Topical Erythropoietin Treatment Accelerates the Healingof Cutaneous Burn Wounds in Diabetic Pigs Through anAquaporin-3–Dependent MechanismSaher Hamed,1 Yehuda Ullmann,2 Dana Egozi,2 Aviad Keren,3 Essam Daod,2 Omer Anis,2 Hoda Kabha,1

Mark Belokopytov,1 Manal Ashkar,1 Rona Shofti,3 Asaph Zaretsky,3 Michal Schlesinger,3 Luc Teot,4 andPaul Y. Liu5

Diabetes 2017;66:2254–2265 | https://doi.org/10.2337/db16-1205

We have previously reported that the topical application oferythropoietin (EPO) to cutaneous wounds in rats and micewith experimentally induced diabetes accelerates theirhealing by stimulating angiogenesis, reepithelialization, andcollagen deposition, and by suppressing the inflammatoryresponse and apoptosis. Aquaporins (AQPs) are integralmembrane proteins whose function is to regulate intracel-lular fluid hemostasis by enabling the transport of water andglycerol. AQP3 is the AQP that is expressed in the skinwhere it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, weprovide the results of an investigation that examined thecontribution of AQP3 to the mechanism of EPO action onthe healing of burn wounds in the skin of pigs with experi-mentally induced type 1 diabetes. We found that topical EPOtreatment of the burns accelerated their healing through anAQP3-dependent mechanism that activates angiogenesis,triggers collagen and hyaluronic acid synthesis and the for-mation of the extracellular matrix (ECM), and stimulatesreepithelialization by keratinocytes. We also found thatincorporating fibronectin, a crucial constituent of the ECM,into the topical EPO-containing gel, can potentiate the accel-erating action of EPO on the healing of the burn injury.

According to the U.S. Centers for Disease Control and Pre-vention,.400 million people worldwide have diabetes. TheCenters for Disease Control and Prevention also estimatesthat 5% of these individuals will develop a diabetic skin

ulcer (DSU) and that 1% will require a lower-extremity am-putation. Despite many advances in wound care and man-agement (1), wound healing in diabetes is delayed becauseall phases of the orchestrated cascade of cellular and bio-chemical events of wound healing are disrupted (2–4).Additionally, the healing of a DSU is delayed because ofimpaired angiogenesis, insufficient blood flow, increased in-flammation, diminished proliferation of fibroblasts (5), andreduced reepithelialization by keratinocytes (6–8).

The glycoprotein hormone erythropoietin (EPO) regulatesred blood cell mass and is an approved drug for treatinganemia. EPO also has nonhematopoietic targets in the skin,and we have shown (9) that these targets participate in thehealing of skin wounds. We previously reported that thehealing of cutaneous wounds in rats and mice with exper-imentally induced diabetes is accelerated after the topicalapplication of recombinant human EPO to the cutaneouswounds by stimulating angiogenesis, reepithelialization,and collagen deposition, and by suppressing the inflamma-tory response and apoptosis (10). The beneficial actions ofEPO on wound healing in diabetes are complemented byfibronectin (FN). FN facilitates the formation of the pro-visional wound matrix and prevents its dissociation (11).

Aquaporins (AQPs) are integral membrane proteinswhose function is to regulate intracellular fluid hemostasisby enabling the transport of water and glycerol. AQPs areexpressed in the plasma membranes of keratinocytes in thebasal layer of the skin and the medullary collecting ducts of

1Department of Research & Development, Remedor Biomed Ltd, Nazareth Illit,Israel2Department of Plastic Surgery, Rambam Health Care Campus, Haifa, Israel3Skin Research Laboratory, Ruth and Bruce Rappaport Faculty of Medicine,Technion-Israel Institute of Technology, Haifa, Israel4Department of Plastic & Reconstructive Surgery and Wound Healing, HopitalLapeyronie, Montpellier, France5Department of Plastic Surgery, Rhode Island Hospital, Warren Alpert MedicalSchool, Brown University, Providence, RI

Corresponding author: Saher Hamed, saher@remedor.com.

Received 4 October 2016 and accepted 24 April 2017.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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the kidney (12). Downregulated expression of AQPs may bethe cause of the reduction in urinary-concentrating abilityin individuals with acute renal failure, and EPO can preventthis downregulation (13). AQP3 is the AQP that is ex-pressed in the skin (14), where it facilitates cell migra-tion and proliferation and reepithelialization during woundhealing (15–18). A positive role for moisture in healing skinwounds was first shown in 1962, when Winter (19) inves-tigated scab formation and the rate of epithelialization ofsuperficial wounds in pig skin and reported that moistwounds heal faster than dry ones. As a corollary, drynessof the skin of the feet correlates with foot ulceration inpatients with diabetes (20). A specific role for AQP3 in di-abetic wound healing was posited when it was found thatAQP3 is downregulated in the regenerating epidermis dur-ing the healing of full-thickness cutaneous wounds in ratswith diabetes (21). The work of these investigators and ourresults indicated that EPO could be used to stimulate thehealing of nonhealing wounds. Such findings also suggestthe existence of a causal relationship between impaired AQP3expression and delayed reepithelialization in diabetes, whichinvolves impaired movement and proliferation of those cellsthat participate in angiogenesis, reduced production of theextracellular matrix (ECM) by fibroblasts, and failure of ker-atinocytes to reepithelialize a cutaneous skin wound.

