BASIC RESEARCH STUDIES
Polydeoxyribonucleotide restores blood flow in an
experimental model of ischemic skin flaps
Francesca Polito, PhD,a,bAlessandra Bitto, MD, PhD,bMariarosaria Galeano, MD,c
Natasha Irrera, JrD,bHerbert Marini, MD,aMargherita Calò, PhD,dFrancesco Squadrito, MD,band
Domenica Altavilla, PhD,bMessina, Italy
Background: Ischemia is a major factor contributing to failure of skin flap surgery, which is routinely used for coverage of
wounds to prevent infection and to restore form and function. An emerging concept is that adenosine A2Areceptors can
improve tissue oxygenation by stimulating angiogenesis, likely through vascular endothelial growth factor (VEGF). This
study assessed the ability of polydeoxyribonucleotide (PDRN) to restore blood flow and improve wound healing, acting
through the A2Areceptor, in a rat model of ischemic skin flaps.
Methods: The H-shaped double-flap model was used in male Sprague-Dawley rats. After surgical procedures, the animals
were randomized to receive intraperitoneal PDRN (8 mg/kg) or vehicle (NaCl 0.9%). Rats were euthanized 3, 5, and 10
days after skin injury, after the evaluation of skin perfusion by laser Doppler. The wounds underwent histologic analysis
and were measured for VEGF messenger RNA and protein expression, hypoxia inducible factor-1-? (HIF-1?), and
inducible nitric oxide synthase (iNOS) protein expression, and nitrite content.
Results: Blood flow markedly increased in blood flow in ischemic flaps treated with PDRN, with a complete recovery
starting from day 5 (ischemic flap ? vehicle, 1.80 ? 0.25; ischemic flap ? PDRN, 2.46 ? 0.25; P < .001). Administration
of PDRN enhanced the expression of VEGF (ischemic flap ? vehicle, 5.3 ? 0.6; ischemic flap ? PDRN, 6.2 ? 0.5; P <
.01) at day 5, and iNOS (ischemic flap ? vehicle, 3.9 ? 0.6; ischemic flap ? PDRN, 5.3 ? 1; P < .01), but reduced
HIF-1? expression (ischemic flap ? vehicle, 7 ? 1.1; ischemic flap ? PDRN, 4.8 ? 0.5; P < .05) at day 3. Histologically,
the PDRN-treated group showed complete re-epithelialization and well-formed granulation tissue rich in fibroblasts.
Conclusions: These results suggest that PDRN restores blood flow and tissue architecture, probably by modulating
HIF-1? and VEGF expression, and may be an effective therapeutic approach in improving healing of ischemic skin flaps.
(J Vasc Surg 2012;55:479-88.)
Clinical Relevance: Future therapies aimed at the enhancement of flap viability in vascular and reconstructive surgery, as well
A2Areceptor. This report provides evidence of an agent that may have clinical use to decrease the risk of flap ischemia.
made in understanding the cellular and biochemical inter-
play that impairs the normal wound healing response.
Ischemia is a major factor contributing to delayed healing
of wounds.1-3Skin ischemia is also a serious clinical prob-
lem in skin flap surgery, which is routinely used for wound
coverage to prevent infection and restore the form and
function of skin.4,5The most common clinical problem in
skin flap surgery is distal ischemic necrosis due to unpre-
dictable vasospasm, thrombosis, and insufficient vascular-
ity.4Wound healing in ischemic tissues, such as flap mar-
gins, is still a source of considerable morbidity in surgical
practice due to inadequate blood supply.6,7
Consequently, local hypoxia, which decreases granula-
tion tissue formation, collagen production, and fibroblast
tion and biomechanical strength parameters in wounds.8-11
Hypoxia is generally recognized as a physiologic cue to
induce angiogenesis, and as mentioned, an impaired reac-
tion to hypoxia could contribute to impaired wound heal-
ing. Hypoxia induces several genes, including those ex-
pressing hypoxia inducible factor-1 (HIF-1), vascular
endothelial growth factor (VEGF), and inducible nitric
oxide synthase (iNOS), which represent a synergistic and
well-orchestrated system involved in angiogenesis.12-14Ni-
From the Department of Biochemical, Physiological and Nutritional Sci-
ences, Section of Physiology and Human Nutrition,athe Department of
Clinical and Experimental Medicine and Pharmacology, Section of Phar-
macology,bthe Department of Surgical Sciences, Section of Plastic Sur-
nology, School of Veterinary Medical Sciences,dUniversity of Messina.
