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Free Radical Biology and Medicine 96 (2016) 374–384
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Free Radical Biology and Medicine
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n CorrE-m
journal homepage: www.elsevier.com/locate/freeradbiomed
NADPH oxidase 4 deficiency leads to impaired wound repair
andreduced dityrosine-crosslinking, but does not affect
myofibroblastformation
Dominik Lévigne a,n, Ali Modarressi a, Karl-Heinz Krause b,
Brigitte Pittet-Cuénod a
a Division of Plastic, Reconstructive & Aesthetic Surgery,
Geneva University Hospitals, Geneva, Switzerlandb Department of
Pathology, Faculty of Medicine, University of Geneva, Geneva,
Switzerland
a r t i c l e i n f o
Article history:Received 30 November 2015Received in revised
form26 April 2016Accepted 28 April 2016Available online 30 April
2016
Keywords:NADPH oxidase 4Dityrosine crosslinkingWound
healingWound repairHIF1alphaCD31MyofibroblastMouse
modelNeovascularizationROSMPONOX2Collagen crosslinkingExtracellular
matrixNOX4VPO1Myeloperoxidase
x.doi.org/10.1016/j.freeradbiomed.2016.04.19449/& 2016 The
Authors. Published by Elsevier
esponding author.ail address: [email protected] (D.
Lévigne).
a b s t r a c t
NADPH oxidases (NOX) mediate redox signaling by generating
superoxide and/or hydrogen peroxide,which are involved in
biosynthetic pathways, e.g. thyroid hormone generation, dityrosine
crosslinking, aswell as bacterial killing. Data investigating the
role of NOX enzymes in cutaneous wound repair is limitedand
specifically their function in skin myofibroblast expression is
unknown. The isoform NOX4 was re-cently shown to be a pre-requisite
for the differentiation of cardiac and pulmonary
myofibroblasts.
In this study we investigate the role of NOX4 in wound repair
using a wound model in NOX4knockout mice (n¼16) and wildtype mice
(n¼16). Wounds were photographed daily until completewound closure.
Mice were sacrificed at day 3, 7, 14; wound tissue was
harvested.
NOX4-deficient mice healed significantly slower (22 days,
SD¼1.9) than wild-type mice (17 days,SD¼1.4, po0.005). However,
there was no difference in myofibroblast expression. Strong
dityrosineformation was observed, but was significantly weaker in
NOX4-/- mice (po0.05). NOX2, HIF1α and CD31expression was
significantly weaker in NOX4-/- mice (po0.05).
In this study we show for the first time that NOX4 plays a role
in cutaneous wound repair. Our datasuggests that NOX4 mediates
HIF1α expression and neoangiogenesis during wound repair. NOX4
dele-tion led to a decreased expression of NOX2, implying a role of
NOX4 in phagocytic cell recruitment. NOX4was required for effective
wound contraction but not myofibroblast expression. We suggest that
myo-fibroblast contraction in NOX4-deficient mice is less effective
in contracting the wound because of in-sufficient
dityrosine-crosslinking of the ECM, providing the first indication
for a physiological function ofdityrosine crosslinking in higher
animals.& 2016 The Authors. Published by Elsevier Inc. This is
an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Wound repair is a complex, dynamic and interactive processthat
involves a great variety of cells, soluble mediators and
ex-tracellular matrix components. In order to guarantee rapid
andeffective wound closure, wound contraction is of primordial
im-portance. When the wound contracts, the uninjured skin
sur-rounding the wound is pulled into the defect, significantly
redu-cing the amount of time and tissue needed to reestablish the
in-tegrity of the skin barrier after injury. The exact mechanisms
un-derlying wound contraction are not yet fully understood.
Cellularcontraction of specialized contractile fibroblasts –
myofibroblasts –constitutes one of the key components of effective
wound
Inc. This is an open access article u
contraction [1,2]. Fibroblast-to-myofibroblast differentiation
oc-curs primarily in response to growth factors (mainly
transforminggrowth factor beta 1 (TGFβ1)) and mechanical stress.
Myofibro-blast are characterized by the expression of α-smooth
muscle actin(αSMA) in stress fibers, which is considered to be the
molecularbasis for their high contractility [1–3].
In recent years it has become increasingly clear that
redoxsignaling is involved in a myriad of physiological cell
functionsincluding differentiation, proliferation, apoptosis and
migration[4–6]. NOX enzymes are membrane-bound complexes that
trans-port electrons across biological membranes to reduce oxygen
tosuperoxide-radical and play a key role in mediating redox
signal-ing [5]. Seven isoforms of NOX enzymes have been
described:NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2.
