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Wound contraction facilitates tissue repair. The correct balance between too little contraction, which leads to non-healing wounds, and too much contraction, which leads to contractures, is important for optimal healing. Thus, understanding which cells cause wound contraction is necessary to optimize repair. Wound contraction is hypothesized to develop from myofibroblast (cells which express alpha-smooth muscle actin; ACTA2) contractility, while the role of fibroblast contractility is unknown. In this study, we utilized ACTA2 null mice to determine what role fibroblasts play in wound contraction. Human scar contractures were immunostained for ACTA2, beta-cytoplasmic actin (ACTB), and gamma-cytoplasmic actin (ACTG1). Full-thickness cutaneous wounds were created on dorsum of ACTA2(+/+) mice and strain-matching ACTA2(+/-) and ACTA2(-/-) mice. Wound contraction was quantified. Tissue was harvested for histologic, immunohistochemical and protein analysis. Compared with surrounding unwounded skin, human scar tissue showed increased expression of ACTA2, ACTB, and ACTG1. ACTA2 was focally expressed in clusters. ACTB and ACTG1 were widely, highly expressed throughout scar tissue. Wound contraction was significantly retarded in ACTA2(-/-) mice, as compared to ACTA2(+/+) controls. Control mice had increased epithelialization, cell proliferation, and neovascularization. ACTA2(-/-) mice had lower levels of apoptosis, and fewer total numbers of cells. Smaller amount of collagen deposition and immature collagen organization in ACTA2(-/-) mice demonstrate that wounds were more immature. These data demonstrate that myofibroblasts contribute to but are not necessary for wound contraction. Mechanisms by which fibroblasts promote wound contraction may include activation of contractile signaling pathways, which promote interaction between non-muscle myosin II and ACTB and ACTG1.Laboratory Investigation advance online publication, 14 September 2015; doi:10.1038/labinvest.2015.116.
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Myofibroblasts contribute to but are not necessary for
wound contraction
Mohamed M Ibrahim
1,5
, Lei Chen
2,5
, Jennifer E Bond
1
, Manuel A Medina
1
, Licheng Ren
1,3
, George Kokosis
1
,
Angelica M Selim
4
and Howard Levinson
1,4
Wound contraction facilitates tissue repair. The correct balance between too little contraction, which leads to non-healing
wounds, and too much contraction, which leads to contractures, is important for optimal healing. Thus, understanding
which cells cause wound contraction is necessary to optimize repair. Wound contraction is hypothesized to develop from
myofibroblast (cells which express alpha-smooth muscle actin; ACTA2) contractility, while the role of fibroblast contractility
is unknown. In this study, we utilized ACTA2 null mice to determine what role fibroblasts play in wound contraction.
Human scar contractures were immunostained for ACTA2, beta-cytoplasmic actin (ACTB), and gamma-cytoplasmic actin
(ACTG1). Full-thickness cutaneous wounds were created on dorsum of ACTA2
+/+
mice and strain-matching ACTA2
+/
and ACTA2
/
mice. Wound contraction was quantified. Tissue was harvested for histologic, immunohistochemical and
protein analysis. Compared with surrounding unwounded skin, human scar tissue showed increased expression of ACTA2,
ACTB, and ACTG1. ACTA2 was focally expressed in clusters. ACTB and ACTG1 were widely, highly expressed throughout
scar tissue. Wound contraction was significantly retarded in ACTA2
/
mice, as compared to ACTA2
+/+
controls. Control
mice had increased epithelialization, cell proliferation, and neovascularization. ACTA2
/
mice had lower levels of
apoptosis, and fewer total numbers of cells. Smaller amount of collagen deposition and immature collagen organization in
ACTA2
/
mice demonstrate that wounds were more immature. These data demonstrate that myofibroblasts contribute
to but are not necessary for wound contraction. Mechanisms by which fibroblasts promote wound contraction may
include activation of contractile signaling pathways, which promote interaction between non-muscle myosin II and
ACTB and ACTG1.
Laboratory Investigation advance online publication, 14 September 2015; doi:10.1038/labinvest.2015.116
Wound contraction facilitates tissue repair. The correct
balance between too little contraction, which leads to
non-healing wounds, and too much contraction, which leads
to contractures, is important for optimal healing. Thus,
understanding which cells cause wound contraction is
necessary to optimize repair. Modified fibroblasts with
smooth muscle (SM)-like features, including the expression
of alpha-SM actin (ACTA2), were first observed by Gabbiani
and colleagues in granulation tissue of healing wounds. This
led him to suggest that myofibroblasts promote wound
contraction and collagen production.1,2 Subsequent in vitro
studies by Hinz et al demonstrated that myofibroblasts
generate increased contractile force, as compared to fibro-
blasts, and that the mechanism is related to ACTA2
incorporation into stress fibers and increased focal adhesion
size.36ACTA2 levels were found to associate with a
fibroblast's ability to wrinkle a deformable substrate and
enhance collagen gel contraction.7
The compelling hypothesis that myofibroblasts are essential
for wound contraction is balanced by a counter hypothesis
that myofibroblasts are not essential for wound contraction.