Competition of interest: none.
Reprint requests: Prof Francesco Squadrito, Department of Clinical and
Experimental Medicine and Pharmacology, Section of Pharmacology,
Torre Biologica 5thFlr, AOU Policlinico “G. Martino” Via C, Valeria
Gazzi, 98125 Messina, Italy (e-mail: firstname.lastname@example.org).
The editors and reviewers of this article have no relevant financial relationships
to disclose per the JVS policy that requires reviewers to decline review of any
manuscript for which they may have a competition of interest.
Copyright © 2012 by the Society for Vascular Surgery.
tric oxide (NO) upregulates VEGF; indeed, VEGF pro-
motes, in turn, NO production and also induces iNOS
expression in vascular endothelial cells, suggesting a con-
tinuous interplay between these angiogenic molecules.15
In light of this background, many potential therapies
have been investigated to improve angiogenesis in ischemic
skin flaps by using pharmacologic and physical agents.16-19
A large body of literature suggests the therapeutic role of
adenosine and A2Areceptor agonists in the promotion of
wound healing.20-22Adenosine A2Areceptors, specifically,
are expressed on most cell types involved in wound healing,
including macrophages, fibroblasts, and endothelial cells;
A2Astimulation in these cells induces VEGF produc-
tion.20-22Moreover, some of the cytokines released during
the inflammatory phase of wound healing, such as tumor
necrosis factor (TNF)-? and interleukin (IL)-1, upregulate
adenosine A2Areceptor expression.20-23Stimulation of the
adenosine receptor results in an increase in VEGF produc-
tion and also in fibroblast differentiation and maturation,
increasing the rate of granulation tissue and, consequently,
accelerating the repair process.24-26
Polydeoxyribonucleotide (PDRN) is a mixture of nu-
cleotides, acting through adenosine receptors, that is able
to stimulate VEGF production during pathologic condi-
tions of low tissue perfusion such as diabetes mellitus,
thermal injury, and hind limb ischemia.27-29PDRN in-
creases VEGF,29most likely through the activation of
HIF-1?. The drug, which is extracted from the sperm of
therapeutically used in humans as an agent to stimulate
In vitro, PDRN enhances the growth rate of human
fibroblasts in primary cultures at therapeutic concentra-
tions.32,33Other clinical studies have also pointed out that
PDRN promotes a more rapid healing of autologous skin
graft at donor sites and stimulates corneal epithelium re-
generation after photorefractive keratectomy.34-36The fa-
vorable effect on cell proliferation appears to be mediated
by the activation of purinergic A2Areceptor.32,37There-
fore, in light of this evidence, we aimed to assess the ability
of PDRN to restore blood flow in a rat model of ischemic
skin flaps after systemic administration.
MATERIALS AND METHODS
All animal procedures in this study complied with the
standards for care and use of animal subjects as stated in the
of Laboratory Animal Resources, Commission on Life Sci-
ences, National Research Council, National Academy
Press, Washington DC, revised 1996). The protocol was
evaluated and accepted by the Ethics Committee of the
University of Messina.
Animals. The study used 42 male Sprague-Dawley
rats, maintained on 12-hour dark/light cycle at 21°C and
allowed free access to water and standard rodent diet.
