closure (48). HO1 and EETs

were found to influence each other expression and to attenuate diabetes and metabolic

syndrome complications (49). Our study for the first time showed that hyperglycemia

suppressed the woundinduced expression of HO1, suggesting a potential contribution of this

defect to delayed wound healing in diabetic corneas. Importantly, application of CoPP induced

robust expression of HO1, resulting in significant improvement of corneal epitheliumwound

closure. Whether EETs and HO1 work synergistically, independently, or mutual exclusively in

promoting corneal wound healing in diabetic corneas remains elusive and warrants further

investigation.

Our previous studies showed that diabetes caused defects in sensory nerve structure

and function in the corneas. Using an Ephx2deficient mouse model, we showed that although no differences in induction of diabetes by STZ were found between WT and Ephx2-/- mice, the hyperglycemiainduced sensory nerve degeneration was prevented in Ephx2/ mice. In humans, almost half the patients who undergo laser in situ keratomileusis (LASIK) experience Page 17 of 34 Diabetes

Sun et al 17

dry eye following the procedure (50). It is believed that the alteration of corneal nerves after

LASIK is the most likely cause of the subjective symptoms of LASIKinduced dry eye. The

corneal sensitivity and the clinical indicators of dry eye usually starts to improve over the first

postoperative month but requires a year to normalize due to the partial recovery of the corneal

nerve plexus (50). Hence, regeneration of the subbasal nerve plexus is a slow process. To that

end, we examined the subbasal nerve plexus at the center of the cornea 1 month post

epithelium debridement and found slower recovery of sensory nerve endings in the diabetic

mouse corneas compared to normal controls in WT but not Ephx2-/- mice, suggesting sustained

positive effects of EETs on nerve regeneration under pathogenic conditions. Thus, exogenous

EETs or inhibition of sEH activity may employed as longterm therapy to help functional

recovery of sensory nerve in diabetic patients.

Diabetes Page 18 of 34

Sun et al 18

Contribution Statement: H.S., P.L., C.Y. performed laboratory testing and edited and checked accuracy of the manuscript. N.G. performed laboratory testing. J.W. and X. F. contributed to the discussion, reviewed and revised manuscript. F.Y. was responsible for study design and recruitment, contributed to sample collection and data analysis, and reviewed and edited the manuscript. F.Y. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Acknowledgments: FuShin X. Yu is the guarantor of this manuscript. We acknowledge support from NIH/NEI R01EY10869, EY17960 (to FSY), p30 EY04078 (NEI core to WSU),

Research to Prevent Blindness (to Kresge Eye Institute). The WSU Lipidomics Core Facility is supported by a grant from NIH National Center for Research Resources (S10RR027926).

Duality of interest. The authors declare that there is no duality of interest associated with this manuscript.

