2254
Topical Erythropoietin Treatment Accelerates the Healing
of Cutaneous Burn Wounds in Diabetic Pigs Through an
Aquaporin-3–Dependent Mechanism
Saher 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 and
Paul Y. Liu5
Diabetes 2017;66:2254–2265 | https://doi.org/10.2337/db16-1205
We have previously reported that the topical application of
erythropoietin (EPO) to cutaneous wounds in rats and mice
with experimentally induced diabetes accelerates their
healing by stimulating angiogenesis, reepithelialization, and
collagen deposition, and by suppressing the inflammatory
response and apoptosis. Aquaporins (AQPs) are integral
COMPLICATIONS
membrane proteins whose function is to regulate intracel-
lular fluid hemostasis by enabling the transport of water and
glycerol. AQP3 is the AQP that is expressed in the skin
where it facilitates cell migration and proliferation and re-
epithelialization during wound healing. In this report, we
provide the results of an investigation that examined the
contribution of AQP3 to the mechanism of EPO action on
the healing of burn wounds in the skin of pigs with experi-
mentally induced type 1 diabetes. We found that topical EPO
treatment of the burns accelerated their healing through an
AQP3-dependent mechanism that activates angiogenesis,
triggers collagen and hyaluronic acid synthesis and the for-
mation of the extracellular matrix (ECM), and stimulates
reepithelialization by keratinocytes. We also found that
incorporating 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. The
Centers for Disease Control and Prevention also estimates
that 5% of these individuals will develop a diabetic skin
1Department of Research & Development, Remedor Biomed Ltd, Nazareth Illit,
Israel
2Department of Plastic Surgery, Rambam Health Care Campus, Haifa, Israel
3Skin Research Laboratory, Ruth and Bruce Rappaport Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa, Israel
4Department of Plastic & Reconstructive Surgery and Wound Healing, Hopital
Lapeyronie, Montpellier, France
5Department of Plastic Surgery, Rhode Island Hospital, Warren Alpert Medical
School, Brown University, Providence, RI
Diabetes Volume 66, August 2017
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 because
all 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 of
impaired angiogenesis, insufficient blood flow, increased in-
flammation, diminished proliferation of fibroblasts (5), and
reduced reepithelialization by keratinocytes (6–8).
The glycoprotein hormone erythropoietin (EPO) regulates
red blood cell mass and is an approved drug for treating
anemia. EPO also has nonhematopoietic targets in the skin,
and we have shown (9) that these targets participate in the
healing of skin wounds. We previously reported that the
healing of cutaneous wounds in rats and mice with exper-
imentally induced diabetes is accelerated after the topical
application of recombinant human EPO to the cutaneous
wounds by stimulating angiogenesis, reepithelialization,
and collagen deposition, and by suppressing the inflamma-
tory response and apoptosis (10). The beneficial actions of
EPO on wound healing in diabetes are complemented by
fibronectin (FN). FN facilitates the formation of the pro-
visional wound matrix and prevents its dissociation (11).
Aquaporins (AQPs) are integral membrane proteins
whose function is to regulate intracellular fluid hemostasis
by enabling the transport of water and glycerol. AQPs are
expressed in the plasma membranes of keratinocytes in the
basal layer of the skin and the medullary collecting ducts of
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 as
long as the work is properly cited, the use is educational and not for profit, and the
work is not altered. More information is available at http://www.diabetesjournals
.org/content/license.
diabetes.diabetesjournals.org
the kidney (12). Downregulated expression of AQPs may be
the cause of the reduction in urinary-concentrating ability
in individuals with acute renal failure, and EPO can prevent
this 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 wound
healing (15–18). A positive role for moisture in healing skin
wounds was first shown in 1962, when Winter (19) inves-
tigated scab formation and the rate of epithelialization of
superficial wounds in pig skin and reported that moist
wounds heal faster than dry ones. As a corollary, dryness
of the skin of the feet correlates with foot ulceration in
patients with diabetes (20). A specific role for AQP3 in di-
abetic wound healing was posited when it was found that
AQP3 is downregulated in the regenerating epidermis dur-
ing the healing of full-thickness cutaneous wounds in rats
with diabetes (21). The work of these investigators and our
results indicated that EPO could be used to stimulate the
healing of nonhealing wounds. Such findings also suggest
the existence of a causal relationship between impaired AQP3
expression and delayed reepithelialization in diabetes, which
involves impaired movement and proliferation of those cells
that participate in angiogenesis, reduced production of the
extracellular matrix (ECM) by fibroblasts, and failure of ker-
atinocytes to reepithelialize a cutaneous skin wound.
