A inibição seletiva da COX 2 melhora a cicatrização cutânea de úlceras por pressão em camundongos através da redução da expressão da iNOS.

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Selective inhibition of COX-2 improves cutaneous wound healing of pressure
ulcers in mice through reduction of iNOS expression
Bruna Romana-Souza, Jeanine Salles dos Santos, Luana Graziella Ban-
deira, Andre´a Monte-Alto-Costa
PII: S0024-3205(16)30238-7
DOI: doi: 10.1016/j.lfs.2016.04.017
Reference: LFS 14858
To appear in: Life Sciences
Received date: 4 December 2015
Revised date: 12 April 2016
Accepted date: 13 April 2016
Please cite this article as: Romana-Souza Bruna, Santos Jeanine Salles dos, Bandeira
Luana Graziella, Monte-Alto-Costa Andre´a, Selective inhibition of COX-2 improves cu-
taneous wound healing of pressure ulcers in mice through reduction of iNOS expression,
Life Sciences (2016), doi: 10.1016/j.lfs.2016.04.017
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Romana-Souza et al. 1 
Selective inhibition of COX-2 improves cutaneous wound healing of pressure ulcers in 
mice through reduction of iNOS expression 
 
Authors’ names: 
Bruna Romana-Souza 
a
, 
Jeanine Salles dos Santos 
b
, 
Luana Graziella Bandeira 
a
, 
Andréa Monte-Alto-Costa 
a
 
 
Authors’ affiliation: 
a 
Department of Histology and Embryology, State University of Rio de Janeiro, Rio de 
Janeiro, Brazil. 
b
 Histocompatibility and Cryopreservation Laboratory, State University of Rio de Janeiro, Rio 
de Janeiro, Brazil. 
 
Corresponding author’s address: 
Dr. Bruna Romana-Souza 
State University of Rio de Janeiro (UERJ) 
Department of Histology and Embryology, 
Av. Marechal Rondom, 381, 2° andar, 
20950-003. Rio de Janeiro, RJ - BRAZIL. 
Telephone: +55 21 2334 2421 Fax: +55 21 2334 2426 
E-mail address: bruna.souza@uerj.br 
 
Running title: Cyclooxygenase-2 inhibition in pressure ulcers 
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Romana-Souza et al. 2 
 
Word count: 4,363 words (Introduction 500 words; Discussion 1,451 words; Conclusion 49 
words) 
 
Figure count: 5 
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Romana-Souza et al. 3 
Structured abstract 
 
Aims: Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are involved in chronic 
inflammation observed in chronic lesions. Nonetheless, neither study demonstrated if 
decreased COX-2 activation could promote the wound healing of pressure ulcers. Therefore, 
this study investigated the effect of the administration of celecoxib (a selective COX-2 
inhibitor) in wound healing of pressure ulcers. 
Materials and Methods: Male mice were treated daily with celecoxib until euthanasia. One 
day after the beginning of treatment, two cycles of ischemia–reperfusion by external 
application of two magnetic plates were performed in skin to induce pressure ulcer formation. 
Key findings: Celecoxib administration reduced the protein expression of inducible nitric 
oxide synthase (iNOS), COX-2 and PGE2. The hydroperoxide levels, neutrophil and 
macrophage number, and protein elastase and matrix metalloproteinase-1 levels were reduced 
in celecoxib-treated group when compared to control group. Celecoxib administration 
increased myofibroblastic differentiation, re-epithelialization and wound contraction, and 
decreased the skin necrosis and angiogenesis. Celecoxib administration also stimulated the 
formation of a more organized and mature scar increasing collagen deposition and reducing 
tenascin-C expression. 
Significance: Celecoxib administration improves the wound healing of pressure ulcers 
through decreased expression of iNOS and COX-2, which reduces wound inflammation and 
promotes dermal reconstruction and scar formation. 
 
Keywords: cyclooxygenase-2; pressure ulcer; mice; celecoxib; prostaglandin E2. 
 
 
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Romana-Souza et al. 4 
Chemical compounds studied in this study: 
Celecoxib (PubChem CID: 2662); Ketamine (PubChem CID: 3821); Xylazine 
(PubChem CID: 5707); Formalin (PubChem CID: 712); Hematoxylin (PubChem CID: 
442514); Eosin yellowish (PubChem CID: 11048); Hydrochloric acid (PubChem CID: 313); 
Sodium hydroxide (PubChem CID: 14798); Xylenol orange (PubChem CID: 16220156); 
Sodium dodecylsulfate (PubChem CID: 3423265); Polyacrylamide (PubChem CID: 6579). 
 
