Inhibition of Pathological Phenotype of Hypertrophic Scar...

of 32 Tissue Engineering

© Mary Ann Liebert, Inc.

DOI: 10.1089/ten.TEA.2016.0550

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Inhibition of pathological phenotype of hypertrophic scar fibroblasts via co‐

culture with adipose derived stem cells

Jingcheng Deng, MD,1† Yuan Shi, MD,1† Zhen Gao, PhD,1 Wenjie Zhang, PhD1, Xiaoli Wu, MD,

PhD,1 Weigang Cao, MD, PhD,1* and Wei Liu, MD, PhD1*

1Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao

Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, 639 Zhi Zao Ju

Road, Shanghai 200011, PR China

†Authors who contributed equally

*Corresponding authors: Dr. Wei Liu and Dr. Weigang Cao

Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao

Tong University School of Medicine

639 Zhi Zao Ju Road,

Shanghai 200011, PR China.

Tel: +86‐21‐23271699, Ext 5061

Fax: +86‐21‐53078128

E‐mail address:

Wei Liu: [email protected]

Weigang Cao: [email protected]

Running title: hASCs inhibit pathological phenotype of hypertrophic scar fibroblasts

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Abstract

Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by fibroblast

over‐proliferation, overproduction and deposition of the extracellular matrix (ECM). Growing

evidence demonstrated that adipose derived stem cells (ASCs) secrete a plethora of trophic and

anti‐fibrotic factors, which suppress inflammation and ameliorate fibrosis of different tissues.

However, few studies investigate their effect on repressing HS activity. This study evaluated the

suppressing effect of ASCs on HS fibroblast bioactivity and the possible mechanism via a co‐culture

model. HS‐derived fibroblasts (HSFs) and ASCs were isolated from individual patients. HSFs or HSFs

treated with transforming growth factor‐β1 (TGF‐β1) were co‐cultured with ASCs and the change

of HSF cellular behaviors, such as cell proliferation, migration, contractility and gene/protein

expression of scar‐related molecules, were evaluated by cell counting assay, cell cycle analysis,

scratch wound assay, fibroblast‐populated collagen lattice (FPCL) contractility assay, real‐time

quantitative polymerase chain reaction (RT‐qPCR), ELISA, and western blotting assay. After 5 days

of ASC co‐culture treatment, the expression levels of collagen I (Col 1), collagen III (Col 3),

fibronectin (FN), TGF‐β1, interleukin‐6 (IL‐6), interleukin‐8(IL‐8), connective tissue growth factor

(CTGF) and alpha‐smooth muscle actin (α‐SMA) in HSFs decreased significantly while the

expression levels of decorin (DCN) and MMP1/TIMP1 ratio increased significantly. Besides, after 5

days of exogenous TGF‐β1 stimulation, the expression levels of collagen 1, fibronectin, TGF‐β1, IL‐

6, CTGF and α‐SMA in HSFs increased significantly. Impressively, all these increased gene

expression levels were reversed by 5 days of ASCs co‐culture treatment. Additionally, the

proliferation, migration and contractility of HSFs were all significantly reduced by ASC co‐culture

treatment. Furthermore, the protein levels of TGF‐β1 and intracellular signal pathway related

molecules, such as p‐smad2, p‐smad3, p‐Stat3 and p‐ERK, were down‐regulated significantly in

HSFs after 5 days of ASCs co‐culture treatment.This study demonstrated that co‐culture of HSFs

with ASCs not only inhibited proliferation, migration and contractility of HSFs but also decreased

the expression levels of HSF‐related or TGF‐β1‐induced molecules. Additionally, the anti‐fibrotic

effect on HSFs was likely mediated by the inhibition of multiple intracellular signaling. The results

of this study suggest the therapeutic potential of ASCs for HS treatment, which is worth of further

investigation.

Keywords: Hypertrophic scar fibroblasts; Adipose‐derived stem cells; Co‐culture; TGF‐1;

Pathological phenotype; Inhibitory effect

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Introduction

Hypertrophic scar (HS) is a common disease characterized by overproduction and

deposition of extracellular matrices (ECM), cell overgrowth and irregular distribution, enhanced

angiogenesis, and enhanced transformation of fibroblasts to myofibroblasts, 1‐3 which is associated

with excessive wound healing response.4 It is usually secondary to massive skin trauma, burns, or

surgical procedures.5 Patients with hypertrophic scar experienced physical, psychological and social

discomfort because of functional disability and facial organ disfigurement caused by tissue

hypertrophy and severe contracture.6, 7 To date, the main therapeutic approaches of HS include

surgical excision, corticosteroid injection, laser therapy and pressure therapy.8 However, due to the

lack of consensus curative effect and severe adverse effects of current therapies, HS treatment

remains a great challenge to clinicians. Studies have confirmed that aberrant proliferation and

activation of fibroblasts and excessively deposited ECM components are recognized as the main

characteristics of HS.9 Thus, it is reasonable to assume that inhibition of fibroblast bioactivity and

their extensive ECM production may become a pivotal therapeutic strategy for HS.