Environmental conditions (light, temperature, and humid-
ity) were constantly maintained in the Animal Facility for
Ischemic flap model and experimental design. For
the experimental model of ischemic skin wound healing,
H-shaped flaps were used as originally described by
Quirinia et al.38,39After general anesthesia with sodium
pentobarbital (50 mg/kg), hair on the back was removed
skin, and the skin was washed with povidone-iodine solu-
tion and wiped with sterile water.
The H-shaped double flap, consisting of a cranially and
a caudally based flap (2 cm wide and 4 cm long), was
marked with a black marker on the dorsal skin (Fig 1). The
ischemic test wound was the horizontal incision in the H.
The skin and panniculus carnosus were both incised to
produce a full-thickness wound. After incision, the flaps
were raised, perforating branches of the flaps were cut, and
the flaps were sutured back in position with 4-0 nylon
suture in an interrupted fashion.
After the surgical procedures, the animals were ran-
domized (21 rats in each group) to receive daily intraperi-
toneal PDRN (8 mg/kg; Mastelli SRL, Sanremo, Italy) or
vehicle (NaCl 0.9%). The PDRN dose was chosen accord-
ing to a previous investigation in our laboratory demon-
strating that the 8 mg/kg dose was able to improve blood
Seven animals in each group were euthanized with an
overdose of anesthetic after 3, 5, and 10 days, respectively,
and the wounds were divided into three segments used for
histologic, biochemical, and molecular analysis. The skin
sample was harvested from the center of the wound joining
used for laser Doppler analysis.
Laser Doppler perfusion imaging. Skin perfusion
was evaluated in the center of the flap, as shown in Fig 1,
using a PIM 1.0 laser Doppler perfusion imager (Perimed/
Periscan, Stockholm, Sweden). Measurements were ob-
tained over the same regions of interest on the day of
surgery (baseline) and at 3, 5, and 10 days after surgery.
The laser source was mounted on a movable rack exactly 20
cm above the skin. The laser beam (780 nm) penetrates the
tissue and part of the incident light is scattered by moving
red blood cells in the vessels, which is detected and pro-
cessed to provide computerized color-coded photographs.
Low or no perfusion is displayed as dark blue, whereas the
highest perfusion interval is displayed as red. The average
skin perfusion at the test sites was estimated by the mea-
surement of perfusion values of a region of interest (ROI)
of approximately 70 individual sampling sites. Once a ROI
was identified, the same ROI was searched in all the other
Isolation of cytoplasmic and nuclear proteins. Briefly,
skin samples were homogenized in 1-mL lysis buffer (25
mM Tris/HCL [pH 7.4], 1.0 mM ethyleneglycotet-
raacetic acid, 1.0 mM ethylenediaminetetraacetic acid, 0.5
phenyl methylsulfonal fluoride, aprotinin, and leupeptin
[10 ?g/mL each]) with an Ultra Turrax (IKA, Staufen,
JOURNAL OF VASCULAR SURGERY
480 Polito et al
Germany) homogenizer. The homogenate was centrifuged
at 15,000 g for 15 minutes at 4°C. The supernatant was
collected and used for protein determination using a pro-
tein assay kit (Bio-Rad, Richmond, Calif).
Determination of VEGF, HIF-1?, and iNOS by
Western blot analysis. Protein samples (30 ?g) were
denatured in reducing buffer (62 mM Tris [pH 6.8], 10%
glycerol, 2% sodium dodecyl sulfate [SDS], 5% ?-mercap-
toethanol, and 0.003% bromophenol blue) and separated
by electrophoresis on a SDS (12%) polyacrylamide gel. The
separated proteins were transferred on to a nitrocellulose
membrane, then blocked with 5% nonfat dry milk in Tris-
buffered saline (TBS)-0.1% Tween for 1 hour at room
temperature, washed three times for 10 minutes each in
TBS-0.1% Tween, and incubated with a primary VEGF or
iNOS (Abcam, Cambridge, United Kingdom) or HIF-1?
(Chemicon, Temecula, Calif) antibody in TBS-0.1%
Tween overnight at 4°C.
