Page 1 of 34 Diabetes
Sun et al 1
Inhibition of Soluble Epoxide Hydrolase 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 Epoxide Hydrolase 2, wound healing,
epoxyeicosatrienoic acids
Number of words in text: 3922
Number of figures: 8
Number of Tables: 0
Correspondence to: Fu Shin X. Yu, Ph.D., Kresge Eye Institute, Wayne State University
School of Medicine, 4717 St. Antoine Blvd, Detroit, MI, 48201
Tel: (313) 577 1657; E mail: [email protected]
Diabetes Publish Ahead of Print, published online April 3, 2018 Diabetes Page 2 of 34
Sun et al 2
ABSTRACT
EPHX2 (soluble Epoxide Hydrolase 2, sEH) converts biologically active epoxyeicosatrienoic
acids (EETs), anti inflammatory and pro fibrinolytic 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 type 2, mouse
type 1 (STZ induced) and/or type 2 diabetes. Ephx2 depletion had no detectable effects on
STZ induced hyperglycemia but prevented the development of tear deficiency. Ephx2-/- mice
showed an acceleration of hyperglycemia delayed epithelium wound healing. Moreover,
inhibition of sEH increased the rate of epithelium wound closure and restored hyperglycemia
suppressed STAT3 activation and heme oxygenase-1 (HO 1) expression in the diabetic
corneas. Treatment of diabetic corneas with Cobalt protoporphyrin, a well known HO 1 inducer,
restored wound induced HO 1 upregulation and accelerated delayed wound healing. Finally,
Ephx2 depletion 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 sight threatening complications such as stromal opacification, surface
irregularity, and microbial keratitis (5). Chronic low grade 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 epithelium debridement is an ideal model to study re epithelialization, 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 genes (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 omega 6 fatty acid and is the precursor that is metabolized to a wide range of biologically and clinically important eicosanoid molecules by various enzymes, including enzymes of the COX, lipoxygenase and cytochrome P450 (CYP) monooxygenase pathways (8). The epoxygenase CYP enzymes generate four bioactive regioisomeric 1 acids (EETs) by metabolizing arachidonic acid: EETs: 5,6 , 8,9 , 11,12 and
14,15 EET. EETs contribute to the regulation of vascular tone, cardiovascular homeostasis, nociception, inflammatory response, angiogenesis, and cell proliferation (9 11). All EETs are then further metabolized by sEH, (10; 12) which is encoded by the gene EPHX2 and are converted into inactive or less active 1,2 diols, 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, hypertension, pain and diabetes (14 17). 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 NF κB related manner (20). Moreover, the gain of function 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 diabetes associated expression of EPXH2 in CECs.
We assessed its role during diabetes impaired epithelium wound 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 oxygenase 1 (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
Six week old 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 blood glucose levels >300 mg/dl within four weeks post injection 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 age matched 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 snap frozen 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 SDS PAGE,
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 non muscle β actin (Sigma, A1978), followed by incubation
with HRP conjugated 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 Tissue Tek OCT compound, and frozen
in liquid nitrogen. Human Six micrometer thick sections were cut and mounted to poly lysine