It is against this background that we posited that thetherapeutically beneficial action of EPO on the healing of aDSU is due in part to the ability of EPO to stimulate AQP3expression in skin. We tested this hypothesis in pigs withexperimentally induced type 1 diabetes and a partial thicknessskin burn.

RESEARCH DESIGN AND METHODS

Pig Model of Type 1 DiabetesThe study comprised four 60-kg healthy female pigs (Susdomesticus) purchased from Lahav Institute (Kibbutz Lahav,Israel). The pigs were singly housed in pens in a room withan artificial 12-h light/dark cycle and had free access to astandard laboratory chow and water. All procedures wereperformed in the Technion according to the Israeli nationallegislation on the use of animals for experimental purposes.

Diabetes induction, creation of the burn wounds, woundtreatment, dressing changes, and data collection were per-formed under general anesthesia. An intravenous catheterwas permanently placed in the right jugular vein in the fourpigs for blood sampling during the investigation. Heart rate,blood oxygen saturation, and the body temperature of thepig were monitored. On day 14, the last day of the exper-iment, specimens were collected and the pigs were then hu-manely killed.

Diabetes was induced in two pigs using streptozotocin(200 mg/kg; Alexis Biochemicals) according to a previouslydescribed protocol (22). Blood glucose levels were checkedevery 15 min for 6 h after streptozotocin administration andat least twice daily during the study period using a glucom-eter (FreeStyle FREEDOM Lite; Abbott). Long-acting insulin(24 international units [IU] Lantus; Aventis Pharmaceuticals)

was injected intravenously to maintain the fasting bloodglucose levels of the diabetic pigs between 300 and400 mg/dL.

Creation of Partial Thickness Skin Burn WoundsDiabetes was maintained for 1 month before partial thick-ness skin burn wounds were created. The bristles of the dorsalskin on each side of the vertebral column of each pig wereremoved using VEET depilatory cream (Reckitt Benckiser)before creating 12-cm2 circular partial thickness skin burnwounds using an aseptic technique. The burn wounds werecreated using a previously described method (23). Briefly,four cylindrical brass rods, each with an ;3.8-cm diameterand weighing 358 g, were placed in hot water (92°C) for2 min. Four groups of six partial thickness burn woundswere created by placing of the rod perpendicular on thedorsal skin of each pig for 20 s with no additional pressure“producing partial thickness third-degree burns.” In the twodiabetic pigs, an additional group of six partial thicknessburn wounds was created.

Topical FormulationsSix different gels for topical wound treatment were pre-pared in the Remedor Biomed Ltd. laboratory in accordancewith the following recommendations by the U.S. Pharma-copeial Convention: 1) a gel that contained no active ingre-dients (vehicle gel); 2) a gel that contained 2,000 IU/g EPO(high-dose EPO); 3) a gel that contained 500 IU/g EPO (low-dose EPO); 4) a gel that contained 300 mg/g FN; 5) a gelthat contained 2,000 IU/g EPO and 300 mg/g FN; and 6) agel that contained 0.1 mmol/L mercuric chloride (HgCl2)and 2,000 IU/g EPO. Recombinant human EPO was pur-chased as an injection (EPREX 40,000 IU; Janssen-Cilag Ltd.,High Wycombe, U.K.). FN was purchased as a 1 mg/mLsolution (EMD Millipore). HgCl2 (0.1 mmol/L) was incor-porated into the gel that contained 2,000 IU/g EPO in orderto block cutaneous AQP3 activity in the wounds (negativecontrol) and was purchased from Sigma-Aldrich. The resultsof the stability testing of the gels established that EPO andFN are stable in the gel for at least 6 months at 4°C, asdetermined by ELISA.

Treatment of WoundsEach group of the six partial thickness burn wounds wasrandomly assigned to be treated with one of the followingtopical gels: the vehicle-containing gel; the high-dose EPO-containing gel; the FN-containing gel; and the EPO/FN-containing gel. The additional group of the burn wounds inone diabetic pig was treated with the EPO/HgCl2-containinggel, and the additional group of burn wounds in the seconddiabetic pig was treated with a low-dose EPO-containing gel(low-dose EPO). A simple randomization sequence was gen-erated by computer software. After allocation of the sixpartial thickness burn wounds in four different groups ineach pig, gel (3 g) was topically applied to each wound every2 days of the 14-day study period. In order to prevent re-moval of the gel after its application by rubbing and toprotect the burn wounds between treatments, the treated

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wounds were covered with nonadherent gauze pads, whichwere stabilized by Tensoplast Elastic Adhesive Bandaging(Smith & Nephew).

Study ParametersOn each treatment day, pigs were weighed; each wound wasphotographed using a 12-megapixel digital camera (Olympus);and a blood sample was collected for determining the redblood cell, leukocyte, and platelet counts, and the plasmahemoglobin and glycated hemoglobin (HbA1c) levels.Punch biopsy specimens from randomly selected areas ofthe regenerating skin of the vehicle-treated and treatedburn wounds and the uninjured skin of the pigs were col-lected on days 2, 7, and 14 using a 6-mm circular blade.Samples of each biopsy specimen were fixed immediately in10% neutral buffered formalin for histological determina-tion of the microvascular density (MVD) or stored in liquidnitrogen for immunohistochemistry, Western blot analysis,ELISA, and PCR. The slides were examined under a NikonEclipse E800 Upright Microscope, and images of the sec-tions were captured and analyzed by Metamorph ImageAnalysis Software (Nikon Instruments Inc.).