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1. Introduction 
Pressure ulcers are one of the chronic healing problems most difficult to treat and its 
number is expected to increase in an aging society, as Brazilian population [1-5]. The 
ischemia-reperfusion (IR) seems to be the major initialing factor of the tissue injury in the 
pressure ulcers [6,7]. In addition, the toxic concentrations of reactive oxygen species (ROS) 
associated to exacerbated inflammatory response are key events of tissue destruction in IR-
induced chronic lesions [8]. 
The ROS are short-lived entities and electron acceptors that are continuously produced 
by all cells at low levels during the course of normal aerobic metabolism [9,10]. In wounded 
and inflamed tissues, the synthesis of ROS by inflammatory cells contributes to the defense 
against invading pathogens and mediates intracellular pathways [10]. However, excessive 
amounts of ROS have deleterious effects on lipids, proteins and nucleic acids of cells 
involved in skin repair leading to tissue damage [10]. It has been demonstrated that fluid from 
chronic leg ulcers presents elevated levels of 8-isoprostane (a product of lipid peroxidation) 
and high ROS levels when compared to that of acute lesions [11,12]. Nonetheless, other 
mediators may also participate in chronic inflammation of ulcers as cyclooxygenase-2 (COX-
2). The COX is present in three isoforms: COX-1, COX-2 and COX-3. The COX-1 is 
normally expressed in the body and has many physiological functions as thromboxane A2 
synthesis in platelets [13]. The COX-3 was recently identified in canine and human cortex 
and it has been involved in a central mechanism of pain and fever [14]. The COX-2 is not 
normally expressed in the most cells, but is rapidly induced in response to inflammatory 
stimuli producing prostaglandins, such as prostaglandin E2 (PGE2) [13]. In chronic venous leg 
ulcers, the excessive expression of inducible nitric oxide synthase (iNOS), COX-2 and high 
PGE2 levels on wound bed contributes to chronic inflammation observed in these lesions 
[15,16]. Thus, the persistent infiltration of inflammatory cells associated to the increase in the 
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ROS production and prostaglandins may contribute to non-healing of chronic lesions. 
Therefore, the administration of anti-inflammatory and antioxidant compounds may be a good 
therapeutic strategy to promote the wound healing of pressure ulcers. 
The celecoxib is a nonsteroidal anti-inflammatory drug (NSAID) that specifically 
inhibits COX-2 having a significant anti-inflammatory property, but lesser toxicity than other 
NSAID such as ibuprofen [17]. The effect of decreased COX-2 activation on cutaneous 
wound healing of acute lesions is still controversial. Some studies propose that decreased 
COX-2 activation decreases inflammatory responsein sponge implants and promotes the 
closure of excisional lesions, while others propose that celecoxib administration reduces the 
wound closure and scar formation in incisional and excisional lesions of rodents [18-20]. In 
addition, other studies also suggest that celecoxib administration does not alter cutaneous 
wound healing of rat acute incisional lesions in mice sponge implants [21]. In experimental 
pressure ulcers, the inhibition of both COX-1 and COX-2 by ibuprofen administration does 
not have a significant effect on wound healing [22]. Nevertheless, neither study demonstrated 
whether the decreased COX-2 activation may reduce oxidative damage and inflammatory 
response improving the cutaneous wound healing of pressure ulcers. 
Therefore, this study investigated the effect of celecoxib administration on wound 
healing of pressure ulcers using a murine model of IR-induced skin injury. 
 
2. Material and Methods 
 
2.1. Animals 
All procedures were carried out in strict accordance with the Brazilian Legislation 
regarding Animal Experimentation (nº 11.794, from October 8, 2008). All experiments in this 
study were approved by the Ethical Committee for Animal Use of the State University of Rio 
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de Janeiro (n° 026/2014). Male Swiss mice (8-12 weeks) were kept in groups (5 animals per 
cage) under controlled conditions with 12 hour light/dark cycle. 
 
2.2. Experimental design 
Mice (n=45) were daily treated by gavage with 5 mg/kg of celecoxib (a selective 
COX-2 inhibitor) (Laboratórios Pfizer Ltda., São Paulo, Brazil) dissolved in water, until 
euthanasia [23]. Another group (n=45) was treated by gavage with vehicle. One day after 
beginning of celecoxib administration, all animals were intraperitoneally anesthetized with 
ketamine (150 mg/kg) and xylazine (15 mg/kg) and their dorsum were shaved. Dorsal skin 
was gently pulled up and placed between a pair of magnet disks with 8-mm diameter (Eudes 
Angelo de Almeida Produtos ME, São Paulo, Brazil). Epidermis, dermis and hypodermis 
were pinched between the magnet plates [7,24]. Two IR cycles were performed in each mouse 
to initiate chronic ulcer formation. A single IR cycle consisted of a 16 hours period of magnet 
placement, followed by a release period of 8 hours. After magnet application, animals were 
left to emerge from anesthesia and individually housed. After the second IR cycle, all mice 
developed two circular ulcers separated by a bridge of normal skin and located 2 cm from the 
occipital bone of the cranium, and this point was considered day 0. Thus, IR injury model was 
used to create chronic lesion similar to pressure ulcer of stage II as previously described 
[7,24]. To create acute lesion, another group of animals (n=5) was intraperitoneally 
anesthetized as described above. After shaving the dorsum, two circular full-thickness 
excisional wounds were created using a biopsy punch with 8 mm diameter as described [25]. 
These lesions were excised 7 days after wounding and used in COX-1 and COX-2 
immunoblotting. 
To investigate the role of iNOS on benefic effects of celecoxib in wound healing of 
mice pressure ulcers, another group of mice (n=6) was daily treated with 50 mg/kg of the N
G
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nitro-L-arginase methyl ester (L-NAME), a non-selective NOS inhibitor, in drinking water 
until euthanasia [26]. The control group (n=6) received only water (in the same volume of 
experimental group). One day after beginning of L-NAME administration, two pressure 
ulcers were created in mice dorsum as described above and collected 7 days after only. In 
these groups, the wound closure, re-epithelialized wound area and iNOS protein expression 
were analyzed. 
 