Ehrlich et al found that human sacrococcygeal pilonidal sinus
wounds contract without a high density of myofibroblasts
being present, ACTA2 is absent in free-floating collagen
lattice contraction, full thickness excisional wounds in rats
are capable of unwounded wound contraction in the absence
of myofibroblasts, and prevention of ACTA2 expression in
rodents treated with vanadate does not alter wound
1
Division of Plastic and Reconstructive Surgery, Departments of Surgery and Pathology, Duke University Medical Center, Durham, NC, USA;
2
Department of Burn Surgery,
First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China;
3
Department of Burns and Reconstructive Surgery, Xiangya Hospital, Central South
University, Changsha, Hunan, China and
4
Department of Pathology, Duke University Medical Center, Durham, NC, USA
Correspondence: Dr H Levinson, MD, Division of Plastic and Reconstructive Surgery, Departments of Surgery and Pathology, Duke University Medical Center, Durham, NC
27710, USA.
E-mail: howard.levinson@duke.edu
5
These authors contributed equally to this work.
Received 13 January 2015; revised 1 July 2015; accepted 28 July 2015; published online 14 September 2015
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 00 2015 1
Laboratory Investigation (2015), 110
©
2015 USCAP, Inc All rights reserved 0023-6837/15
contraction.812 In 2002, Wrobel et al13 demonstrated that
fibroblasts and myofibroblasts produce similar contractile
forces.
To determine the role of ACTA2 in wound contraction, we
used human scar tissue, Acta2 null (Acta2
/
) mice and
murine open wound contraction model.14 Human tissue was
immunostained for ACTA2, beta-cytoplasmic actin (ACTB),
and gamma-cytoplasmic actin (ACTG1). Human scar tissue
showed increased expression of ACTA2, ACTB, and ACTG1.
ACTA2 was focally expressed in clusters. ACTB and ACTG1
were widely, highly expressed throughout scar tissue. Wound
contraction was significantly retarded in ACTA2
/
mice, as
compared to ACTA2
+/+
controls. Control mice had increased
epithelialization, cell proliferation, and neovascularization.
ACTA2
/
mice had lower levels of apoptosis, and fewer total
numbers of cells. The smaller amount of collagen deposition
and immature collagen organization in ACTA2
/
mice
demonstrate that the wounds were more immature. We
conclude that the expression of ACTA2 is beneficial but not
essential for wound contraction.
MATERIALS AND METHODS
Human Tissue
A total of 18 human scar samples with surrounding
unwounded tissue were obtained following approved
IRB protocols: 12 samples were obtained from First
Affiliated Hospital of Sun Yat-sen University in accordance
with IRB protocol, and 6 samples were obtained from
Duke University Medical Center (DUMC) Department of
Pathology. Samples were collected within 1 year of cicatriza-
tion. All the included samples exhibited obvious contrac-
ture (Supplementary Figure 1). Samples were categorized
according to patientsrace, gender, age, and scar location
(Table 1).
Animals
Male and female Acta2
+/+
,Acta2
+/
, and Acta2
/
mice
(1012 weeks old and weighing from 18 to 23 g) were used
in this study. The Acta2
/
mice used in this study were
established by inserting the Pol2NeobpA cassette15 into the
+1 start site of the Acta2 gene.16 Acta2
/
,Acta2
+/+
, and
Acta2
+
mice from the same breeding colony were obtained
from Warren E Zimmer, PhD at Texas A&M Health Science
Center. All mice were monitored for signs of toxicity.
The mice were housed under protocols approved by the
Institutional Animal Care and Use Committee (IACUC) of
Duke University.
Excisional Wounds and Gross Examination
All procedures were performed in accordance with a protocol
approved by Duke University IACUC. Mice were anesthetized
using gas anesthesia (oxygen, 2 l/min, isoflurane, 2%). The
back of the mouse was shaved with metallic clippers. The back
was then sterilized using alcohol. Full-thickness excisional
wounds were created using 8-mm biopsy punches (Miltex,
York, Gibbstown, PA, USA) in the area between mices
scapular angles. The wounds were then covered by Tegaderm
(Transparent Film Dressing Frame Style, 3M Health Care, St
Paul, MN, USA). Dressings were changed daily for the first
4 days and then removed. Wounds were measured and
photographed daily using Canon PowerShot A470 digital
camera. Wound contraction was measured by gravitational
planimetry and expressed as percentage of original
wound size.