It is against this background that we posited that the
therapeutically beneficial action of EPO on the healing of a
DSU is due in part to the ability of EPO to stimulate AQP3
expression in skin. We tested this hypothesis in pigs with
experimentally induced type 1 diabetes and a partial thickness
skin burn.
RESEARCH DESIGN AND METHODS
Pig Model of Type 1 Diabetes
The study comprised four 60-kg healthy female pigs (Sus
domesticus) purchased from Lahav Institute (Kibbutz Lahav,
Israel). The pigs were singly housed in pens in a room with
an artificial 12-h light/dark cycle and had free access to a
standard laboratory chow and water. All procedures were
performed in the Technion according to the Israeli national
legislation on the use of animals for experimental purposes.
Diabetes induction, creation of the burn wounds, wound
treatment, dressing changes, and data collection were per-
formed under general anesthesia. An intravenous catheter
was permanently placed in the right jugular vein in the four
pigs for blood sampling during the investigation. Heart rate,
blood oxygen saturation, and the body temperature of the
pig 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 previously
described protocol (22). Blood glucose levels were checked
every 15 min for 6 h after streptozotocin administration and
at 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)
Hamed and Associates 2255
was injected intravenously to maintain the fasting blood
glucose levels of the diabetic pigs between 300 and
400 mg/dL.
Creation of Partial Thickness Skin Burn Wounds
Diabetes was maintained for 1 month before partial thick-
ness skin burn wounds were created. The bristles of the dorsal
skin on each side of the vertebral column of each pig were
removed using VEET depilatory cream (Reckitt Benckiser)
before creating 12-cm2 circular partial thickness skin burn
wounds using an aseptic technique. The burn wounds were
created using a previously described method (23). Briefly,
four cylindrical brass rods, each with an ;3.8-cm diameter
and weighing 358 g, were placed in hot water (92°C) for
2 min. Four groups of six partial thickness burn wounds
were created by placing of the rod perpendicular on the
dorsal skin of each pig for 20 s with no additional pressure
“producing partial thickness third-degree burns.” In the two
diabetic pigs, an additional group of six partial thickness
burn wounds was created.
Topical Formulations
Six different gels for topical wound treatment were pre-
pared in the Remedor Biomed Ltd. laboratory in accordance
with 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 gel
that contained 2,000 IU/g EPO and 300 mg/g FN; and 6) a
gel 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/mL
solution (EMD Millipore). HgCl2 (0.1 mmol/L) was incor-
porated into the gel that contained 2,000 IU/g EPO in order
to block cutaneous AQP3 activity in the wounds (negative
control) and was purchased from Sigma-Aldrich. The results
of the stability testing of the gels established that EPO and
FN are stable in the gel for at least 6 months at 4°C, as
determined by ELISA.
Treatment of Wounds
Each group of the six partial thickness burn wounds was
randomly assigned to be treated with one of the following
topical 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 in
one diabetic pig was treated with the EPO/HgCl2-containing
gel, and the additional group of burn wounds in the second
diabetic 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 six
partial thickness burn wounds in four different groups in
each pig, gel (3 g) was topically applied to each wound every
2 days of the 14-day study period. In order to prevent re-
moval of the gel after its application by rubbing and to
protect the burn wounds between treatments, the treated
2256 EPO Accelerates Burn Wound Healing
wounds were covered with nonadherent gauze pads, which
were stabilized by Tensoplast Elastic Adhesive Bandaging
(Smith & Nephew).
Study Parameters
On each treatment day, pigs were weighed; each wound was
photographed using a 12-megapixel digital camera (Olympus);
and a blood sample was collected for determining the red
blood cell, leukocyte, and platelet counts, and the plasma
hemoglobin and glycated hemoglobin (HbA 1c) levels.