Despitetheir similar structure, they differ in function and
mechanism ofactivation. During wound repair, they could play
important roles inkey elements such as coagulation, inflammation,
fibroplasia,
nder the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
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D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384 375
angiogenesis, re-epithelialization and contraction [6,7]. The
role ofNOX2 during wound repair is long known in microbial killing
as itguarantees the production of large amounts of reactive
oxygenspecies (ROS) by immune cells, which is of great importance
foreffective host defense [8–11]. In fact, patients with chronic
gran-ulomatous disease, a rare congenital abnormality of the
NOX2system, show impaired wound repair and are more prone towound
infection [12]. Our knowledge concerning the role of NOX-mediated
redox signaling in wound repair, especially concerningwound
contraction, is still limited and relies mainly on studiesconducted
in other fields than skin wound repair.
Recent literature shows that TGFβ1 induces hydrogen
peroxideproduction in human fibroblasts [13]. In fact, NOX4 has
been re-ported to mediate TGFβ1-induced myofibroblast
differentiation ofcardiac [14] and pulmonary fibroblasts [15] as
well as the pro-liferation of pulmonary smooth muscle cells [16].
Carnesecchi et alinvestigated the role of NOX4 in
fibroblast-to-myofibroblast dif-ferentiation during
bleomycin-induced pulmonary fibrosis, theaccumulation of
myofibroblasts being a hallmark in advanced andprogressive
pulmonary fibrosis. They showed that NOX4 was aprerequisite for
myofibroblast differentiation [17]. The accumula-tion of
myofibroblasts during lung fibrosis is mainly driven byTGFβ1
signaling [18,19], in line with what has been observed incutaneous
wound repair studies [20].
The extracellular matrix (ECM) plays a primordial role inwound
repair, not only for myofibroblast differentiation but alsofor
wound contraction itself. The crosslinking of ECM proteins
iscrucial for the development of a mechanically resistant
granula-tion tissue. Dimerization of the phenolic amino acid
L-tyrosine hasbeen suggested as a possible mechanism of
crosslinking ECMproteins. Both collagen and elastin have been shown
to undergodityrosine crosslinking when H2O2 is added exogenously
[21,22].In culture, it has been observed that TGFβ1-activated
fibroblastsrelease ROS in the extracellular space in a NOX-mediated
manner[23]. Larios et al reported that H2O2 generated by
TGFβ1-activatedfibroblasts are capable of mediating dimerization of
L-tyrosine inthe ECM [24]. A physiological function for
NOX/ROS-dependentcross-linking of dityrosine residues in the ECM
has so far only bedemonstrated in primitive organisms:
stabilization of cutaneoustissue in Caenorhabditis elegans [25];
hardening of the fertilizationenvelope in sea urchin eggs [26]; and
stabilization of drosophilawings [27]. We hypothesize that NOX4
plays a role in the devel-opment of the granulation tissue during
wound repair by med-iating ROS-induced crosslinking of tyrosine
residues in the ECM.
NOX4 may also play a role in other processes of wound
repair,possibly during angiogenesis as it was previously reported
thatgrowth of blood vessels into sponges is abolished in
NOX4knockout mice [28]. Hypoxia is arguably the most
importanttrigger for angiogenesis during wound repair. NOX4 could
play akey role in cell response to hypoxia. Pulmonary artery
smoothmuscle cells cultured under hypoxic conditions showed
increasedexpression of NOX4 [4,29] and their proliferation was
NOX4-de-pended [30]. There is increasing evidence that the
stabilization ofhypoxia-inducible factor (HIF) can also be directly
induced by ROS[31,32]. In fact, there appears to be a positive
feed-forward loopinvolving NOX4 and HIF1α: ROS generated by NOX4
activateHIF1α [33] and HIF1α activates the expression of NOX4 [34].
Thenotion that redox signaling plays an important role in
angiogen-esis is supported by evidence suggesting that a large
number ofantioxidants limit angiogenesis [35–37].
In this study we aimed to understand 1) whether NOX4 plays arole
in cutaneous wound repair, 2) whether NOX4 is necessary
foreffective contraction studying myofibroblast expression,
collagendeposition and dityrosine crosslinking and 3) whether NOX4
playsa role in HIF1α expression and angiogenesis during wound
repair.
2. Material and methods
2.1. Animal wound model
In this study, 16 wild type mice (C57BL/6J) and 16 NOX4knockout
mice (B6/129S9-NOX4) were used. We created circularfull thickness
wounds of 1.5 cm diameter (1.8 cm2) on the back ofNOX4-deficient
knockout mice and of wild type mice, using acircular template. All
animal experiments were approved by thelocal veterinary authority
(“Direction générale de la santé deGenève”, authorization number
G65/3878).
2.2. Wound repair assessment
Wound size and aspect was documented immediately afterwounding
and on day 3, 7, 10, 14, 17 and 21. Wounds that wereclose to
complete wound closure at any of these time points werephotographed
daily from there on in order to determine the day ofcomplete wound
closure. Wounds were photographed at a con-stant distance with a
ruler next to the wound for scaling. Thewound surface was
calculated on photos using a computer-as-sisted image analysis
system (Image J).
At complete wound closure (i.e., full epithelialization),
thesurface of hairless skin of the scar was measured and considered
tocorrespond to the area of the wound healed by
epithelialization.The surface of the wound healed by contraction
was then esti-mated by subtraction of the epithelialized surface
from the woundsurface measured at day 0.