Tissue Collection
Mice were euthanized and tissue was collected. Collected
tissue was cut into equal halves. One half was preserved in
10% formalin for histological (HIS) analyses and the other
half was immediately frozen in liquid nitrogen for additional
analyses. Prior to staining, tissue sections were deparaffinized
by warming at 65 °C overnight, immersed in xylene for
15 min, and rehydrated with decreasing concentrations of
ethanol in distilled water.
Table 1 Patient demographic of selected scar samples
Group N
All 18
Race
Caucasian 6
Asian 12
Gender
Male 12
Female 6
Age (years)
o20 8
420 10
Scar location
Head and trunk 9
Upper extremities 3
Lower extremities 6
Scar ages
15 months 7
610 months 6
410 months 5
A total number of 18 skin lesions with typical contraction scar and surround-
ing unwounded skin between 1 and 12 months were matched according to
patientsrace, gender, age, scar location, and scar ages.
Myofibroblasts and wound contraction
MM Ibrahim et al
2Laboratory Investigation | Volume 00 2015 | www.laboratoryinvestigation.org
HIS, Immunohistochemical, and Immunofluorescence
Analysis
For HIS analysis, hematoxylin and eosin (H&E,
Sigma-Aldrich, St Louis, MO, USA) staining was performed
following standard techniques. Massons Trichrome staining
was performed by use of Trichrome Stain kit
(Sigma-Aldrich), for collagen evaluation. The collagen index
value was calculated as collagen index =B+G/2 R+B+G for
each pixel within the image (where R, G, and B represent the
red, blue, and green pixel values, respectively). The value
of the collagen index ranged from 0 for extremely red
objects to 1 for completely bluegreen objects. The average
collagen index of three (HPF) images for each time point was
graphed.
For immunohistochemical (IHC) analysis, immunostain-
ing was done. Briefly, to inhibit endogenous peroxidase
activity, rehydrated sections were immersed in 3% hydrogen
peroxide (H2O2) for 10 min to block endogenous peroxidase.
Slides were rinsed with deionized water and then placed
under retrieval solution (Target Retrieval Solution, Dako
North America Carpinteria, CA, USA) in a water bath (98 °C)
to unmask antigens. After further tris-buffered saline (TBS,
TBS Automation Washing Buffer, Biocare Medical, Concord,
CA, USA) rinsing, sections were treated with 10% goat serum
(Normal Goat Serum, Vector Laboratories, Burlingame,
CA, USA) for 1 h to block non-specific antibody binding.
The primary antibody was incubated overnight at 4 °C. The
primary antibodies used included (1) for human scar tissue
IHC: rabbit anti ACTA2 polyclonal antibody (1:100 dilution,
Abcam, Cambridge, MA, USA), mouse anti ACTB
monoclonal antibody (1:5000 dilution, Abcam), mouse
anti-ACTG1 monoclonal antibody (1:1000 dilution, Santa
Cruz Biotechnology, Santa Cruz, CA, USA); and (2) for mice
wound tissue IHC: rabbit anti-Ki67 monoclonal antibody
(1:400 dilution, Thermo Scientific, Rockford, IL, USA), rabbit
anti-CD31 polyclonal antibody (1:200 dilution, Abcam). After
washing with TBS, the slides were incubated with appropriate
secondary antibody. The secondary antibodies used were:
biotinylated goat anti-rabbit IgG (1:200 dilution, Vector
Laboratories), goat anti-mouse IgG (1:200 dilution, Vector
Laboratories), and horse anti-mouse IgG (1:200 dilution,
Vector Laboratories). The reactions were developed with an
avidinbiotin complex reaction (Vector Laboratories).
For immunofluorescence analysis, Terminal deoxynucleo-
tidyltransferase (TdT) dUTP nick-end labeling (TUNEL)
staining was performed by using in situ Cell Death Detection
POD kit (Roche, IN, USA) according to the manufacturers
instructions. 40,6-diamidino-2-phenylindole (DAPI) counter
stain was performed with Vectashield Mounting Medium
(Vector Laboratories, Burlingame, CA, USA). Labeled cells
were visualized by use of a Nikon eclipse E600 microscope
and images were captured with a Nikon DXM 1200 digital
camera under the same setting. Morphometric evaluations
were done from sections through the center of wounds in
order to obtain the maximum wound area for evaluation.
