Xanthine Oxidoreductase Function Contributes to Normal Wound … · 2018-10-04 · Xanthine Oxidoreductase Function Contributes to Normal Wound Healing Michael C Madigan,1,2 Ryan

INTRODUCTIONChronic, nonhealing wounds arise

from diverse etiologies such as diabetes,venous stasis and pressure. Woundsoccur in 12–25% of diabetic patients andcontribute to a high incidence of limbloss and death (1). Despite improve-ments in glycemic control, antibiotics,and wound care adjuvants, nonhealingwounds continue to present a formidableproblem. The underlying molecularmechanisms of impaired wound healingare poorly understood despite extensive

knowledge of the normal wound repairprocess. Efficient wound repair involvesmany cytokines, chemokines and growthfactors that regulate the inflammatoryand regenerative phases of wound heal-ing (2). Unfortunately, novel therapiestargeting these factors, such as topicalplatelet-derived growth factor, have hadlimited success (3).

Inflammation is a necessary response totissue injury, leading to the production ofnitric oxide (NO) and reactive oxygenspecies (ROS) and reactive nitrogen

species (RNS) that mediate oxidativestress (4). Recent evidence demonstratesthat physiologic oxidative stress is essen-tial for normal wound healing (5,6).Redox signaling has been reported to playan important role in antibiosis, hemosta-sis, inflammation, re-epithelialization, an-giogenesis and growth factor modulation(7,8). However, an overabundance of in-flammatory by-products has deleteriouseffects on the wound (4). For example,ROS contributed to the failure of woundhealing in a rodent ischemic flap model(9) and high doses of topical hydrogenperoxide (H2O2) delayed wound repair byreducing collagen deposition and angio-genesis (10). Achieving the ideal balanceof ROS/RNS in damaged tissues may de-termine wound healing efficiency.

Wound repair also depends on NO.NO synthesis in wounds occurs throughthe arginine-NO synthase (NOS) path-way (11) and modulates inflammation,chemotaxis, antibacterial defenses, colla-gen production and angiogenesis

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Xanthine Oxidoreductase Function Contributes to NormalWound Healing

Michael C Madigan,1,2 Ryan M McEnaney,1,2 Ankur J Shukla,1,2 Guiying Hong,1,2 Eric E Kelley,3

Margaret M Tarpey,1,3 Mark Gladwin,4 Brian S Zuckerbraun,1,2 and Edith Tzeng1,2

1Surgery Services, Department of Veterans Affairs Medical Center, Pittsburgh, Pennsylvania, United States of America; Departmentsof 2Surgery, 3Anesthesia, and 4Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) areboth required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) hasthe unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneouswounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2,0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 toassess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocytecell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edgesas well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impairedhealing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tung-sten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing buttopical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fedmice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the firsttime that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2014.00191

Address correspondence to Edith Tzeng, Chief of Vascular Surgery, VA Pittsburgh Health-

care System, Professor of Surgery, Department of Surgery, University of Pittsburgh, A1010

PUH, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-802-3025; Fax: 412-291-1669;

E-mail: [email protected].

Submitted September 23, 2014; Accepted for publication April 14, 2015; Published Online

(www.molmed.org) April 14, 2015.

(12–14). NO deficiency has been impli-cated in the delayed wound healing indiabetic rodent models (15,16), whereNOS was downregulated. In addition,arginase I uses L-arginine to producepolyamines and proline, both essentialfor collagen synthesis and cell prolifera-tion, and is induced and competes forthe NOS substrate (16–18). InducibleNOS (iNOS) or endothelial NOS (eNOS)deficiency both delayed wound closurein normal mice, whereas restoration ofNOS activity in these mice improvedhealing (19,20), supporting an essentialrole of NO in wound repair.

Xanthine oxidoreductase (XOR) is a ho-modimeric enzyme that metabolizes xan-thine and generates ROS. It is also a po-tent nitrite reductase, converting nitriteback to NO (21,22). This pathway is espe-cially efficient in hypoxia, a conditionthat reduces NOS-dependent NO produc-tion. We and others have reported that ni-trite can serve as a source of NO throughXOR in models of vascular injury andpulmonary hypertension (23,24). XORhas been detected in skin, where it is in-volved in ROS production in response tolipopolysaccharide (LPS) (25) and ultravi-olet irradiation (26). Thus, we hypothe-size that XOR participates in normalwound healing through ROS/RNS andNO production. We used a murine exci-sional wound-healing model and manip-ulated XOR activity (27,28) to examine itsrole in normal wound repair.