Page 19 of 34 Diabetes

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References

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Sun et al 20 ureido)cyclohexyloxy]benzoic acid is neuroprotective in rat model of ischemic stroke. Am J Physiol Heart Circ Physiol 2013;305:H16051613 20. Elmarakby AA, Faulkner J, AlShabrawey M, Wang MH, Maddipati KR, Imig JD: Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocininduced type 1 diabetes. American journal of physiology Regulatory, integrative and comparative physiology 2011;301:R13071317 21. Shuey MM, Billings FTt, Wei S, Milne GL, Nian H, Yu C, Brown NJ: Association of gainof function EPHX2 polymorphism Lys55Arg with acute kidney injury following cardiac surgery. PLoS One 2017;12:e0175292 22. Bikbova G, Oshitari T, Tawada A, Yamamoto S: Corneal changes in diabetes mellitus. Current diabetes reviews 2012;8:294302 23. Xu K, Yu FS: Impaired Epithelial Wound Healing and EGFR Signaling Pathways in the Corneas of Diabetic Rats. Invest Ophthalmol Vis Sci 2011;52:33013308 24. Yin J, Huang J, Chen C, Gao N, Wang F, Yu FS: Corneal complications in streptozocin induced type I diabetic rats. Invest Ophthalmol Vis Sci 2011;52:65896596 25. Gao N, Yan C, Lee P, Sun H, Yu FS: Dendritic cell dysfunction and diabetic sensory neuropathy in the cornea. J Clin Invest 2016;126:19982011 26. Maddipati KR, Romero R, Chaiworapongsa T, Chaemsaithong P, Zhou SL, Xu Z, Tarca AL, Kusanovic JP, Gomez R, Docheva N, Honn KV: Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome. J Lipid Res 2016;57:19061916 27. Zhu Y, Blum M, Hoff U, Wesser T, Fechner M, Westphal C, Gurgen D, Catar RA, Philippe A, Wu K, Bubalo G, Rothe M, Weldon SM, Dragun D, Schunck WH: Renal / in Soluble Epoxide HydrolaseDeficient Mice. PLoS One 2016;11:e0145645 28. Manhiani M, Quigley JE, Knight SF, Tasoobshirazi S, Moore T, Brands MW, Hammock BD, Imig JD: Soluble epoxide hydrolase gene deletion attenuates renal injury and inflammation with DOCAsalt hypertension. Am J Physiol Renal Physiol 2009;297:F740748 29. Luo P, Chang HH, Zhou Y, Zhang S, Hwang SH, Morisseau C, Wang CY, Inscho EW, Hammock BD, Wang MH: Inhibition or deletion of soluble epoxide hydrolase prevents hyperglycemia, promotes insulin secretion, and reduces islet apoptosis. The Journal of pharmacology and experimental therapeutics 2010;334:430438 30. ArranzValsero I, SorianoRomani L, GarciaPosadas L, LopezGarcia A, Diebold Y: IL6 as a corneal wound healing mediator in an in vitro scratch assay. Exp Eye Res 2014;125:183192 31. Merkel MJ, Liu L, Cao Z, Packwood W, Young J, Alkayed NJ, Van Winkle DM: Inhibition of soluble epoxide hydrolase preserves cardiomyocytes: role of STAT3 signaling. Am J Physiol Heart Circ Physiol 2010;298:H679687 32. Tan Q, Wang H, Hu Y, Hu M, Li X, Aodengqimuge, Ma Y, Wei C, Song L: Src/STAT3 dependent heme oxygenase1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy. Cancer Sci 2015;106:10231032 33. Shen L, Peng H, Zhao S, Xu D: A potent soluble epoxide hydrolase inhibitor, tAUCB, modulates cholesterol balance and oxidized low density lipoprotein metabolism in adipocytes in vitro. Biol Chem 2014;395:443451 34. Podolin PL, Bolognese BJ, Foley JF, Long E, 3rd, Peck B, Umbrecht S, Zhang X, Zhu P, Schwartz B, Xie W, Quinn C, Qi H, Sweitzer S, Chen S, Galop M, Ding Y, Belyanskaya SL, Israel DI, Morgan BA, Behm DJ, Marino JP, Jr., Kurali E, Barnette MS, Mayer RJ, BoothGenthe Page 21 of 34 Diabetes

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CL, Callahan JF: In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor. Prostaglandins Other Lipid Mediat 2013;104105:2531 35. Cao J, Vecoli C, Neglia D, Tavazzi B, Lazzarino G, Novelli M, Masiello P, Wang YT, Puri N, Paolocci N, L'Abbate A, Abraham NG: CobaltProtoporphyrin Improves Heart Function by Blunting Oxidative Stress and Restoring NO Synthase Equilibrium in an Animal Model of Experimental Diabetes. Front Physiol 2012;3:160 36. Oguro A, Fujita N, Imaoka S: Regulation of soluble epoxide hydrolase (sEH) in mice with diabetes: high glucose suppresses sEH expression. Drug metabolism and pharmacokinetics 2009;24:438445 37. Bettaieb A, Koike S, Hsu MF, Ito Y, Chahed S, Bachaalany S, Gruzdev A, CalvoRubio M, Lee KSS, Inceoglu B, Imig JD, Villalba JM, Zeldin DC, Hammock BD, Haj FG: Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia. Biochim Biophys Acta 2017;1861:27582765 38. Chen G, Xu R, Wang Y, Wang P, Zhao G, Xu X, Gruzdev A, Zeldin DC, Wang DW: Genetic Disruption of Soluble Epoxide Hydrolase is Protective Against Streptozotocininduced Diabetic Nephropathy. American journal of physiology Endocrinology and metabolism 2012; 39. Abdelhamid G, ElKadi AO: Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: roles of the NFkappaB and MAPK signaling pathways. Free Radic Biol Med 2015;82:112 40. Bracalente C, Ibanez IL, Berenstein A, Notcovich C, Cerda MB, Klamt F, Chernomoretz A, Duran H: Reprogramming human A375 amelanotic melanoma cells by catalase overexpression: Upregulation of antioxidant genes correlates with regression of melanoma malignancy and with malignant progression when downregulated. Oncotarget 2016; 7: 4115441171 41. Panigrahy D, Kalish BT, Huang S, Bielenberg DR, Le HD, Yang J, Edin ML, Lee CR, Benny O, Mudge DK, Butterfield CE, Mammoto A, Mammoto T, Inceoglu B, Jenkins RL, Simpson MA, Akino T, Lih FB, Tomer KB, Ingber DE, Hammock BD, Falck JR, Manthati VL, Kaipainen A, D'Amore PA, Puder M, Zeldin DC, Kieran MW: Epoxyeicosanoids promote organ and tissue regeneration. Proc Natl Acad Sci U S A 2013;110:1352813533 42. Sander AL, Jakob H, Sommer K, Sadler C, Fleming I, Marzi I, Frank J: Cytochrome P450 derived epoxyeicosatrienoic acids accelerate wound epithelialization and neovascularization in the hairless mouse ear wound model. Langenbecks Arch Surg 2011;396:12451253 43. Zhao TT, Wasti B, Xu DY, Shen L, Du JQ, Zhao SP: Soluble epoxide hydrolase and ischemic cardiomyopathy. Int J Cardiol 2012;155:181187 44. Simpkins AN, Rudic RD, Roy S, Tsai HJ, Hammock BD, Imig JD: Soluble epoxide hydrolase inhibition modulates vascular remodeling. Am J Physiol Heart Circ Physiol 2010;298:H795806 45. Li L, Li N, Pang W, Zhang X, Hammock BD, Ai D, Zhu Y: Opposite effects of gene deficiency and pharmacological inhibition of soluble epoxide hydrolase on cardiac fibrosis. PLoS One 2014;9:e94092 46. Zhang B, Xie S, Su Z, Song S, Xu H, Chen G, Cao W, Yin S, Gao Q, Wang H: Heme oxygenase1 induction attenuates imiquimodinduced psoriasiform inflammation by negative regulation of Stat3 signaling. Sci Rep 2016;6:21132 47. Dulak J, Jozkowicz A: Novel faces of heme oxygenase1: mechanisms and therapeutic potentials. Antioxid Redox Signal 2014;20:16731676 Diabetes Page 22 of 34