It has been reported that mesenchymal stem cells (MSCs) could inhibit fibrotic tissue

formation by secreting a number of cytokines and anti‐fibrotic factors.10, 11 Adipose derived stem

cells (ASCs) are one type of MSCs, which have the advantages of autologous, non‐immunogenic,

self‐renewal, multi‐differentiation potential, easy access, available for large quantity and less

donor site morbidity and thus are an ideal therapeutic cell source.12, 13 Recently, increasing

evidence demonstrates that ASCs have potential of serving as an anti‐scarring or anti‐fibrotic

agent.

For example, implantation of autologous fat graft for the treatment of gluteal muscle

contracture could achieve satisfactory aesthetic results.14 It was also shown that intralesional

injection of ASCs could suppress hypertrophic scar in a rabbit ear model.15 Another study

demonstrated that ASC transplantation acquired smaller infarct size and less scar formation in a

mouse model of acute myocardial infarction.16 ASCs could also attenuate pulmonary fibrosis

induced by repetitive bleomycin administration.17 Furthermore, in vitro studies confirmed that

ASCs could attenuate collagen production and enhance dermal fibroblast functions.18, 19

TGF‐β1 is a well‐known profibrotic cytokine widely involved in fibroblast activation, proliferation

and ECM production in hypertrophic scar formation.20 HS and their derived fibroblasts express

excessive TGF‐β.21, 22 and its receptors23 when compared with normal skin and normal fibroblasts.

Additionally, TGF‐β1‐related signaling pathway plays an important role in HS pathogenesis

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involving a multitude of cytokines, which regulate a broad array of intracellular responses in HS

including ECM production and deposition, fibroblast proliferation, angiogenesis, and inflammatory

cell infiltration.24, 25 Because of this, manipulation of TGF‐β function or blocking its associated signal

transduction may hold putative therapeutic strategy for HS.26

To date, the inhibitory effects of ASCs on HSF activity and its potential molecular mechanisms are

not well defined. Therefore, in this study, we investigated whether co‐culture of ASCs with HSFs

could potentially inhibit HSF functions and the possible biochemical mechanism that may involve in

the inhibitory effect of ASCs on various cellular behaviors such as cell proliferation, migration,

contractility, and gene/protein expression of scar related molecules.

Materials and Methods

Harvest and culture of human ASCs

Protocols for the handling of human tissue and cells were approved by the Ethics Committee

of Shanghai Jiao Tong University School of Medicine. Human adipose tissues were donated by the

patients for research purposes only with written informed consent. Human adipose tissue was

harvested from abdomen or inner thigh portion of patients by standard liposuction procedures.

The samples were washed extensively with chloramphenicol once and phosphate‐buffered saline

(PBS) twice and then digested by 0.075 % collagenase type I (Sigma‐Aldrich, St. Louis, MO, USA)

dissolved in Dulbecco’s Modified Eagle Medium (DMEM, Hyclone, Logan City, UT) containing 10 %

fetal bovine serum (FBS, Biosum, South America) at 37°C for 1 h with vigorous shaking. Equal

volume of DMEM containing 10 % FBS was added to neutralize the collagenase activity. The cell

suspension was centrifuged at 1200×g for 10 min. The pellet was resuspended in DMEM containing

10 % FBS, penicillin (100 U/ml) and streptomycin (0.1 mg/ml), and then incubated at 37°C in a

humidified atmosphere containing 95 % air and 5% CO2. These primary cells were defined as

passage 0 and were passaged with 0.05 % trypsin‐EDTA (Gibco, Grand Island, NY) once 80‐90

percentage of confluency reached. Cells were cultured to passage three for the use of all

experiments. To reduce individual variation, ASCs were respectively extracted from each of 9

patients’ fat tissues, three cell samples were randomly selected and mixed as one pooled cell

sample and total three pooled cell samples were generated for various experiments.

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Harvest and culture of HSFs

Protocols for the handling of human tissue and cells were approved by the Ethics Committee

of Shanghai Jiao Tong University School of Medicine. Excised human hypertrophic scar samples

were donated by patients who received scar resection with informed consent. For HSF isolation,

the tissue samples were washed extensively with chloramphenicol once and phosphate‐buffered

saline (PBS) twice, cut into 2×2×2 mm3 fragments followed by enzyme digestion with 0.2 %

collagenase type II (SERVA, GER) dissolved in DMEM containing 10% FBS at 37°C for 4 h with

vigorous shaking. After centrifugation, the pellet was resuspended in DMEM containing 10 % FBS,

penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Then, cells were seeded onto 10 cm culture

dishes at a density of 1×104 cells/cm2 and incubated at 37°C in a humidified atmosphere containing

95 % air and 5 % CO2. The cells were passaged once 80‐90 percentage of confluency reached and

then subcultured at the same density for two more passages. Passage three cells were harvested

for the use of all experiments. To reduce individual variation, hHSFs were respectively extracted

from each of 9 patients’ fat tissues, three cell samples were randomly selected and mixed as one

pooled cell samples and total three pooled cell samples were generated for various experiments.