MATERIALS AND METHODS

Excisional Wound Healing ModelAll procedures conformed to the Guide

for the Care and Use of Laboratory Animals(29) and the policies of the InstitutionalAnimal Use and Care Committee of theUniversity of Pittsburgh (protocol#1104675A). Male C57BL/6 mice (8–12wks old; The Jackson Laboratory, BarHarbor, ME, USA) were anesthetizedwith Nembutal (70 mg/kg, Abbott Labs,Chicago, IL, USA) and isoflurane. Aftershaving, a 1.5 × 1.5–cm excisional woundwas created on the back of each mouseand then covered with bio-occlusive

dressings (Systagenix, Quincy, MA,USA). Wound area was measured by ac-etate tracings every other day untilwound closure. The areas were calcu-lated using MetaMorph® (Version7.7.5.0; Molecular Devices, Inc., Sunny-vale, CA, USA). Wounds were also col-lected at earlier time points for proteinand immunohistochemical analyses.

Dietary and Topical WoundTreatments

Tungsten-enriched diet (#960350; MPBiomedicals, Irvine, CA, USA) wasstarted 2 wks before wounding to opti-mize molybdenum replacement in XORand maintained thereafter. Allopurinol(100 mg/kg/day; Sigma-Aldrich, St.Louis, MO, USA) in drinking water,sodium nitrite (300 mg/L in deionizedwater; Sigma-Aldrich) or nitrite-free diet(Harlan Teklad amino acid diet, TD99366; Harlan, Indianapolis, IN, USA)was initiated 1 wk before wounding andcontinued.

Topical H2O2 was applied to thewound as a 0.15% H2O2 solution(Thermo Fisher Scientific Inc., Waltham,MA, USA) in normal saline, and thewound was covered. Topical allopurinol(30 μg/wound) was similarly applied toeach wound. Treatment was initiated im-mediately after wounding and continuedevery other day.

Western Blot AnalysisWound samples were collected and di-

vided into the granulation tissue and thewound edge. Skin adjacent to the woundwas also collected. Samples were homog-enized in lysis buffer (Cell SignalingTechnology, Danvers, MA, USA) andquantified using a Pierce® BCA Proteinassay (Thermo Fisher Scientific). Westernblot analysis for XOR (rabbit mono-clonal, 1:5,000; ab109235; Abcam, Cam-bridge, MA, USA), iNOS (rabbit poly-clonal, 1:200; ab15323; Abcam) orarginase I (mouse monoclonal, 1:2,000;BD Biosciences, San Jose, CA, USA) wasperformed using horseradish peroxi-dase–linked goat anti-rabbit or anti-mouse secondary antibody (1:10,000;

Thermo Fisher Scientific). The mem-branes were developed by using Super-Signal® West Pico Chemiluminescent#34080 (Thermo Fisher Scientific).

Wound ImmunohistochemistryWounds were collected on d 7 or at

wound closure and fixed in 2% parafor -maldehyde, cryoprotected in 30% sucrose,embedded in OCT (Tissue Tek®; SakuraFinetek, Torrance, CA, USA) and sectioned(7 μm). Sections were treated with rabbitpolyclonal anti-XOR (1:100; Santa CruzBiotechnology, Santa Cruz, CA, USA),rabbit polyclonal anti- collagen I (1:200;Abcam), monoclonal anti-Ki67 (1:200;Abcam) or rat monoclonal anti-CD31(1:50; BD Biosciences) antibody followedby goat anti-rabbit 488 or goat anti-ratCy5 at 1:1,000 (Invitrogen [Thermo FisherScientific]). Nuclei were counterstainedwith Hoechst 33325 (2 μg/mL, Sigma-Aldrich). Images were collected using theFluoview® FV1000 confocal microscope(Olympus, Center Valley, PA, USA).

Wound AngiogenesisWound sections were stained with

CD31, and two confocal images of thewound granulation tissue were obtainedfor each section. Wound angiogenesiswas calculated as the number of CD31-stained lumens with ImageJ (version1.45s; National Institutes of Health, Be-thesda, MD, USA) and as the percent areaof CD31 staining using MetaMorph®.

XOR and Aldehyde Oxidase ActivityXOR activity was quantified as de-

scribed (23) via HPLC with electrochemi-cal detection. Briefly, endogenous uricacid (UA) was removed by using aSephadex G-25 column (GE Healthcare,Waukesha, WI, USA). Samples were thentreated with oxonic acid (2 mmol/L) to inhibit uricase. XOR activity wasquantified by UA production after addi-tion of xanthine (75 μmol/L). Total XDHactivity was assessed by exposure toNAD+ (0.5 mmol/L) and pyruvic acid(5 mmol/L). The specificity for XOR activ-ity was verified by allopurinol inhibitableUA formation. Aldehyde oxidase (AO) ac-

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tivity was measured by incubating tissuehomogenates with the AO substrate 4-(dimethylamino)cinnamaldehyde(DMAC) (25 μmol/L in potassium phos-phate [KPi], pH 7.8, and at 25°C) andmonitored for a decrease in absorbanceat 398 nm.