2.3. Macroscopic analyses 
To evaluate wound closure, lesions were measured soon after application of second IR 
cycle (day 0), 3, 7 and 14 days later without scab removal as described [24]. The results are 
expressed as percentage of the original wound area. 
To measure necrotic area, a transparent plastic sheet was placed over the ulcer and the 
margins of total ulcer and necrotic area were traced soon after application of second IR cycle 
(day 0) and 7 days later [27]. After digitalization, wound area was measured using ImageJ 
software (National Institute of Mental Health, Bethesda, MD, USA). Results are expressed as 
percentage of necrotic area. 
 
2.4. Tissue harvesting 
Mice (15 animals per day) were intraperitoneally anesthetized with ketamine (150 
mg/kg) and xylazine (15 mg/kg) and killed by carbon dioxide inhalation 3, 7 and 14 days after 
ulceration. Ten lesions (two in each animal) and adjacent normal skin per group were 
formalin-fixed (pH 7.2) and paraffin-embedded and destined to histological analyses. Ten 
lesions (two in each animal) per group were frozen at -70ºC and destined to perform 
hydroxyproline levels. Ten lesions (two in each animal) per group were macerated in lysis 
buffer and total protein concentration was determined using the bicinchoninic acid protein 
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assay (Thermo Fisher Scientific, Rockwood, TN, USA). This lysate was used to perform lipid 
hydroperoxide levels, ELISA and immunoblotting. 
 
2.5. Histological analyses 
Sections (5 µm) were stained with hematoxylin-eosin to quantify, microscopically, the 
length of migrating epithelial tongue, re-epithelialized wound area, neo-epidermis thickness 
and the volume density of blood vessels. To measure the length of migrating epithelial 
tongue, re-epithelialized wound area and neo-epidermis thickness, slides were digitalized 
using Pannoramic Digital Slide Scanner (3DHistech Ltd., Budapest, Hungary), and the 
measurements were performed using Pannoramic Viewer software (3DHistech Ltd.) as 
previously described [24,28]. The results are presented in m. The volume density of blood 
vessels was evaluated using point counting as described [24,29,30]. The results are presented 
as volume density of blood vessels (Vv[blood vessels]%). 
Sections were also stained with Sirius red, and observed under polarization, to 
evaluate collagen fiber organization. To measure microscopically the necrotic area, sections 
were stained with Masson trichrome and digitalized using Pannoramic Digital Slide Scanner 
(3DHistech Ltd.) and necrotic area was measured using Pannoramic Viewer software 
(3DHistech Ltd.). The necrotic area was determined based on the different staining pattern of 
normal skin and necrotic skin, mainly the cells of epidermis and dermis with intense 
eosinophilia of cytoplasm and nuclear pyknosis, and compacted fibers in the dermis [7]. 
 
2.6. Immunohistochemistry and quantification 
Immunohistochemistry was used to investigate the number of neutrophils 
(myeloperoxidase), macrophages (F4/80), cellular apoptosis (cleaved caspase-3) and tenascin-
C-positive fibroblastic-like cells. The antibodies were used: rat monoclonal against 
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myeloperoxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:500), rat monoclonal 
against F4/80 (Serotec Inc., Raleigh, NC; 1:500), rabbit polyclonal against cleaved caspase-3 
(Cell Signaling Technology, Danvers, MA, USA; 1:200) and goat polyclonal antibody against 
tenascin-C (Santa Cruz Biotechnology; 1:50) as described [24]. To quantify the number of 
immunostained cells, five random fields per lesion (14,689 µm
2
) were analyzed as previously 
described [24]. The results are presentedas cells per mm
2
. 
The quantification of cellular proliferation was performed using sections 
immunolabelled with mouse monoclonal antibody against proliferating cell nuclear antigen 
(PCNA) (DAKO; 1:500) plus EnVision (DAKO; 1:20) as described [31]. Cellular 
proliferation was evaluated in the neo-epidermis and granulation tissue as described [31]. 
The quantification of myofibroblasts was performed using sections immunolabelled 
with mouse monoclonal antibody against -smooth muscle actin (DAKO; 1:100) plus anti-
mouse EnVision System (DAKO; 1:20) as described [32]. The volume density of 
myofibroblasts (Vv[myofibroblasts]%) was evaluated using point counting [24,29,30]. 
In addition, sections from control group were immunolabelled with rabbit polyclonal 
antibody against COX-2 to determine the expression of the COX-2 in the wound area 7 days 
after ulceration (Santa Cruz Biotechnology; 1:200). 
 
2.7. Biochemical analyses 
To estimate the collagen deposition, the hydroxyproline levels were measured [33]. 
For this, dry and defatted samples were hydrolyzed in hydrochloric acid (Vetec, Rio de 
Janeiro, Brazil) for 18h at 110°C and neutralized with sodium hydroxide (Vetec). 
Hydroxyproline levels were measured as described [33]. Results are expressed as ng of 
hydroxyproline per mg of tissue. 
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Lipid peroxidation was used to investigate the oxidative damage in pressure ulcer of 
mice. To evaluate the lipid peroxidation, the levels of lipid hydroperoxides were measured in 
wound lysate using the ferrous oxidation in xylenol orange method as described [34]. The 
results are expressed as nM of lipid hydroperoxides per mg total protein. 
 
