A synthetic uric acid analog accelerates cutaneous wound healing in mice.

Abstract

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.

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A Synthetic Uric Acid Analog Accelerates Cutaneous
Wound Healing in Mice
Srinivasulu Chigurupati1,2,3., Mohamed R. Mughal1., Sic L. Chan3, Thiruma V. Arumugam1, Akanksha
Baharani3, Sung-Chun Tang1, Qian-Sheng Yu1, Harold W. Holloway1, Ross Wheeler4, Suresh Poosala2,
Nigel H. Greig1, Mark P. Mattson1,5*
1Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, Maryland, United States of America, 2Research Resources Branch,
National Institute on Aging Intramural Research Program, Baltimore, Maryland, United States of America, 3Biomolecular Science, University of Central Florida, Orlando,
Florida, United States of America, 4Department of Pathology and Medical Education, University of Central Florida, Orlando, Florida, United States of America,
5Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Abstract
Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and
leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants
have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free
radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with
increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells
against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA
or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways
consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA
significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice.
Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to
vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results
support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development
of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.
Citation: Chigurupati S, Mughal MR, Chan SL, Arumugam TV, Baharani A, et al. (2010) A Synthetic Uric Acid Analog Accelerates Cutaneous Wound Healing in
Mice. PLoS ONE 5(4): e10044. doi:10.1371/journal.pone.0010044
Editor: Joanna Mary Bridger, Brunel University, United Kingdom
Received August 19, 2009; Accepted February 26, 2010; Published April 6, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This research was supported by the National Institute on Aging Intramural Research Program of the National Institutes of Helath (NIH). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mattsonm@grc.nia.nih.gov
. These authors contributed equally to this work.
Introduction
The rapid and coordinated responses of several different cell
types, including circulating platelets and immune cells, and
intrinsic keratinocytes, fibroblasts and vascular endothelial cells,
are required for the proper healing of full-thickness wounds [1,2].
Within seconds to minutes of the injury, platelets are recruited to
the wound site to aid in clot formation, and inflammatory
processes mediated by innate molecular cascades and infiltrating
leukocytes occur. These rapid responses limit blood loss and guard
against infectious agents. During the ensuing days keratinocytes
migrate over the injured dermis and the granulation tissue and
proliferate to restore the barrier function of the skin. Concomi-
tantly, fibroblasts migrate into the clot and proliferate and
angiogenesis occurs at the wound edge. Then, in a slower process
that occurs over a period of months to several years, tissue
remodeling occurs and (in mammals) a scar is formed by collagen-
producing fibroblasts.
The cellular signaling mechanisms that regulate wound healing
are complex and poorly understood, but recent findings suggest
key roles for growth factors such as EGF, FGF2 and HGF, the
cytokine TGFb and the cell fate regulator Notch [2–7]. From a
clinical perspective, an attractive feature of full-thickness wounds is
that treatments can be applied topically, thus reducing or
eliminating adverse effects on other organs. Thus, topical
application of ligands for several growth factors have been
reported to enhance wound healing in animal models [3,4,7]
and, in some cases, in human subjects [8,9].
A rapid increase in oxygen free radical production and oxidative
damage to proteins, DNA and lipid occurs in cells within and
adjacent to the wound site [10–12]. Some reactive oxygen species
appear to serve beneficial signaling roles in the recruitment of
immune cells and clearance of cellular debris, for example [13].
However, oxidative stress is detrimental to multiple cellular
processes that occur during the period of tissue healing and
remodeling that occurs over a period of days to weeks after the
injury [14–16]. Reactive oxygen species (ROS) generated in
wounded dermal cells include superoxide, hydrogen peroxide,
hydroxyl radical (formed by the interaction of hydrogen peroxide
with Fe2+) and peroxynitrite (formed by the interaction of
superoxide with nitric oxide) [17]. These ROS result in membrane
lipid peroxidation, protein oxidation and damage to nucleic acids,
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any of which may impair cellular processes involved in wound
healing including proliferation and migration of epidermal cells,
and angiogenesis [18]. The increased ROS levels experienced by
cells in wounded tissue may be exacerbated by the depletion of
antioxidant enzymes including Cu/Zn superoxide dismutase
(SOD1) and glutathione peroxidase [19].