Wound ROS/RNS MeasurementTotal ROS/RNS was quantified by

using the OxiSelect™ In Vitro ROS/RNSAssay Kit (Cell Biolabs, Inc., San Diego,CA, USA) per instructions. Homoge-nized wound tissues (50 μg) were as-sayed for ROS/RNS by the conversion ofdichlorodihydrofluorescein to 2′,7′-dichlorodihydrofluorescein diacetate.

In Vitro Studies in Keratinocyte Cellsand Endothelial Cells

Human epidermal keratinocyte cells(KCs) (PCS-200-011; ATCC, Manassas,VA, USA) were cultured in Dermal CellBasal Media (PCS-200-030, ATCC) plusKeratinocyte Growth Kit (PCS-200-040,ATCC). KCs were differentiated with 1.2 mmol/L of calcium. Whole cell lysateswere used for Western blot analysis forXOR (1:100; Santa Cruz). For immunocy-tochemistry, KCs were cultured on glassslides, fixed with 2% paraformaldehyde,blocked in 2% BSA in PBS and then incu-bated with rabbit polyclonal anti-XOR(1:100; Santa Cruz) and mouse mono-clonal anti-K10 (1:100; Abcam) followedby secondary goat anti-rabbit or anti-mouse antibody (1:1,000). Nuclei werecounterstained with Hoechst 33325 (2 μg/mL) (Sigma-Aldrich). Images wereacquired on the Fluoview® FV1000 con-focal microscope (Olympus).

Human dermal microvascular endothe-lial cells (ECs) (VEC Technologies; Rensse-laer, NY, USA) were cultured as described(30) and low passage was used. Briefly,cells were grown in a 1:1 mix of MCDB131(VEC Technologies) and Dulbecco modi-fied Eagle medium (DMEM) with 5%fetal bovine serum (FBS). In proliferationassays, ECs were cultured in DMEM with1% FBS overnight. Proliferation was mea-sured by using 3H-thymidine as described(31) in 1% FBS medium with nitrite,

H2O2, catalase or allopurinol (Sigma-Aldrich). Migration was measured usingthe scratch assay at 6 h (32) and quanti-fied with ImageJ. For in vitro angiogene-sis, ECs were cultured on Matrigel™ (BD Biosciences) as described (30) withand without nitrite, allopurinol, H2O2 orcatalase. Tube formation was examined at6 h, quantifying boxes and number oflong tubes (>110 pixels in length) as amarker of healthy tubes.

Statistical AnalysisData are presented as mean ± standard

error of the mean (SEM). Statistical anal-ysis was performed with a Student t testor analysis of variance by using the Sig-maStat 11.0 software (Systat SoftwareInc., San Jose, CA), and significance wasdetermined at P < 0.05. All pairwise mul-tiple comparisons were performed byusing the Holm-Sidak method.

RESULTS

XOR, iNOS and Arginase I Expressionin Wound Tissue

C57BL/6 mouse skin had easily detect-able XOR expression by Western blot(Figure 1A). XOR expression was not al-tered by allopurinol, tungsten and nitritediets (data not shown). The wound edgehad less XOR expression, whereas thegranulation tissue had the lowest. In con-trast, iNOS expression was minimal inthe skin but was upregulated in thewound edge and granulation tissue (Fig-ure 1A). Arginase I had a similar expres-sion pattern to iNOS (Figure 1A). Im-munostaining of wounds at d 7 and afterhealing at d 14 revealed that the epider-mal KCs expressed high levels of XOR(Figure 1B). Much less XOR was detectedin the dermis or subcutaneous tissues.XOR expression was most pronounced inthe thick, proliferating layer of KCs sur-rounding the healing wound as well asin the thinner layer of KCs in normalskin. At 2 d after wounding (Figure 1C),the upregulation of XOR expression inthe wound edge was evident comparedwith the surrounding skin. In addition,there were cells in the peri-wound der-

mis and subcutaneous tissues thatstained for XOR and are consistent withneutrophils by the appearance of the nu-clear morphology. This finding indicatesthat XOR is highly expressed in thewound throughout the healing processand early inflammatory infiltrates alsoexpress XOR. Cultured KCs also ex-pressed XOR by immunostaining andWestern blot (Figures 1D, E). Differentia-tion of the KCs with calcium, as indi-cated by the expression of keratin-10, didnot affect XOR expression.