Membranes were washed three times for 10 minutes
each in TBS-0.1% Tween and incubated with a second
antibody peroxidase-conjugated goat antirabbit immuno-
globulin G (Pierce, Rockford, Ill) for 1 hour at room
the enhanced chemiluminescence system according to the
manufacturer’s protocol (Amersham, Little Chalfont,
United Kingdom). The protein signal was quantified by
scanning densitometry using a bio-image analysis system
tal group were expressed as relative integrated intensity
compared with ?-actin (Cell Signaling, Danvers, Mass) as a
Real-time polymerase chain reaction. Total RNA
was extracted from the skin by using a commercial reagent
(Trizol; Invitrogen, Carlsbad, Calif) according to the man-
ufacturer’s instruction and was spectrophotometrically
quantified (Biophotometer; Eppendorf, Amburg, Ger-
many). After reverse transcription (cDNA Archive; Applied
Biosystems, Monza, Italy), complementary DNA (cDNA)
was used to quantify the amount of VEGF cDNA by
real-time polymerase chain reaction (PCR) using ABI
Prism 7300 (Applied Biosystems), as well as ?-actin cDNA
as the endogenous control. The results for the target gene
were expressed as the n-fold difference relative to the
endogenous control (relative expression levels).
Fig 1. A, In this schematic representation of the H-shaped double-flap model, the rectangle represents the ischemic
test wound and the area investigated by laser Doppler, and the square inside the rectangle represents the area of region
of interest calculation. Laser Doppler images are shown for (B) intact skin and in ischemic flaps at (C) 10 minutes, in
ischemic flaps ? vehicle at (D) 3, (E) 5, and (F) 10 days after wounding, and in ischemic flaps ? polydeoxyribonucle-
perfusion interval is displayed as red.
JOURNAL OF VASCULAR SURGERY
Volume 55, Number 2
Polito et al 481
Determination of NO2?/NO3?. NO products were
determined in wound lysates using the Griess reaction.
Samples of skin wounds were homogenized in 2? lysis
buffer (1? lysis buffer: 1% Triton X-100, 20 mM Tris/
HCL [pH 8.0], 137 mM NaCl, 10% glycerol, 5 mM
ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfo-
nyl fluoride, 15 ?g/mL leupeptin). Lysates (1 mL) were
cleared by centrifugation at 10,000 g for 30 minutes.
Cleared wound lysates (200 ?L) were mixed with 20 ?L
sulfanilamide (dissolved in 1.2 M HCl) and 20 ?L N-
naphtylethylendiamine dihydrochloride. After 5 minutes at
room temperature, the absorbance was measured at 540
nm with reference wavelength at 690 nm. Combined
nicotinamide adenine dinucleotide phosphate (NADPH).
For this, 80 ?L of samples were incubated with 10 ?L of
Aspergillus nitrate reductase (1 U/mL, Sigma, St. Louis,
MO) and 10 ?L of NADPH (1 mmol/L) for 1 hour at
27°C before the addition of the Griess reagent.
All samples and standards were assayed in triplicate.
Data are expressed as mean ? standard deviation of
nmol/g of tissue. Phenylmethylsulfonyl fluoride, leupep-
tin, and N-naphtylethylendiamine dihydrochloride, were
obtained from Sigma Biochemicals (St. Louis, Mo).
Histologic and immunohistochemical analysis.
Histologic evaluations took place on coded samples after 5
and 10 days of treatment with vehicle or PDRN by a
pathologist blinded to the treatment. The skin sample was
harvested from the center of the wound joining the tips of
the flaps. The specimens were immediately fixed in 10%
neutral buffered formalin. Sections were washed in tap
water, dehydrated in graded ethanol, cleared in xylene, and
embedded in paraffin according to routine procedures.
Sections of paraffin-embedded tissue (5 ?m thick) were
mounted on glass slides, hydrated in distilled water, and
stained with hematoxylin and eosin.