2.3. Histology and immunohistochemistry
Mice were sacrificed at day 3, 7 and 14 (n¼2 per time pointand
per group) and the entire wound with the souring uninjuredskin was
removed. The tissue was fixed in 4% bufferedformaldehyde.
4 μm thick tissue sections were analyzed by
im-munohistochemistry using with the Ventana Discovery
automatedstaining system (Ventana Medical Systems, Tucson, AZ,
USA).Ventana reagents were used for the entire procedure.
For αSMA (anti-alpha-smooth muscle actin, Dako, #M0851,70mg/ml)
and DT (anti dityrosine, JaICA, MDT-020P, 100ug/ml)mouse monoclonal
antibodies, no antigen retrieval pre-treatmentwas required. After
automatic deparaffinization, slides were in-cubated 30 minutes at
37 °C with primary antibodies diluted at1/300 and 1/400
respectively for αSMA and DT. Then secondaryantibodies were applied
at dilution 1/250 (anti-mouse Ig-G1þ IgG2aþ IgG3, abcam, ab133469,
2.03 mg/ml). Detection ofsecondary antibodies was carried out using
the rabbit OmniMapkit (Ventana Medical Systems), based on
conversion of diamino-benzidine to a dye with multimeric
horseradish peroxidase (HRP).
For HIF1α (Novusbio, NB100-479, 1 mg/ml), antigen retrievalwas
performed by heating slides 36 mns in standard CC2 EDTAsolution pH
8.4, dilution 1/400, detection Rabbit-OmniM detectionkit.
For CD31 (Abcam, ab28364), antigen retrieval was performedby
heating slides 52 min in standard CC1 citrate solution pH
6.0,dilution 1/50, detection Rabbit-OmniM detection kit.
MPO staining was performed using a rabbit polyclonal
antibody(Dako,#A0398, 3.2 g/L). Slides were incubated 12 min in CC1
buf-fer for antigen retrieval and primary antibody was incubated30
min at 37 °C at dilution 1/1000. As a negative control, spinalcord
tissue of wildtype mice was used. As a positive control, weused
spinal cord tissue from an amyotrophic lateral sclerosismouse model
which is known to have activated microglia thatexpress MPO
[38].
NOX2 staining was performed using a mouse monoclonal
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D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384376
antibody (Santa Cruz, #sc-130548, 200 μg/ml). Slides were
in-cubated 36 min in CC1 buffer for antigen retrieval. First the
pri-mary antibody was incubated 30 min at 37 °C at dilution
1/100and then a secondary antibody was applied at dilution 1/250
(anti-mouse IgG1þ IgG2aþ IgG3, abcam, ab133469, 2.03 mg/ml).
Detection was carried out using the rabbit OmniMap kit(Ventana
Medical Systems), based on conversion of diamino-benzidine to a dye
with multimeric horseradish peroxidase (HRP).
NOX2 antibody control staining was performed using the
sameantibody in immunofluorescence on cell cultures of mouse
bone-marrow-derived dendritic cells fixed in PFA 4% at a 1:100
dilutionof either wildtype or NOX2-/- mice (data kindly provided by
DrTamara Seredenina, Department of Pathology and
Immunology,University of Geneva).
Standard Masson’s trichrome staining was used to
quantifycollagen expression.
2.4. Image analysis
Sections were scanned with a Mirax Widefield scanner(brightfield
detector: Marlin F-146C IRF Medical). Pictures werethen processed
using Definiens Tissue Studio software (DefiniensAG, Munich,
Germany). Regions of interest (ROIs) were defined inthe granulation
tissue and in the epithelium. To evaluate α-SMAexpression, vessels
were manually excluded from the image toexclude pericytes. Results
were given as % of ROI area stained.
2.5. Statistical methods
All values are expressed as mean7standard deviation (SD).Data
was analyzed with Prism6 software (GraphPad Software Inc.,La Jolla,
USA). Statistical analysis consisted in a comparison of datafrom
wild type versus NOX4-deficient mice, using two-tailed
Fig. 1. A) Wound aspect in wildtype and NOX4-/- mice: Wounds in
both groups healewound closure, scale bar¼1 cm. B) Percentage of
mice with open wounds over time: wh24 days in the NOX4-/- group,
Mantel-cox test comparing the two curves showed a p
valsignificantly slower compared to wildtype mice (white par bar),
whiskers indicate min
Student t-tests for unpaired comparisons between groups.
Differ-ences were considered significant at po0.05. In cases of
multiplecomparisons, a post hoc correction with the Bonferroni
procedurewas performed. The Mantel-cox test was used for comparing
thedifference between the control and NOX4-deficient group in
per-centage of wounds closed at different time points.
3. Results
3.1. Macroscopic analysis
We observed a significant delay in wound closure in
NOX4-deficient mice compared to the control group. The
macroscopicwound aspect showed no obvious difference with regards
togranulation tissue formation or fibrin deposition (see Fig.