Co‐culture of hASCs and HSFs

A 6‐well transwell culture dish (0.4 μm inserts, Corning, USA) was used to develop the co‐

culture system. Passage three ASCs were harvested with 0.25 % trypsin‐EDTA treatment,

resuspended in DMEM containing 10 % FBS and antibiotics (penicillin/100 U/ml, streptomycin/0.1

mg/ml), and then inoculated on the upper chamber of the transwell culture dishes at a density of

1×104 cells/cm2. Passage three HSFs were similarly harvested and inoculated on the lower chamber

with the same density and culture medium as those of ASCs. Culture media of both chambers were

changed every 2 days. The co‐cultured cells were set as an experimental group. As a control group,

passage three HSFs were inoculated on the lower chamber of 6‐well culture dishes at a density of

1×104 cells/cm2 in DMEM plus 10 % FBS and antibiotics.

To further explore the effect of ASCs on attenuating TGF‐, at day 1 of the experiment, both

experimental and control cells were cultured without or with TGF‐β1 at a concentration of 2 ng/ml

of culture medium.

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Cell proliferation assay

During the culture process as above described, HSFs were harvested with 0.25 % trypsin‐EDTA

treatment at days 1, 3, 5 and 7, centrifuged at 1000×g for 5 min and then resuspended in DMEM.

Ten microliters of cell suspension solution were added to a hematocytometer for cell counting

under an inverted‐phase contrast microscope (Olympus, Japan). The assay was performed in

triplicate and repeated in four pooled cell samples at each desired time point.

Cell cycle analysis

At the 5th day of cell culture, HSFs of both co‐culture group and control group were

respectively harvested with the treatment of 0.25 % trypsin‐EDTA. The cells were then centrifuged

at 1200×g for 5 min, washed with PBS twice and centrifuged again. The pellet was resuspended in a

mixed solution containing 0.3 ml of PBS and 0.7 ml of ice‐cold absolute ethyl alcohol with gentle

agitation for fixation and then the samples were incubated at 4°C overnight. Thereafter, the cells

were centrifuged and stained in 1 ml ice‐cold PBS solution containing 20μl of propidium iodide (PI,

1μg/ml, Sigma, USA), 1μl of Triton X‐100 (0.1 %, Sigma, USA), 0.2mg of RNase (1mg/ml, Sigma,

USA) for 30 min. The cells were then analyzed using a flow cytometer (Beckman Coulter) equipped

with ModiFit LT v2.0 software. The analysis was repeated in three pooled cell samples.

RNA isolation and real‐time quantitative polymerase chain reaction (qPCR)

Total RNA extraction and reverse transcription were performed as previously described.27 Briefly,

total RNA was extracted from the cells of co‐culture group and control group at day 5 and day 10

or from the cells of both groups without or with TGF‐β1 treatment at day 2 and day 5 using a Trizol

Reagent (Invitrogen, Carlsbad, CA). cDNA was reversely transcribed from 2μg of total RNA per

sample with the use of AMV reverse transcriptase (Promega, Madison, WI). A 20μl reaction

solution composed of 2 μg total RNA, 4 μl 5 buffer, 2 μl dNTP, 1μl oligo‐(dT), 0.5 μl AMV reverse

transcriptase, 0.5 μl RNase inhibitor and ddH2O was filled up to total volume of 20 μl for the

reaction. The mixture was incubated at 30°C for 10min, 45°C for 60min, 98°C for 5 min and 5°C for

5 min.

The designed primers for qPCR analysis were listed in Table 1. cDNA was amplified using a

Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) in a real‐time thermal

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cycler (Stratagene) and measurement was conducted in triplicates for each sample. qPCR reaction

conditions were set as: denaturation at 95°C for 30 s, primer annealing at temperatures listed in

Table 1 for 30 s, and elongation at 72°C for 45 s with total 40 cycles. GAPDH gene was used as an

internal control. qPCR assay was performed in triplicate and repeated in three pooled cell

samples.

In vitro scratch wound assay

As previously described,28 an in vitro scratch wound assay was used to evaluate cell

migration. Briefly, HSFs derived from co‐culture group and control group were cultured in 6 well

culture plate along with DMEM containing 10 % FBS until 100 % confluency, then a scratch was

created on each well using a 200 l pipette tip (PipetTipFinder, LLC, Knoxville, TN) to make a

scratch in the middle of the culture dish. The scratched wound was about 0.45‐0.50 mm in width

per well. Afterwards, the cultures were switched to serum‐free medium for 24 h and 48 h. Digital

photograph of each wound was acquired under an inverted‐phase contrast microscope (Olympus,

Japan) immediately, 24 h and 48 h after the scraping. Wound closure (cell migration) was

investigated by measuring the wound area using the commercial software Image pro‐plus version

6.0 (Media Cybernetics, Silver Spring, MD) and public domain image processing program. Results

were presented as the percentage of the initial wound area using the following formula: Cell

migration rate (%) = (Gap24 h (or Gap48 h) – Gap0h)/Gap0h × 100 %. Photographs of each wound

were acquired in three random views and the mean cell migration rate of each sample plus

standard deviation was presented as the final result. The assay was repeated in three pooled cell

samples.