Punch biopsy specimens from randomly selected areas of
the regenerating skin of the vehicle-treated and treated
burn 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 in
10% neutral buffered formalin for histological determina-
tion of the microvascular density (MVD) or stored in liquid
nitrogen for immunohistochemistry, Western blot analysis,
ELISA, and PCR. The slides were examined under a Nikon
Eclipse E800 Upright Microscope, and images of the sec-
tions were captured and analyzed by Metamorph Image
Analysis Software (Nikon Instruments Inc.).
Wound Closure Rate
The wound closure rate was calculated from the area of
reepithelialized tissue in the burn wound. In order to calcu-
late the rate, the area of each burn wound was categorized
into the following three areas: 1) a scab area where the
burnt skin becomes a rigid crust after creation of the burn
injury; 2) a red area where the scab can be detached but has
no epithelial cells; and 3) a white area where the scab
can be detached and contains new epithelial cells. The
wound closure rate was calculated by measuring the white
area on days 2, 4, 7, 9, 11, and 14. To this end, transpar-
ent paper was placed over each wound, and the shape of
the white area was drawn on the paper. The transparent
paper was then superimposed onto a 1-mm2 graph paper
in order to measure the white area in the wound. The rate
of time-dependent changes in the size of the white area
were calculated using the following formula:
White  area  on  day  X
%  White  areaðwound  closureÞ5 3100
Wound  area  on  day  0
Measurement of Blood Flow
Blood flow in the wounds was measured noninvasively by a
laser Doppler perfusion imaging system (PeriScan PIM
2 System; Perimed) during the study period according to
the manufacturer instructions. Perfusion in a region of
interest (ROI) is measured on a scale of six colors in which
dark blue depicts the lowest perfusion rate and red depicts
the highest perfusion rate using PIMSoft software for blood
perfusion imaging (Lisca Development AB). For each burn
wound, the ROI was a 12-cm2 circle that was drawn around
the initial burn wound, and the blood flow in the wound is
the average value of all colors in the ROI.
Diabetes Volume 66, August 2017
Determination of Angiogenesis
The MVD and the extent of angiogenesis in the regenerat-
ing skin of the healing wounds was determined by staining
5-mm thick sections of the formalin-maintained samples
punch biopsies of the wounds with CD31 (R&D Systems).
The number of capillaries in the regenerating skin in each
wound site was counted in five random microscopic fields
(320 magnification).
Detection of AQP3
AQP3 was detected in wound-free healthy and diabetic pig
skin 1 month after diabetes induction and 1 day before
creation of the burn wounds and in the regenerating
skin of the wounds during the study period by immu-
nohistochemistry and immunofluorescence. For AQP3
immunohistochemistry, 5-mm-thick sections were stained
with rabbit polyclonal AQP3 primary antibody (Santa Cruz
Biotechnology). For confirming the presence of AQP3 by
immunofluorescence, 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 Collagen
The 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 fibers
stained blue, nuclei stained black, and cytoplasm and muscle
fibers stained red.
Determination of the Amount of Collagen
Hydroxyproline (HP), an amino acid constituent of type I
collagen, was used as a marker and an indicator of the
amount of collagen in the skin specimens from the regen-
erating skin of the burn wounds. The amount of HP was
determined using a previously described protocol (10).
Amount of Hyaluronic Acid
Since hyaluronic acid (HA) is linked to skin strength and
hydration, the HA amount in the regenerating skin of the
burn wound tissues was determined in specimens that were
collected from the nondiabetic and diabetic pigs using the
Hyaluronan Quantikine ELISA Kit (R&D Systems) accord-
ing to the manufacturer protocol.
Western Blot Analysis
The expression levels of AQP3, endothelial nitric oxide
synthase (eNOS), HA synthase (HAS) 1, and HAS2 were
determined in the regenerating skin of the burn wound
tissues. Briefly, tissue extracts were prepared using radio-
immunoprecipitation assay buffer (Elpis-Biotech). Proteins
from tissue lysates were separated by 10% SDS-PAGE and
then transferred to nitrocellulose membranes. The mem-
branes were probed with indicated antibodies. Protein
expression levels were detected by densitometry using the
Immun-Star horseradish peroxidase chemiluminescence
detection system (BIO-RAD). The results were expressed
as a percentage of the average protein level of duplicate
diabetes.diabetesjournals.org Hamed and Associates 2257

readings in the vehicle-treated burn wounds of the healthy
pigs (100%).