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48. Patil K, Bellner L, Cullaro G, Gotlinger KH, Dunn MW, Schwartzman ML: Heme oxygenase 1 induction attenuates corneal inflammation and accelerates wound healing after epithelial injury. Invest Ophthalmol Vis Sci 2008;49:33793386 49. Burgess A, Vanella L, Bellner L, Schwartzman ML, Abraham NG: Epoxyeicosatrienoic acids and heme oxygenase1 interaction attenuates diabetes and metabolic syndrome complications. Prostaglandins Other Lipid Mediat 2012;97:116 50. Chao C, Golebiowski B, Stapleton F: The role of corneal innervation in LASIKinduced neuropathic dry eye. Ocul Surf 2014;12:3245

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FIGURE LEGENDS

Figure 1. EPHX2 expression was increased in diabetic mouse cornea. Eightweekold

C57BL/6 (WT) mice were intraperitoneally injected with 5 doses of 50 mg/kg STZ diluted in

citrate buffer, IP (DM). (A) Immunohistochemistry showing EPHX2 expression (green) in NL and

DM weeks after STZ injection. The cryostat sections were also counterstained with DAPI

showing nuclei (Blue). Inserts in STZ 8 week, primary antibody absorbed with mouse

recombinant EPHX2. E, epithelium; S, stroma. (B) WB analysis of EPHX2 expression in mouse

CECs, 2 weeks, 4 weeks and 8 weeks postSTZtreatment. For each sample, 20 g total protein

from collected CECs were analyzed by WB with EPHX2 antibody. The numbers above EPHX2

bands (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

a sEH CellBased Assay Kit from Cayman. CECs were collected and 50 g of total protein lysis

was loaded for the assay, according the protocol of the Kit. Stars on top of columns are P value

results comparing with the control. *P < 0.05 and **P < 0.01 (Oneway 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 years old male

patient without Diabetes; DM, a donor cornea from 53 years old female patient without severe

diabetic retinopathy. The Figure, except 1D, is the representative of three independent

experiments.

Figure 2. Hyperglycemia, growth and tear secretion in WT and Ephx2-/- mice treated with

STZ. Eightweekold wildtype B6 (WT) and Ephx2-/- mice were intraperitoneally injected with 5

doses of 70 mg/kg STZ diluted in citrate buffer (DM). Blood glucose (A) and body weight (B)

were measured at 11:00 am at 5 days interval starting from the next day of last STZinjection.

(C) Western blotting showing EPHX2 expression in cornea epithelial cells extracted from WT

and Ephx2-/- mice at 8 weeks post STZ injection with βactin staining as internal controls for Diabetes Page 24 of 34

Sun et al 24 protein loading. Left side number indicate protein molecular weights (the predicted molecular weight: 63 kDa for EPHX2). (D) Schirmer’s test measurements of tear secretion we in NL and

DM mice using phenol red–impregnated cotton threads; red color, indicative of wet threads. (E)

Tear secretion was quantitated and represented as the length (mm) red threads (mean + SD; unpaired Ttest, N=5, *P < 0.05). Three independent experiments were performed, 1 representative image for each condition was presented.