2.8 ELISA 
The protein levels of tumor necrosis factor- (TNF-) (BD Biosciences Pharmingen, 
San Diego, CA, USA) and PGE2 (Cayman, Ann Arbor, MI, USA) were measured in wound 
lysate using an ELISA assay. The assay was performed according to the manufacturers’ 
instructions. The results are presented as pg of TNF- or PGE2 per mg total protein. 
 
2.9. Western-blotting 
Proteins of wound lysate were separated by sodium dodecylsulfate-polyacrylamide, 
transferred to polyvinylidene fluoride membrane and probed with antibodies against: goat 
polyclonal to COX-1 (72 kDa) (Santa Cruz Biotechnology; 1:200), goat polyclonal to COX-2 
(70-72 kDa) (Santa Cruz Biotechnology; 1:200), rabbit polyclonal to iNOS (100-150 kDa) 
(Santa Cruz Biotechnology; 1:200), rabbit polyclonal to nitrotyrosine (85 kDa) (Santa Cruz 
Biotechnology; 1:200), rabbit polyclonal to neutrophil elastase (29 kDa) (Santa Cruz 
Biotechnology; 1:500), mouse monoclonal to matrix metaloproteinase-1 (MMP-1) (52 kDa) 
(Santa Cruz Biotechnology; 1:200), vascular endothelial growth factor-A (VEGF-A), rabbit 
polyclonal to latent transforming growth factor-β (TGF-β)-1/2/3 (47 KDa) (Santa Cruz 
Biotechnology; 1:200) or mouse monoclonal to β-actin (42 kDa) (Sigma-Aldrich; 1:1,000). 
Following incubation with the appropriate horseradish peroxidase-conjugated secondary 
antibodies, immune complexes were detected using enhanced chemiluminescence (Santa Cruz 
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Biotechnology). The β-actin was used as a loading control protein and the results are 
expressed as arbitrary units. 
 
2.10. Statistical analysis 
All data are presented as mean ± standard error of the mean (SEM). Statistical analysis 
was performed using unpaired Student’s t-test or Mann-Whitney test and GraphPad Prism 
software was used to perform the statistical analyses (GraphPad Prism version 6.0, San Diego, 
CA, USA). The values of p<0.05 were considered statistically significant for all tests. 
 
3. Results 
 
3.1. Celecoxib administration reduces the lipid peroxidation and COX-2, PGE2, iNOS 
expression in mice pressure ulcers. 
Both COX-1 and COX-2 are up-regulated in chronic venous ulcers; however, the 
COX-2 is most likely responsible for the persistent inflammation in chronic venous leg ulcers 
[15]. We verified if our model of pressure ulcer could increase the COX-1 and COX-2 
expression and subsequently if celecoxib administration could reverse the increase of these 
COX isoforms. The pressure ulcers of control and celecoxib-treated groups presented an 
increase in the protein levels of COX-1 and COX-2 when compared to acute lesion the same 
day of wounding (7 days) (Figs. 1A, 1B). However, celecoxib administration reduced only the 
protein levels of COX-2 when compared to control group and acute lesion (Figs. 1A, 1B). In 
chronic venous ulcers, pro-inflammatory PGE2 is the main product of COX-2 activation [15]. 
Thus, we investigated if celecoxib administration could alter the PGE2 levels, which is a 
marker of COX-2 activation. Celecoxib administration reduced the protein levels of PGE2 in 
the ulcers 3 and 7 days after ulceration (Fig. 1C). The upregulation of iNOS/nitric oxide (NO) 
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increases COX-2 production and peroxynitrite synthesis leading to tissue injury [16,27]. We 
also determined if celecoxib administration could alter the protein expression of iNOS and 
oxidative damage in lipids. Celecoxib administration reduced the protein levels of iNOS and 
the lipid hydroperoxides (a marker of lipid peroxidation) in ulcers when compared to control 
group 3 and 7 days after ulceration (Fig. 1D, 1E). In addition, the protein levels of 
nitrotyrosine (a stable product of the formation of peroxynitrites) were higher in the ulcers of 
celecoxib-treated group than in that of the control group 3 days after ulceration, but lower 7 
days later (Fig. 1F). 
To investigate the role of iNOS on wound healing of mice pressure ulcers, mice were 
treated with L-NAME. The L-NAME administration improved the wound closure 3 and 7 
days after ulceration and re-epithelialization 7 days after ulceration (Fig. 1G, 1H). In addition, 
the protein levels of iNOS were reduced in the ulcers of the L-NAME-treated group when 
compared to that of the control group (Fig. 1I). 
To observe the COX-2 distribution in experimental pressure ulcers, sections were 
immunostained. The pressure ulcers of control group contained COX-2-positive 
keratinocytes, endothelial cells, inflammatory cells and fibroblasts (Fig. 1J). 
 
3.2. Celecoxib administration decreases the inflammatory cell infiltration and proteases 
levels in the pressure ulcer of mice. 
It has been reported that the wound bed of pressure ulcers presents a massive 
infiltration of inflammatory cells (mainly macrophages and neutrophils) and theirs proteolytic 
enzymes (as elastase and MMP-1) [1,34,35]. We investigated if celecoxib administration 
could alter the inflammatory cell infiltration and their protease levels in mice pressure ulcers. 
Celecoxib administration reduced the neutrophil number and protein neutrophil elastase levels 
in the ulcers when compared to control group 3 days after ulceration (Figs. 2A, 2B). There 
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was no protein expression of neutrophil elastase 7 days after ulceration in both studied groups 
(Fig. 2B). In addition, the macrophage number and protein MMP-1 levels were reduced in the 
celecoxib-treated group when compared to control group 3 and 7 days after ulceration (Figs. 
2C, 2D). Celecoxib administration also decreased the protein levels of TNF- in the ulcers 
when compared to control group 7 days after ulceration (Fig. 2E). 
 