Uric acid (UA) is perhaps best known for its central role in gout,
a disorder characterized by elevated levels of UA resulting in its
precipitation to form crystals that are deposited in joint tissues
where they cause inflammation and pain [20]. On the other hand,
low levels of uric acid are associated with several major disorders
including Alzheimer’s and Parkinson’s diseases, and multiple
sclerosis [21]. Soluble UA functions as a free radical scavenger of
hydroxyl radical and peroxynitrite and, in fact, UA is the most
prominent antioxidant in the blood of humans and birds [22,23].
Previous findings have demonstrated a benefit of intraperitoneal or
intravenous administration of UA in experimental models of
several disorders that involve increased oxidative stress including
multiple sclerosis [24], Alzheimer’s disease [25], stroke [26] and
spinal cord injury [27]. However, the relative insolubility of UA
and its ability to form toxic crystals reduces its clinical utility. We
recently reported on the development of novel UA analogs with
greatly increased solubility and potent antioxidant acitivity [28]. In
vitro and cell culture screening identified 1, 7-dimethyluric acid
and 6, 8-dithiouric acid as two analogs with high antioxidant and
neuroprotective activities. When administered intravenously in
mice, both UA analogs lessened damage to the brain and
improved functional outcome in an ischemia–reperfusion mouse
model of stroke [28]. In the present study we provide evidence that
topical administration of 6, 8-dithiouric acid accelerates wound
healing in mice by a mechanism that may involve actions of the
UA analog on fibroblasts, keratinocytes and vascular endothelial
cells. These findings show that soluble UA analogs can improve
wound healing and suggest novel therapeutic uses for UA analogs
in clinical settings.
Results
6, 8 dithio-uric acid enhances the motility and
proliferation of vascular endothelial cells
By producing microvessels that provide nutrients and oxygen to
growing dermal cells, angiogenesis plays a critical role in wound
healing [29]. We therefore determined whether UA analogs affect
angiogenic behaviors of cultured vascular endothelial cells.
Human microvascular endothelial cells (HMEC-1 cells) were
treated with vehicle (control), uric acid, 1, 7 dimethyl-uric acid
(UA1) or 6, 8 dithio-uric acid (UA2) and cell migration was
evaluated using a 24 well Transwell chamber chemoatraction
assay. The concentration of UA and UA analogs used (15 mM) was
chosen based on our previous studies [28] and preliminary dose-
findings experiments. UA2, but not UA or UA1, significantly
enhance vascular endothelial cell migration rate toward the
chemoatractant medium (Fig. 1A). We next employed a scrape
wound assay in which monolayers of cultured vascular endothelial
cells were mechanically wounded with a pipette tip. The migration
of cells across the substrate in the wound chasm was significantly
enhanced in cells treated with UA2 compared to those treated
with vehicle, UA or UA1 (Fig. 1B). The proliferation rate of the
endothelial cells during a 3 day period was significantly greater in
endothelial cells treated with UA2 than controls or cells treated
with UA or UA1 (Fig. 1C). These results suggest that UA2 can
enhance two behaviors of endothelial cells, proliferation and
directed cell migration, that are critical for angiogenesis in wound
healing. Blood vessel formation requires that endothelial cells
interact with each other to form tubes [30]. We found that the
ability of endothelial cells to form three dimensional tubes when
grown in matrigel was significantly increased by more than two-
fold in the presence of U2 (Fig. 1D, E). In contrast, UA had no
significant effect on endothelial cell tube formation and UA1
increased tube formation by only 25%.
6, 8 dithio-uric acid enhances the motility and
proliferation of fibroblasts and keratinocytes
The reformation of a functional germ and toxin-resistant dermis
and epidermis in a wound requires the proliferation and migration
of both fibroblasts and keratinocytes [31]. We first evaluated the
migration of keratinocytes and skin fibroblasts using the cell
monolayer/scratch wound assay. Treatment with UA2, but not
UA or UA2, significantly increased the rate of migration of both
keratinocytes and fibroblasts into the wound area compared to the
migration rates of these cells in control cultures (Fig. 2A, B). We
also found that U2 enhanced the proliferation of keratinocytes and
fibroblasts, whereas UA and UA1 did not affect cell proliferation
significantly (Fig. 2C, D).