Real-time PCR
Total RNA was extracted from homogenates prepared from
regenerating skin of the burn wound tissues using the
MasterPure RNA Purification Kit (EPICENTRE Biotechnol-
ogies). cDNA was generated by absolute qPCR mixes reverse
transcription reagents (ABgene) and analyzed by qPCR
using SYBR Green PCR Master Mix (Molecular Probes) in a
Rotor-Gene 6000 Cycler (Corbett Life Science). The results
were expressed as a percentage change from control after
normalization to an endogenous reference gene (GAPDH).
Statistics
All statistical analyses were performed using a computerized
statistical program (GraphPad Prism version 5.0; GraphPad
Software Inc.), and all data are presented as the mean or
percentage 6 SD. Statistical significance was set at 5%. A
two-tailed Student t test was used to compare study param-
eters of the healthy and diabetic pigs, and a two-way ANOVA
with Bonferroni correction to control for type I error was
used for multiple comparisons. The wound closure rates
among groups were compared and analyzed using a one-
way ANOVA with Tukey post hoc test and a two-way ANOVA
with Bonferroni correction to control for type I error in the
multiple comparisons.
RESULTS
Topical treatment of the burns with the vehicle-, FN-, EPO-
and EPO/FN-containing gels did not change 1) the red
blood cell, leukocyte, or platelet counts and the elevated
blood glucose levels in the diabetic pigs and 2) the blood
hemoglobin and HbA1c levels in the control and diabetic
pigs (Table 1).
Topical EPO Accelerates Wound Closure and Increases
Blood Flow in the Regenerating Skin of Diabetic Wounds
in a Dose-Dependent Manner, and This Effect Is
Potentiated by FN
The 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 wounds
were significantly greater than those of the vehicle-treated
diabetic wounds. The most significant differences in wound
closure rates were detected between the vehicle-treated and
the EPO/FN-treated diabetic wounds. From day 4 onward,
the wound closure rate of the EPO/FN-treated wounds was
significantly faster than those of the FN-treated and the
EPO-treated diabetic wounds. The blood flow in the EPO-
treated diabetic wounds was not significantly different from
that of the EPO/FN-treated diabetic wounds, and these
two blood flows were significantly higher than those in the
vehicle-treated and FN-treated diabetic wounds (Fig. 1A–C).
The wound closure and blood flow rates of the low-dose
EPO-treated diabetic wounds were significantly greater than
those of the vehicle-treated diabetic wounds, and the wound
closure and blood flow rates of the high-dose EPO-treated
diabetic wounds were significantly greater than those of the
low-dose EPO-treated diabetic wounds from day 9 onward
(Fig. 1D–F).
Topical FN Does Not Affect Wound Closure and Blood
Flow in the Regenerating Skin of Nondiabetic Wounds
The wound closure and blood flow rates of the vehicle-
treated and FN-treated wounds of healthy pigs were very
similar. From day 4 onward, the wound closure and blood
flow rates of the EPO-treated nondiabetic wounds were
significantly greater than those of the vehicle-treated and
FN-treated nondiabetic wounds. The most significant dif-
ferences in wound closure and blood flow rates were
detected between the EPO/FN-treated wounds and the
vehicle-treated and the FN-treated wounds in the healthy
pigs over the period from day 4 to day 11 (Fig. 1G–I).
Topical EPO Increases the MVD and eNOS Expression
Levels in the Regenerating Skin of Diabetic Wounds, and
FN Potentiates These Effects
The MVD and eNOS expression levels in the vehicle-treated
diabetic 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 (10 /mL)
6
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 the
significance of the difference between the two groups at days 0 and 7 and day 14.