3.3. Celecoxib administration improves re-epithelialization and decreasesnecrotic area in 
mice pressure ulcer. 
Chronic lesions present a reduction of re-epithelialization due to delay in the 
keratinocyte migration [36]. In addition, necrotic tissue loss and re-epithelialization are 
crucial steps to tissue repair of experimental pressure ulcers [37,38]. Thus, we verified if 
celecoxib administration could promote the re-epithelialization of mice pressure ulcers and 
the reduction of necrotic area. Celecoxib administration increased the length of migrating 
epithelial tongue when compared to control group 7 days after ulceration (Fig. 3A). The re-
epithelialized wound area was higher in the celecoxib-treated group when compared to 
control group 3 and 7 days after ulceration (Fig. 3B). In macroscopic analysis, the necrotic 
area was reduced in the celecoxib-treated group when compared to control group (Fig. 3C). 
To confirm this observation, the necrotic area also measured in Masson’s trichrome-stained 
sections by light microscopy. A morphometric analysis using image analysis software 
demonstrated that necrotic area was lower in the celecoxib-treated group than in the control 
group 7 days after wounding (Fig. 3D). 
 
3.4. The dermal reconstruction and wound closure are improved by celecoxib administration. 
The proteolytic environment of pressure ulcers causes rapid destruction of growth 
factors and excessive degradation of extracellular matrix impairing wound closure [1]. We 
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determined if celecoxib administration could improve dermal reconstruction and wound 
closure. Celecoxib administration decreased cellular proliferation in granulation tissue when 
compared to control group 3 and 7 days after ulceration (Fig. 4A). The cellular apoptosis in 
the granulation tissue was lower in the celecoxib-treated group when compared to control 
group only 7 days after ulceration (Fig. 4A). Celecoxib administration also reduced blood 
vessel density and protein levels of VEGF-A when compared to control group 7 days after 
ulceration (Fig. 4B, 4C). The myofibroblast density and protein levels of latent TGF-β 1/2/3 
were higher in the celecoxib-treated group when compared to control group 3 days after 
ulceration, but they were lower 7 days later (Fig. 4D, 4E). In addition, celecoxib 
administration increased the wound closure of pressure ulcers when compared to control 
group 3, 7 and 14 days after ulceration (Fig. 4F). 
 