Tungsten-Inhibited XOR Activity andDelayed Wound Healing in C57BL/6Mice

Tungsten substitutes for the molybde-num (Mb) in XOR, inhibiting it irre-versibly. Tungsten diet significantly in-hibited skin XOR activity compared withmice fed allopurinol (18.5 ± 15.2 versus147.6 ± 4.8 μU/mg protein, P < 0.001) ora chow diet (165.3 ± 10.7 μU/mg protein,P < 0.001) (Figure 2A). XOR activity wassimilar between allopurinol and controlmice. XOR activity at the wound edgewas moderately reduced in allopurinol-treated versus control mice (100.9 ± 15.5versus 178.7 ± 12.1 μU/mg protein, P <0.001) but tungsten-treated mice hadnear complete inhibition of XOR activity(6.6 ± 4.1 μU/mg protein, P < 0.001 ver-sus control or allopurinol). Both allopuri-nol and tungsten-treated mice (35.3 ±14.4 and 4.6 ± 2.9 μU/mg protein, re-spectively) had significantly less XOR ac-tivity in granulation tissue than controlmice (107.6 ± 8.3 μU/mg protein, P <0.01 versus tungsten or allopurinol).Wound healing was significantly delayedin mice receiving the tungsten-enricheddiet versus mice on chow (Figure 2B;21.2 ± 0.7 versus 16.4 ± 0.2 d, P < 0.001).Dietary allopurinol did not delay woundhealing (Figure 2B).

Because oral allopurinol was much lesseffective in blocking XOR activity inwounds and skin than tungsten, we ex-amined the effect of locally applied allo -purinol on wound healing in mice. Topi-cal allopurinol significantly impairedwound healing at nearly all time points

R E S E A R C H A R T I C L E


compared with wounds from controlmice (Figure 2C). Measurement of XORactivity showed that allopurinol appliedto the wounds markedly reduced XORactivity by over 70% compared with con-trol wound tissues (156.2 ± 90.5 μU/mgversus 547.4 ± 15.9, respectively; n = 3/group; P = 0.013).

Minimal Expression of Other Mb-Containing Oxidases WasDetected in Wound Tissue

AO and sulfite oxidase (SO) both con-tain Mb and can be inhibited by tungsten.They can also function as nitrite reduc-tases. Very low levels of AO were detectedin the skin and wound tissues by Western

blot (Figure 2D). SO, which is primarilymitochondrial, was not detectible in thewounds as well (data not shown). Assayof wound tissues for AO activity showedno detectable oxidation of DMAC (n = 3),supporting the absence of AO activity inwounds. These findings support that thepredominant effect of tungsten on woundhealing is mediated through XOR.

XOR Inhibition with Tungsten ReducedROS/RNS in Granulation Tissue

To determine the effect of dietary tung-sten or allopurinol on wound levels ofROS/RNS, granulation tissue was col-lected at d 5. By using the OxiSelect InVitro ROS/RNS Assay, granulation tissuefrom control mice had similar levels ofROS/RNS as wounds from mice treatedwith dietary allopurinol (Figure 3A).However, tungsten reduced wound ROS/RNS by ~60%, confirming the efficient in-hibition of XOR by tungsten but not di-etary allopurinol (n = 7/group; P < 0.01versus control, P < 0.05 versus allopuri-nol). In a separate experiment, topical al-lopurinol did significantly reduce woundROS/RNS production compared with un-treated wounds (n = 3/ group, 0.55 ± 0.10versus 1.00 ± 0.09-fold change; P = 0.029).

XOR Inhibition with Tungsten ReducedWound Angiogenesis and KCProliferation

Tissue sections from healed woundswere stained for CD31 as a marker for ECs(Figure 3B). Wounds from tungsten-treatedmice had 25.3% fewer CD31-positive lumi-nal structures versus wounds from controlmice (Figure 3C; P < 0.05; n = 6). Woundproliferation was measured by Ki67 stain-ing at d 7. Ki67 was most prominent in thewound edge basilar KCs in control mice(30.7 ± 5.3%, Figure 3D). However, tung-sten-treated mice (Figure 3D) had signifi-cantly fewer proliferating basilar KCs (9.1 ± 4.8%, P < 0.05).

Topical H2O2 Reversed Tungsten-Induced Delay in Wound Healing andImproved Angiogenesis

To determine if the role of XOR inwound healing was mediated through



Figure 1. Examination of XOR expression in wounds and keratinocytes. (A) Western blot anal-ysis for XOR, iNOS and arginase I expression in mouse skin, wound edge and granulation tis-sue is shown. Wound samples at d 7 from three separate animals are represented. β-Actinwas used as a loading control. (B) XOR expression in healing wounds at d 14 (left) and nor-mal skin (right) was examined by immunostaining (green = XOR, blue = nuclei, red = colla-gen; magnification of 100×). The area of the healing wound that is shown is the healed areaadjacent to the wound bed. (C) XOR expression in early wounds (d 2) is demonstrated on apanoramic view showing normal skin, wound edge and the wound (green = XOR, blue = nu-clei). (D) Immunostaining of cultured undifferentiated (left) and differentiated (right) humankeratinocytes (200× magnification; green = XOR, blue = nuclei, red = keratin; representativephotomicrographs, n = 3/group). (D) Western blot analysis for XOR expression in culturedhuman keratinocytes. Treatment with Ca2+ differentiated the cells to mature keratinocytes.Positive control for XOR was mouse liver.