Measurements of epidermal thickness,17,18
collagen content,19 positive immunostaining intensity,20 and
counting2123 were performed with NIH ImageJ software,
and all the analyses were run as triplicates. To quantify
non-muscle myosin II (isoform a, b, c, and ASMA) level of
expression in scar vs normal tissues by immunochemical
stained image analysis, the 24-bit RGB images per region
(300 p.p.i.) were converted to 8-bit gray value images with
pixel intensity values ranging from 0 (black) to 255 (white)
using NIH ImageJ software. An 8 × 8 mm sample region
containing both scar and normal tissue was selected for
analysis. The sample image was divided into an 8 ×8 grid
(each box =1mm
2
). A mean pixel intensity value for each
box was measured using ImageJ. The middle four columns
were excluded from analysis since the area contains both
scar and normal tissues. As a result, 16-sample pixel
intensity values were each obtained for normal and
scar tissue. Statistical analysis was performed using
JMP software (SAS Institute ver. 7). The means of the two
samples were compared via Studentst-test. Differences
were considered to be statistically significant at values of
Po0.01.
Statistical Analysis
All data are presented as the mean and s.e.m. of three
independent experiments. Statistical differences were
determined using Studentst-test or one-way ANOVA with
Bonferronispost hoc test. The difference was considered
significant when the P-value was 0.05 or less.
RESULTS
Expression of ACTA2, ACTB, and ACTG1 in Unwounded
Human Skin and Human Contracture Scar
In unwounded skin, expression of ACTA2 was absent in the
epidermis and dermal fibroblasts, but expression was detected
in SM cells of blood vessels and skin appendages, whereas
ACTB and ACTG1 were expressed both in the epidermis and
all the dermis including dermal fibroblasts (Figure 1a, upper
row) of unwounded skin. In contracted scar, the epidermis
was thickened and flattened, and the appendages were
missing. ACTA2 expression in scar epidermis was absent.
The expression of ACTA2 in scar dermis was detected in
vascular SM cells and in cells located in the nodular structure
in the deeper layers of the scar. ACTB and ACTG1 expression
in scar epidermis was present similar to that in the
unwounded skin epidermis, and the expression in the scar
dermis was increased in comparison with ACTA2 (Figure 1b,
upper row). The overall expression of ACTA2, ACTB, and
ACTG1 (Figure 1c) was found to be significantly increased in
contracted scar tissue when compared with unwounded
human skin tissue (1.8-fold for ACTA2, 2.5-fold for ACTB
and ACTG1; P0.01). Variation caused by patientsrace,
gender, age, scar location, and staining intensity of ACTA2,
ACTB, and ACTG1 in unwounded tissue and scar were
detected and summarized (Table 2).
Myofibroblasts and wound contraction
MM Ibrahim et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 00 2015 3
The Effect of ACTA2 Deficiency on Murine Wound
Contraction
On day 2, compared with Acta2
+/
mice (85.5 ±1.9%),
relative wound areas were significantly larger in Acta2
/
mice
(92.1 ±2.0%). There is no significant difference between
Acta2
+/+
and Acta2
+/
groups at days 3 and 4 after injury;
however, the impaired wound contraction in Acta2
/
mice
in comparison with that of Acta2
+/
mice still presented
significant difference afterwards except day 9. In comparison
with the wound size of the Acta2
+/+
mice (70.4 ±1.7%) the
relative wound size on day 3 was significantly lager in Acta2
/
mice (78.9 ±2.1%) and the delay in wound contraction in
Acta2
/
mice group was observed up to day 11. Finally,
11 days after injury, the wounds of Acta2
/
mice healed to
the same degree as those of Acta2
+/+
and Acta2
/
mice
(Figure 2). Collectively, the gross wound contraction was
delayed in the absence of ACTA2.
The Effect of ACTA2 Deficiency on Wound Evaluation
Microscopic assessment using H&E of wounds demonstrated
that fibroblasts were the dominant cell type on days 7 and 11
in the wound area (Figure 3a). All the wounds in the mice
Figure 1 ACTA2, ACTB, and ACTG1 expression in the remodeling phase of repair. (a) Representative human unwounded skin. (b) Scar specimens
investigated by IHC staining are shown to reveal the expression pattern of ACTA2, ACTB, and ACTG1. Scale bars, 400 μm for × 4 (upper panel of (a) and
(b)) and 100 μm for ×40 (lower panel of (a) and (b)) images; e, epidermis; d, dermis. (c) The staining intensity of human unwounded skin tissue and
contraction scar over time was quantified. Data are expressed as mean ± s.e.m. *Po0.05, tested by one-way ANOVA with Bonferronispost hoc test,
performed on 6- to 10-month vs 410-month scar for ACTB expression. ANOVA, analysis of variance; IHC, immunohistochemistry.
Myofibroblasts and wound contraction
MM Ibrahim et al
4Laboratory Investigation | Volume 00 2015 | www.laboratoryinvestigation.org
were re-epithelialized by day 11. Increased numbers of
fibroblasts were arranged in parallel to the epidermis and
were observed in the scar area. Follicular sebaceous glands
were present at the wound edge and juxtaposed unwounded
tissue, but absent in the scar. The scar showed increased
epidermal thickness compared to surrounding unwounded
tissue (Figure 3b).