Fibroblast‐populated collagen lattice (FPCL) contractility assay

FPCL contractility assay was performed as previously described.26 Briefly, according to the

manufacturer’s protocol, collagen lattices were polymerized in a 24‐well transwell culture dish. To

form the collagen solution, 200 μl of rat tail tendon collagen solution (5mg/ml, Shenyou

Biotechnology Co., Ltd, Zhejiang, China) were added to 12 μl of NaOH (0.1 mol/L) and mixed

immediately by pipetting. Then, 23 μl 10PBS was added into the mixture solution. Thereafter, 760

μl of cell suspension (containing 3×104 cells) were added to the mixture solution, gently mixed and

added into the lower chamber of the 24‐well transwell culture dish with 500 μl volume per well.

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Collagen lattices were placed at room temperature for 30 min to form the gel, and then 1 ml of

DMEM containing 10 % FBS and antibiotics was added to each well. Afterwards, the cell‐contained

gels were detached from the culture dishes as the floating state for self‐contraction in each well. At

the same time, for co‐culture group, ASCs of passage 3 were inoculated on the upper chamber of

the 24‐well transwell culture dishes at a density of 1×104 cells/cm2 with 1.5 ml of DMEM containing

10 % FBS and antibiotics. For the blank control group, 1.5 ml of DMEM containing 10 % FBS and

antibiotics (without cell) was added to the upper chamber of the 24‐well transwell culture dishes.

Digital images of the floating lattices were acquired immediately, 24 h, 48 h, and 72 h post‐

detaching. To quantify the contracture ability, the surface areas of the gels were measured using

image software (Macropath 5, PRO, Guangzhou, China) at each time point. Contraction rate of

FPCL was normalized to the bottom area of the well. The assay was repeated in three pooled cell

samples.

ELISA analysis for TGF‐β1 protein expression

HSFs from co‐culture group and control group were cultured in DMEM containing 10% FBS

until the 5th day. Cells were then serum starved for 12 h, and then cultured in serum free medium

for 24 h. Thereafter, the medium was collected for ELISA analysis of TGF β1 (Excell bio, China)

according to manufacturer’s protocol. Absorbance was measured at 450nm. ELISA analysis was

performed in triplicate and repeated in three pooled cell samples.

Western blotting analysis

After 5 days of co‐culture with ASCs as indicated, total protein was extracted from HSFs of

both groups with RIPA lysis buffer as described previously.29 Protein extracts were denatured by

heat at 100°C for 5 min and electrophoretically separated on a 12 % SDS‐PAGE (Bio‐Rad, USA).

Proteins were transferred to PVDF membrane, blocked with 5 % milk/Tris buffered saline,

incubated with primary antibodies as below described, followed by incubation with appropriate

HRP‐conjugated secondary antibodies (Jackson ImmunoResearch). The protein bands were

eventually visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham, USA).

The primary antibodies of rabbit against human Phospho‐Erk1/2 (Thr202/Tyr204), rabbit against

human Erk1/2, mouse against human Phospho‐Stat3‐Y705, rabbit against human Stat, rabbit

against human Phospho‐Smad2 (Ser465/467), rabbit against human Phospho‐Smad3 (Ser423/425), rabbit

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against human Smad2/3 (BD Biosciences) and rabbit against human β‐actin (Sigma‐Aldrich) were

purchased from Cell Signaling Technology. The experiment was repeated in three pooled cell

samples.

Statistical analysis

All data are presented as means ± standard derivation. The differences between co‐culture

and control groups were analyzed with Student‐t test. P value less than 0.05 was considered

statistically significant. SPSS software, version 19.0 (SPSS Inc., Chicago, IL) was applied in this

statistical analysis.

Results

ASCs inhibited proliferation and blocked cell cycle of co‐cultured HSFs

With co‐culture of ASCs, the cell proliferation rate of HSFs became apparently slower when

compare with that of blank control HSFs. As shown in Figure 1A, the cell density was relatively

lower in experimental group than in control group at days 3, 5 and 7.

To further quantitatively analyze, cells were planted into 24‐well plates at a density of

5000 cells/well. As shown in Figure 1B, a logarithmic phase appeared from day 1 to day 7. At day 3,

the mount of HSFs in the control group (3.5 ± 0.21104) was significantly more than that in the co‐

culture group (2.7 ± 0.12104) (p < 0.05). At day 5, the mount of HSFs in the blank control and co‐

culture groups was 6.5 ± 0.24104 and 4.9 ± 0.19104 respectively with significant difference

between two groups (p<0.01). At day 7, total cell number reached 8.2 ± 0.22104 and 5.9 ±

0.21104 respectively in control and co‐culture groups with significant difference between them

(p<0.01).