6, 8 dithio-uric acid accelerates the healing of full-
thickness wounds in mice
Because UA2, but not UA or UA1, affected the behaviors of
cultured fibroblasts, keratinocytes and vascular endothelial cells in
ways that would be expected to enhance wound healing in vivo.
We therefore employed a mouse model to determine whether
topical application of UA2 would modify the healing of full-
thickness dermal wounds. Two full-thickness wounds were induced
in young adult male C57BL/6 mice and then UA2 or vehicle was
applied topically to the wounds once daily. Images of the wounds
were acquired on post-injury days 1, 3, 5, 8 and 13 and wound
sizes were quantified. On post-injury day 1 the size of wounds in
UA2-treated mice was approximately 15% smaller than wound
size in control mice (Fig. 3A, B). Subsequently, there was a rapid
acceleration of wound healing in the UA2-treated mice such that
on days 3 and 5 the wounds were approximately 50% and 80%
smaller than controls, respectively. By day 8 the wounds of UA2-
treated mice were completely closed, whereas the wounds of
control mice had not yet healed completely (Fig. 3B). We observed
no adverse effects of topical UA2 treatment on the body weight,
general health or behavior of the mice.
In a parallel experiment, we euthanized mice in UA2-treated and
control groups at post-injury days 1, 3, 5, 8 and 13 and then
performed a histological evaluation of skin tissue sections stained
with hematoxylin and eosin. UA-treated mice exhibited enhanced
restoration of dermal and epidermal tissues in the wound (Figs. 4, 5
and see File S1 for a detailed description of histological changes in
the different groups of mice). Examination of the skin tissue sections
revealed that, in addition to accelerating the closure of the wounds,
UA2 treatment resulted in restoration of a near-normal dermis and
epidermis by post-injury days 8 and 13 (Figs. 4, 5). In contrast, the
skin tissue in the closed wounds of control mice (post-injury day 13)
had not been restored and exhibited acellularity, vacuolation and
accumulations of cell debris. Based on our histological evaluation it
is clear that at days 8 and 13 denser and extended granulation tissue
is seen in the UA2-treated group compared to the control group.
While this is clearly beneficial for the healing process, it remains to
be seen whether this may also result in temporary hypertrophic scar
formation. Since enhanced myofibroblast differentiation may
explain the acceleration of wound closure in UA2-treated mice,
we stained wound tissues at post injury days 5, 8 and 13 with an
antibody against alpha smooth muscle actin, a differentiation
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marker of smooth muscle cells (Fig. 4B, C). The results revealed that
enhancedmyofibroblastdifferentiationoccurred inthe UA2-treated
wounds. Histomorphometricanalysis(Fig. 5A, B) showedthat UA2-
treated wounds on day 5 had more infiltrated mononuclear
inflammatory cells and new blood vessels compared to vehicle-
treated wounds (Fig. 5A, B).
UA2 treatment results in increased levels of SOD1, and
decreased accumulation of protein carbonyls and
nitrated proteins, in the wound tissue
Elevated levels of oxidative stress in cells within and surrounding
the damaged tissue in a wound are believed to impair the wound
healing process [10] which may result, in part, from depletion of
antioxidant enzymes [11]. We therefore measured relative levels of
Cu/Zn-superoxide dismutase (SOD1) in dermal wound tissue
from control and UA2-treated mice. SOD1 levels were two-fold
greater in the wound tissue of UA2-treated mice on all post-injury
days examined (days, 1, 3, 5, 8 and 13) (Figs. 6, 7). In addition to
SOD-1 levels, to measure severity of oxidative stress at the wound
site, we also performed oxyblot analysis with protein lysates from
wound tissue from control and UA2-treated mice to determine the
levels of oxidized proteins, which are characterized by the
presence of carbonyl groups. In four independent experiments
with different wound lysates, at days 1, 3, 5, 8 and 13, wound
samples from control mice had a consistently higher protein
carbonyl content compared with samples from UA2-treated mice
(Fig. 6C, D). As another indicator of oxidative damage, we
immunostained wound tissue sections with an antibody against
nitrotyrosine, which reflects interaction of proteins with peroxini-
trite, a toxic reactive oxygen species formed by the interaction of
nitric oxide with superoxide anion radicals. The analysis showed
that there were significantly more nitrotyrosine immunoreactive
cells in the wounds of mice in the control group compared to those
in the UA2-treated group (Fig. 7A, B).