1a).While it took 18 days until 90% of the wounds were closed in
thecontrol group, it took 24 days in the NOX4-/- group (see Fig.
1b).Wounds in the control group took on average 17 days until
com-plete wound closure (SD¼1.4) compared to 22 days in the
NOX4-/-group (SD¼1.9, po0.005, see Fig. 1c).
When looking at the fraction of the wound area closed by
re-epithelialization (hairless skin at the time of complete
woundclosure), we observed that wounds healed almost exclusively
bycontraction, as expected for this kind of wound repair mousemodel
(6.172.3% and 6.772.4% of the surface healed by
re-epi-thelialization in wildtype and NOX4-/- mice respectively).
Thedifference in the ratio between epithelialization and
contractionbetween the two groups was not statistically
significant.
3.2. Myofibroblast expression
Staining for αSMA revealed no statistically significant
d predominantly through contraction. NOX4-/- mice showed a
significant delay inile it took 18 days until 90% of the wounds
were closed in the control group, it tookue of o0.005. C) Time to
complete wound closure: NOX4 -/- mice (gray bar) healedand max
values, n¼10 per group, po0.005.
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Fig. 2. Myofibroblast expression. Immunohistochemistry showing
αSMA expression in the granulation tissue at day 3, 7 and 14 after
wound infliction in wildtype (leftcolumn) and NOX4-/- mice (middle
column). E¼epithelium; G¼granulation tissue, V¼vessel. A strong
increase in expression of αSMA was observed at day 7 with a
furtherincrease at day 14. Computer-assisted quantification (right
column) showed no significant difference between wildtype and
NOX4-/- mice in αSMA expression in thegranulation tissue. Values
are expressed as % area of the region of interest stained by
immunohistochemistry and as mean7SD.
D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384 377
difference between wildtype and NOX4-/- mice. We observed aweak
expression of αSMA at day 3 (0.570.47% vs 0.570.2% of ROIstained
respectively) and a strongly increased expression at day
7(13.271.6% vs 12.074.3%), which was again increased at day
14(20.071.8% vs 18.374.0%) (see Fig. 2).
3.3. Collagen deposition
Masson’s trichrome staining showed less methyl blue
staining(collagen fibers) in the granulation tissue of NOX4-/- mice
com-pared to the wildtype group. A tendency toward a decreased
Fig. 3. Masson’s trichrome staining. Pictures of wound edges at
day 3, 7 and 14 afterE¼epithelium; G¼granulation tissue.
Computer-assisted quantification of methyl blue ingroup compared to
control mice. Values are expressed as % area of the region of
intere
expression was found at day 3 and 7, a significant difference
wasseen at day 14 (65.375.4 % of ROI stained in control animals
vs19.9716.5% in NOX4-/- animals; po0.05) (see Fig. 3).
3.4. Dityrosine formation
Dityrosine staining revealed a strong expression in wildtypemice
in the granulation tissue at day 3 (23.676.6% of ROI stained)while
NOX4-deficient mice showed a significantly weaker ex-pression
(4.171.3% of ROI stained, po0.05). At day 7, dityrosineexpression
increased in wildtype and NOX4-/- mice (34.873.6% vs
wound infliction in wildtype (left column) and NOX4-/- mice
(middle column).the granulation tissue showed significantly lower
values at day 14 in the NOX4 -/-
st stained by methyl blue and as mean7SD; *po0.05.
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Fig. 4. Dityrosine formation. Immunohistochemistry showing
dityrosine formation at day 3, 7 and 14 after wound infliction in
wildtype (left column) and NOX4-/- mice(middle column).
E¼epithelium; G¼granulation tissue. Dityrosine formation appeared
weaker in the NOX4-/- group at all time points in the granulation
tissue. No dif-ference was observed in the epithelium at the wound
edge. Computer-assisted quantification (right column) showed a
significant impair of dityrosine formation in NOX4-/-mice in the
granulation tissue at day 3, 7 and 14 as compared to wildtype mice.
Values are expressed as % area of the region of interest and as
mean7SD; *po0.05.
D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384378
20.474.6% of ROI stained respectively), but the expression
re-mained significantly lower in NOX4-/- mice (po0.05). At day
14,the dityrosine expression slightly decreased in both
groups(34.178.6% vs 13.576.7% of ROI stained for wildtype and
NOX4-/-respectively), the difference between the two groups being
againstatistically significant (po0.05) (see Fig. 4).
In the epithelium, dityrosine formation was generally strongeras
compared to the granulation tissue. At day 3, wildtype miceshowed
54.5720.5% and NOX4-/- mice 24.8713.4% of ROIstained. At day 7,
48.4711.9% and 67.971.0% of the ROI wasstained and at day 14,
68.878.4% and 47.470.3% respectively forwildtype and NOX4-/- mice.
None of the results in the epitheliumwere statistically different
between wildtype and NOX4-/- mice.