Cell cycle analysis demonstrated that co‐culture group presented less percentage of HSFs in

G2/M phases at day 2 (3.16 ± 0.56 %) and day 5 (4.13 ± 0.49 %) compared to day 2 (5.93 ± 0.69 %)

and day 5 (7.38 ± 0.54 %) of control group with significant difference (p<0.05). In addition, co‐

culture group also presented less percentage of HSFs in S phase at day 5 (2.91 ± 0.21 %) than that

of control group (5.29 ± 0.68 %) with significant difference (p<0.05). Additionally, cell percentage

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(92.8 ± 0.69 %) of co‐culture group in the G0/G1 phases was also significantly higher than that of

control group (87.5 ± 1.2 %) at day 5 with significant difference (p<0.05).

HASCs inhibited cell migration of co‐cultured HSFs

As shown in Figure 2, at 24 h, 61.7 ± 1.7 % of the scratched area was filled by migrated HSFs

of control group. By contrast, 36.9 ± 3.1 % of the scratched area was filled by the migrated HSFs of

co‐culture group, which was significantly different from that of control group (p<0.01, Fig. 2B).

After 48 h of culture, 84.5 ± 1.1 % of the area was filled by the migrated HSFs of control group,

whereas 43.2 ± 3.2 % of the area was filled by the migrated HSFs of co‐culture group with

significant difference between two groups (p<0.01, Fig. 2B), suggesting that co‐cultured with ASCs

could significantly inhibit the migration of HSFs.

ASCs attenuated the contraction of fibroblast‐populated collagen lattice

A FPCL model was used to evaluate the inhibitory effect of ASCs on HSF contractility. As

shown in Figure 3, at 24 h post‐release of the gel, cell containing floating collagen lattices self‐

contracted, which led to area reduction to 72.3 ± 2.9 % of original area in control group. By

contrast, co‐culture with ASCs led to less contraction of floating lattice with 95.7 ± 0.9 % of original

area, which is significantly different from that of control group (p<0.01, Fig. 3B). At 48 h, the

collagen gels contracted more and the average areas were 61.4 ± 3.5 % and 93.2 ± 0.7 % of original

area respectively for control and co‐culture groups with significant difference between them

(p<0.01, Fig. 3B). At 72 h, the average areas of control and co‐culture groups respectively reached

45.6 ± 2.5 % and 79.3 ± 1.3 % of original area with significant difference (p<0.01, Fig. 3B).

ASCs inhibited ECM gene expression in co‐cultured HSFs

After 5 days of co‐culture with ASCs, the gene expression levels of ECM molecules were

significantly inhibited for Col 1 (0.8 ± 0.07‐fold of control, p<0.05, Fig. 4A), Col 3 (0.62 ± 0.13‐fold of

control, p<0.05, Fig. 4B), and FN (0.45 ± 0.09‐fold of control, p<0.01, Fig. 4C). No significant

difference in DCN gene expression was found (p>0.05, Fig. 4F). Besides, the significant inhibitory

effect was also found in FN at day 10 (0.73 ± 0.09‐fold of control, p<0.05, Fig. 4C). Additionally,

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increased MMP‐1/TIMP‐1 ratio of their gene expression levels (1.18 ± 0.06‐folds of control, p<0.05,

Fig. 4D) was also observed at day 5. ‐SMA, a marker for myofibroblasts, was also significantly

inhibited for its gene expression in the co‐culture group (0.49 ± 0.13‐fold of control, p<0.05, Fig.

4E) at day 5.

ASCs inhibited gene expression of cytokines/growth factors in co‐cultured HSFs

After 5 days of co‐culture with ASCs, the expression levels of pro‐fibrotic genes of HSFs were

significantly reduced compared to those of control HSFs, including TGF‐β1 (0.51 ± 0.15‐fold of

control, p<0.05, Fig. 4G), IL‐6 (0.23 ± 0.08‐fold of control, p<0.01, Fig. 4H), IL‐8 (0.58 ± 0.1‐fold of

control, p<0.01, Fig. 4I) and CTGF (0.87 ± 0.04‐fold of control, p<0.05, Fig. 5D). Similar inhibitory

effect was also observed after 10 days of co‐culture with ASCs, including TGF‐β1 (0.81 ± 0.05‐fold

of control, p<0.01, Fig. 4G), IL‐6 (0.59 ± 0.06‐fold of control, p<0.01, Fig. 4H) and IL‐8 (0.76 ± 0.05‐

fold of control, p<0.01, Fig. 4I).

ASCs attenuated TGF‐ induced fibrotic response of HSFs

To further evaluate the possible anti‐TGF‐β effect mediated by co‐cultured ASCs, HSFs were

cultured with or without exogenous TGF‐β1. As shown in Figure 5, after 5 days of culture,

exogenous TGF‐β1 (2ng/ml) could significantly induce the gene expression levels of Col1 (Fig. 5A,

p<0.05), FN (Fig. 5C, p<0.05), CTGF (Fig. 5D, p<0.05), ‐SMA (Fig. 5E, p<0.05), TGF‐β1 (Fig. 5G,

p<0.05) and IL‐6 (Fig. 5H, p<0.05). Importantly, co‐cultured ASCs could inhibit the expression of all

these TGF‐β1 induced genes with significant difference (p<0.05). In addition, co‐cultured ASCs also

inhibited the expression of other TGF‐β1 induced genes including Col3 (Fig. 5B, p<0.05) and IL‐8

with significance (Fig. 5I, p<0.05). However, this inhibitory effect was not significant for most of the

examined genes (p>0.05) at time point day 2, except for TGF‐β1 (Fig. 5G, p<0.05) and FN (Fig. 5C,

p<0.05).