The following parameters were evaluated and scored:
re-epithelialization, dermal matrix deposition and regener-
ation, granulation tissue formation, and remodeling. The
edges of the wound in each of the sections were used as
comparison for scoring. The histologic score used in this
study was performed and evaluated according to the liter-
els.40The criteria used as histologic scores of wound heal-
ing are summarized in the Table.
Paraffin-embedded tissues were sectioned (5 ?m), and
antigen retrieval was performed using 0.05 M sodium
citrate buffer. Tissues were treated with primary antibody
against platelet cell adhesion molecule-1 (PECAM-1)/
CD31 (Santa Cruz Biotechnology, Santa Cruz, Calif).
Secondary antibody was provided by Innovex (Richmond,
Calif), and the location of the reaction was visualized with
3,30-diaminobenzidine tetrahydrochloride (Sigma). Slides
were counterstained with hematoxylin and mounted with
To assess the angiogenic response, microvessel density
was estimated after PECAM-1 staining. Briefly, three “hot
?with nitrate reductase in the presence of reduced
?levels were determined by reducing NO2
spots,” or areas with the highest visible blood vessel density
(marked by the vessel marker) per section were selected,
and the blood vessels with a visible lumen were counted per
high-power field (original magnification ?40) by two pa-
thologists blinded to the samples. For each group, nine to
18 fields in three to six randomly chosen sections were
Drugs. PDRN was a gift of Mastelli Srl, Sanremo,
Italy. PDRN is extracted from the sperm of trout bred for
process with purifying and high temperature sterilizing
procedures to obtain an ?95% pure active principle with-
out pharmacologically active proteins and peptides (Regis-
tration Dossier, Italian Ministry of Health). PDRN was
freshly prepared, dissolved in 0.9% saline solution, and
administered immediately after surgical procedures.
SD. The data were analyzed by two-way analysis of variance
test, followed by the Tukey multiple comparison test. The
level for statistical significance was set at P?.05.Graphswere
drawn using Graph Pad Prism 4.0 software (GraphPad Soft-
ware, La Jolla, Calif).
PDRN improves tissue perfusion of ischemic flaps.
Fig 1 shows laser Doppler perfusion images of tissue perfu-
sion of the study groups after 10 minutes, and at 3, 5, and
10 days after the surgical procedures. A dramatic fall in
of the entire flap demonstrates lack of blood supply, with a
marked ischemia in the center of the flap (Fig 1, C). At 5
and 10 days after surgery, flow gradually and significantly
increased in untreated ischemic flaps (Fig 1, E and F). In
contrast, a marked increase in blood flow was observed in
days and gradually over time, with a complete recovery
starting from day 5 (Fig 1, G–I).
Fig 2 shows the quantitative analysis of skin perfusion
extrapolate by ROI calculation. Skin perfusion significantly
Table. Criteria to evaluate histologic scores of wound
1Little epidermal and
Thin granulation layer
4Very thick granulation
JOURNAL OF VASCULAR SURGERY
482 Polito et al
diminished in the ischemic flap 10 minutes after surgery
(intact skin. 2.88 ? 0.3; ischemic flap, 0.8 ? 0.24; Fig 2, A
and B). The ischemic flap in untreated ischemic animals
showed a slight increase in blood flow evaluated at 3 (1.23 ?
0.20), 5 (1.80 ? 0.25), and 10 days (2 ? 0.3) after surgery
(Fig 2, A and B). In contrast, a significant increase in blood
flow was noticed in ischemic flaps treated with PDRN after
3 days and gradually over time (P ? .001 at 3, 5, and 10
days vs ischemic flap ? vehicle; Fig 2, A and B).
PDRN enhances VEGF expression in ischemic
flaps. To assess the effect of PDRN on the repair process,
we examined the expression of VEGF mRNA (Fig 3, A)
Western blotting. Ischemic flaps showed an increase in
VEGF mRNA and protein expression at day 3 (3.3 ? 0.6)
0.5). The vehicle animals treated with PDRN showed higher
amounts of VEGF mRNA and protein at day 3 (5.3 ? 0.8),
with a further increase at day 5 (6.2 ? 0.5) and a decline at
day 10 (3 ? 0.4 for ischemic flap ? PDRN; P ? .001 vs
ischemic flap; Fig 3, A and B).