The Effect of ACTA2 Deficiency on Collagen Deposition
in Wound Sites
Massons Trichrome staining showed more collagen
surrounding the fibroblasts in the granulation tissue area on
day 11 compared to day 7 in all groups. Wounds in Acta2
+/
and Acta2
/
mice showed scant collagen deposition
(Figure 4a). Our analysis19 has demonstrated a decrease in
collagen accumulation in Acta2
+/
and Acta2
/
mice
compared to that observed in Acta2
+/+
mice (Figure 4b).
The Effect of ACTA2 Deficiency on Cellularity
To further assess the granulation tissue area in the different
mice, Ki67, a nuclear antigen expressed in proliferating cells,
was used to evaluate the proliferative activity of the
fibroblastic area (Figure 5a). The number of Ki67-positive
cells in the dermis of Acta2
/
mice wound was increased on
days 7 and 11 compared to mice expressing ACTA2
(Figure 5b). Cellular apoptosis was assayed using TUNEL
staining (Figure 6a), as apoptosis is a mechanism of
myofibroblast resolution. The cellular apoptosis observed in
the granulation tissue area of Acta2
/
mice was approxi-
mately half that compared to that observed in the granulation
tissue area of Acta2
+/+
mice on days 7 and 11 (Figure 6b).
DAPI staining was used to quantify cellularity. Acta2
/
mice
had decreased number of cells in the granulation tissue area
only on day 7 compared to that in mice expressing ACTA2
(Figure 6c). In summary, ACTA2
+/+
mice had a greater
number of cells (as determined by DAPI) earlier in the wound
Table 2 ACTA2, ACTB, and ACTG1 expression comparison
among groups
Group P-value
ACTA2 ACTB ACTG1
All 52.0 ± 1.7* 107.5 ± 3.7 104.3 ± 2.9*
Race
Caucasian 49.5 ± 4.0 100.7 ± 7.7 98.2 ± 5.7
Asian 53.3 ± 1.6 110.8 ± 3.9 107.4 ± 3.0
Gender
Male 51.6 ± 2.2 110.1 ± 4.5 103.6 ± 3.1
Female 52.8 ± 2.8 102.2 ± 6.5 105.8 ± 6.4
Age (years)
o20 52.9 ± 1.9 111.7 ± 5.2 105.5 ± 4.1
420 51.3 ± 2.7 104.1 ± 5.2 103.4 ± 4.2
Scar location
Head and trunk 50.8 ± 4.5 103.7 ± 8.9 103.8 ± 6.9
Upper extremities 52.1 ± 3.2 114.6 ± 2.3 103.4 ± 9.1
Lower extremities 53.8 ± 2.2 109.5 ± 6.8 105.6 ± 4.1
Expression levels of ACTA2, ACTB, and ACTG1 in human contraction scar
were measured by IHC staining intensity analysis. Values are indicated as
mean ± s.e.m., for groups matched according to patientsrace, gender, age,
scar location, and scar ages, *Po0.01 tested by one-way ANOVA with
Bonferronispost hoc test, performed on ACTA2 vs ACTB, respectively. For
groups matched according to tissue type, *Po0.01 tested by Studentst-test,
performed on unwounded skin tissue vs scar tissue. ANOVA, analysis of var-
iance; IHC, immunohistochemistry.
Figure 2 Skin wound healing of Acta2
+/+
,Acta2
+/
, and Acta2
/
mice.
(a) Representative macroscopic views from wounded mice are shown at
days 0, 3, 7, and 11 post wounding. All pictures were taken at the same
distance. (b) The fraction of wound area at indicated time point in
comparison to the original wound area (quantified as described in
Material and Methods section) was plotted and shown. Values are
represented as mean ± s.e.m. (n= 25, 31 and 38 for Acta2
+/+
,Acta2
+/
,
and Acta2
/
mice group). *Po0.05, tested by one-way ANOVA with
Bonferronispost hoc test, performed on Acta2
+/+
vs Acta2
+/
,Acta2
+/
vs
Acta2
/
, and Acta2
+/+
vs Acta2
/
, respectively. ANOVA, analysis of
variance. A full color version of this figure is available at the Laboratory
Investigation journal online.
Myofibroblasts and wound contraction
MM Ibrahim et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 00 2015 5
repair process; however, due to the increased proliferation
and decreased apoptosis in ACTA2
+/
and ACTA2
/
mice,
as observed on day 7, and relative reduction of proliferation
and increase in apoptosis in ACTA2
+/+
mice, as observed on
day 7, the level of cellularity in ACTA2
+/
and ACTA2
/
mice eventually equaled that of ACTA2
+/+
mice on day 11.