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ASCs blocked intracellular signaling cascades in HSFs

As shown in Figure 6A, co‐culture with ASCs could significantly decrease TGF‐β1 protein

release from the HSFs (2.49 ± 0.43 pg/ml) when compared to that of control HSFs (6.02 ± 1.46

pg/ml, p<0.05).

To further investigate the potential mechanism, western blot was employed to examine the

protein levels of related signaling molecules. As shown in Figure 6B, co‐culture with ASCs could

significantly reduce protein levels of phosphorylated Smad2 and Smad3, whereas the level of total

Smad2/3 protein was not significantly reduced (Fig. 6B). In addition, reduced protein production of

phosphorylated Stat3 (Fig.6C) and phosphorylated Erk1/2 (Fig. 6D) were also observed without

significant change of total protein levels of Stat3 and Erk1/2. These data indicate that ASCs may

exert its anti‐fibrotic effect via blocking related signaling process and reducing TGF‐β autocrine

production.

Discussion

Hypertrophic scar is the result of abnormal growth of fibrous tissue characterized by over

proliferation of fibroblasts, excessive ECM deposition and severe contracture.1‐3 Over deposition of

extracellular matrices in HS not only results from the overproduction, but also from reduced matrix

degradation via aberrant expression of MMPs and their inhibitors (TIMPs).30

Although not fully defined, the mechanism of HS may involve over production of pro‐fibrotic

factors, which leads to fibroblast proliferation and ECM production. These potential factors include

TGF‐β1 and its down‐stream molecule like CTGF, IL‐6 and IL‐8.28

The clinical treatment of HS remains a challenge due to the conventional therapies such as

pressure 8, silicone 9 or steroid injection 5 are still relatively less effective for control scar prevention

and contracture. Emerging therapy such as stem cell based treatment may provide helpful solution

in addition to current therapies.

MSC based therapy is an emerging strategy for anti‐scarring and anti‐fibrosis treatment via

secreting a number of trophic functions.10, 31 In this area, ASCs are a particularly preferable MSC

source because they are more widely available and more easily obtained by less traumatic

methods such as liposuction, and meanwhile they have fewer ethical issues and lower

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immunogenicity compared with other stem cell types.12, 13 Recent studies have demonstrated that

ASCs were potent in promoting wound healing and limiting scar formation,17, 18, 32 which make

them an attractive therapeutic cell source for treating HS.

Despite increasing emerging evidence of ASC mediated repressing effect on HS or tissue

fibrosis in both animal study and clinical therapy,15‐17, 33, 34 little effort has been made to understand

the mechanism, particularly in molecule aspects of its efficacy on HSFs. Therefore, we performed

this experiment by co‐culturing ASCs with HSFs to observe the effect of ASC on the proliferative

and profibrotic phenotypes associated with HSFs.

The results of this study showed ASCs possessed specific anti‐HS therapeutic effect by

inhibiting HSF proliferation and partially blocking cell cycle (Fig.1), inhibiting matrix gene

expression (Fig.4 and Fig.5) and substantially inhibiting collagen lattice contraction (Fig.3) in an in

vitro experimental setting. It is also likely to enhance ECM degradation as they could modulate the

MMP1/TIMP‐1 ratio (Fig. 4D). This ASC‐mediated specific inhibition on HSF pathological phenotype

will be crucial for the treatment of HS.9, 24, 35

In addition to inhibiting cell proliferation and matrix production, ASCs also significantly

inhibited the expression of pro‐fibrotic factors and inflammatory cytokines such as TGF‐β1, IL‐6 and

CTGF as shown in Figure 4 and Figure 5. Additionally, α‐SMA, a biomarker of HS was also

significantly down regulated by co‐cultured ASCs (Fig.4E), suggesting that ASCs may inhibit

myofibroblast transformation mediated by TGF‐β1 as indicated by previous studies.20, 36 One of the

HS clinical characters is tissue contracture majorly mediated by TGF‐β induced myofibroblast

transformation. As shown in Figure 3, using a FPCL contraction assay,37 co‐culturing of HSFs with

ASCs could significantly inhibit the contractility of HSFs and the effect became more obvious with

prolonged culture time period. Previous study showed that myofibroblast transformation was the

major cause of HS tissue contraction,38 which was enhanced by TGF‐β1 with characterized

enhanced expression of α‐SMA.39 Truly, implantation of ASCs or fat tissue was shown to decrease

scar contracture,14 our cellular experimental results (Fig. 4 and Fig.5) further support this reported

clinical phenomenon.

As multiple intracellular signaling are involved in HS development including Smad, Stat3, ERK3,

21, 40‐43 and TGF‐β/Smad is the most important one for HS.40, 41 The reduced phosphorylation of

Smad2 and Smad3 (Fig. 6) may partially explain ASC mediated reduction of TGF‐ autocrine

production, which in return reduce matrix expression, cell proliferation and collagen contraction.