3.5. Celecoxib administration improves the scar formation of mice pressure ulcers. 
To evaluate the effects of celecoxib administration on scar formation in mice pressure 
ulcer, we evaluate collagen deposition, keratinocyte proliferation, tenascin-C expression and 
neo-epidermis thickness. The organization of collagen fibers was more similar to normal skin 
(reddish and thick collagen fibers arranged basket-like) in the celecoxib-treated group than in 
the control group 14 days after ulceration (Fig. 5A). In addition, the hydroxyproline levels 
were higher in the celecoxib-treated group when compared to control group 14 days after 
ulceration (Fig. 5B). The number of tenascin-C-positive fibroblast-like cells was lower in the 
celecoxib-treated group than in the control group 14 days after ulceration (Fig. 5C). In 
addition, celecoxib administration did not alter keratinocyte proliferation (Fig. 5D). However, 
the neo-epidermis of celecoxib-treated group presented the beginning of cutaneous appendage 
formation when compared to control group (Fig. 5E). Celecoxib administration also decreased 
neo-epidermis thickness when compared to control group 14 days after ulceration (Fig. 5E). 
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4. Discussion 
The reperfusion phase of the IR cycle is an important component of the injury 
observed in pressure ulcers [39]. The restoration of blood flow after ischemia increases the 
ROS production to cytotoxic levels, resulting in tissue damage and necrosis [6,39,40]. The 
COX-2 and PGE2 are also involved in inflammatory response observed in chronic lesions 
through iNOS/NO production [15,16,27,41]. Epithelial cells, blood vessels, inflammatory 
cells, fibroblasts, and mast cells of chronic venous ulcers present higher COX-2 
immunoreactivity than normal human skin [15]. In addition, fluids from chronic venous ulcers 
presents high levels of COX-2 and this isoform may produce tissue damage through the 
production of prostanoides, such as PGE2 [15]. The cytotoxic ROS levels-stimulated iNOS 
production may promote the production of COX-2 and contribute to chronic inflammation in 
mice pressure ulcers [27]. This mechanism was confirmed in model of carrageenan-induced 
rat paw inflammation where the high levels of iNOS/NO activates COX-2 resulting in 
prostanoides production, such as PGE2 [41]. In this study, we investigated if iNOS and COX-
2 are involved in tissue damage and necrosis of pressure ulcers in mice. In addition, we tested 
if the inhibition of COX-2 by the celecoxib administration could accelerate the wound healing 
of pressure ulcer in mice. The protein expression of COX-1 and COX-2 was elevated in 
pressure ulcer of mice comparing to acute lesions. In addition, celecoxib reduced protein 
levels of COX-2 and PGE2, suggesting that celecoxib not only reduces COX-2 activation, but 
also decreases protein expression of this COX isoform. Celecoxib administration also 
decreased the protein levels of iNOS and lipid peroxidation. In rodents, celecoxib 
administration reduces PGE2 levels in acute cutaneous lesions and carrageenan-airpouch 
model [19,20,23]. We also observed that pressure ulcers in mice presented high levels of NO 
and ROS when compared to normal skin [42]. Thus, we suggest that the reduction of protein 
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COX-2 and iNOS expression induced by celecoxib may be involved to improvement of the 
wound healing of experimental pressure ulcers. This hypothesis is reinforced since L-NAME 
administration also decreased iNOS protein expression and accelerated the wound closure and 
re-epithelialization of mice pressure ulcers. We also observed that keratinocytes, endothelial 
cells, inflammatory cells and fibroblasts were COX-2-positive in experimental pressure ulcers 
indicating that these cells were the target of celecoxib administration. Moreover, the reaction 
of the NO with ROS (as superoxide anion) forms peroxynitrites, which elicit lipid 
peroxidation and cellular damage [43]. In chronic venous ulcers, high expression of iNOS 
may be involved in peroxynitrite production, which contributes to cell death by apoptosis or 
tissue necrosis [16]. Thus, celecoxib-induced reduction of the protein iNOS and peroxynitrite 
levels may also have decreased oxidative damage promoting the closure of pressure ulcers. 
The reperfusion causes an inflammatory response characterized mainly by the 
leukocyte migration, pro-inflammatory cytokine production and protease synthesis [39,40]. 
Fluids from human and mice pressure ulcers present a massive infiltration of neutrophils and 
high levels of elastases and proteases which cause an increase in the cell death, and a 
destruction of extracellular matrix and growth factors delaying wound closure [1,2,24,35,44]. 
The synthesis of COX-2-induced PGE2 may contribute to chronic inflammation and cell death 
in chronic venous ulcers [15]. In this study, celecoxib administration reduced neutrophil and 
macrophage infiltration and protein levels of neutrophil elastase, MMP-1 and TNF- in 
experimental pressure ulcers. In acute incisional lesions, some studies showed that decreasedCOX-2 activation reduces neutrophil number, while others demonstrated that the 
administration of COX-2 inhibitors does not alter inflammatory cell infiltration [20,21]. Thus, 
we propose that the reduction of PGE2 protein expression by celecoxib may have contributed 
to reduction of exacerbate inflammatory response in ulcers promoting the subsequent phases 
of wound healing. This hypothesis may be reinforced by the effect of celecoxib on dermal 
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reconstruction and wound closure. Celecoxib administration decreased the cellular 
proliferation and apoptosis and accelerated protein expression of TGF-β and myofibroblast 