Discussion
Impaired wound healing continues to be a major health
problem that predisposes to infections, long-term morbidity and
Figure 1. A uric acid analog enhances the motility and proliferation of human vascular endothelial cells. A. Cultured HMEC-1 were
treated with vehicle (control), uric acid, 1, 7 dimethyl-uric acid (UA1) or 6, 8 dithio-uric acid (UA2) (15 mM) in conditioned medium and plated into
chemo-attractant medium consisting of regular growth medium supplemented with 10% fetal bovine serum and other growth factors, and cell
migration was evaluated using a 24 well Transwell chamber assay. (values are the mean and SEM for cells per 1006field; n=3–4). **p,0.01 compared
to control values. B. HMEC-1 monolayers were mechanically wounded with a sterile tip of 20–200 ml pipette tip following treatment without
(control) or with UA, UA1 or UA2 (15 mM). Values are the mean and SEM (n=3 separate experiments). *p,0.001. C. Cultured HMEC-1 were treated with
vehicle, UA, UA1 or UA2 (15 mM) for the indicated time relative cell numbers were quantified by O.D readings (n=4–6 experiments), *p,0.01. D&E.
HMEC-1 cells were seeded on Matrigel-precoated wells and cultured in the presence of low-serum medium with vehicle (control), UA, UA1 or UA2
(15 mM). Tube formation, designated as the number of branch points/100X field) was evaluated 18 h after cell plating. Representative images are
shown in D and quantitative data in E. Values are the mean and SEM (n=12–16 cultures). *p,0.05; *p,0.001. Scale bars represent 100 mm.
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mortality, particularly in high risk patients including those with
diabetes or suppressed immune function, and the elderly [32–34].
In a previous study we screened a panel of soluble UA analogs to
establish their antioxidant and neuroprotective properties and
identified 1, 7 dimethyl-uric acid (UA1) and 6, 8 dithio-uric acid
(UA2) as being the most effective [28]. In the present study we
found that UA2 was much more effective than UA or UA1 in
promoting the proliferation and migration of dermal fibroblasts,
keratinocytes and vascular endothelial cells. We then tested the
therapeutic potential of UA2 in a mouse model of wound healing
and found that, indeed, topical application of UA greatly
accelerated wound healing and enhanced the restoration of
normal dermal and epidermal tissue structure in the wound area.
These findings suggest a potential use of UA2 or related UA
analogs in the treatment of wounds humans.
Previous findings have suggested a potential for the use of
antioxidants to treat wounds. For example, Serarslan et al. [35]
reported that caffeic acid phenethyl ester reduces oxidative stress
and accelerates cutaneous wound healing in a rat model, and
Alleva et al. [36] reported that dietary supplementation with
alpha-lipoic acid enhanced wound healing in human subjects
undergoing hyperbaric oxygen treatment. In addition, overex-
pression of manganese SOD enhanced wound healing in diabetic
mice [37] and a recent study showed that application of a wound
dressing with curcumin-loaded nanofibers enhanced diabetic
wound healing [38]. The free radical-scavenging property of
UA2 likely contributes to its beneficial effects in the in vivo and cell
culture models of wound healing. Indeed, we found that levels of
protein carbonyls and nitrated proteins were significantly lower in
wounded tissue from UA2-treated mice compared to wounded
tissue from vehicle-treated control mice. UA and some UA
analogs, including UA2, have been shown to scavenge free radicals
including hydroxyl radical and peroxynitrite [22,23,28]. Consis-
tent with this mechanism, we previously found that UA2 was more
effective than UA or UA1 in scavenging free radicals [28].
However, in the latter study UA1 and UA2 were similarly effective
in reducing brain damage and improving functional outcome in a
mouse model of stroke. It will therefore be of interest to evaluate
the efficacy of a range of doses of UA and more soluble UA
analogs in wound healing models.
We found that UA2 enhanced the proliferation and migration of
vascularendothelialcells,andalsoacceleratedtheformationofvessel-
like tubes by endothelial cells grown in a three-dimensional matrix.