2258 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

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 wound
closure 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 of
the vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds in the diabetic pigs. Representative set of photographic images (D) and
the 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 days
0, 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 diabetic
pigs. Representative set of photographic images (G) and the corresponding laser Doppler scans (H) of a vehicle-, an FN-, an EPO-, and an
EPO/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 wound
area on day 0. The dark blue color represents nonvascularized regions, and the yellow and red colors represent vascularized regions with the
red-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 burn
wounds and the other treatments according to the results of a two-way ANOVA with Bonferroni correction. †P < 0.05, significance of the
difference between 1) the EPO-treated or EPO/FN-treated burn wounds and the FN-treated burn wounds and 2) the high-dose EPO-treated burn
wounds 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.

in the diabetic and nondiabetic wounds. On the other hand, nondiabetic wounds (Fig. 2A–E). In the EPO/FN-treated
topical EPO treatment for 14 days significantly increases nondiabetic wounds, the MVD and eNOS expression lev-
the MVD and eNOS expression levels in the diabetic and els were not significantly different from those in the
diabetes.diabetesjournals.org Hamed and Associates 2259

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

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 than
those in the EPO-treated diabetic wounds (Fig. 2D and E).
Topical EPO Increases Collagen Deposition and HA
Synthesis in the Regenerating Skin of Diabetic Wounds,
and FN Potentiates This Effect
The amounts of HP and HA and the expression levels of the
two HASs, HAS1 and HAS2, in the vehicle-treated diabetic
wounds were lower than those in the vehicle-treated non-
diabetic wounds. Topical FN treatment for 14 days did not
change the HP and HA amounts and the HAS1 and HAS2
expression levels in the diabetic and nondiabetic wounds.
On the other hand, topical EPO treatment significantly
increased the HP and HA amounts and the HAS1 and HAS2
expression levels in diabetic and nondiabetic wounds (Fig.
3A–G). HP amounts in the EPO/FN-treated nondiabetic and
diabetic wounds were significantly higher than those in the
EPO-treated nondiabetic and diabetic wounds, respectively
(Fig. 3B and C). The HA amount and the HAS1 and HAS2
expression levels in the EPO/FN-treated nondiabetic wounds
were similar to those of the EPO-treated nondiabetic wounds
(Fig. 3D and F). In contrast, the HA amount and the HAS1
and HAS2 expression levels in the EPO/FN-treated diabetic
wounds were significantly higher than those in the EPO-
treated diabetic wounds (Fig. 3E and G).
2260 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

Figure 3—Topical EPO increases the amount of HP and HA synthesis in the regenerating skin of diabetic wounds, and this effect is potentiated
by FN. A: Representative set of micrographs that shows immunohistochemical staining for the amount of HP by Masson trichrome staining in
the 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 the
EPO/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 the
vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs (D) and of the diabetic pigs (E). Representative
Western blots of HAS1 and HAS2 expression levels in tissue samples that were collected on day 14 and then measured in lysates that were
prepared from the regenerating skin of the vehicle-, the FN-, the EPO-, and the EPO/FN-treated burn wounds of the nondiabetic pigs (F) and of
the diabetic pigs (G). a-Actin was used to normalize protein loading, and the blots were derived from samples that were analyzed concomitantly
and run on a separate gel. The sample size of each treatment group was 12, and the data are expressed as the average of duplicate
measurements of the amount of HP or HA 6 SD. *P < 0.05; **P < 0.01, significance of the difference between the vehicle-treated burn wounds
and the other treatments according to the results of the one-way ANOVA with Tukey post hoc test. †P < 0.05, significance of the difference
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.

AQP3 Expression Is Decreased in Wound-Free
Diabetic Skin
AQP3 protein and mRNA expression levels in wound-free
diabetic 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 the
Regenerating Skin of Diabetic Wounds, and FN
Potentiates This Effect
After 14 days, AQP3 protein and mRNA expression levels in
the vehicle-treated diabetic wounds were significantly lower
than those in the vehicle-treated nondiabetic wounds. In
the diabetic and control pigs, AQP3 protein and mRNA
expression 1) in the EPO-treated wounds were significantly
higher than those of the vehicle-treated and FN-treated
wounds, 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 not
different from each other (Fig. 4J–O).