H2O2 production, wounds were treatedtopically with 0.15% H2O2 or saline everyother day until complete wound closure.H2O2 did not alter wound healing ratesin control mice (Figure 3E), but it signifi-cantly improved healing in tungsten-fedmice versus tungsten diet alone (18.5 ±0.7 versus 21.0 ± 0.7 d; P < 0.01), ap-proaching the rates in control mice. H2O2

treatment of wounds in tungsten-treatedmice showed a trend toward improvedangiogenesis (Figure 3F; P = 0.067).

Dietary Nitrite Manipulations Did NotAlter Systemic Levels of Nitrite orWound Healing

Mice receiving supplemental sodiumnitrite showed no improvement in woundhealing compared with controls (17.4 ±0.3 versus 16.7 ± 0.3 d, P = nonsignificant[NS], n = 5). Similarly, mice on a nitrite-free diet showed no change in wound

healing versus controls (17.2 ± 0.3 versus16.7 ± 0.3 d, respectively; P = NS). How-ever, serum nitrite levels remained un-changed in mice receiving nitrite supple-mentation or depletion compared withregular chow (282.5 ± 35.5 versus 254.8 ±64.0 versus 199.3 ± 53.9 nmol/L, respec-tively; P = 0.54, n = 4). The inability tochange serum nitrite levels with diet mayexplain the lack of effect observed.

XOR Mediates KC and EC FunctionKCs are the predominant cell type in

the wounds expressing XOR and tung-sten inhibition of XOR reduced basilarKC proliferation. Thus, we evaluated theeffect of XOR activity on KCs in vitro. In-hibition of XOR with allopurinol did notaffect proliferation in cultured humanKCs (Figure 4A). Nitrite treatment signif-icantly increased KC proliferation, whichwas reversed by allopurinol, indicating

nitrite functioned through XOR to pro-mote proliferation. H2O2 also increasedKC proliferation.

Because tungsten also reduced woundangiogenesis, the role of XOR in EC func-tion was also examined. By Western blot,cultured human ECs express XOR atbaseline (data not shown). There was atrend toward reduced proliferation whenECs were treated with allopurinol (P =0.064) and catalase (P = 0.073) (Figure4B), suggesting XOR and H2O2 contributeto baseline EC proliferation. Nitrite sig-nificantly increased EC proliferation, andthis was reversed with allopurinol, indi-cating that nitrite effects were mediatedby XOR. Treatment with H2O2 also in-creased proliferation. Similarly, nitriteand H2O2 both increased cell migration(Figure 4C). Allopurinol did not alter mi-gration but nitrite could enhance XOR-mediated NO production to increase mi-gration, suggesting that XOR did notregulate baseline migration but could beused to improve migration with nitrite.EC tubing, a measure of angiogenic activ-ity, was inhibited by allopurinol, whichreduced box formation and reduced thenumber of long tubes (Figures 4D, E). Ni-trite significantly increased tubing com-plexity and tube lengths in an allopurinolreversible fashion.

DISCUSSIONOur investigations identified a novel

role for XOR in the normal wound heal-ing process. High levels of XOR expres-sion were detected in the skin andwound edge of normal mice, with mostof the expression located within the basi-lar KCs. This expression was upregu-lated shortly after wounding and likelycoincides with the proliferative activityof the KCs at the wound edge. This en-hanced XOR expression is observedthroughout the healing process andlikely subsides as the KCs stop prolifer-ating and the epidermis matures. We alsoobserved XOR expression in the early in-flammatory infiltrates in the wound.XOR inhibition with tungsten signifi-cantly delayed wound closure. Localwound inhibition of XOR with topical al-



Figure 2. Effect of XOR inhibition on excisional wound-healing rates. (A) XOR activity wasmeasured in the skin, wound edge and granulation tissue in control, tungsten-fed or allop-urinol-fed mice (n = 3 mice/group; *P < 0.05 versus control and allopurinol groups, ‡P < 0.05versus control and tungsten groups). (B) Wound healing rates in control, tungsten-fed andallopurinol-fed mice as reported as days to reach 25%, 50%, 75% and 100% wound closure(*P < 0.05 and †P < 0.001 versus regular or allopurinol diet). (C) Effect of topical allopurinolon wound healing (n = 5–6 mice/group; P < 0.05 at all wound sizes). (E) Western blot for AOexpression in the skin and wound tissue of control mice with hepatocytes serving as thepositive control.

lopurinol also significantly delayedwound healing. Both of these findingssupport an important role for XOR inwound repair. The ability to reverse ef-fect of tungsten with topical H2O2 sug-gests that XOR contributes to woundH2O2 production and is required for nor-mal healing. In vitro studies demon-strated the ability of XOR to mediatepro-angiogenic functions in ECs and pro-liferation in KCs. These findings togetherstrongly support the contribution of XORin the normal wound healing process.