The importance of angiogenesis in wound healing [29] and our
Figure 2. A uric acid analog enhances the motility and proliferation of keratinocytes and fibroblasts. A–D. Monolayers of cultured
keratinocytes (A) or fibroblasts (B) were treated with vehicle (Control), UA, UA1 or UA2 (15 mM) and were then subjected to scratch wounding.
Eighteen hours after wounding, images of the wound area were acquired and the number of cells per field that had migrated into the cell-free
wound zone was determined for each culture. Quantitative data A&B are shown. Values are the mean and SEM (n=3 separate experiments). *p,0.05.
C and D. Cultured keratinocytes (C) and fibroblasts (D) were treated with vehicle (Control), UA, UA1 or UA2 (15 mM) for the indicated time periods and
relative cell numbers were estimated by O.D readings, p,0.01. Values are the mean and SEM (n=4–6 experiments).
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findings in the present study, suggest that similar actions of UA2 on
vascular endothelial cells play an important role in the accelerated
and cytoarchitecturally superior healing of full-thickness wounds
treated with UA2. It remains to be determined whether antioxidant
actions of UA2 account for its ability to enhance angiogenesis, or
whether UA2 has other biological activities that enhance vessel
formation by endothelial cells. It will be of considerable interest to
determine whether UA2 might also have beneficial effects in other
clinical settings where angiogenesis is impaired.
Materials and Methods
Uric acid analogues
In a previous study (28) we described the process for the
synthesis of water soluble uric acid analogues, and characterization
and preclinical development of several different UA analogs
including dtUA (6, 8-dithiouricacid). In vitro and cell culture
screening showed that this dtUA has high a antioxidant activity
and is cytoprotective in cell culture and in vivo.
Cell Cultures
Keratinocytes.
from ATCC (# CRL-2309TM) and grown in keratinocyte
complete growth mediumcontaining
pituitary extract (BPE) and 5 ng/ml epidermal growth factor
(EGF) (GIBCO, Invitrogen USA). Cells were grown as a
monolayer.
Human Micro VascularEndothelial Cells (HMEC-1). Human
microvascularendothelialcells(HMEC-1)areSimianvacuolatingvirus
40 Tag (SV40 LT) transformed stable cell line provided by Dr.
Fransisco Candal (Center for Disease Control, Atlanta, GA), were
maintained in MCDB 131 formula (GIBCO, Invitrogen, San Diego,
CA) supplemented with 10% fetal bovine serum (FBS), epidermal
growth factor (EGF, 10 ng/mL), hydrocortisone (1 mg/mL), and L-
glutamine (10 mmol/L).
Primary culture of fibroblasts.
skin of young adult mice were used to harvest fibroblasts. Dermal
tissue specimens were cut into ,5 mm pieces. These fragments
were placed on the surface of 100 mm Petri dishes for 40–50
Human keratinocyte cells were obtained
0.05 mg/ml bovine
Dermal explants from the
Figure 3. Topical application of 6, 8 dithio-uric acid accelerates the healing of full-thickness wounds in mice. Two full-thickness
wounds were induced in vehicle- and UA2-treated (100 mM solution) mice. A. Images of a representative mouse from each group taken on post-injury
days 1, 3, 5, 8 and 13 are shown. B. Wound sizes at the indicated time points in Control and UA2 (15 mM) topical treated mice. Values are the mean
and SEM (n=6 mice per group). ***p,0.001, **p,0.01, *p,0.05 #p,0.01 compared to the control value. Scale bar=4 mm.
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minutes to allow adherence of the tissue to the culture surface.
10 ml of DMEM with 20% fetal bovine serum, penicillin (100 UI/
ml) and streptomycin (100 mg/ml) (pH 7.6), at 37uC, was gently
added to the culture dishes.
Cultures were maintained in a humidified incubator at 37uC in
a 5% CO2/95% air atmosphere.
Cultures were passaged on reaching 80% confluence, using
0.05% trypsin/EDTA (GIBCO, Invitrogen) and the media was
changed every two days, for this rate enables the maintenance of
ideal conditions of pH between 7.6 and 7.8 without non-
physiologic upheavals. Cells were used at passage 4 or 5 for cell
migration or proliferation assays in order to minimize the
influence of genetic alterations and senescent changes in the
cellular morphology.
Full-thickness wounds and quantification of healing
These methods were similar to those described previously [4].