PDRN reduces HIF-1? expression in ischemic
flaps. To study the effects of PDRN on HIF-1? expres-
sion, skin samples from each group were evaluated by
Western blot. Ischemia caused a marked increase in
HIF-1? expression at day 3 and 5, whereas HIF-1? expres-
sion was barely detectable at day 10 (Fig 3, C). The
administration of PDRN significantly reduced HIF-1? ex-
Fig 2. A, Quantitative analysis of blood flow evaluated by laser Doppler is shown at 10 minutes, and at 3, 5, and 10
days after surgical procedures in the various groups. Rats were administered vehicle or polydeoxyribonucleotide
(PDRN) starting immediately after surgery. The blood flow of the ischemic flap is expressed as a relative amount compared
with the intact skin.*P ? .001 vs ischemic flap ? vehicle, #P ? .01 vs ischemic flap ? vehicle. B, Percentage of blood flow
vs intact skin evaluated by laser Doppler at 3, 5, and 10 days after surgical procedures. Rats were administered vehicle or
PDRN starting immediately after surgery. The blood flow of ischemic flaps is expressed as a relative amount compared with
the intact skin.*P ? .001 vs ischemic flap ? vehicle. The error bars show the standard deviation.
JOURNAL OF VASCULAR SURGERY
Volume 55, Number 2
Polito et al 483
pression in the ischemic flaps at days 3 (4.8 ? 0.5) and 5
(3.2 ? 0.9, ischemic flap ? PDRN; Fig 3, C).
PDRN enhances iNOS expression and NO2
during wound healing and angiogenesis in response to
tissue ischemia, and protein expression of iNOS and
increase in iNOS expression at day 3 (3.9 ? 0.6) and day 5
(4.2 ? 0.6) and NO2
and day 5 (35 ? 5), with a decline at day 10 after wounding
(Fig 4, A and B). In contrast, the administration of PDRN
significantly enhanced iNOS expression (5.3 ? 1) and
with a marked reduction at day 5 and 10 (Fig 4, A and B).
PDRN improves histologic alterations and vascu-
larity in ischemic flaps. Skin samples obtained from un-
treated ischemic flaps showed a poor healing process, char-
acterized by the persisting presence at day 10 of the crust, a
sustained inflammatory infiltrate with a very low organized
granulation tissue (Fig 5). By contrast, PDRN administra-
?in ischemic flaps. Inducible NOS is important
?content was assessed in the wounded skin at
?content at day 3 (28 ? 4)
?(45 ? 6) content in ischemic flaps at day 3,
architecture starting from day 5 (Fig 5). In agreement with
these findings, the histologic score healing indicated that
PDRN qualitatively and quantitatively improved wound
healing at day 10 (Fig 5). Staining for CD31 revealed an
increase in neovessel formation in ischemic flaps treated
with PDRN compared with vehicle, supporting the results
obtained on VEGF expression (Fig 5).
Skin flap failure, due principally to distal necrosis,
represents a major complication in plastic and recon-
structive surgery.4Several therapeutic approaches have
been used, in various experimental models, to enhance
skin viability by improving angiogenesis and blood flow,
although the results are controversial and make some
drugs impractical for clinical use.16-19In light of the
promising role of adenosine and adenosine receptors in
improving blood supply, angiogenesis, and wound heal-
ing,41,42we hypothesized that PDRN, an A2Areceptor
agonist, might exert protective effects in the ischemic
flap. PDRN is commonly used in clinical setting by
plastic and vascular surgeons in presurgical cutaneous
Fig 3. A, Expression of vascular endothelial growth factor (VEGF) messenger RNA is shown at 3, 5, and 10 days after
surgery. Bars represent the mean ? standard deviation (error bars) of seven animals.*P ? .01 vs ischemic flap ? vehicle.