3.5. NOX2 expression
NOX2 staining showed no difference between wildtype andNOX4-/-
mice at day 3 (2.270.6% and 1.770.2% of ROI stainedrespectively).
However, a significantly weaker signal was seen atday 7 in NOX4-/-
mice (1.670.3% of ROI stained) compared towildtype mice (5.271.5%
of ROI stained). At day 14, there wasagain no significant
difference found between wildtype andNOX4-/-, as NOX2 expression
decreased in wildtype mice to3.070.4% of ROI stained, being similar
to NOX4-/- mice at thistime point (1.470.6% of ROI stained).
Specificity of the NOX2antibody was tested by staining cultured
bone-marrow-deriveddendritic cells from either wildtype or NOX2-/-
mice (see Fig. 5).
3.6. MPO expression
No significant difference was found between NOX4-/- mice andwild
type mice when staining granulation tissues for MPO. Thesignal was
strongest at day 3 (5.674.6% in wildtype and 4.871.1%of ROI stained
in NOX4-/- mice) and gradually decreased. At day 7,4.071.4% of ROI
was stained in wildtype mice and 2.171.2% inNOX4-/- mice. At day
14, it was 2.371.5% and 1.770.1%respectively.
0.1% of ROI was stained in our negative control (spinal
cordtissue of wildtype mice). 4.1% of ROI was stained in our
positivecontrol (spinal cord from an amyotrophic lateral sclerosis
mousemodel). Our isotype control showed no staining (see Fig.
6).
3.7. Hypoxia inducible factor
HIF1α staining of the granulation tissue at day 3
showed24.877.4% of ROI stained in the control group compared
to5.170.2 in the NOX4-/- group (po0.05). At day 14,
expressionincreased in both groups, but remained significantly
higher in thecontrol group (33.274.6%) as compared to the NOX4-/-
group(13.574.2% of ROI stained, po0.05). At day 14, expressionwas
thehighest in both groups being 35.470.4% in the control group
and27.771.3% in the NOX4-/- group. The difference at day 14 was
notstatistically significant (see Fig. 7).
HIF1α expression was stronger in the epithelium at the woundedge
as compared to the granulation tissue. The highest expres-sion was
found at day 14 with 52.277.6% in the control group and52.8710.9%
in the NOX4-/- group. None of the results in theepithelium were
statistically different between the control andNOX4-/- group.
3.8. Neoangiogenesis and CD31 expression
CD31 staining was used to study wound angiogenesis. At day 3,low
levels of CD31 expression were found (2.171.3 wildtype vs1.771.0
NOX4-/-). A strong increase was observed in control an-imals at day
7, while this increase was less pronounced in NOX4-/-mice (6.670.7%
vs 3.370.4% respectively, po0.05). However,CD31 expression strongly
increased between day 7 and 14 inNOX4-/- mice while control animals
showed only a slight increasein the same period. In fact, NOX4-/-
wounds reached similar levelsof expression by day 14 as compared to
the control group (7.770.5% control vs 6.37 0.8% NOX4-/-; ns) (see
Fig. 8).
-
Fig. 5. NOX2 expression. Immunohistochemistry showing NOX2
expression at day 3, 7 and 14 after wound infliction in wildtype
(left column) and NOX4-/- mice (middlecolumn). E¼epithelium;
G¼granulation tissue. NOX2 expression appeared weaker in the
NOX4-/- group at day 7. Computer-assisted quantification (right
column) showed asignificant impair of dityrosine expression in
NOX4-/- mice in the granulation tissue at day 7 as compared to
wildtype mice. Isotype control staining showed no signal
(rightcolumn, middle picture). NOX2 antibody control staining on
cultured bone-marrow-derived dendritic cells from wildtype and
NOX2-/- mice showed specific staining (rightlower corner). Values
are expressed as % area of the region of interest and as mean7SD;
*po0.05.
D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384 379
4. Discussion
Our study shows for the first time that NOX4-deficiency leadsto
significantly impaired cutaneous wound repair. Wounds in
bothwildtype and NOX4-deficient mice healed predominately
Fig. 6. Myeloperoxidase. Immunohistochemistry showing
myeloperoxidase presence at(middle column). E¼epithelium;
G¼granulation tissue, a¼ representative view of cell ac3 and
continuously decreased over time. Computer-assisted quantification
(right columnthe granulation tissue at day 3, 7 and 14 as compared
to wildtype mice. The negative cowas stained in positive control
tissues (PCO). Isotype control staining was negative (rig
(9372%) through wound contraction, suggesting that the delay
inhealing associated with NOX4-deficiency in our model is mainlydue
to impaired wound contraction. However, in our study NOX4was not
required for myofibroblast differentiation during woundcontraction
and αSMA expression patterns were very similar
day 3, 7 and 14 after wound infliction in wildtype (left column)
and NOX4-/- micecumulations observed in both groups.
Myeloperoxidase was detected as early as day) showed no significant
difference in myeloperoxidase formation in NOX4-/- mice inntrol
(NCO) showed a signal in 0.1% of the region of interest (ROI) while
4.1% of ROIht lower corner). Values are expressed as % area of ROI
and as mean7SD.