These observations relate to wound size and may simply
reflect the need to heal the wound.
Neovascularization in Acta2
/
Mice
To assess neovascularization, CD31 immunostaining was
performed (Figure 7a). Neovascularization relies upon SM
Figure 3 Histological evaluation of wounded skin. (a) H&E staining was performed at days 4, 7, and 11 after injury. Representative results from Acta2
+/+
,
Acta2
+/
, and Acta2
/
mice are shown. The dotted lines indicate the margin areas of the wound. Scale bars, 200 μm for × 10 images; e, epidermis; d,
dermis. (b) Epidermal thickness was measured in Acta2
+/+
,Acta2
+/
, and Acta2
/
mice at day 11 after injury. Data are expressed as mean ± s.e.m. (n=3
per genotype). **Po0.05 tested by one-way ANOVA with Bonferronispost hoc test, performed on Acta2
+/+
vs Acta2
+/
and Acta2
+/+
vs Acta2
/
,
respectively. ANOVA, analysis of variance; H&E, hematoxylin and eosin. A full color version of this figure is available at the Laboratory Investigation
journal online.
Myofibroblasts and wound contraction
MM Ibrahim et al
6Laboratory Investigation | Volume 00 2015 | www.laboratoryinvestigation.org
cells, so mice deficient in ACTA2 could have defects in blood
vessel formation that would affect healing. Compared to
Acta2
+/+
mice, the granulation tissue areas of Acta2
+/
and
Acta2
/
mice had a surprisingly significantly greater number
of vessels (Figure 7b). This result is consistent with previous
mice wound contraction data and suggested that mice lack
of ACTA2 expression have significantly enhanced level of
angiogenesis than that of Acta2
+/+
mice.
DISCUSSION
In our current study, the use of Acta2
/
mice allowed us to
directly test the role of alpha-SM actin-expressing myofibro-
blasts in wound contraction. Wound contraction started after
surgery, and the contraction observed in Acta2
/
mice was
significantly slower than that of Acta2
+/+
mice at all time
points except day 2. The wound contraction difference
between Acta2
+/+
and Acta2
/
mice peaked around day 7
post injury and then slowed down afterwards. Nonetheless,
control mice healed their wounds. Collectively, these results
identify a role for ACTA2 in wound closure but not an
absolute necessity.
The wound healing process is a stepwise sequence of
overlapping events, which often results in scar.24 Persistent
wound contraction after a wound has epithelialized leads to
contractures, whereas delayed wound contraction leads to
chronic non-healing wounds.24 Current theories posit that
Figure 4 Representative wound section following Massons Trichrome
staining. (a) Collagen deposition and organization in days 7 and 11. Blue
area represents collagen. (b) Collagen deposition results are shown as
mean ± s.e.m. (n= 3 per genotype). *Po0.05 tested by one-way ANOVA
with Bonferronispost hoc test, performed on Acta2
+/+
vs Acta2
/
for
day 7 mice tissue, on Acta2
+/+
vs Acta2
+/
and Acta2
+/+
vs Acta2
/
for
day 11 mice tissue, respectively. Scale bars, 100 μm for × 40 images.
ANOVA, analysis of variance.
Figure 5 Proliferation activity in wound dermis. (a) Days 7 and 11
Acta2
+/+
,Acta2
+/
, and Acta2
/
mice wound specimens were evaluated
by staining with anti-Ki67 antibody. Representative photos are shown and
Ki67-positive cells are indicated by black arrows. Level of cell proliferation
was determined by the number of positive-stained nuclei in wound
dermis. Scale bars, 100 μm for × 40 images. (b) Quantified data are
represented as means ± s.e.m. (n= 3 per genotype). *Po0.01 tested by
one-way ANOVA with Bonferronispost hoc test, performed on Acta2
+/+
vs
Acta2
+/
,Acta2
+/
vs Acta2
/
, and Acta2
+/+
vs Acta2
/
, respectively.
ANOVA, analysis of variance.