AQP3 Protein Expression Correlates Positively With the
Extent of Angiogenesis and HP and HA Amounts in the
Regenerating Skin of Diabetic Burn Wounds
We used the Pearson correlation to investigate the rela-
tionships among AQP3 protein expression levels, the extent
of angiogenesis, and the amounts of HP and HA in the burn
wounds of healthy and diabetic pigs. In the diabetic and
healthy pigs, AQP3 protein expression levels correlated
positively 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 wounds
of the diabetic pigs than those found in the regenerating
skin of the burn wounds of the healthy pigs.
diabetes.diabetesjournals.org Hamed and Associates 2261

Figure 4—AQP3 expression is decreased in wound-free diabetic skin. Topical EPO application stimulates AQP3 expression in the regenerating
skin of diabetic wounds, and this effect is potentiated by FN. Representative set of micrographs of immunohistochemical staining for AQP3 in
the 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 of
immunofluorescence staining for AQP3 in the wound-free skin of a nondiabetic pig (C) and of a diabetic pig (D). E and G: Quantification (E) and
representative Western blots (G) of AQP3 expression in the wound-free skin of diabetic pigs 30 days after diabetes induction in wound-free skin
of 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 mean 6 SD of triplicate measurements of AQP3 protein expression and mRNA expression and as a percentage of
the expression in wound-free nondiabetic skin (100%). Representative set of micrographs of immunohistochemical staining for AQP3 in the
regenerating 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 protein
expression 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 an
EPO/FN-containing gel. Values are expressed as the mean 6 SD of triplicate measurements of AQP3 protein expression in vehicle-treated burn
wounds of the nondiabetic pigs (100%). Representative Western blots of AQP3 protein expression in lysates of the regenerating skin of burn
wounds from the nondiabetic pigs (L) and the diabetic pigs (M) after 14 days of treatment. a-Actin was used to normalize protein loading, and the
blots were derived from samples that were concomitantly run on a separate gel. AQP3 mRNA levels of total RNA that was isolated from lysates
of the regenerating skin of vehicle-, FN-, EPO-, and EPO/FN-treated burn wounds of the nondiabetic pigs (N) and the diabetic pigs (O). Values
are expressed as the average 6 SD of duplicate measurements of AQP3 mRNA expression and as a percentage of the expression in wound-free
2262 EPO Accelerates Burn Wound Healing
AQP3 Inhibition Reduces the Effect of Topical EPO on
the Wound Closure Rate, the Extent of Angiogenesis,
and the HP and HA Amounts in Diabetic Burn Wounds
When 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 was
accompanied by a reduced blood flow (Fig. 6A and B), low
eNOS expression levels, low HAS1 and HAS2 expression lev-
els (Fig. 6C), a low extent of angiogenesis, a reduced HP
amount, and a reduced HA amount. Moreover, the wound
closure rates; the expression levels of eNOS, HAS1, and
HAS2; and the amounts of HP and HA in the EPO/HgCl2-
treated diabetic wounds were significantly lower than those
in the EPO-treated diabetic wounds (Fig. 6D–F).
DISCUSSION
The healing of a DSU is delayed because of impaired
angiogenesis, reduced cutaneous cellular activity, and in-
creased inflammatory response. In this report, we describe
a new mechanism by which topical EPO accelerates the
healing of a diabetic skin wound. We found that topical
EPO treatment of the burns in the diabetic pigs accelerated
their healing through an AQP3-dependent mechanism by
stimulating angiogenesis and ECM production.
Angiogenesis, the synthesis of ECM constituents such as
collagen and HA, and proper cell hydration in the wound
bed are indispensable for normal wound healing. EPO
stimulates endothelial cell proliferation and the secre-
tion of angiogenic cytokines and growth factors, such as
vascular endothelial growth factor, fibroblast growth
factor, and IGF-I from endothelial cells and keratinocytes,
to cause the sprouting of new blood vessels into the
wound bed (24). In this investigation, we found that top-
ical EPO treatment of a wound substantially increases blood
flow in the regenerating skin of diabetic burn wounds as
measured by laser Doppler scanning. We confirmed this
effect when we measured the MVD and eNOS expression
levels 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 burn
wounds. Vedrenne et al. (25) reported the existence of a
close relationship between the ECM and the synthesis of
molecules that regulate attachment between cells and the
ECM, angiogenesis, skin wound healing, and turnover of
resident dermal fibroblasts. Collagen and HA have many
functions in the ECM, one of which is to be a tissue scaffold
for maintaining cellular shape and differentiation, support-
ing cellular movement and migration, and enabling the ECM
in the dermal layer to resist compression.