Wound Closure RateThe wound closure rate was calculated from the area ofreepithelialized tissue in the burn wound. In order to calcu-late the rate, the area of each burn wound was categorizedinto the following three areas: 1) a scab area where theburnt skin becomes a rigid crust after creation of the burninjury; 2) a red area where the scab can be detached but hasno epithelial cells; and 3) a white area where the scabcan be detached and contains new epithelial cells. Thewound closure rate was calculated by measuring the whitearea on days 2, 4, 7, 9, 11, and 14. To this end, transpar-ent paper was placed over each wound, and the shape ofthe white area was drawn on the paper. The transparentpaper was then superimposed onto a 1-mm2 graph paperin order to measure the white area in the wound. The rateof time-dependent changes in the size of the white areawere calculated using the following formula:

% White  areaðwound  closureÞ5White  area  on  day  XWound  area  on  day  0

3100

Measurement of Blood FlowBlood flow in the wounds was measured noninvasively by alaser Doppler perfusion imaging system (PeriScan PIM2 System; Perimed) during the study period according tothe manufacturer instructions. Perfusion in a region ofinterest (ROI) is measured on a scale of six colors in whichdark blue depicts the lowest perfusion rate and red depictsthe highest perfusion rate using PIMSoft software for bloodperfusion imaging (Lisca Development AB). For each burnwound, the ROI was a 12-cm2 circle that was drawn aroundthe initial burn wound, and the blood flow in the wound isthe average value of all colors in the ROI.

Determination of AngiogenesisThe MVD and the extent of angiogenesis in the regenerat-ing skin of the healing wounds was determined by staining5-mm thick sections of the formalin-maintained samplespunch biopsies of the wounds with CD31 (R&D Systems).The number of capillaries in the regenerating skin in eachwound site was counted in five random microscopic fields(320 magnification).

Detection of AQP3AQP3 was detected in wound-free healthy and diabetic pigskin 1 month after diabetes induction and 1 day beforecreation of the burn wounds and in the regeneratingskin of the wounds during the study period by immu-nohistochemistry and immunofluorescence. For AQP3immunohistochemistry, 5-mm-thick sections were stainedwith rabbit polyclonal AQP3 primary antibody (Santa CruzBiotechnology). For confirming the presence of AQP3 byimmunofluorescence, another set of the AQP3-stained sec-tions were counterstained with the nuclear stain TOPRO-3(Invitrogen) and examined under a confocal microscope(BIO-RAD).

Detection of CollagenThe amount of collagen in the regenerating skin was deter-mined in specimens that were stained with Masson’s tri-chrome (Sigma-Aldrich). Using this method, collagen fibersstained blue, nuclei stained black, and cytoplasm and musclefibers stained red.

Determination of the Amount of CollagenHydroxyproline (HP), an amino acid constituent of type Icollagen, was used as a marker and an indicator of theamount of collagen in the skin specimens from the regen-erating skin of the burn wounds. The amount of HP wasdetermined using a previously described protocol (10).

Amount of Hyaluronic AcidSince hyaluronic acid (HA) is linked to skin strength andhydration, the HA amount in the regenerating skin of theburn wound tissues was determined in specimens that werecollected from the nondiabetic and diabetic pigs using theHyaluronan Quantikine ELISA Kit (R&D Systems) accord-ing to the manufacturer protocol.

Western Blot AnalysisThe expression levels of AQP3, endothelial nitric oxidesynthase (eNOS), HA synthase (HAS) 1, and HAS2 weredetermined in the regenerating skin of the burn woundtissues. Briefly, tissue extracts were prepared using radio-immunoprecipitation assay buffer (Elpis-Biotech). Proteinsfrom tissue lysates were separated by 10% SDS-PAGE andthen transferred to nitrocellulose membranes. The mem-branes were probed with indicated antibodies. Proteinexpression levels were detected by densitometry using theImmun-Star horseradish peroxidase chemiluminescencedetection system (BIO-RAD). The results were expressedas a percentage of the average protein level of duplicate

2256 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

readings in the vehicle-treated burn wounds of the healthypigs (100%).

Real-time PCRTotal RNA was extracted from homogenates prepared fromregenerating skin of the burn wound tissues using theMasterPure RNA Purification Kit (EPICENTRE Biotechnol-ogies). cDNA was generated by absolute qPCR mixes reversetranscription reagents (ABgene) and analyzed by qPCRusing SYBR Green PCR Master Mix (Molecular Probes) in aRotor-Gene 6000 Cycler (Corbett Life Science). The resultswere expressed as a percentage change from control afternormalization to an endogenous reference gene (GAPDH).

StatisticsAll statistical analyses were performed using a computerizedstatistical program (GraphPad Prism version 5.0; GraphPadSoftware Inc.), and all data are presented as the mean orpercentage 6 SD. Statistical significance was set at 5%. Atwo-tailed Student t test was used to compare study param-eters of the healthy and diabetic pigs, and a two-way ANOVAwith Bonferroni correction to control for type I error wasused for multiple comparisons. The wound closure ratesamong groups were compared and analyzed using a one-way ANOVA with Tukey post hoc test and a two-way ANOVAwith Bonferroni correction to control for type I error in themultiple comparisons.