B, Western blots show VEGF in ischemic flaps at 3, 5, and 10 days after surgery. Upper panel, Representative
autoradiography highlights VEGF expression. Lower panel, Quantitative data represent the mean ? standard
deviation (error bars) of seven animals.*P ? .05 vs ischemic flap ? vehicle. C, Western blots show hypoxia inducible
factor-1 (HIF-1?) at 3, 5, and 10 days after surgery. Upper panel, Representative autoradiography highlights HIF-1?
expression. Lower panel, Quantitative data represent the mean ? standard deviation (error bars) of seven animals.
*P ? .05 vs ischemic flap ? vehicle. PDRN, Polydeoxyribonucleotide.
JOURNAL OF VASCULAR SURGERY
484 Polito et al
treatments and venous ulcers because it stimulates fibro-
blast metabolism and promotes an increase in the num-
ber of fibroblasts and in dermal matrix component pro-
duction.32,43Consequently, the present study was done
to evaluate the efficacy of PDRN in ischemic wound
We first demonstrated, using a well-known experimen-
tal model of ischemic skin flap, that PDRN efficiently
improves blood flow by stimulating VEGF and leading in
turn to an improvement of skin healing. Specifically, we
used an H-shaped double-flap model as previously de-
scribed by Quirinia et al.38,39This model resembles many
of the molecular abnormalities that characterize the envi-
ronment of ischemic chronic wounds in humans. Indeed, it
is associated with a moderate transient ischemia that causes
delayed healing of full-thickness skin wounds, leading in
turn to a reduction in the biomechanical properties of
are tightly associated with an accelerated reperfusion of the
flap, confirmed by laser Doppler analysis that showed this
effect was extremely evident during the first days of sys-
temic administration, when blood flow tended to be slower
in untreated flaps. Under our experimental conditions,
flaps recovered from ischemia from day 10 on, thus show-
ing that PDRN treatment improves blood flow and is also
able to promote skin survival and grafting.
The molecular mechanism(s) by which PDRN re-
duces ischemia is likely related to stimulation of the
adenosine A2Areceptors and the consequent upregula-
tion in VEGF expression and release. VEGF is a key
mediator of angiogenesis and is released from hypoxic
tissues in physiologic and pathologic conditions.44In
this context, previous studies showed that the adminis-
tration of adenosine or adenosine agonists, as well as the
upregulation of endogenous adenosine, can increase the
expression of VEGF.45This effect is probably mediated
by HIF-1?, a transcription factor that is able to regulate
HIF-1? accumulates under hypoxic conditions and acti-
vates VEGF transcription by binding to specific pro-
In our study, we found a higher expression of VEGF in
PDRN-treated rats, and surprisingly, a lower expression of
HIF-1? compared with untreated animals. This latter
might be explained by the enhancement in skin perfusion
and reduction of hypoxia; consequently, at the intervals
examined in our experiment, the observed decrease of
HIF-1? to basal levels suggests that tissue oxygenation
returns to adequate volume. HIF-1? also regulates the
expression of other hypoxia-responsive genes, such as
iNOS.48,49NO has several biologic roles in the process of
cutaneous wound healing,50including regulation of vaso-
dilatation, VEGF-induced neoangiogenesis, and protec-
tion against invading pathogens.50,51In agreement with
these data, hypoxia induced a marked expression of iNOS
and nitrate production in ischemic skin flaps. In contrast,
PDRN treatment caused, in a first step of healing, an
Fig 4. A, Western blots show inducible nitric oxide synthase (iNOS) at 3, 5, and 10 days after surgery. Upper panel,
Representative autoradiography highlights iNOS expression. Lower panel, Quantitative data represent the mean ?
standard deviation (error bars) of seven animals.*P ? .01 vs ischemic flap ? vehicle. B, Quantitative data of NO
products in ischemic flaps are shown at 3, 5, and 10 days after surgery. Data represent the mean ? standard deviation
(error bars) of seven animals. #P ? .001 vs ischemic flap ? vehicle. PDRN, Polydeoxyribonucleotide.