of nondiabetic skin (100%). **P < 0.01, significance of the difference between the vehicle-treated burn wounds and the other treatments
according to the results of a two-way ANOVA with Bonferroni correction.†P < 0.05, significance of the difference between EPO/FN-treated burn
wounds and the EPO-treated burn wounds according to the results of a two-tailed Student t test.
Diabetes Volume 66, August 2017
AQP3 is abundant in native and reconstructed human
skin epidermis, where it is primarily localized to keratino-
cyte plasma membranes in the epidermis and is consistent
with water distribution in this tissue. In addition, water
transport and water permeability studies demonstrated that
AQP3 is functional in human epidermis and confers a high
water permeability to viable layers of the epidermis. These
results suggested that AQP3 can play a significant role in the
hydration of the epidermis by preventing the formation of
an osmotic gradient across viable layers of this tissue (14,26).
Growing evidence demonstrated that AQP3 facilitates cell
migration and proliferation and reepithelialization during
wound healing (17,18), and skin restoration after an injury
is boosted when AQP3 is stimulated (27). Cell hydration
and a moist environment are critical for facilitating fibro-
blast turnover, angiogenesis, and reepithelialization by ker-
atinocytes during wound healing. Therefore, any factor that
prevents or limits local AQP3 protein expression and/or
activation probably reduces the level of cell hydration and
impair wound healing. It was demonstrated that AQP3 is
downregulated in the regenerating epidermis during the
healing of full-thickness cutaneous wounds in rats with di-
abetes (21). Here, we found that AQP3 expression levels
were reduced in the wound-free pigs and in the regenerat-
ing skin of the diabetic pigs compared with those in the
healthy pigs.
In this study, we found that the slow wound closure rate
of the diabetic wounds is associated with reduced angio-
genesis and low HP and HA amounts in the wound bed. HA
is a very hydrophilic molecule, and this property enables it
to regulate tissue hydration because it attracts and binds
water. We also found that topical EPO treatment signifi-
cantly increased angiogenesis and the amounts of HP and
HA in the diabetic wounds and that these increases were
correlated with a significant increase in AQP3 expression
levels. Furthermore, we found that these correlations were
stronger in the EPO-treated and EPO/FN-treated burn
wounds of the diabetic pigs than those in the EPO-treated
and EPO/FN-treated burn wounds of the healthy pigs.
Expectedly, the inhibition of AQP3 by HgCl2 in the burn
wounds of diabetic pigs antagonized the positive actions of
EPO, and this result implies that EPO-mediated stimulation
of AQP3 can stimulate wound healing in diabetes. Alto-
gether, suggest that EPO-induced acceleration of healing
is mediated through AQP3-dependent mechanisms. When
these mechanisms are activated, the key events of the
wound-healing process, namely angiogenesis, collagen and
HA synthesis, and reepithelialization, are stimulated, and
the wound closure rate is accelerated.
diabetes.diabetesjournals.org Hamed and Associates 2263

Figure 5—AQP3 protein expression correlates positively with the extent of angiogenesis and the amounts of HP and HA in the regenerating skin
of diabetic wounds. AQP3 protein expression significantly correlates with the MVD (r = 0.61) (A), the amount of HP (r = 0.79) (C), and the amount
of 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 of
AQP3 protein expression, the MVD, and the amounts of HP and HA in six wounds.