RESULTS

Topical treatment of the burns with the vehicle-, FN-, EPO-and EPO/FN-containing gels did not change 1) the redblood cell, leukocyte, or platelet counts and the elevatedblood glucose levels in the diabetic pigs and 2) the bloodhemoglobin and HbA1c levels in the control and diabeticpigs (Table 1).

Topical EPO Accelerates Wound Closure and IncreasesBlood Flow in the Regenerating Skin of Diabetic Woundsin a Dose-Dependent Manner, and This Effect IsPotentiated by FNThe wound closure and blood flow rates of the vehicle-treated diabetic wounds were significantly lower than those

of the vehicle-treated healthy wounds from day 2 onward.The wound closure and blood flow rates of the EPO-treated,the FN-treated, and the EPO/FN-treated diabetic woundswere significantly greater than those of the vehicle-treateddiabetic wounds. The most significant differences in woundclosure rates were detected between the vehicle-treated andthe EPO/FN-treated diabetic wounds. From day 4 onward,the wound closure rate of the EPO/FN-treated wounds wassignificantly faster than those of the FN-treated and theEPO-treated diabetic wounds. The blood flow in the EPO-treated diabetic wounds was not significantly different fromthat of the EPO/FN-treated diabetic wounds, and thesetwo blood flows were significantly higher than those in thevehicle-treated and FN-treated diabetic wounds (Fig. 1A–C).

The wound closure and blood flow rates of the low-doseEPO-treated diabetic wounds were significantly greater thanthose of the vehicle-treated diabetic wounds, and the woundclosure and blood flow rates of the high-dose EPO-treateddiabetic wounds were significantly greater than those of thelow-dose EPO-treated diabetic wounds from day 9 onward(Fig. 1D–F).

Topical FN Does Not Affect Wound Closure and BloodFlow in the Regenerating Skin of Nondiabetic WoundsThe wound closure and blood flow rates of the vehicle-treated and FN-treated wounds of healthy pigs were verysimilar. From day 4 onward, the wound closure and bloodflow rates of the EPO-treated nondiabetic wounds weresignificantly greater than those of the vehicle-treated andFN-treated nondiabetic wounds. The most significant dif-ferences in wound closure and blood flow rates weredetected between the EPO/FN-treated wounds and thevehicle-treated and the FN-treated wounds in the healthypigs over the period from day 4 to day 11 (Fig. 1G–I).

Topical EPO Increases the MVD and eNOS ExpressionLevels in the Regenerating Skin of Diabetic Wounds, andFN Potentiates These EffectsThe MVD and eNOS expression levels in the vehicle-treateddiabetic wounds were lower than those in the vehicle-treated nondiabetic wounds after 14 days. Topical FN treat-ment had no effect on the MVD and eNOS expression levels

Table 1—Effect of diabetes on the weight of the body of the pig and topical wound treatment on hematology

Nondiabetic pigs (n = 2) Diabetic pigs (n = 2)

Day 0 Day 7 Day 14 Day 0 Day 7 Day 14

Body weight (kg) 68 6 3 73 6 4 78 6 4 54 6 3* 53 6 3* 52 6 3*

Blood glucose levels (mg/dL) 109 6 8 99 6 10 102 6 7 587 6 123* 601 6 134* 569 6 187*

HbA1c (%) 5.1 6 0.3 4.9 6 0.3 5.0 6 0.3 5.3 6 0.4 5.2 6 0.4 5.5 6 0.4

RBC count (106/mL) 7.2 6 0.4 6.9 6 0.3 7.4 6 0.5 6.8 6 0.4 6.6 6 0.5 7.0 6 0.7

Leukocyte count (103/mL) 17.6 6 0.7 18.2 6 0.9 18.0 6 0.8 19.6 6 1.1 17.5 6 0.8 18.3 6 0.9

Platelet count (103/mL) 407 6 71 461 6 92 387 6 69 419 6 73 428 6 82 456 6 101

Hemoglobin levels (g/dL) 9.6 6 0.6 10.7 6 0.9 10.7 6 1.1 8.8 6 0.7 10.9 6 1.2 10.8 6 0.9

Values are presented as the mean 6 SD. Statistical significance is set at 5%. n, number of pigs; RBC, red blood cell. *P , 0.05 is thesignificance of the difference between the two groups at days 0 and 7 and day 14.

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in the diabetic and nondiabetic wounds. On the other hand,topical EPO treatment for 14 days significantly increasesthe MVD and eNOS expression levels in the diabetic and

nondiabetic wounds (Fig. 2A–E). In the EPO/FN-treatednondiabetic wounds, the MVD and eNOS expression lev-els were not significantly different from those in the