differentiation increasing collagen deposition and wound contraction. Thus, these results also 
suggest that the reduction of inflammatory response induced by celecoxib have promoted the 
dermal reconstruction and accelerated the closure of pressure ulcers in mice. However, in 
acute cutaneous lesions, the reduction of COX-2 activity and PGE2 production by celecoxib 
reduces -smooth muscle actin expression, TGF-β levels, dermal proliferation, re-
epithelialization and wound contraction in mice [19,20]. In addition, naproxen (a selective 
COX-2 inhibitor) administration, but not celecoxib, diminishes hydroxyproline levels in 
sponge implants in rats [23]. Thus, the reduction of the COX-2 activation in acute lesions may 
impair normal inflammatory response delaying the development of subsequent phases of 
wound healing. However, the reduction of COX-2 expression may have a positive effect in 
lesions where the inflammatory response is chronic, as in pressure ulcers. 
Angiogenesis is another important event of dermal reconstruction. The pressure ulcers, 
using IR model, presents an increase in blood vessel formation mainly by high VEGF levels, 
since VEGF is the most important angiogenesis stimulant [24,45]. Moreover, the production 
of ROS during reperfusion phase of IR cycle may promote wound angiogenesis through 
VEGF expression in chronic ulcers [46]. In this study, celecoxib reduced the blood vessel 
density and VEGF protein in pressure ulcer. The administration of COX-2 inhibitors in acute 
lesions has a similar effect observed in this study [19,21]. Our findings also indicated that 
celecoxib administration have reduced the blood vessel formation due to decreased VEGF 
expression. 
The loss of necrotic tissue is essential to the closure of pressure ulcers. In pressure 
ulcers, the delay in the keratinocyte migration compromises re-epithelization impairing 
necrotic skin loss and subsequent phases of wound healing. Celecoxib administration 
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increased re-epithelialization and reduced the necrotic area. These results indicate that the 
acceleration of migrating epithelial tongue formation by celecoxib have promoted re-
epithelialization and reduced the necrotic area. In addition, the acceleration of re-
epithelialization and necrotic area loss have also contributed to the closure of pressure ulcers 
in mice and scar formation. In in vitro assay, the administration of COX-2 inhibitors reduce 
human keratinocyte proliferation; however, our study demonstrated that celecoxib 
administration did not alter keratinocyte proliferation in mice pressure ulcers [47]. A 
complete re-epithelialization is necessary to consider a scar formation [24]. To evaluate the 
scar formation, we evaluated the neo-epidermis thickness, collagen fiber organization and 
deposition, and tenascin-C expression [24]. Celecoxib administration reduced tenascin-C 
expression and stimulated the development of a mature and organized collagenous matrix due 
to increase in collagen deposition. Tenascin stimulates myofibroblast-performed wound 
closure and disappears soon after re-epithelialization [48]. Thus, the reduction of tenascin-C 
expression stimulated by celecoxib have contributed to development of a mature and 
organized collagenous scar. 
COX-1 and COX-2 are up-regulated in fluids from chronic venous ulcers; however, 
COX-2 is the main isoform involved in the chronic inflammation of chronic lesions [15]. It 
was demonstrated that the reduction of COX-1 and COX-2 activation by ibuprofen 
administration is not capable to alter the wound healing of pressure ulcer in rats [22]. 
Nonetheless, we demonstrated that the reduction of COX-2 protein expression have a 
beneficial effect on mice pressure ulcers probably through the reduction of iNOS and PGE2 
protein expression. These findings demonstrate that the oral administration of celecoxib might 
be a good therapeutic strategy to promote tissue repair of pressure ulcers. Nonetheless, it is 
necessary to consider the adverse effects of selective COX-2 inhibitor administration on 
cardiovascular system [49,50]. 
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The role of iNOS expression on cutaneous wound healing of acute lesions seems to be 
controversial. The selective iNOS inhibition does not affect wound healing of full-thickness 
incisional lesions in hairless SKH-1 mice [51]. Nonetheless, some compounds, such as 
propionyl-L-carnitine, are capable of stimulating angiogenesis in acute excisional lesions 
through the increase in iNOS and VEGF expression in capillaries [52]. Different types of 
chronic lesions present exacerbate iNOS expression. In chronically stressed mice, the increase 
in iNOS-positive cells and inflammatory cell infiltration on granulation tissue contributes to 
impair cutaneous wound healing [53]. In diabetic animals, the increase in iNOS expression by 
macrophages is involved with chronic inflammation and delayed skin wounds closure [54]. In 
chronic venous leg ulcers, iNOS expression in wound bed is increased when compared to 
normal skin [16], leading to increase in NO synthesis until cytotoxic levels [15,16]. In 
experimental pressure ulcers, iNOS gene expression is increased when compared to normal 
skin [55]. In this study, the inhibition of all NOS isoforms accelerated wound closure and re-
epithelialization, and reduced iNOS protein expression. Reinforcing that iNOS expression 
reduction in experimental pressure ulcers is beneficial for wound healing. However, it was 
demonstrated that high levels of iNOS could be associated to faster healing rate of chronic leg 
ulcers [56]. That study presents some problems in methodology, such as, the authors did not 
compare iNOS protein levels of chronic venous ulcers with that of a normal control group (as 
normal skin or acute lesion), which does not allow conclusion or comparison with other 
studies. 
 