All experiments were performed using 3–4 month-old male
C57BL/6 mice. Mice were anesthetized using 2 to 2.5% vaporized
inhaled isoflurane and the dorsal skin was cleansed with Betadine.
Two full-thickness wounds were created in the skin on the back of
each mouse using a 4 mm diameter biopsy punch (Miltex
Instrument, York, PA, USA) and a biotome (Acu Punch, Acuderm
Inc., Fort Lauderdale, FL, USA). Mice were treated with vehicle
(10 ml of dimethylsulfoxide) or 100 mM of either UA, UA2 or UA2
applied directly to the wound site once daily in a blinded manner.
Some mice in each group were euthanized on days 1, 3, 5, 8 and
13 post wounding, and skin tissue samples from the wound site
were collected from all of the mice for histological and biochemical
analyses. Some mice from each genotype/treatment group (n=6–
8) were evaluated daily for 13 days following wounding. Digital
photographs of the injury site were taken with a standard-sized dot
placed beside the wound; wound size was expressed as the ratio of
the wound area to the dot measurement.
Measurement of wound healing rate
Measurements of length and width were done using a caliper.
The first post-incision wound measurement was made on day 0.
The measurements were done without knowledge of the treatment
history of the mice. Wound area was calculated using digital
planimetry. Linear healing progress (LHP) was determined using
the following formula [39]
Figure 4. Histological features of wound healing in mice treated with UA2 or vehicle. A. Images of skin tissue sections stained with
hematoxylin and eosin showing histological changes during the wound healing process in control mice, with uric acid analog at post-injury days 1, 3,
5, 8 and 13. UA2 treated mice exhibited enhanced restoration of dermal and epidermal tissues in the wound. See File S1 for a detailed description of
histological changes in the different groups of mice. Scale bar=1 mm. These images are representative of 12 wounds in 6 mice for each treatment
group. B. Immunostaining for a smooth muscle actin showing wound healing tissues on days 5, 8 and 13 in mice treated with UA2 or vehicle.
Pictures showing enhanced myofibroblast differentiation in UA2 treated groups compared to controls in days 5, 8 (**p,0.01) and 13 ((*p,0.05).
Scale bar=25 mm. C. Quantification of alpha smooth muscle actin positive staining.
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LHP~DA?Pavg
where, DA represents change in wound area between first and last
days of healing period and Pavg stands for the mean wound
perimeter at the same days:
DA~ A2{A1
ð
Pavg~ P1zP2
ð
Þ
Þ=2
Linear healing rate (LHR, mm/day) was measured using the next
equation:
LHR~LHP=t
where, t represents the healing period in days [39].
Tissue preparation and examination
The biopsy specimens involving the central part of the wounds
(Days 1, 3 5, 8 and 13) were obtained perpendicularly to the
dorsal midline from mice for light microscopy. Skin specimens
were fixed in formalin, dehydrated through a graded series of
ethanols, cleared in xylene, and embedded in paraffin wax. 5 mm
thick sections were prepared and stained with hematoxylin-
eosin. The histomorphometric method was an adaptation of the
point-counting procedure. The counting procedure in each
section was performed at a total magnification of 200 in 3
random fields per section limited to the wounded area. Two
components of each section were examined in specimens
associatedwith 4 mice per
inflammatory cells (MICs) and blood vessels on days 5. After
transferring the images to the computer, a 252-square graticule
was superimposed on the screen over the wounded site to
facilitate counting. The epithelium was assessed on days 5
standard histologic grading system.
time interval: mononuclear
Histology
To assess cellular infiltration into the wounded area, samples
from three mice per group were collected on days 1, 3, 5, 8 and 13
during the healing process. To obtain skin samples from the
biopsied areas, mice were euthanized with an overdose of sodium
pentobarbital and the tissues were subsequently removed by
dissection. Formalin-fixed samples were sectioned at 4 mm and
stained with hematoxylin and eosin. All the slides were evaluated
by two veterinary pathologists (S. C. and S. P.) in blinded manner.
Immunoblot analysis
Tissue proteinwasextracted using T-PER tissue protein extraction
buffer with protease inhibitor cocktail (Sigma). Methods for protein
quantitation, electrophoretic separation, and transfer to nitrocellulose
membranes were as described previously [40]. Membranes were
incubated in blocking solution (5% milk in Tween Tris-buffered
saline; TTBS) overnight at 4uC followed by a 1 h incubation in
primary antibody diluted in blocking solution at room temperature.