Similar effect was also observed in ASC mediated TGF‐/Smad signaling in vocal fold fibroblasts.44

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In addition to Smad signaling, the mitogen‐activated protein kinase (MAPK) pathway was also

found to be widely involved in HS pathogenesis.3, 45 Extracellular signal regulated kinases (ERK) was

one of the MAPK members, which can be activated by TGF‐β1 and participates in cell proliferation,

differentiation and apoptosis.43, 46 This study showed that co‐culture with ASCs could also

significantly reduce the phosphorylation of ERK in cultured HSFs (Fig. 6).

Stat3 is a transcription factor activated by tyrosine phosphorylation in human HS tissue

sections3 and Stat3 phosphorylation led to the activation of downstream targeting genes that

control ECM production and cellular proliferation in HS lesions.3, 47 Evidenced in Figure 6, reduced

phosphorylation of Stat3 was also observed in HSFs co‐cultured with ASCs. Additionally, recent

studies also showed that Stat3 could be activated by IL‐6 trans‐signaling pathways in HS, which

mediated ECM production and dermal fibroblast proliferation.42 Therefore, reduced expression of

IL‐6 by co‐cultured ASCs may also contribute to the reduced Stat3 signaling in HSFs.

In this study, a transwell co‐culture system is used, indicating that paracrine factors play an

essential role. Whether cell‐cell contact may have more important effect remains to be

investigated. Although not tested, the possible candidate factors for anti‐inflammatory and anti‐

fibrotic effect observed in this study may include hepatocyte growth factor (HGF), IL‐10, and

Prostaglandin E2 as reported in the literature.30, 48‐53

HGF is regarded as an anti‐fibrotic facilitator contributing to ASCs’ anti‐fibrosis function because

it was found to be able to up‐regulate the expressions of MMP‐1, ‐3, and ‐9 in injured sites in a

PI3K/Akt/p70‐ dependent manner to promote apoptosis of myofibroblasts.54, 55 Besides, the

presence of HGF antibody could block the inhibitory effect of ASCs on myofibroblasts.48

IL‐10 is also likely the candidate as it was found to predominantly inhibit the synthesis of pro‐

inflammatory cytokines, such as IL‐6 and IL‐8.56, 57 It was also suggested that expressions of pro‐

and anti‐inflammatory cytokines are closely related to the development of HS. Therefore, ASC

mediated reduction of pro‐/anti‐inflammatory cytokine ratio may also contribute its anti‐

inflammatory and anti‐fibrotic effects. ASCs may also exert anti‐inflammatory effect via secreting

Prostaglandin E2,30, 53, 58 which plays a key role in the immunosuppressive properties of ASCs.

Accumulating evidence has suggested that PGE2 inhibits TGF‐β1‐induced activation and fibroblast

proliferation, thereby reducing the production of ‐SMA and collagens by elevating intracellular

cAMP levels and promoting apoptosis in myofibroblasts by increasing the activity of the PTEN

protein, which blocks the PI3K/Aktsignalling pathway.59 In addition to these potential mechanisms,

a recent study also demonstrated that exosomes derived from human ASCs could accelerates

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cutaneous wound healing via optimizing the characteristics of fibroblasts,60 suggesting that micro

RNA, a common part of exosome contained molecules, may also involve in this particular

regulation.

Conclusion

This study demonstrated that paracrine factors released from ASCs could significantly inhibit

pathogenic phenotype of HSFs, including the reduction of cell proliferation and cell migration,

down‐regulated gene expression of matrix molecules, growth factors and inflammatory cytokines

as well as reduced cell contractility. These observed phenomena are likely mediated by attenuated

multiple intracellular signaling. Further definition of the key released paracrine factors via

proteomic or HPLC analysis and other techniques may hold potential therapeutic hope of ASCs in

HS treatment.

Acknowledgements

This work was supported by National Natural Science Foundation (31470943) and Innovation

Program of Shanghai Municipal Education Commission (13ZZ090).

Disclosures

No conflicts of interest, financial or otherwise, are declared by the authors.

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of 32

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Address correspondence to:

Wei Liu

Department of Plastic and Reconstructive Surgery

Shanghai 9th People’s Hospital

Shanghai Jiao Tong University School of Medicine

639 Zhi Zao Ju Road

Shanghai 200011

PR China

E‐mail: [email protected]

Weigang Cao

Department of Plastic and Reconstructive Surgery

Shanghai 9th People’s Hospital

Shanghai Jiao Tong University School of Medicine

639 Zhi Zao Ju Road

Shanghai 200011

PR China

E‐mail: [email protected]

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Table 1. Primers used in qPCR analysis.