5. Conclusion 
Celecoxib administration promotes the cutaneous wound healing of pressure ulcers in 
mice due to the reduction of protein iNOS, COX-2 and PGE2 expression. The reduction of 
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Romana-Souza et al. 21 
inflammatory response and oxidative damage induced by celecoxib promotes dermal 
reconstruction, wound closure and re-epithelialization resulting in a scar with high quality. 
 
Acknowledgements 
We thank Thatiana L. Assis de Brito for assistance with animal handling and 
histological analyses. This study was supported by the Carlos Chagas Filho Foundation for 
Research Support in the State of Rio de Janeiro (FAPERJ) and National Council of Research 
(CNPq). 
 
Conflict of interest statement 
The authors declare that there are no conflicts of interest. 
 
 
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FIGURE LEGENDS 
 
Fig. 1. Effect of celecoxib on inflammatory response and oxidative damage in pressure ulcers 
of mice. (A) The protein levels of cyclooxygenase-1 (COX-1) (72 kDa) in the ulcer are 
similar in control and celecoxib-treated groups 3 and 7 days after ulceration, but lower in 
acute lesion. (B) The protein levels of cyclooxygenase-2 (COX-2) (70-72 kDa) in the ulcer 
are lower in the celecoxib-treated group than in the control group 3 and 7 days after 
ulceration, and lower than in acute lesion 7 days after ulceration. (C) The protein levels of 
prostaglandin E2 (PGE2) in the ulcer are lower in the celecoxib-treated group than in the 
control group 3 and 7 days after ulceration. (D) The protein levels of inducible nitric oxide 
synthase (iNOS) (150-100 kDa) in the ulcer are lower in the celecoxib-treated group than in 
the control group 3 and 7 days after ulceration. (E) Levels of lipid hydroperoxides in the ulcer 
are lower in the celecoxib-treated group than in the control group 3 and 7 days after 
ulceration. (F) The protein levels of nitrotyrosine (85 kDa) in the ulcer are higher in the 
celecoxib-treated group than in the control group 3 days after ulceration, but lower 7 days 
after ulceration. (G) The N
G
-nitro-L-arginase methyl ester (L-NAME) administration 
improved the wound closure of pressure ulcers 3 and 7 days after ulceration. (H) The re-
epithelialized wound area was increased by L-NAME administration 7 days after ulceration. 
(I) The levels of inducible nitric oxide synthase (iNOS) (150-100 kDa) were reduced by L-
NAME administration 7 days after ulceration. (J) The cyclooxygenase-2 (COX-2)-positive 
cells (arrows) in the wound area of control group 7 days after ulceration. Bars are equal to 50 
µm. The densitometry is expressed as arbitrary units (a. u.) for all immunoblottings. The -
actin (42 kDa) was used as a loading control protein for all immunoblottings. Data (n=10 
lesions per group) are expressed as the mean ± SEM. *p<0.05 vs. control group. #p<0.05 vs. 
celecoxib-treated group. 
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Fig. 2. Effects of celecoxib on inflammatory cell infiltration and protease production in 
pressure ulcers of mice. (A) The number of myeloperoxidase (MPO)-positive neutrophils in 
the ulcer is lower in the celecoxib-treated group than in the control group 3 days after 
ulceration. (B) The protein levels of neutrophil elastase (29 kDa) in the ulcer are lower in the 
celecoxib-treated group than in the control group 3 days after ulceration. (C) The number of 
F4/80-positive macrophages in the ulcer is lower in the celecoxib-treated group than in the 
control group 3 and 7 days after ulceration. (D) The protein levels of matrix metaloproteinase-
1 (MMP-1) (52 kDa) in the ulcer are lower in the celecoxib-treated group than in the control 
group 3 and 7 days after ulceration. (E) The protein levels of tumor necrosis factor- (TNF-) 
in the ulcer are lower in the celecoxib-treated group than in the control group 7 days after 
ulceration. The densitometry is expressed as arbitrary units (a. u.) for all immunoblottings. 
The -actin (42 kDa) was used as a loading control protein for all immunoblottings. Data 
(n=10 lesions per group) are expressed as the mean ± SEM. *p<0.05 vs. control group. 
 
Fig. 3. Effects of celecoxib on re-epithelization and skin necrosis in pressure ulcer of mice. 
(A) Length of migrating epithelial tongue is longer in the celecoxib-treated group than in the 
control group 7 days after ulceration (7d). Yellow line shows the length of migrating 
epithelial tongue, asterisk show normal skin and sections are stained with hematoxylin-eosin. 
Bar is equal to 200 m (B) The re-epithelialized wound area is higher in the celecoxib-treated 
group than in the control group 3 and 7 days after ulceration (7d). Epithelial gap is indicated 
by yellow arrows, asterisks show normal skin and sections are stained with hematoxylin-
eosin. Bar is equal to 1,000 µm. (C) In macroscopic analysis, the necroticarea is lower in the 
celecoxib-treated group than in the control group 7 days after ulceration (7d). Yellow line 
surrounds the necrotic skin. (D) In microscopic analysis, the necrotic area is lower in the 
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celecoxib-treated group than in the control group 7 days after ulceration (7d). Yellow line 
surrounds the necrotic tissue, asterisks show normal skin and sections is stained with 
Masson’s trichrome. Bar is equal to 1,000 µm. Data (n=10 lesions per group) are expressed as 
the mean ± SEM. *p<0.05 vs. control group. 
 
Fig. 4. Effect of celecoxib on dermal reconstruction in pressure ulcer of mice. (A) Cellular 
proliferation is lower in the celecoxib-treated group 3 and 7 days after ulceration, but cellular 
apoptosis only 7 days, than in the control group. (B) The volume density of blood vessels 
(Vv[blood vessels]%) in the ulcer is lower in the celecoxib-treated group than in the control group 
7 days after ulceration. (C) The protein levels of vascular endothelial growth factor-A 
(VEGF-A) (45 kDa) in the ulcer is lower in the celecoxib-treated group than in the control 
group 7 days after ulceration. (D) The volume density of myofibroblast (Vv[myofibroblast]%) is 
higher in the celecoxib-treated group 3 days after ulceration, but lower 7 days later, than in 
the control group. (E) The protein levels of latent transforming growth factor (TGF-) 1/2/3 
(47 kDa) are increased in the celecoxib-treated group 3 days after ulceration, but reduced 7 
days later. (F) Celecoxib administration increases the wound closure 3, 7 and 14 days after 
ulceration. The densitometry is expressed as arbitrary units (a. u.) for all immunoblottings. 
The -actin (42 kDa) was used as a loading control protein for all immunoblottings. Data 
(n=10 lesions per group) are expressed as the mean ± SEM. *p<0.05 vs. control group. 
 
Fig. 5. Effect of celecoxib on scar formation in pressure ulcer of mice. (A) In Sirius red-
stained sections, collagen fiber organization is more similar to normal skin in celecoxib-
treated group than in control group 14 days after ulceration. Bar is equal to 50 m. (B) 
Celecoxib administration increases hydroxyproline levels 14 days after ulceration. (c) The 
number of tenascin-C-positive fibroblastic-like cells is reduced in the celecoxib-treated group 
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14 days after ulceration. (D) Keratinocyte proliferation in neo-epidermis is similar in control 
and celecoxib-treated groups 14 days after ulceration. (E) The neo-epidermis thickness is 
higher in the celecoxib-treated group than in the control group 14 days after ulceration. The 
neo-epidermis of celecoxib-treated group presents the beginning of appendage formation. 
Sections are stained with hematoxylin-eosin and bar is equal to 50 m. Data (n=10 lesions per 
group) are expressed as the mean ± SEM. *p<0.05 vs. control group. 
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Figure 2.
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Graphical abstract