Figure 1—Topical EPO application accelerates wound closure and increases blood flow in the regenerating skin of diabetic wounds in a dose-dependent manner; an effect potentiated by FN. In the regenerating skin of nondiabetic wounds, topical application of FN does not affect woundclosure and blood flow rates. Representative set of photographic images (A) and the corresponding laser Doppler scans (B) of a vehicle-, an FN-,an EPO-, and an EPO/FN-treated burn wound in a nondiabetic and diabetic pig on days (d) 0, 2, 4, 7, 9, 11, and 14. C: Wound closure rates ofthe vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds in the diabetic pigs. Representative set of photographic images (D) andthe corresponding laser Doppler scans (E) of a vehicle-, a low-dose EPO-, and a high-dose EPO-treated burn wound in a diabetic pig on days0, 4, 9, and 14. F: Wound closure rates of the vehicle-, the low-dose EPO-, and the high-dose EPO-treated burn wounds in the diabeticpigs. Representative set of photographic images (G) and the corresponding laser Doppler scans (H) of a vehicle-, an FN-, an EPO-, and anEPO/FN-treated burn wound in a nondiabetic pig on days 0, 4, 9, and 14. The white circles in the laser Doppler scans represent the burn woundarea on day 0. The dark blue color represents nonvascularized regions, and the yellow and red colors represent vascularized regions with thered-colored regions depicting regions that are more vascularized than the yellow-colored regions. I: Wound closure rates of the vehicle-, FN-,EPO-, and EPO/FN-treated burn wounds in the nondiabetic pigs. The sample size in each treatment group was 12 except in the low-dose EPO-treated burn wound group, where the sample was 6. *P < 0.05; **P < 0.01, significance of the difference between the vehicle-treated burnwounds and the other treatments according to the results of a two-way ANOVA with Bonferroni correction. †P < 0.05, significance of thedifference between 1) the EPO-treated or EPO/FN-treated burn wounds and the FN-treated burn wounds and 2) the high-dose EPO-treated burnwounds and the low-dose EPO-treated burn wounds according to the results of a two-way ANOVA with Bonferroni correction. EPO/H, high-dose EPO-treated burn wounds; EPO/L, low-dose EPO-treated burn wounds.

2258 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

EPO-treated nondiabetic wounds (Fig. 2B and C). In con-trast, the MVD and eNOS expression levels of the EPO/FN-treated diabetic wounds were significantly higher thanthose in the EPO-treated diabetic wounds (Fig. 2D and E).

Topical EPO Increases Collagen Deposition and HASynthesis in the Regenerating Skin of Diabetic Wounds,and FN Potentiates This EffectThe amounts of HP and HA and the expression levels of thetwo HASs, HAS1 and HAS2, in the vehicle-treated diabeticwounds were lower than those in the vehicle-treated non-diabetic wounds. Topical FN treatment for 14 days did notchange the HP and HA amounts and the HAS1 and HAS2expression levels in the diabetic and nondiabetic wounds.

On the other hand, topical EPO treatment significantlyincreased the HP and HA amounts and the HAS1 and HAS2expression levels in diabetic and nondiabetic wounds (Fig.3A–G). HP amounts in the EPO/FN-treated nondiabetic anddiabetic wounds were significantly higher than those in theEPO-treated nondiabetic and diabetic wounds, respectively(Fig. 3B and C). The HA amount and the HAS1 and HAS2expression levels in the EPO/FN-treated nondiabetic woundswere similar to those of the EPO-treated nondiabetic wounds(Fig. 3D and F). In contrast, the HA amount and the HAS1and HAS2 expression levels in the EPO/FN-treated diabeticwounds were significantly higher than those in the EPO-treated diabetic wounds (Fig. 3E and G).

Figure 2—Topical EPO application increases MVD and eNOS expression in the regenerating skin of diabetic wounds, and these effects arepotentiated by FN. A: Representative set of micrographs that show immunohistochemical staining for CD31 expression by vascular endothelialcells of the capillaries in the regenerating skin of the vehicle-, FN-, EPO-, and EPO/FN-treated burn wounds of the nondiabetic pigs (left panel) andof the diabetic pigs (right panel) that were collected after 14 days of treatment. The MVD in the regenerating skin was determined by counting thenumber of capillaries in five random microscopic fields (320 magnification) under a light microscope at each wound site. B: The MVD in theregenerating skin of the vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs. C: Representative Westernblots of eNOS expression in tissues that were collected on day 14 and then measured in lysates that were prepared from the regenerating skin ofvehicle-, FN-, EPO-, and EPO/FN-treated burn wounds of the nondiabetic pigs. D: The MVD in the regenerating skin of the vehicle-, the FN-, theEPO-, and the EPO/FN-treated burn wounds of the diabetic pigs. E: Representative Western blots of eNOS expression in tissues that werecollected on day 14 and then measured in lysates that were prepared from the regenerating skin of vehicle-, FN-, EPO-, and EPO/FN-treated burnwounds of the diabetic pigs. a-Actin was used to normalize protein loading, and the blots were derived from samples that were analyzedconcomitantly on a separate gel. The sample size of each treatment group was 12, and data are expressed as the average of duplicatemeasurements 6 SD for each treatment group. *P < 0.05 and **P < 0.01, significance of the difference between the vehicle-treated burnwounds and the other treatments according to the results of a one-way ANOVA with a Tukey post hoc test. †P < 0.05, significance of thedifference between EPO/FN-treated and EPO-treated burn wounds according to the results of a one-way ANOVA with a Tukey post hoc test.Scale bars: 200 mm.

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AQP3 Expression Is Decreased in Wound-FreeDiabetic SkinAQP3 protein and mRNA expression levels in wound-freediabetic pig skin were significantly lower (P, 0.01 for both)than those in wound-free healthy pig skin (Fig. 4A–G).

Topical EPO Stimulates AQP3 Expression in theRegenerating Skin of Diabetic Wounds, and FNPotentiates This EffectAfter 14 days, AQP3 protein and mRNA expression levels inthe vehicle-treated diabetic wounds were significantly lowerthan those in the vehicle-treated nondiabetic wounds. Inthe diabetic and control pigs, AQP3 protein and mRNAexpression 1) in the EPO-treated wounds were significantlyhigher than those of the vehicle-treated and FN-treatedwounds, 2) in the EPO/FN-treated wounds were signifi-cantly higher than that of the EPO-treated wounds, and

3) in the vehicle-treated and FN-treated wounds were notdifferent from each other (Fig. 4J–O).