Membranes were then incubated for 1 h in secondary antibody
conjugated to horseradish peroxidase and bands were visualized
using a chemiluminiscence detection kit (ECL, Amersham). The
primary antibodies were. SOD1 (Abcam, Cambridge, MA USA) and
an actin antibody (Sigma St. Louis, MO USA).
Detection of Protein Carbonyls by Oxyblot
Protein carbonyls in tissue protein were assayed with a protein
oxidation detection kit (Oxyblot; Cell Biolabs, San Diego, CA).
The tissue protein samples were prepared for electrophoresis with
4X reducing SDS sample buffer. The gel proteins were transferred
to a PVDF membrane. The membrane was immersed in 100%
methanol and dried at room temperature and then equilibrated
with TBS containing 20% methanol. After washing with 2 N HCl
the derivatization of the carbonyl groups of proteins by
Dinitrophenyl hydrazine (DNPH) was performed on 20 mg of
tissue proteins for 5 minutes at room temperature. The reaction
was stopped with 2 N HCl and the membrane was washed two
times with 100% methanol. The blocking was one hour with 5%
Figure 5. Wounds treated with a uric acid analog exhibit enhanced infiltration of immune cells and enhanced growth of blood
vessels. A. Numbers of mononuclear immune cells in wound tissue 5 days after the injury in control and UA2-treated mice. B. Numbers of blood
vessels in wound tissue 5 days after the injury in control and UA2-treated mice. Values are the mean and SEM (n=6 mice). *p,0.05.
doi:10.1371/journal.pone.0010044.g005
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non fat dry milk in TBST and incubated with Rabbit anti-DNP
antibody (1:1000) at 4uC overnight. The membrane was washed
three times and then incubated with secondary antibody goat anti-
rabbit IgG, HRP-conjugate (1:3000) for one hour at room
temperature and then washed with TBST. The oxidized proteins
were detected by chemiluminescence (ECL Thermoscientific).
Immunofluorescence
4-mm punch biopsy wounds, including the wound edge, were
harvested on days 1, 3, 5, 8, and 13 from control and UA2 treated
mice. Skin tissue was embedded in Optimal Cutting Temperature
(OCT) compound and frozen. Sections (6 mm) were cut with a
cryostat and fixed in acetone. Subsequently sections were blocked
with10% goat serum before being incubated with rabbit anti- alpha
smooth muscle actin (1:200; Abcam) and mouse anti- nitrotyrosine
(1:200; Zymed) overnight at 4uC. After being washed, the sections
were incubated in anti-rabbit and anti mouse IgG conjugated to
Alexa 568 and 488 respectively for 45 min at room temperature
(both 1:200). Sections were counterstained with Hoechst 33342
(Invitrogen) visualized under a Nikon Eclipse 80i microscope.
Quantification of immunohistochemistry
Using spatially calibrated images with the automated measure-
ment tools in IP lab software (BD Biosciences Bio-imaging,
Rockville, MD) total area of positive pixel intensity was measured
and analyzed with two-way ANOVA using GraphPad Prism
version 5.00 for Windows, GraphPad Software, San Diego
California USA.
Endothelial cell scratch wound healing assay
Human Microvascular Endothelial Cells (HMEC-1 cells), human
keratinocytes and mouse fibroblasts were seeded into 60 mm plates
and grown to confluency. After 24 hours of serum starvation
(DMEM supplemented with 1% FBS), cells were treated with either
Figure 6. Levels of Cu/Zn superoxide dismutase (SOD1) are increased in wound tissue from mice treated with UA2 (n=4). A. Mice
were treated with vehicle (control) or UA2 for the indicated number of days post-injury along with unwounded skin tissue. Wound tissue samples
were then removed and were subjected to immunoblot analysis (40 mg protein/lane) using antibodies to SOD1, and actin (44 kDa). B. Densitometric
analysis of band intensity (Image J, NIH) of immunoblots showed a significantly increased level of SOD in the skin tissue at 1, 3, 5, 8, 13 days in UA2
treated mice compared to control mice. * P,0.01 compared to values for each of the other groups. Statistical comparisons were made using ANOVA
with Newman-Keuls post hoc tests for pair wise comparisons using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego
California USA, www.graphpad.com. C. Enhanced oxidative stress in control wound tissues compared to UA2 treated at days 1,3,5,8 and 13. Lysates
(20 mg of total protein) were analyzed for the presence of oxidized proteins by oxyblot analysis. The membrane was re-probed with an antibody to b-
actin (n=4). D. Densitometric analysis of band intensity (Image J, NIH) of oxyblot showing significant decrease in carbonyl protein groups in UA2
treated group, ** P,0.01, * P,0.05.