-
Fig. 7. HIF1α expression. Immunohistochemistry showing HIF1α
expression at day 3, 7 and 14 after wound infliction in wildtype
(left column) and NOX4-/- mice (middlecolumn). E¼epithelium;
G¼granulation tissue. HIF1α expression appeared weaker in the
NOX4-/- group at all time points in the granulation tissue. No
difference was seenin the epithelium at the wound edge.
Computer-assisted quantification (right column) showed a
significantly lower expression at day 3 and day 7 in the
granulation tissue inthe NOX4-/- group compared the control group,
no significant difference was seen at day 14. Values are expressed
as % area of the region of interest and as mean7SD;*po0.05.
D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384380
between NOX4-deficient and wildtype mice. This is in contrast
toother studies where NOX4 was shown to be a prerequisite
formyofibroblast differentiation of cardiac [14] and pulmonary
fi-broblasts [15]. Our results suggest that NOX4 is required
formyofibroblast differentiation in a situation- and/or
tissue-specificmanner.
Fig. 8. CD31 expression. Immunohistochemistry showing CD31
expression at day 3, 7 acolumn). E¼epithelium; G¼granulation
tissue; S¼scab. Similar levels of CD31 expresscontrol group which
appeared weaker in the NOX4-/- group. Computer-assisted
quangranulation tissue in the NOX4-/- group compared the control
group. At day 14, woundssimilar levels to the control group.
Quantification showed no significant difference at dayand as
mean7SD; *po0.05.
The fact that wound contraction was significantly
impaireddespite seemingly unchanged myofibroblast expression raises
aseries of questions regarding the role of the myofibroblast as
themain cell orchestrating wound contraction and regarding the
exactmechanisms that lead to the generation of contractile
forceswithin the wound bed.
nd 14 after wound infliction in wildtype (left column) and
NOX4-/- mice (middleion were seen at day 3 in both groups. A strong
increase was seen at day 7 in thetification (right column) showed a
significantly lower expression at day 7 in thein NOX4-/- mice
showed a strong increase of CD31 expression and had now reached14
between the two groups. Values are expressed as % area of the
region of interest
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D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384 381
In order to understand why wound contraction is
significantlyimpaired in NOX4-deficient mice, we looked for
possible ex-planations why myofibroblast contraction could be less
effectivedespite preserved αSMA expression. Fully differentiated
myofi-broblasts show a strong expression of stress fibers and
fibronectin[1,39–41] and are traditionally associated with the
expression ofαSMA [42]. The incorporation of αSMA into stress
fibers is be-lieved to be the backbone of their contractile
activity [43,44].However, recent data suggests that αSMA expression
is not a pre-requisite for myofibroblast contraction during wound
repair andthat smooth muscle γ-actin and skeletal muscle α-actin
cancompletely compensate for a lack of αSMA [45,46]. Possibly,
NOX4plays a role in the expression of other muscle actins than
αSMA,such as smooth muscle γ-actin or skeletal muscle α-actin
inmyofibroblast function during wound repair. If this is the
case,NOX4-deficient myofibroblasts could be less contractile
despiteexpressing normal amounts of αSMA. Further (in vitro)
studiesshould investigate whether NOX4-deficient skin-derived
myofi-broblasts show a different pattern of actin expression.
The mechanical properties of the ECM are of primordial
im-portance to translate the cellular contraction of
myofibroblastsinto a contraction of the wound tissue. While the
provisionalmatrix of early granulation tissue is highly compliant,
the maturegranulation tissue becomes increasingly stiff over time
[47]. Mi-grating fibroblasts remodel the ECM, synthesizing a
variety of ECMcomponents, such as collagen and fibronectins
[48,49]. It is cur-rently thought that traction forces induced by
fibroblast migrationwithin the provisional matrix and their
ECM-remodeling activitygradually increase the stiffness of the
granulation tissue [1,50–52].NOX4 has been shown to be required for
ECM component pro-duction in lung-derived fibroblasts in vitro
[15,53]. Targeting NOX4results in attenuation of an established
fibrotic response, withreductions in gene transcripts for the
extracellular matrix com-ponents collagen 1α1, collagen 3α1, and
fibronectin [53]. Accord-ingly, in this study we found that
collagen deposition was sig-nificantly reduced in NOX4-deficient
mice. A less dense collagenmatrix could explain a lack of
translation of the cellular forces intoa contraction of the tissue
as a whole. However, a lack of rigidityshould also lead to a
reduction in myofibroblast expression, whichis not the case in our
study.