AQP3 Protein Expression Correlates Positively With theExtent of Angiogenesis and HP and HA Amounts in theRegenerating Skin of Diabetic Burn WoundsWe used the Pearson correlation to investigate the rela-tionships among AQP3 protein expression levels, the extentof angiogenesis, and the amounts of HP and HA in the burnwounds of healthy and diabetic pigs. In the diabetic andhealthy pigs, AQP3 protein expression levels correlatedpositively with the extent of angiogenesis (Fig. 5A and B),the HP amount (Fig. 5C and D), and the HA amount (Fig.5E and F). Interestingly, these correlations were signifi-cantly stronger in the regenerating skin of the burn woundsof the diabetic pigs than those found in the regeneratingskin of the burn wounds of the healthy pigs.

Figure 3—Topical EPO increases the amount of HP and HA synthesis in the regenerating skin of diabetic wounds, and this effect is potentiatedby FN. A: Representative set of micrographs that shows immunohistochemical staining for the amount of HP by Masson trichrome staining inthe regenerating skin of the vehicle-, FN-, EPO-, and EPO/FN-treated burn wounds of the nondiabetic pigs (left panel) and of the diabetic pigs(right panel) that were collected after 14 days of treatment. The HP content in regenerating skin of the vehicle-, the FN-, the EPO-, and theEPO/FN-treated burn wounds of the nondiabetic pigs (B) and of the diabetic pigs (C) on day 14. The HA amount in the regenerating skin of thevehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs (D) and of the diabetic pigs (E). RepresentativeWestern blots of HAS1 and HAS2 expression levels in tissue samples that were collected on day 14 and then measured in lysates that wereprepared from the regenerating skin of the vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs (F) and ofthe diabetic pigs (G). a-Actin was used to normalize protein loading, and the blots were derived from samples that were analyzed concomitantlyand run on a separate gel. The sample size of each treatment group was 12, and the data are expressed as the average of duplicatemeasurements of the amount of HP or HA6 SD. *P< 0.05; **P< 0.01, significance of the difference between the vehicle-treated burn woundsand the other treatments according to the results of the one-way ANOVA with Tukey post hoc test. †P < 0.05, significance of the differencebetween EPO/FN-treated and EPO-treated burn wounds according to the results of a one-way ANOVA with a Tukey post hoc test. Scale bars:200 mm.

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Figure 4—AQP3 expression is decreased in wound-free diabetic skin. Topical EPO application stimulates AQP3 expression in the regeneratingskin of diabetic wounds, and this effect is potentiated by FN. Representative set of micrographs of immunohistochemical staining for AQP3 inthe wound-free skin of a nondiabetic pig (A) and of a diabetic pig (B). Scale bars: left, 50 mm; right, 200 mm. Representative set of micrographs ofimmunofluorescence staining for AQP3 in the wound-free skin of a nondiabetic pig (C) and of a diabetic pig (D). E and G: Quantification (E) andrepresentative Western blots (G) of AQP3 expression in the wound-free skin of diabetic pigs 30 days after diabetes induction in wound-free skinof the nondiabetic pigs. F: AQP3 mRNA levels of total RNA that was isolated from wound-free skin collected on day 30 after diabetes induction.Values are expressed as the mean6 SD of triplicate measurements of AQP3 protein expression and mRNA expression and as a percentage ofthe expression in wound-free nondiabetic skin (100%). Representative set of micrographs of immunohistochemical staining for AQP3 in theregenerating skin of the vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs (H) and the diabetic pigs (I),which were collected after 14 days of treatment. Scale bars: left, 50 mm; right, 200 mm. Quantification of Western blot levels of AQP3 proteinexpression after a 14-day treatment of burn wounds in the nondiabetic pigs (J) and the diabetic pigs (K) with a vehicle-, an FN-, an EPO-, and anEPO/FN-containing gel. Values are expressed as the mean6 SD of triplicate measurements of AQP3 protein expression in vehicle-treated burnwounds of the nondiabetic pigs (100%). Representative Western blots of AQP3 protein expression in lysates of the regenerating skin of burnwounds from the nondiabetic pigs (L) and the diabetic pigs (M) after 14 days of treatment. a-Actin was used to normalize protein loading, and theblots were derived from samples that were concomitantly run on a separate gel. AQP3 mRNA levels of total RNA that was isolated from lysatesof the regenerating skin of vehicle-, FN-, EPO-, and EPO/FN-treated burn wounds of the nondiabetic pigs (N) and the diabetic pigs (O). Valuesare expressed as the average6 SD of duplicate measurements of AQP3 mRNA expression and as a percentage of the expression in wound-free

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AQP3 Inhibition Reduces the Effect of Topical EPO onthe Wound Closure Rate, the Extent of Angiogenesis,and the HP and HA Amounts in Diabetic Burn WoundsWhen AQP3 activity was blocked by HgCl2, the wound clo-sure rates of EPO/HgCl2-treated diabetic wounds were sig-nificantly reduced. This reduction in wound closure rate wasaccompanied by a reduced blood flow (Fig. 6A and B), loweNOS expression levels, low HAS1 and HAS2 expression lev-els (Fig. 6C), a low extent of angiogenesis, a reduced HPamount, and a reduced HA amount. Moreover, the woundclosure rates; the expression levels of eNOS, HAS1, andHAS2; and the amounts of HP and HA in the EPO/HgCl2-treated diabetic wounds were significantly lower than thosein the EPO-treated diabetic wounds (Fig. 6D–F).