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vehicle, UA, UA1 or UA2 (15 mM). The cell monolayer was then
subjected to a mechanical scratch-wound induced using a sterile
pipette tip. Cells were then cultured for additional period of
24 hoursin a serum-free basal medium in the continued presence of
vehicle, UA, UA1 or UA2. Cells were then fixed in a solution of 4%
paraformaldehyde in PBS and stained with crystal violet. Cells in
the injury area were visualized under phase-contrast optics (10X
objective) and the number of cells which had migrated into the
initially cell-free scratch area was counted.
Endothelial tube formation and chemotaxis cell
migration assays
HMEC-1 cells (16103cells/well) were dispensed to Matrigel-
coated 8-well chamber slides (Lab-Tek, Nalge Nunc International,
Rochester,NY,USA)in125 mlofEGM-2containingeithervehicle,
UA, UA1 or UA2 (15 mM) and incubated for 18 hours. The cells
were then visualized by microscopy and tube formation was scored
as described previously [4,41]. Cell migration analysis was
performed using Transwell membrane filters (Corning, Costar)
containing a polycarbonate filter with 8 mm pores. The bottom
chamber was filled with complete growth medium containing
chemoattractant growth factors. Cells (56104in 100 ml) were
seeded into each transwell with EGM containing 0.2% fetal bovine
serum with vehicle, UA, UA1 or UA2 (15 mM) and allowed to
migrate for 6 hours. At the end of the incubation, non-migrated
cells remaining in the transwell insert were removed. The migrated
cells (on the outer bottom of the transwell) were fixed with methanol
and stained with hematoxylin and eosin, and the stained cells were
counted in 5 or more random 100X fields. Each experiment was
performed in triplicate, and the experiment was repeated twice.
Growth correction was not applied because no increase in the cell
number was observed during the incubation period of 6 hours.
Quantification of cell proliferation
The proliferation of cultured endothelial cells, keratinocytes and
fibroblasts was measured using a colorimetric assay. Cells (16104)
were incubated with either vehicle, UA, UA1 or UA2 (15 mM) for
24, 48 and 72 hours. Then 10 ml of 3-(4, 5dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (R&D
Systems Inc. Minneapolis, MN) was added to each well and the
cells were incubated for a further 4 hours at 37uC. After the cells
were washed 3 times with PBS (pH 7.4), the insoluble formazan
product was dissolved by incubation with 100 ml detergent for
2 hours. The absorbance of each well was measured on an
Figure 7. Eight wound halves, control vs. UA2 treated were analyzed by immunofluorescence for the presence of nitrotyrosine
positive cells in indicated number of days post-injury. A. Representative images of staining. Nitrotyrosine-positive cells are green in color.
Nuclei were counterstained with Hoechst 33342. Scale ba r=50 mm. B. Graph showing the quantification of immunostaining. The percentage of
nitrotyrosine positive cells was determined using the IP lab software (BD Biosciences). Statistical analysis was performed two- way ANOVA using
GraphPad Prism version 5.00. D. (*P,0.001).
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enzyme-linked immunosorbent assay (ELISA) micro-plate reader
at 570 nm. Each experiment was performed in quadruplicate. The
proliferation rate was calculated as follows: (absorbance experi-
mental/Absorbance control-1) 6100.
Supporting Information
File S1
Found at: doi:10.1371/journal.pone.0010044.s001 (0.02 MB
RTF)
Author Contributions
Conceived and designed the experiments: SP MPM. Performed the
experiments: SC MRM SLC TA AB SCT. Analyzed the data: SC MRM
SLC TA RW. Contributed reagents/materials/analysis tools: QSY HH
NHG. Wrote the paper: RW MPM. Synthesized, characterized and
developed the uric acid analogues for the study: QSY HH NHG.
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