In addition to the expression of ECM components, their
cross-linking is likely to play an important role in the
transduction ofcontractile forces [47,54,55]. This is a little
understood processwhere NOX enzymes are particularly likely to play
a crucial role. Asoutlined before, we hypothesized that
NOX-mediated dityrosinecrosslinking of the ECM is a possible player
in wound contraction.In fact, dityrosine formation significantly
increased during thewound repair process in our study, suggesting
it has a physiolo-gical role. To the best of our knowledge, this
study provides thefirst data suggestive of a physiological function
of dityrosinecrosslinking in mammals. We could also show that
NOX4-defi-ciency leads to significant impairment of dityrosine
expression,providing a possible explanation as to how myofibroblast
con-traction in NOX4-deficient mice might be less effective in
con-tracting the wound. It is conceivable that myofibroblast
contrac-tion is only effectively translated into tissue contraction
if cells areimbedded in a well cross-linked ECM [56–58].
Tyrosine dimerization is thought to require the presence of
ROS(i.e. H2O2) and of a peroxidase [24,59]. In fact, there is
strongevidence that peroxidases mediate ECM crosslinking [25,60].
Onepossible source of peroxidase during physiological wound repair
ismacrophages and neutrophils as they are known to secrete
mye-loperoxidase (MPO), which is mainly known for its role in
hostdefense. However, in our study we observe no correlation
betweendityrosine formation and MPO expression. Also, there was
nodifference in expression between wildtype and NOX4-/- mice,
suggesting that MPO is not the main peroxidase catalyzing
dityr-osine formation during wound repair.
A promising candidate for a major role in mediating
ECMcrosslinking is vascular peroxidase 1 (VPO1, a.k.a.
peroxidasin,PXDN) as it contains, besides its peroxidase domain,
modules thatare characteristic of the ECM [61]. VPO1 has also been
shown to besecreted by myofibroblasts into the ECM, where it
organizes into afibril-like network colocalizing with fibronectin
[62]. In fact, Lázáret al. have found that VPO1 mediates the
crosslinking of collagenIV in hot spots near the cell surface [63].
It has also been suggestedthat VPO1 catalyzes tyrosyl radical
formation and promotes di-tyrosine cross-linking [64]. VPO1
requires H2O2 to function, whichis supplied to the enzyme by a
currently unknown cellular source.In a model of hypoxia-induced
pulmonary hypertension, Liu et alpropose NOX4 as a provider of
hydrogen peroxidase for VPO1during inflammatory reaction [65] but
to our knowledge the re-lation between NOX4 and VPO1 is yet to be
established. We thinkNOX4 is a promising candidate as a provider of
H2O2 for VPO1-mediated collagen crosslinking through tyrosine
dimerization,which could be an important mechanism of granulation
tissuestiffening during skin wound repair.
Besides tyrosine formation, peroxidases also catalyze
otherprotein cross-links, which might also participate along with
di-tyrosine to the stiffening of the ECM. For instance,
peroxidase-catalyzed cross-link is formed from the deamination of
proteinlysyl ϵ-amino groups to form lysyl aldehydes, which then
reactwith amino acid residues of adjacent molecules [66,67].
In order to understand whether NOX4 is directly involved inthe
dityrosine cross-linking reaction or whether the presence
ofdityrosine in the wound could also be explained by a
NOX4-de-pendent recruitment and/or activation of phagocytic cells,
welooked at NOX2 expression in the granulation tissue. We found
asignificantly weaker expression of NOX2 at day 7 in the wounds
ofNOX4-/- mice, suggesting that NOX4 plays a role in the
recruit-ment of phagocytic cells into the wound. This recruitment
couldtake place through NOX4 dependent interleukin-6 (IL-6)
expres-sion, similar to what has been shown in human microglia and
non-small cell lung cancer cells [56,57]. IL-6 plays an essential
role inskin wound repair as evidenced by delayed wound healing in
IL-6-deficient mice [58]. These results suggest that NOX4
deficiencyleads to a reduction of dityrosine formation at least
partly througha reduction in NOX2-induced ROS in the granulation
tissue. NOX2has also been associated with VPO1 expression before
and it ispossible that NOX2 provides ROS for VPO1-mediated
dityrosineformation [65,68].
As discussed above, HIF1α has been repeatedly associated
withNOX4 [53]. In this study we show that NOX4-deficiency is in
factlinked to a significant delay in HIF1α expression in the
granulationtissue. However, it appears that other pathways can
compensatefor the lack of NOX4 as expression was first delayed in
theknockout group but eventually reached levels similar to the
con-trol group at day 14. Accordingly, we found that angiogenesis
asvisualized by CD31 staining was delayed in NOX4-deficient
micewith a significantly lower expression at day 7 that
eventuallyreached similar levels to control at day 14. This is in
line withearlier studies that showed that NOX4 is required for
effectiveangiogenesis [28]. NOX4 has also been extensively studied
for itsrole as an oxygen sensor and it has been demonstrated that
NOX4is capable of generating hydrogen peroxide as a function of
oxygenconcentration throughout a physiological range of pO2 values
andto respond rapidly to changes in pO₂ [69].