DISCUSSION

The healing of a DSU is delayed because of impairedangiogenesis, reduced cutaneous cellular activity, and in-creased inflammatory response. In this report, we describea new mechanism by which topical EPO accelerates thehealing of a diabetic skin wound. We found that topicalEPO treatment of the burns in the diabetic pigs acceleratedtheir healing through an AQP3-dependent mechanism bystimulating angiogenesis and ECM production.

Angiogenesis, the synthesis of ECM constituents such ascollagen and HA, and proper cell hydration in the woundbed are indispensable for normal wound healing. EPOstimulates endothelial cell proliferation and the secre-tion of angiogenic cytokines and growth factors, such asvascular endothelial growth factor, fibroblast growthfactor, and IGF-I from endothelial cells and keratinocytes,to cause the sprouting of new blood vessels into thewound bed (24). In this investigation, we found that top-ical EPO treatment of a wound substantially increases bloodflow in the regenerating skin of diabetic burn wounds asmeasured by laser Doppler scanning. We confirmed thiseffect when we measured the MVD and eNOS expressionlevels in the regenerating skin of diabetic burn wounds.Topical EPO treatment also resulted in significantly in-creased amounts of HP and HA in the diabetic burnwounds. Vedrenne et al. (25) reported the existence of aclose relationship between the ECM and the synthesis ofmolecules that regulate attachment between cells and theECM, angiogenesis, skin wound healing, and turnover ofresident dermal fibroblasts. Collagen and HA have manyfunctions in the ECM, one of which is to be a tissue scaffoldfor maintaining cellular shape and differentiation, support-ing cellular movement and migration, and enabling the ECMin the dermal layer to resist compression.

AQP3 is abundant in native and reconstructed humanskin epidermis, where it is primarily localized to keratino-cyte plasma membranes in the epidermis and is consistentwith water distribution in this tissue. In addition, watertransport and water permeability studies demonstrated thatAQP3 is functional in human epidermis and confers a highwater permeability to viable layers of the epidermis. Theseresults suggested that AQP3 can play a significant role in thehydration of the epidermis by preventing the formation ofan osmotic gradient across viable layers of this tissue (14,26).Growing evidence demonstrated that AQP3 facilitates cellmigration and proliferation and reepithelialization duringwound healing (17,18), and skin restoration after an injuryis boosted when AQP3 is stimulated (27). Cell hydrationand a moist environment are critical for facilitating fibro-blast turnover, angiogenesis, and reepithelialization by ker-atinocytes during wound healing. Therefore, any factor thatprevents or limits local AQP3 protein expression and/oractivation probably reduces the level of cell hydration andimpair wound healing. It was demonstrated that AQP3 isdownregulated in the regenerating epidermis during thehealing of full-thickness cutaneous wounds in rats with di-abetes (21). Here, we found that AQP3 expression levelswere reduced in the wound-free pigs and in the regenerat-ing skin of the diabetic pigs compared with those in thehealthy pigs.

In this study, we found that the slow wound closure rateof the diabetic wounds is associated with reduced angio-genesis and low HP and HA amounts in the wound bed. HAis a very hydrophilic molecule, and this property enables itto regulate tissue hydration because it attracts and bindswater. We also found that topical EPO treatment signifi-cantly increased angiogenesis and the amounts of HP andHA in the diabetic wounds and that these increases werecorrelated with a significant increase in AQP3 expressionlevels. Furthermore, we found that these correlations werestronger in the EPO-treated and EPO/FN-treated burnwounds of the diabetic pigs than those in the EPO-treatedand EPO/FN-treated burn wounds of the healthy pigs.Expectedly, the inhibition of AQP3 by HgCl2 in the burnwounds of diabetic pigs antagonized the positive actions ofEPO, and this result implies that EPO-mediated stimulationof AQP3 can stimulate wound healing in diabetes. Alto-gether, suggest that EPO-induced acceleration of healingis mediated through AQP3-dependent mechanisms. Whenthese mechanisms are activated, the key events of thewound-healing process, namely angiogenesis, collagen andHA synthesis, and reepithelialization, are stimulated, andthe wound closure rate is accelerated.

of nondiabetic skin (100%). **P < 0.01, significance of the difference between the vehicle-treated burn wounds and the other treatmentsaccording to the results of a two-way ANOVA with Bonferroni correction.†P< 0.05, significance of the difference between EPO/FN-treated burnwounds and the EPO-treated burn wounds according to the results of a two-tailed Student t test.

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Another interesting finding in our study was that AQP3expression levels in the EPO/FN-treated diabetic woundswere four times higher than those in the EPO-treateddiabetic wounds. This substantial increase in AQP3 ex-pression levels in the EPO/FN-treated diabetic woundsis associated with rapid wound closure rates and elevatedblood flow and a twofold increase in the MVD and theamounts of HP and HA in the wound tissues. FN has anessential function in the formation of granulation tissueduring the proliferative phase of wound healing (7,15). In di-abetes, a deficiency in FN and/or its degradation by prote-ases leads to disintegration of the provisional matrix, and

reepithelialization does not occur or is delayed (11). Wefound that exogenous FN alone has no effect on woundclosure rate and does not potentiate the accelerating actionof EPO on the healing of burn wounds of healthy pigsbecause endogenous FN is present in normal levels andis not degraded in healthy pigs. Since endogenous FN isdegraded in a DSU, we found that incorporating FN intoan EPO-containing gel is desirable because FN potenti-ates the salutary actions of an EPO-containing gel.