Figure 3. EPHX2 deficiency changed corneal epithelial wound healing rate and gene expression in corneal epithelial cells. The corneas of WT and Ephx2-/- mice with (DM) or without (NL) STZ treatment were wounded by epitheliumdebridement (1.5mm diameter). (A)

The remaining wounds at 24 hpw were visualized by fluorescein staining under slit lamp. (B)

Analysis of fluorescentstained areas using Adobe Photoshop software. The denuded area was

measured as the numbers of pixels; the results were 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 DM corneas before (0) and 24 h

post wounding (24). The cryostat sections were also counterstained with DAPI showing nuclei.

Inserts in CDM (24) panels were 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.

Figure 4. Impaired STAT3 signaling in WT but not Ephx2-/- DM corneas during epithelial wound healing. CECs were harvested from one cornea of WT (WT) or Ephx2/ (KO) mice with

(D) or without diabetes (N) during epithelium debridement (0) or 24 h post wounding (24 h) and subjected to Western botting with STAT3 phosphorylation site specific antibodies (T705 or

S727) or panSTAT3 antibody to normalize the loading. Two independent experiments were Page 25 of 34 Diabetes

Sun et al 25

performed (n=3), 1 representative image for each condition was presented. The number under

each lane is the number of pixels determined using ImageJ and numbers in () were those

normalized with total STAT3 reading; the numbers following () are relative increases/decreases

with WT, unwounded, nondiabetic corneas as 1.

Figure 5. Expression and distribution of HO-1 in unwounded and healing corneas of WT

and Ephx2-/- mice with or without diabetes. CECs were harvested as described in Figure 4

and subjected to realtime PCR (A) and Western blotting (B) analyses for the expression of HO1

in unwounded (0) and healing (24 h) corneas of WT (W or WT) or Ephx2-/- (K or KO) mice with

(N) or without diabetes (D). The results in panel A were first normalized with the levels of βactin

and then compared with the levels of WT, NL unwounded (value 1), presented as fold changes,

n=5, **p<0.01 (twoway ANOVA with Bonferroni posttest among the healing CECs). (C)

Immunohistochemistry showing HO1 expression and distribution in unwounded (0 h) and

healing (24 h) corneas of WT and Ephx2-/- 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.

Figure 6. Effects of sEH inhibitors on diabetic epithelial wound healing and HO1

expression. NL and DM mice were subconjunctivally injected with 5 l control (PBS) or EPHX2

inhibitor tAUCB (10nM, Cayman), GSK 2256294A (10 nM, Medchemexpress) 4 h before

epitheliumdebridement. Corneas were photographed at 24 hpw (A, B) and the wound sizes

were fluorescence stained and calculated. Results were presented as the mean of the

remaining wound area (C and D), **p<0.01 (twoway ANOVA with Bonferroni posttest). (E, F)

Realtime PCR analysis of HO1 expression in CECs. The results were first normalized with the

levels of βactin and then compared with the levels of WT, NL unwounded (value 1, C1 and

C1’), presented as fold changes, n=5, **p<0.01 (twoway ANOVA with Bonferroni posttest

among the healing CECs). Diabetes Page 26 of 34

Sun et al 26

Figure 7. Effects of HO-1 induction on diabetic epithelial wound healing. DM mice were subconjunctivally injected with 5 l control (PBS) or HO1 inducer, CoPP, (1g/l, Sigma) 4 h before epitheliumdebridement. (A) At 24 hpw, the remaining wounds were fluorescentstained and photographed. (B) The wound sizes were calculated and results were presented as the mean of the remaining wound area, **p<0.01 (unpaired student’s T test, N=5). (C) CECs were collected and subjected to Western blotting with HO1 antibody with actin as the loading control.

Figure 8. Effects of EPHX2-deficiency on sensory nerve innervation and regeneration in

B6 mouse corneas. (A) A 1.5 mm epitheliumdebridement wound was created in the corneas of WT and Ephx2 mice with (DM) or without diabetes (NL). One month post wounding, unwounded and 30 days post wound corneas of WT and Ephx2-/- (EPHX2 KO) mice were excised, stained with tubulin III for sensory nerves, and visualized with whole mount confocal microscopy. The center of the cornea was photographed and the innervation was calculated as the percent area positive for βtubulin III by Image J (B). The results are representative of two independent experiments (N=3 each) and indicated p values were generated using twoway

ANOVA with Bonferroni posttest, ** p<0.01.

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