XOR represents a mix of xanthine oxi-dase (XO) and dehydrogenase (XDH)(both are involved in purine metabolism[21]) and is clinically notable for its rolein gout, where metabolism of xanthine touric acid (UA) results in joint depositionand inflammation. This pathway alsoyields H2O2 and superoxide, both ofwhich are associated with tissue injuryduring ischemia/reperfusion (I/R)(33,34). Inhibition of XOR reduces ROS-mediated I/R injury (28). In the settingof ischemia and hypoxia, XO predomi-nates and more efficiently generates su-peroxide, although XDH also possessespartial oxidase activity (35). While detri-mental in excess, physiologic levels ofROS are essential in cell signaling (36)and tissue homeostasis and repair (6,7).Most tissue production of ROS has beenattributed to nicotinamide adenine dinu-cleotide phosphate (NADPH) oxidase,and the role of XOR in the generation ofphysiologic ROS has been underappreci-ated. More recently, XOR gained atten-tion for its ability to convert nitrite toNO to mediate cytoprotection (23,37). Weand others have shown that XOR is re-quired for the beneficial actions of sup-plemental nitrite in the inhibition of inti-mal hyperplasia after vascular injury (23)and in reversing pulmonary hyperten-sion (24). XOR expression in skin hasbeen previously reported, linked to thepathogenesis of sunburn and skin in-flammation (24,25). Beyond that, little isknown about the role of XOR in the skin.

XOR inhibition with dietary tungstensignificantly delayed wound closure.Tungsten replaces the Mb within XOR,



Figure 3. Effect of XOR inhibition on wound angiogenesis and proliferation. (A) Effect of tung-sten and allopurinol diet on wound ROS/RNS production. Wound tissue was collected frommice at 5 d after wounding. ROS/RNS production was measured by using the OxiSelect In VitroROS/RNS Assay Kit, which detects H2O2, peroxyl radical, NO and peroxynitrite anion. Levels ofROS/RNS are normalized to the levels measured in mice fed regular diets (n = 5 mice/group, *P = 0.006 versus regular diet and P = 0.016 versus allopurinol diet). (B) Effect of XOR inhibitionon wound angiogenesis, as indicated by CD31-positive structures, was examined in wound tis-sues collected from control mice and tungsten fed mice immediately upon wound closure(CD31 for ECs, red; Hoechst 33325 for nuclei, blue; collagen, green). Representative photomi-crographs of wounds from mice fed regular and tungsten diets are shown (100× magnifica-tion). (C) Quantification of wound angiogenesis was performed by counting luminal structures(two sections per wound, n = 6/group; *P < 0.05 versus control diet). (D) Effect of XOR inhibitionwith tungsten on wound keratinocyte proliferation. Wound tissue was collected from controland tungsten diet–fed mice at d 7 after wounding. Tissues were stained for cell proliferationwith Ki67 (red) and for cell nuclei with Hoechst 33325 (blue). Representative photomicrographs(100× magnification) are shown. Red arrows indicate proliferating basilar KCs at the woundedge. Magnified views of the red box areas are provided. (E) Effect of H2O2 on wound healingin tungsten-treated mice. C57BL/6 mice were fed either regular diet or a tungsten-enricheddiet starting 2 wks before wounding and then maintained. Wounds were treated with eithertopical H2O2 (0.15%) or saline application every other day. Wounds were measured everyother day until complete closure. The time required to achieve 25%, 50%, 75% and 100% closurefor each treatment group is presented graphically (*P < 0.01 versus H2O2 and tungsten + H2O2).(F) Quantification of wound angiogenesis was performed in wounds treated with and withoutH2O2. Data are presented as number of CD31-positive lumen structures per high-power field (n = 6/treatment; *P < 0.02 versus all other groups; †P = 0.067 versus tungsten + H2O2).

irreversibly inhibiting its function (21).However, Mb is also essential for otheroxidoreductases such as AO and SO. Wefound low levels of these enzymes in

wound tissues. The other inhibitor ofXOR that we used was allopurinol, adrug used to clinically treat gout and asan investigative tool in other experi-

ments (23,24). In our studies, dietary al-lopurinol did not alter wound healingrates and raised concerns that tungsteneffects may be mediated by actions onAO or SO. However, we identified essen-tially no AO or SO expression and nomeasurable AO activity as well in un-treated wounds. We did find that tung-sten dramatically inhibited skin andwound XOR activity, while dietary allop-urinol only reduced it modestly in thewound edge and granulation tissue. Al-lopurinol has been used to block XOR ac-tivity in other tissues, such as in carotidand pulmonary arteries, but was muchless effective in our model. The reducedability of allopurinol to inhibit XOR inwounds may be due to poor biodistribu-tion in the relatively hypoperfusedwound bed and skin. Its bioavailabilitymay also be diminished by renal clear-ance and the requirement for conversionto its active form, oxypurinol (38,39). Inaddition, the binding of XOR to endothe-lial glycosaminoglycans results in resist-ance to allopurinol (38,39). In contrast,topical allopurinol significantly delayedwound closure and reduced XOR activityand wound ROS/RNS levels. These find-ings confirm the role of wound XORfunction in mediating normal wound re-pair. They also illustrate the potentialability to target wound XOR by topicaltreatments.