Myofibroblasts and wound contraction
MM Ibrahim et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 00 2015 7
alpha-SM actin-expressing myofibroblasts are necessary for
wound contraction.1Although myofibroblasts are clearly
present in granulation tissue during wound closure and in
pathological contracture tissues, questions have arisen as to
whether they are essential for collagen/granulation tissue
contraction.25 It is clear that traction forces that are generated
by fibroblasts as they migrate on a compliant substratum
can reorganize collagen matrices.26 In fact, in free-floating
collagen lattices the resulting reduction in lattice diameter is
entirely due to fibroblast traction forces, which are generated
by fibroblast migration and contractility.27 This is sufficient
to result in wound closure, this makes the involvement of a
specialized contractile cell unnecessary.9
The gross evaluation from our study showed that the
wound contraction rate of Acta2
/
mice do not match that of
Acta2
+/+
and Acta2
+/
mice but the wounds still contracted
and closed. This actin expression defect disturbs the balance
of wound contraction and regeneration, which sequentially
upregulates the proportion of dermal regeneration in wound
closure process.28 As present results demonstrate, although
Acta2
/
mice showed impaired wound contraction, they have
increased neovascularization and cell proliferation in the
wound bed as compared to Acta2
+/
and Acta2
+/+
mice. The
increase level of neovascularization in Acta2
/
mice implies
that the absence of ACTA2 did not intervene the development
and formation of new blood vessels, which complies with the
previous finding of Ehrlich et al12 that the development of
ACTA2 in fibroblasts requires signals different from those
required for these structures to appear in SM cells.
Wound contraction and scar contracture share a similar
mechanism of action, namely, the contraction of the
granulation bed.3Our current study started with IHC analysis
of human contracture scar. Except blood vessels, ACTA2 was
detected in the nodular structure found in contracture scar
Figure 6 Apoptosis at wound sites. (a) TUNEL staining of wound sections were used for apoptosis detection (green), and DAPI staining was performed
to visualize the total cell number (blue). Scale bars, 75μm for × 20 images. Cell counting was performed at ×20 magnification. Quantification of
apoptosis ratio was achieved by plotting the number of TUNEL-positive cell against that of DAPI-positive cells (b). Wound cellularity was quantified by
counting the number of DAPI-positive cells (c). Data are represented as means ± s.e.m. (n= 3 per genotype). *Po0.05 tested by one-way ANOVA with
Bonferronispost hoc test, performed on Acta2
+/+
vs Acta2
/
, respectively. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole;
TUNEL, terminal deoxynucleotidyltransferase (TdT) dUTP nick-end labeling.
Myofibroblasts and wound contraction
MM Ibrahim et al
8Laboratory Investigation | Volume 00 2015 | www.laboratoryinvestigation.org
samples, which is consistent with previous reports.29,30 It
appears that the existence of the particular nodule structure
may have a relationship with the gross contractile characteristic
of these samples.30 The expression of ATCB and ACTG1 in
the scar was significantly higher in comparison with that in
unwounded tissue. In contrast with the local, nodular
expression of ACTA2, ATCB, and ACTG1 was widely
expressed throughout the scar. The proportional difference
in actin isoform expression within scar brought up the same
questions proposed by a sizeable body of studies8,9,11,12,25,3133
concerning the controversial role of ACTA2: (1) is the
contribution of ACTA2 expression necessary for contractile
force generation and tissue remodeling? (2) Will ACTB and
ACTG1 expression sufficiently compensate the function
of ACTA2?
In addition, there are variations in different investigations
concerning the ACTA2 expression level in scar tissue.29,30,34
Possible explanations for this inconsistency are: (1) differences
in the sources of samples related with species, patients with
demographic distribution; (2) differences in the samples'
inclusion and exclusion criteria; (3) percentages of scar in
different stages of repair process; (4) differences in the
evaluation methods; and (5) sizes of sample population and
observer variance. Our current research focuses on determin-
ing the relationship between actin isoforms and wound
contraction. We observed a statistically significant higher
expression of each actin in scar tissue, consistent with reports
of Ehrlich et al,30 our laboratories previous work,20 Tian
et al35 and Wang et al.34
While our manuscript was in preparation, Tomasek et al36
reported on wound healing in the Acta2
/
mouse. Analogous
to our study, he found that ACTA2 expression was not
necessary for wound closure. He reported that alpha-SM actin
null fibroblasts become more contractile in response to
TGF-βand that compensatory increases in actin isoforms,
such as cardiac muscle α-actin, cytoplasmic β-actin,
cytoplasmic γ-actin, skeletal muscle α-actin, and SM γ-actin
could explain normal wound contraction rates. Differential
expression of actin isoforms enabled non-alpha-SM actin
isoform-expressing myofibroblasts to form. He also found
a statistically different rate of wound contraction between
animals, but not at all time points, as we observed. We believe
that the discrepancies can be attributed to: (1) smaller wound
sizes in his study, and (2) fewer time points to closure;
meaning he may have missed statistically significant
differences at more time points. Nevertheless, our work is
in agreement with his that alpha-SM actin is not necessary for
wound closure.