Gene Name Primer Sequence Annealing

Temperature

( )

PCR Product

Size

(bp)

GAPDH F: GACTTCAACAGCAACTCCCAC

R: TCCACCACCCTGTTGCTGTA

60

125

Collagen I F: GGCGGCCAGGGCTCCGACCC

R: AATTCCTGGTCTGGGGCACC

60 320

Collagen III F: CAACCAGTGCAAGTGACCAA

R: GCACCATTGAGACATTTTGAAG

60 174

TGF‐β1 F: GAAGTGGATCCACGAGCCCAAG

R: GCTGCACTTGCAGGAGCGCAC

60 227

Decorin F: CTACCTTCACAACAACAACATCTC

R: GCAGAACGCACATAGACACATC

60 165

IL‐6 F: CCTGACCCAACCACAAATGC

R: ATCTGAGGTGCCCATGCTAC

58 157

IL‐8 F: TCCTGATTTCTGCAGCTCTGTGTG

R: AATTTCTGTGTTGGCGCAGTGTGG

58 161

TIMP1 F: CTTCTGCAATTCCGACCTCGT

R: ACGCTGGTATAAGGTGGTCTG

60 79

MMP1 F: TCTGGGGAAAACCTTTCGACT

R: CACCAACGTATTCAAAAGCACAA

60 80

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α‐SMA F: CCCAGACATCAGGGAGTAATGG

R: TCTATCGGATACTTCAGCGTCA

58 104

CTGF F: CAGCATGGACGTTCGTCTG

R: AACCACGGTTTGGTCCTTGG

60 115

Fibronectin F: GCCACTGGAGTCTTTACCACA

R: CCTCGGTGTTGTAAGGTGGA

58 61

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Figure legends:

FIG. 1. Co‐culture of HSFs with ASCs inhibited HSF proliferation. (A) Cell density observation under

phase contrast microscope, which shows relatively lower cell density in co‐culture group than in

blank control group at days 3, 5 and 7 (40, bar = 100 m). (B) Cell growth curve of both co‐culture

group and control group. (C) Cell cycle analysis with flow cytometry. *p<0.05 between control and

co‐culture groups and **p<0.01 between control and co‐culture groups.

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FIG. 2. Co‐culture of HSFs with ASCs inhibited HSF migration. (A) Photography of in vitro wound

and cell migration immediately after a scratch and 24 h and 48 h post‐scratch. Original

magnifications: ×40. (B) Quantitative analysis of cell migration rate at 24 h and 48 h post‐scratch.

**p<0.01 between control and co‐culture groups.

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FIG. 3. Co‐cultured of HSFs with ASCs attenuated the contraction of HSF‐populated collagen lattice

(FPCL). (A) Gross view of FPCL contraction at 2 h, 24 h, 48 h, and 72 h. (B) Semi‐quantification of

contraction rate of the FPCL of both groups. **p<0.01 between control and co‐culture groups.

Tis

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Eng

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of 32

30

Tis

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Eng

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In

hibi

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of

path

olog

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phe

noty

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f hy

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FIG. 4. Co‐cultured of HSFs with ASCs inhibited the gene expression of extracellular matrix and pro‐

fibrotic factors. Quantitative PCR analysis of various gene expressions in HSFs without or with co‐

culture of ASCs at day 5 and day 10 for collagen I (A, Col 1), collagen III (B, Col 3), fibronectin (C,

FN), MMP1/TIMP1 (D), alpha‐smooth muscle actin (E, ‐SMA), decorin (F, DCN), transforming

growth factor‐1 (G, TGF‐1), interleukin‐6 (H, IL‐6), interleukin‐8 (I, IL‐8). *p<0.05 between

control and co‐culture groups, **p<0.01 between control and co‐culture groups.

Tis

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31

Tis

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In

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phe

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ls (

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.

FIG. 5. Co‐culture of HSFs with ASCs counteracted TGF‐β1‐mediate pro‐fibrotic effect. Quantitative

PCR analysis of various gene expressions in HSFs without or with TGF‐ stimulation of both control

and co‐culture groups at day 2 and day 5 for collagen I (A, Col 1), collagen III (B, Col 3), fibronectin

(C, FN), connective growth factor (D, CTGF), alpha‐smooth muscle actin (E, ‐SMA), decorin (F,

DCN), transforming growth factor‐ (G, TGF‐1), interleukin‐6 (H, IL‐6), interleukin‐8 (I, IL‐8).

*p<0.05 between indicated two groups, **p<0.01 between indicated two groups.

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Par

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32

Tis

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In

hibi

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of

path

olog

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phe

noty

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via

co-c

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re w

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dipo

se d

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tem

cel

ls (

DO

I: 1

0.10

89/t

en.T

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6.05

50)

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per

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FIG. 6. Co‐culture of HSFs with ASCs antagonized intracellular signaling in vitro. Elisa analysis shows

that co‐culture of ASCs reduces TGF‐1 protein release (A). Western blot analysis shows that co‐

culture with ASCs reduces the phosphorylation level of smad2 and smad3 (B), phosphorylation

level of Stat3 (C) and phosphorylation of Erk1/2 (D). *p<0.05 between control and co‐culture

groups.

Tis

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Eng

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Par

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of p

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-cul

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pose

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i: 10

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Thi

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yedi

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f co

rrec

tion.

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fin

al p

ublis

hed

vers

ion

may

dif

fer

from

this

pro

of.

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Inhibition of Pathological Phenotype of Hypertrophic Scar...

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