NO is required for normal tissue repair(12,14), and its deficiency contributes todelayed wound healing, whereas NOSgene transfer restores healing rates tonormal (19). Diabetic wounds have re-duced NOS expression and NO produc-tion (16,18), resulting in delayed woundhealing. Arginase I is also upregulatedduring wound healing (16) for the pro-duction of polyamines that are essentialfor cell proliferation and collagen synthe-sis but competes with NOS for arginineand can reduce NO production (17). Di-etary nitrite can serve as an alternatesource of NO through XOR (21,22). Thispathway has been shown to be biologi-cally important in the setting of vascularinjury and pulmonary hypertensionwhere dietary nitrite reduced the adverse



Figure 4. Effect of XOR function on keratinocyte and endothelial cell behavior in vitro. (A) Human KCs were cultured in 1% serum media with allopurinol (allo; 100 μmol/L), nitrite(100 μmol/L), nitrite + allopurinol and H2O2 (250 μmol/L) for 24 h. Proliferation was measuredby 3H-thymidine uptake, and results are expressed as fold-change over control (mean ±SEM of four experiments; *P < 0.05 nitrite versus control or nitrite + allopurinol). (B) ECs weregrown in 1% serum media with the addition of allopurinol (allo; 100 μmol/L), nitrite (100μmol/L), nitrite + allopurinol, H2O2 (250 μmol/L), catalase (1,000 U) or catalase + H2O2. ECproliferation was quantified by 3H-thymidine incorporation at 24 h (mean ± SEM of 6–11 ex-periments; *P < 0.05 versus control and nitrite + allopurinol; †P < 0.001 versus H2O2 + cata-lase). (C) EC migration was measured using the scratch assay. Cells were treated with ni-trite (100 μmol/L), H2O2 (125 μmol/L) or allopurinol (100 μmol/L), and migration wasquantified at 24 h by measuring the area occupied by cells within the scratch and nor-malizing to that achieved by control ECs (mean ± SEM of three separate experiments; *P <0.01 versus control and allopurinol). (D) EC capillary tubing was performed by culturing theECs on Matrigel™ with allopurinol or nitrite. Photomicrographs are shown from 6 h (repre-sentative of six experiments). (E) Quantification of EC tubing was performed by countingbox formation and total number of tubes measuring >110 pixels in length as an indicatorof long tubes (mean ± SEM of six separate experiments; long tube number *P < 0.05 versusall other groups; box number †P = 0.011 versus allopurinol + nitrite).

vascular remodeling (23,24,37). On thebasis of these reports and the abundanceof XOR in skin, we proposed that woundNO production may be increasedthrough XOR-mediated nitrite conver-sion to NO. Harnessing this local nitritereductase activity may be an attractivemechanism to increase wound NO pro-duction in settings where NOS activity isreduced, such as diabetic wounds(15–18). In our study, neither dietary ni-trite depletion nor supplementation al-tered healing rates. Measurements ofserum nitrite levels revealed that thesedietary manipulations did not changeserum nitrite levels. A potential explana-tion for these findings is that systemic ni-trite production may contribute to signif-icant serum stores, making it difficult toreduce or elevate systemic levels withdiet alone. The poor vascularity of thewounds may also reduce tissue nitritebioavailability. Thus, no conclusion canyet be drawn about the ability to manip-ulate XOR-mediated NO production inwound healing. In addition, normalwound healing is efficient and difficult toimprove. Future studies will examineXOR in models of impaired wound heal-ing such as diabetes.

Other products of XOR that may con-tribute to wound healing are ROS. Whilehigh levels of ROS in the skin are linkedto injury and disease (40,41), physiologiclevels of ROS, particularly H2O2, are es-sential for wound repair (6,8). Overex-pression of catalase, which breaks downH2O2, delayed wound healing, whereasphysiologic levels of H2O2 applied to awound-enhanced healing (6). In ze-brafish, H2O2 production is upregulatedimmediately after wounding and pro-motes the inflammatory phase by rapidleukocyte recruitment (8). The decompo-sition of H2O2 stalled vascular endothe-lial growth factor (VEGF)–VEGF receptorsignaling and impaired angiogenesis andwound healing (42). In all of these stud-ies, NADPH oxidase was determined tobe the source of the H2O2 and other ROS.In the tungsten-fed mice and in topicalallopurinol-treated wounds, ROS/RNSlevels were significantly reduced, sug-

gesting that a deficiency in oxidants con-tributes to poor wound healing. In a hy-poxic environment, such as the woundbed, the predominant ROS generated byXOR is H2O2 (43). Thus, we applied di-lute H2O2 topically on wounds in tung-sten-fed mice and restored healing tonear normal and improved wound an-giogenesis. These findings support thatXOR production of ROS/RNS, likelyH2O2, mediates normal wound healingthat was previously attributed toNADPH oxidase. Our studies do notquantify or discount the role of NADPHoxidase–derived ROS/H2O2 in woundhealing but strongly support the impor-tant contribution of XOR in the produc-tion of oxidants necessary for normal bi-ologic processes. Interestingly, Nam et al.(44) showed that NADPH oxidase inhibi-tion reduced KC H2O2 production atearly time points (30 min) but did notalter production at 20 h, supporting analternate source of H2O2.