JOURNAL OF VASCULAR SURGERY
Volume 55, Number 2
Polito et al 485
upregulation of iNOS and nitrite content, while a marked
reduction of both was observed at day 5 and 10 after
wounding, thus confirming its ability to restore an ade-
quate tissue blood supply and to improve the healing
Overall, our experiments indicate that PDRN in-
duces two main steps of reperfusion in ischemic flaps. In
the early stage, the vasodilator effect of VEGF is medi-
ated especially by NO, which counterbalances the vaso-
spasm caused by surgical procedures. In later stages, the
angiogenic effect of VEGF improves local blood flow.
Indeed, the stimulation of the A2Areceptor subtype has
been shown to induce vasodilation itself, and specific
agonists of this receptor have been developed in recent
years to provide a stronger effect, especially at myocar-
dial level.52As a matter of fact, untreated ischemic flap
showed a still-incomplete re-epithelialization and the
persistence of inflammatory infiltrate, with a very poor
organized granulation tissue. In contrast, a complete
re-epithelialization and well-formed granulation tissue
rich in fibroblasts oriented parallel to the epidermal layer
were observed in flaps treated with PDRN. Our data are
in agreement with previous clinical studies in which
topically applied PDRN positively affected the repair
processes and significantly reduced the time of complete
healing at autologous skin graft donor sites.34,35
A phase 4 double-blind, randomized placebo-con-
trolled, safety and efficacy trial investigating the effic-
acy of PDRN in improving the healing of vascular
and diabetic ulcers (http://clinicaltrials.gov Identifier:
NCT00638872) has recently been completed. This trial
comprised 200 patients, aged 45 to 80 years, who were
randomly allocated to receive the active drug or placebo
in indistinguishable formulations. The primary outcome
measure is the reduction of ulcer rate and secondary
outcomes are safety and tolerability of the compound.
Fig 5. Histologic evaluation (hematoxylin and eosin) of skin wound obtained from ischemic flaps is shown at day 5
and 10 after surgery (original magnification, ?10). A, Ischemic flap ? vehicle at day 5 shows the absence of a healing
process and a marked inflammatory infiltrate. B, Ischemic flap ? polydeoxyribonucleotide (PDRN) at day 5 shows a
still-incomplete healing process. C, Ischemic flap ? vehicle at day 10 shows a poor healing process, with a sustained
inflammatory infiltrate and a marked granulation tissue. D, Ischemic flap ? PDRN at day 10 shows a complete healing
and a normal skin architecture. Histologic score of (E) granulation tissue thickness and (F) epidermal regeneration
were evaluated at day 10 according to the Table criteria. Each bar represents the mean ? standard deviation (error bar)
of seven animals *P ? .001 vs ischemic flap ? vehicle. G, CD31 staining of ischemic flap ? vehicle at day 10 shows
slight staining of vascular structures. H, Ischemic flap ? PDRN at day 10 shows a marked staining of small vessels. I,
Microvascular density shows the number of positive capillaries per microscopic field. Each bar represents the mean ?
standard deviation (error bars) of seven animals. *P ? .05 vs ischemic flap ? vehicle.
JOURNAL OF VASCULAR SURGERY
486 Polito et al
These results confirm the positive effects of PDRN in
improving blood flow, indicating that this compound
viability in vascular and reconstructive surgery and to
achieve a faster wound repair under ischemic conditions.
Conception and design: FP, MG, FS
Analysis and interpretation: FP, AB, HM
Data collection: FP, MG, NI, MC
Writing the article: FP, AB, HM
Critical revision of the article: AB, HM, FS
Final approval of the article: FP, AB, MG, NI, HM, MC,
Statistical analysis: NI, HM
Obtained funding: FS, DA
Overall responsibility: FS
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