In an earlier study investigating αSMA expression on
wrinklingsubstrates over time we showed that hypoxia reduced
myofibro-blast contraction from approximately 70 to 15% [70]. It is
con-ceivable that a decrease of HIF1α leads to a reduction in
woundvascularization and thus oxygenation, triggering the loss
of
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D. Lévigne et al. / Free Radical Biology and Medicine 96 (2016)
374–384382
contractility of myofibroblasts. However, we had also shown that
apersistent state of ischemia significantly reduces αSMA
expressionin the wound bed, which was not the case in the current
study.In vitro, the loss in contractility clearly preceded the loss
in αSMAexpression [70]. Possibly, a light decrease in wound
oxygenation –as opposed to severe ischemia – leads to a loss of
contractility butnot to a loss of αSMA expression in
myofibroblasts. Further studiesshould investigate the effects of
hypoxia on the expression of ac-tins other than αSMA in
myofibroblasts.
Besides wound contraction, re-epithelialization is an
importantprocess for effective wound closure. In this study we did
not detectany difference in re-epithelialization but it is possible
that NOX4also interferes with keratinocyte activity during
re-epithelializa-tion as recent data suggests that keratinocytes
are producing ROSvia NOX enzymes, including NOX4 [71,72]. It has
been shown thatROS at low concentrations induce keratinocyte
migration in vitro[73,74]. Matrix metalloproteinase (MMP)
production is essentialfor keratinocyte migration as it contributes
to cleaving a paththrough the ECM [75,76]. The collagenase MMP-1 is
an importantconstituent of the matrix-degrading apparatus of
keratinocytesand is expressed in a NOX4-mediated, ROS-dependent way
[77].The isoform NOX1 has been studied in epithelial repair of
in-testinal mucosa healing and it has been reported that
NOX1mediates epithelial migration through activation and
modificationof focal adhesion proteins involved in regulating cell
migration[78]. Our analyses of the wound do not indicate any major
im-pairment of re-epithelialization in NOX4-deficient mice but
thewound model used in this study does not seem appropriate tostudy
re-epithelialization as wounds heal overwhelminglythrough
contraction. More specific analysis should be done in or-der to
better understand the repercussions of NOX4 deficiency
onre-epithelialization during wound repair, possibly by using
asplintered wound model.
5. Conclusions
In this study we show for the first time that NOX4 plays a
rolein cutaneous wound repair. We provide evidence that NOX4
pro-motes dityrosine crosslinking of the ECM as well as the
recruit-ment of phagocytic cells, as significantly less
NOX2-positive cellswere detected in NOX4-/-mice. NOX4-deficiency
also led to re-duced HIF1α and CD31 expression, suggesting a role
of NOX4 inneoangiogenesis during wound repair. NOX4 was required
for ef-fective wound contraction but – surprisingly – not for
myofibro-blast expression, challenging the role of the
myofibroblast as theprimordial mediator of wound contraction. We
suggest thatmyofibroblast contraction in NOX4-deficient mice is
less effectivein contracting the wound because of insufficient
dityrosinecrosslinking of the ECM. MPO expression was not
significantlyaltered by NOX4 deletion, suggesting that other
peroxidases areinvolved in dityrosine formation during wound
repair, possiblyVPO1. In fact, this study allows us to propose the
first physiologicalrole of dityrosine crosslinking in higher
animals, as we observed astrong upregulation in wildtype mice
during skin wound repair.
Author declaration
We wish to confirm that there are no known conflicts of
in-terest associated with this publication and there has been
nosignificant financial support for this work that could have
influ-enced its outcome. This research was supported by the
SwissNational Science Foundation (grant # 310030_120571) (to
BrigittePittet-Cuénod).
We confirm that the manuscript has been read and approved
by all named authors and that there are no other persons
whosatisfied the criteria for authorship but are not listed. We
furtherconfirm that the order of authors listed in the manuscript
has beenapproved by all of us.
We confirm that we have given due consideration to the
pro-tection of intellectual property associated with this work and
thatthere are no impediments to publication, including the timing
ofpublication, with respect to intellectual property. In so doing
weconfirm that we have followed the regulations of our
institutionsconcerning intellectual property.
We further confirm that any aspect of the work covered in
thismanuscript that has involved experimental animals has
beenconducted with the ethical approval of all relevant bodies and
thatsuch approvals are acknowledged within the manuscript.
We understand that the Corresponding Author is the solecontact
for the Editorial process (including Editorial Manager anddirect
communications with the office). She is responsible
forcommunicating with the other authors about progress,
submis-sions of revisions and final approval of proofs. We confirm
that wehave provided a current, correct email address which is
accessibleby the Corresponding Author and which has been configured
toaccept email from [email protected].
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NADPH oxidase 4 deficiency leads to impaired wound repair and
reduced dityrosine-crosslinking, but does not
affect...IntroductionMaterial and methodsAnimal wound modelWound
repair assessmentHistology and immunohistochemistryImage
analysisStatistical methods
ResultsMacroscopic analysisMyofibroblast expressionCollagen
depositionDityrosine formationNOX2 expressionMPO expressionHypoxia
inducible factorNeoangiogenesis and CD31 expression
DiscussionConclusionsAuthor declarationReferences