Another product of XOR function isUA. UA has been associated with inflam-mation and has been detected in woundfluids, where its concentrations correlatedwith degree of impaired healing (45). UAmay have direct injurious actions inde-pendent of XOR-derived ROS, but thishas yet to be determined. We hypothesizethat, in the setting of abnormal wound re-pair, such as in diabetes, XOR may be up-regulated and leads to the increased ox-idative stress that results in protractedinflammation and poor healing. NO andH2O2 have been implicated in KC prolif-eration and in the modulation of epider-mal growth factor receptor in lung andhuman foreskin KC (46,47). Similarly,they have also been implicated in woundangiogenesis (5). We found prominentproliferation in the basilar KC lining thewound edge, and tungsten reduced thenumber of proliferating KCs in thewound. We also observed reduced angio-genesis in wounds from tungsten-treatedmice, supporting a role for XOR inwound angiogenesis. Our in vitro studiesconfirm an important role for XOR in me-diating EC proliferation, migration andangiogenic activity. Allopurinol blocked

proliferation and tube formation in vitro,indicating that endogenous XOR activitycontributes to these key EC behaviors.The ability of nitrite and H2O2 to enhanceEC proliferation and migration suggeststhat both NO and H2O2 production likelymediate the wound-healing properties ofXOR in vivo. Kou et al. (48) reported therole of endogenous XO in maintainingVEGF- induced EC survival. It was alsoreported that NO derived from XOR reg-ulates hypoxia-inducible factor 1-α(HIF1α)- and VEGF-dependent angiogen-esis (49). The impaired angiogenesis re-sulting from XOR inhibition may be dueto loss of H2O2-mediated induction of an-giogenic VEGF (42). It was also shownthat VEGF downstream signaling de-pends on ROS production (42), again pre-sumably through NADPH oxidase. Fu-ture studies will isolate the contributionof XOR-derived ROS from that ofNADPH oxidase.

Isolated XOR deficiency in humans israre and is only documented in a few casereports (50,51). However, Mb cofactor de-ficiency has been described and is associ-ated with severe refractory seizure activ-ity with childhood fatality (reviewed inref. [52]). Global XOR deficiency is fatal,and, thus, no XOR-deficient mouse is cur-rently available to assist in the investiga-tion of XOR function in wound healing.The generation of tissue-specific or condi-tional XOR knockouts will be extremelyhelpful and is currently underway.

CONCLUSIONWe provide the first evidence that XOR

contributes to normal wound healing.Our data showed that XOR stimulatesKC proliferation and wound angiogene-sis through the production of ROS. Thein vitro effects of XOR on EC behaviorare mediated by both NO and H2O2. Fu-ture studies are necessary to better definethe role of XOR-mediated NO generationin these effects. In addition, it is impor-tant to determine how XOR activity isregulated in impaired wound healingsuch as in diabetes, venous stasis and is-chemia. Targeting wound XOR may pro-vide a way to manipulate local ROS and



NO production, possibly achievedthrough topical routes that are extremelyattractive in the management of patientswith difficult wounds.

ACKNOWLEDGMENTSWe gratefully acknowledge the excel-

lent technical assistance provided by NJ Hundley, SI Zharikov and N Cantu-Medellin. This material is based on worksupported in part by the Department ofVeterans Affairs, Veterans Health Admin-istration and Office of Biomedical Labo-ratory Research and Development (ET),through funding from the National Insti-tutes of Health (NIH) (NIH T32 HL098036to MC Madigan, RM McEnaney and AJ Shukla and NIH R01 HL058115 toMM Tarpey). M Gladwin received re-search support from NIH grantsR01HL098032, R01HL096973 andPO1HL103455 as well as from the Insti-tute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania.

The contents of this manuscript do notrepresent the views of the Department ofVeterans Affairs or the United StatesGovernment.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

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Cite this article as: Madigan MC, et al. (2015) Xan-thine oxidoreductase function contributes to nor-mal wound healing. Mol. Med. 21:313–22.

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Xanthine Oxidoreductase Function Contributes to Normal Wound … · 2018-10-04 · Xanthine Oxidoreductase Function Contributes to Normal Wound Healing Michael C Madigan,1,2 Ryan