The present study has demonstrated that alpha-SM actin-
expressing myofibroblasts contribute to but are not necessary
for wound contraction. Although Acta2
/
mice showed
impaired wound contraction, they have increased neo-
vascularization and cell proliferation in the wound bed as
compared to Acta2
+/
and Acta2
+/+
mice. Compensatory
increases in actin isoform expression may explain why wound
contraction still occurs until wound closure. Thus, we posit
that targeting contractile elements different than actin
isoforms; yet, shared by myofibroblasts and fibroblasts
(eg, non-muscle myosin II) may be a necessary approach
for promoting healing of chronic wounds or reducing scar
contractures.
Supplementary Information accompanies the paper on the Laboratory
Investigation website (http://www.laboratoryinvestigation.org)
Figure 7 Angiogenesis in mice skin after wounding. (a) Days 7 and 11
Acta2
+/+
,Acta2
+/
, and Acta2
/
mice wound sections were evaluated by
staining with anti-CD31 antibody. Representative micrographs are shown
and CD31-positive vessels were indicated by black arrows. Scale bars,
100 μm for × 40 images. (b) Levels of vascularization were determined by
the number of vessels stained positive for CD31 per high-power field in
wound area. Data are represented as means ± s.e.m. (n= 3 per genotype).
*Po0.05 tested by one-way ANOVA with Bonferronispost hoc test,
performed on Acta2
+/+
vs Acta2
/
for day 7 mice tissue, on Acta2
+/+
vs
Acta2
+/
,Acta2
+/
vs Acta2
/
, and Acta2
+/+
vs Acta2
/
for day 11 mice
tissue, respectively. ANOVA, analysis of variance. A full color version of
this figure is available at the Laboratory Investigation journal online.
Myofibroblasts and wound contraction
MM Ibrahim et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 00 2015 9
ACKNOWLEDGMENTS
This work was supported by a Plastic Surgery Foundation National
Endowment Grant and by a grant from the National Institutes of Health
K08GM085562. We thank Dr Bruce Klitzman for his supervision of animal
experiment, Dr Zuowei Su for his advice and technical assistance for
immunohistochemical experiments, Dr Luisa A DiPietro for her technical
assistance, and Gloria Adcock for her assistance with tissue processing. We
also thank Warren E Zimmer, PhD at Texas A&M Health Science Center for
providing the ACTA2
/
mice used in this study and Dr Shaohai Qi for
providing the human scar samples used in this work.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Myofibroblasts and wound contraction
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10 Laboratory Investigation | Volume 00 2015 | www.laboratoryinvestigation.org
... α-Smooth muscle actin (αSMA) expression is another marker used to characterize wound regeneration as it represents myofibroblast activation and differentiation which is critical for wound healing (43,46). However, excessive myofibroblast activation, which can be quantified through αSMA expression, is indicative of scar tissue development (43,46). ...
... α-Smooth muscle actin (αSMA) expression is another marker used to characterize wound regeneration as it represents myofibroblast activation and differentiation which is critical for wound healing (43,46). However, excessive myofibroblast activation, which can be quantified through αSMA expression, is indicative of scar tissue development (43,46). αSMA expression within the wound was quantified through the analysis of stained frozen skin tissue samples collected on days 7 and 12 after skin biopsy. ...
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... The collagen index ranges from 0 for primary (extremely red) to 1 for fully matured (completely bluegreen) collagen deposition. 87 Picrosirius red staining (PSR) was also used to highlight the types of collagen fibers deposited on the wound site for day 15. To do so, the processed samples were photographed by polarizing OLYMPUS microscope BH-2 BHMPUS microscope, which displayed the stained collagen fibers from bright green to orange red. ...
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We have studied the expression of alpha-smooth muscle actin, smooth muscle myosin, and desmin in granulation tissue during the healing of an open wound in the rat by means of electron microscopy and immunohistochemistry, at the light and electron microscopic levels, using specific antibodies directed against these proteins. Important amounts of the three antigens were always expressed in pericytic and/or smooth muscle cells of neoformed small vessels. In fibroblastic cells, microfilaments were absent 4 days after wounding but accumulated gradually, starting from the 6th up to the 15th day; at this time, they were evident in about 70% of fibroblastic cells (myofibroblasts). They then regressed progressively, and on the 30th day, microfilaments were no longer present in scar fibroblasts. alpha-Smooth muscle actin, but not smooth muscle myosin and desmin, was always present in microfilament bundles of myofibroblasts. The staining intensity was progressive from the 6th to the 15th day and decreased thereafter; no staining was seen at the 30th day. Between the 20th and the 25th day, many apoptotic figures were seen in fibroblastic and endothelial cells, suggesting that apoptosis is the mechanism of their disappearance. We conclude that myofibroblasts develop gradually from granulation tissue fibroblasts and temporarily express a marker of smooth muscle differentiation. These results may be relevant for the understanding of the mechanisms of normal and pathologic wound healing.
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The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts.
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