Another interesting finding in our study was that AQP3
expression levels in the EPO/FN-treated diabetic wounds
were four times higher than those in the EPO-treated
diabetic wounds. This substantial increase in AQP3 ex-
pression levels in the EPO/FN-treated diabetic wounds
is associated with rapid wound closure rates and elevated
blood flow and a twofold increase in the MVD and the
amounts of HP and HA in the wound tissues. FN has an
essential function in the formation of granulation tissue
during 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). We
found that exogenous FN alone has no effect on wound
closure rate and does not potentiate the accelerating action
of EPO on the healing of burn wounds of healthy pigs
because endogenous FN is present in normal levels and
is not degraded in healthy pigs. Since endogenous FN is
degraded in a DSU, we found that incorporating FN into
an 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 supports
our hypothesis that topical EPO treatment of burn injuries
in the skin of diabetic pigs accelerated their healing through
2264 EPO Accelerates Burn Wound Healing Diabetes Volume 66, August 2017

Figure 6—AQP3 inhibition by HgCl2 reduces the effect of topical EPO on the wound closure rate, the extent of angiogenesis, and the amounts of
HP and HA in diabetic wounds. The wound closure and blood flow rates in the regenerating skin of diabetic pigs were determined after 14 days
of treatment with the vehicle-, the high-dose EPO-, and the EPO/HgCl2-containing gels. A: Representative set of photographic images (top
panel) and laser Doppler scans (bottom panel) of a vehicle-, EPO-, and an EPO/HgCl2-treated burn wound in the diabetic pig after 14 days of
treatment. 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-, the
EPO-, and the EPO/HgCl2-treated burn wounds of the diabetic pigs. Burn wounds (n = 6) in each treatment group in one of the diabetic pigs
were assessed and compared. C: Representative Western blots of eNOS, HAS1, and HAS2 expression levels in tissues that were collected on
day 14 and then measured in lysates that were prepared from the regenerating skin of the vehicle-, the EPO-, and the EPO/HgCl2-treated burn
wounds of the diabetic pigs. a-Actin was used to normalize protein loading, and the blots were derived from samples that were concomitantly
run 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-treated
burn wounds of the diabetic pigs. B: *P < 0.05; **P < 0.01, significance of the difference between the vehicle-treated burn wounds and the other
treatments according to the results a two-way ANOVA with Bonferroni correction. D–F: **P < 0.01, significance of the difference between the
vehicle-treated burn wounds and the other treatments according to the results of a one-way ANOVA with Tukey post hoc test; †P < 0.05, significance
of the difference between the EPO/HgCl2-treated and EPO-treated burn wounds according to the results of a two-tailed Student t test.

an AQP3-dependent mechanism by stimulating angiogene-
sis and ECM production. The results of this study can also
expect that stimulating AQP3 expression in a nonhealing
ulcer by EPO further accelerates healing, perhaps by increas-
ing cell hydration and raising the moisture levels of the
wound. Increasing cell hydration and raising moisture levels
facilitate interactions among the various cell types and ECM
components. However, the effect of EPO on the hydration
of regenerating skin cells is still under investigation and has
to be illustrated. Such interactions result in proper cellular
movement, migration, and differentiation and, ultimately,
in the restoration of intact skin. These new and exciting
findings in a diabetic pig present the possibility that the
topical application of EPO may be therapeutically beneficial
for stimulating the healing of a DSU by raising cutaneous
AQP3 expression levels. Additional studies are now required
diabetes.diabetesjournals.org
to validate these findings, and clinical trials are needed to
evaluate the safety and efficacy of topical EPO treatment in
patients 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 of
the Chief Scientist of Israel’s Ministry of Economy.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. S.H. designed, supervised, and carried out the
experiments; performed computational analysis; analyzed all of the data; and wrote
the manuscript. Y.U. designed, supervised, and carried out the experiments and
analyzed all of the data. D.E. designed and carried out the experiments and analyzed
all of the data. A.K. and E.D. carried out the experiments, performed computational
analysis, and supervised experiments with imaging of pig tissues, as well as some
immunological measurements. O.A. carried out the experiments and supervised
experiments 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 to
carry out the experiments. M.S. helped to carry out the experiments and supervised
the experiments with laser Doppler. L.T. designed the experiments, supervised all
experiments, and analyzed all of the data. P.Y.L. supervised all experiments and
wrote the manuscript. S.H. is the guarantor of this work and, as such, had full access
to all the data in the study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
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