The results of this study provide evidence that supportsour hypothesis that topical EPO treatment of burn injuriesin the skin of diabetic pigs accelerated their healing through

Figure 5—AQP3 protein expression correlates positively with the extent of angiogenesis and the amounts of HP and HA in the regenerating skinof diabetic wounds. AQP3 protein expression significantly correlates with the MVD (r = 0.61) (A), the amount of HP (r = 0.79) (C), and the amountof HA (r = 0.88) (E) in the burn wounds of the nondiabetic pigs. AQP3 protein expression also significantly correlates with the MVD (r = 0.88) (B),the amount of HP (r = 0.92) (D), and the amount of HA (r = 0.93) (F) in the burn wounds of the diabetic pigs. Each point represents the average ofAQP3 protein expression, the MVD, and the amounts of HP and HA in six wounds.

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an AQP3-dependent mechanism by stimulating angiogene-sis and ECM production. The results of this study can alsoexpect that stimulating AQP3 expression in a nonhealingulcer by EPO further accelerates healing, perhaps by increas-ing cell hydration and raising the moisture levels of thewound. Increasing cell hydration and raising moisture levelsfacilitate interactions among the various cell types and ECMcomponents. However, the effect of EPO on the hydration

of regenerating skin cells is still under investigation and hasto be illustrated. Such interactions result in proper cellularmovement, migration, and differentiation and, ultimately,in the restoration of intact skin. These new and excitingfindings in a diabetic pig present the possibility that thetopical application of EPO may be therapeutically beneficialfor stimulating the healing of a DSU by raising cutaneousAQP3 expression levels. Additional studies are now required

Figure 6—AQP3 inhibition by HgCl2 reduces the effect of topical EPO on the wound closure rate, the extent of angiogenesis, and the amounts ofHP and HA in diabetic wounds. The wound closure and blood flow rates in the regenerating skin of diabetic pigs were determined after 14 daysof treatment with the vehicle-, the high-dose EPO-, and the EPO/HgCl2-containing gels. A: Representative set of photographic images (toppanel) and laser Doppler scans (bottom panel) of a vehicle-, EPO-, and an EPO/HgCl2-treated burn wound in the diabetic pig after 14 days oftreatment. The dark blue color represents nonvascularized regions, and the yellow and red colors represent vascularized regions with red-colored regions representing regions that are more vascularized than the yellow-colored regions. B: The wound closure rates of the vehicle-, theEPO-, and the EPO/HgCl2-treated burn wounds of the diabetic pigs. Burn wounds (n = 6) in each treatment group in one of the diabetic pigswere assessed and compared. C: Representative Western blots of eNOS, HAS1, and HAS2 expression levels in tissues that were collected onday 14 and then measured in lysates that were prepared from the regenerating skin of the vehicle-, the EPO-, and the EPO/HgCl2-treated burnwounds of the diabetic pigs. a-Actin was used to normalize protein loading, and the blots were derived from samples that were concomitantlyrun on a separate gel. The amount of HA (D), MVD (E), and HP (F) in the regenerating skin of the vehicle-, the EPO-, and the EPO/HgCl2-treatedburn wounds of the diabetic pigs. B: *P< 0.05; **P< 0.01, significance of the difference between the vehicle-treated burn wounds and the othertreatments according to the results a two-way ANOVA with Bonferroni correction. D–F: **P < 0.01, significance of the difference between thevehicle-treated burn wounds and the other treatments according to the results of a one-way ANOVAwith Tukey post hoc test; †P< 0.05, significanceof the difference between the EPO/HgCl2-treated and EPO-treated burn wounds according to the results of a two-tailed Student t test.

2264 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

to validate these findings, and clinical trials are needed toevaluate the safety and efficacy of topical EPO treatment inpatients with diabetes and a DSU.

Acknowledgments. The authors thank Dr. Arieh Bomzon, ConsulWrite(www.consulwrite.com), for his editorial assistance in preparing the manuscript.Funding. This study was supported by Remedor Biomed Ltd. and the Office ofthe Chief Scientist of Israel’s Ministry of Economy.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.H. designed, supervised, and carried out theexperiments; performed computational analysis; analyzed all of the data; and wrotethe manuscript. Y.U. designed, supervised, and carried out the experiments andanalyzed all of the data. D.E. designed and carried out the experiments and analyzedall of the data. A.K. and E.D. carried out the experiments, performed computationalanalysis, and supervised experiments with imaging of pig tissues, as well as someimmunological measurements. O.A. carried out the experiments and supervisedexperiments with imaging of pig tissues, as well as some immunological mea-surements. H.K. and M.A. prepared vehicle-, EPO-, EPO/HgCl2-, FN-, and EPO/FN-containing gels. M.B. performed the computational analysis. R.S. and A.Z. helped tocarry out the experiments. M.S. helped to carry out the experiments and supervisedthe experiments with laser Doppler. L.T. designed the experiments, supervised allexperiments, and analyzed all of the data. P.Y.L. supervised all experiments andwrote the manuscript. S.H. is the guarantor of this work and, as such, had full accessto all the data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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