Adipose-derived mesenchymal stem cells transplantation facilitate experimental

peritoneal fibrosis repair by suppressing epithelial–mesenchymal transition

Keiichi Wakabayashi, Chieko Hamada, Reo Kanda, Takanori Nakano, Hiroaki Io,

Satoshi Horikoshi, and Yasuhiko Tomino

Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty

of Medicine

Name and address for correspondence: Yasuhiko Tomino, PhD, MD

2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

Phone: +81-3-5802-1065, Fax: +81-3-3813-1183, E-mail: yasu@juntendo.ac.jp

Short title: Effects of ASCs transplantation on peritoneal fibrosis

1

Abstract 1

Background. Prevention or reversal of peritoneal damage is critical in peritoneal dialysis. 2

Although autologous cell transplantation has beneficial effects on tissue repair in various 3

organs, few studies have investigated the effects of adipose-derived mesenchymal stem 4

cells (ASCs) transplantation on peritoneal fibrosis (PF). Thus, we examined the mechanism 5

of facilitated peritoneal reconstruction induced by ASCs transplantation on chlorhexidine 6

gluconate (CG)-induced PF in rats. 7

Methods. To induce PF in rats, continuous-infusion pumps containing 8% CG were placed 8

in the abdominal cavity for 21 days. The pumps were removed on day 22 and ASCs were 9

immediately injected into the peritoneal cavity. Morphological alterations and mRNA 10

expression levels of fibrosis-related factors were examined on days 29 and 35. 11

Results. ASCs transplantation significantly facilitated peritoneal repair. mRNA expression 12

of tumor necrosis factor-α, interleukin-1β, monocyte chemotactic protein-1, and 13

epithelial–mesenchymal transition (EMT) markers such as Snail and α-smooth muscle actin 14

were suppressed, whereas that of vascular endothelial growth factor (VEGF) and 15

platelet-derived growth factor-BB (PDGF-BB) overexpressed after ASCs transplantation. 16

Immunofluorescence indicated that some transplanted ASCs expressed VEGF and 17

PDGF-BB and differentiated into vascular cells. 18

Conclusions. ASCs transplantation facilitate peritoneal repair by suppressing EMT and 19

2

modulating inflammation and angiogenesis during the early phase of tissue repair in 1

experimental PF. 2

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Keywords: peritoneal fibrosis, adipose-derived mesenchymal stem cells, 5

epithelial–mesenchymal transition, angiogenesis 6

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Introduction 1

Peritoneal fibrosis (PF) remains a life-threatening complication in peritoneal dialysis [1]. 2

Bioincompatible dialysates and infectious peritonitis are known to upregulate the 3

production of fibrosis-related factors in the peritoneum, resulting in mesothelial cell loss, 4

submesothelial compact (SMC) zone thickening, and abnormal angiogenesis [2]. 5

Excessive production of vascular endothelial growth factor (VEGF) and transforming 6

growth factor-β (TGF-β) induce both abnormal angiogenesis and an 7

epithelial–mesenchymal transition (EMT), resulting in the development of PF [2, 3]. 8

Several studies have shown that TGF-β as well as tumor necrosis factor-α (TNF-α) and 9

hypoxia inducible factor-1α (HIF-1α) are capable of inducing EMT through Snail 10

activation [4]. 11

There are considerable ongoing efforts to examine the therapeutic potential of 12

mesenchymal stem cells (MSCs) in various organs [5, 6]. Since MSCs possess many 13

characteristics, including a multilineage potential [7], immunomodulatory properties [8], 14

and self-renewal capability in vitro [9], they have been found to decrease fibrosis in the 15

heart [10], lungs [11] and kidneys [12]. Moreover, injected bone marrow-derived MSCs 16

(BMSCs) have been shown to differentiate into peritoneal mesothelial cells [13] and repair 17

PF morphologically and functionally in animal models through anti-inflammatory and 18

anti-EMT effects [14-16]. 19

4

The immunomodulatory properties of adipose-derived mesenchymal stem cells (ASCs) 1

may be more potent than those of BMSCs [17]. In addition, the use of ASCs is more 2

advantageous than that of BMSCs because harvesting ASCs is minimally invasive and in 3

vitro culturing yields an unlimited supply [18]. A previous report demonstrated that ASCs 4

treatment decreased kidney damage caused by ischemia-reperfusion injury through 5

suppressing oxidative stress and inflammation [19]. Takahashi et al. [20] also demonstrated 6

that ASCs appeared to have the capacity to differentiate into endothelial cells (ECs) as well 7

as secrete paracrine factors that stimulated ECs repair in a rat femoral artery wire injury 8

model. 9

To determine the effects of intraperitoneal ASCs transplantation, we injected ASCs into 10

rats with chlorhexidine gluconate (CG)-induced PF. 11

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Subjects and Methods 1

Animals 2

35 male Sprague–Dawley rats (body weight, 200–250 g; Sankyo Labo Service Corporation 3

Inc, Tokyo, Japan) were housed in standard rodent cages under conventional laboratory 4

conditions of 22 °C and a 12-h light/dark cycle with free access to laboratory chow and tap 5

water. Our experimental protocol was approved by the Ethics Review Committee of 6

Animal Experimentation at Juntendo University Faculty of Medicine, Tokyo, Japan. 7

8

Animal models of peritoneal fibrosis 9

Rats were continuously infused with 8% CG using infusion pumps as previously described 10

[21, 22]. In brief, infusion pumps were placed in the abdominal cavity for 21 days and the 11

anterior abdominal peritoneum was harvested on days 0. 22, 29 and 35 (CG group). 12

13

ASCs transplantation 14

After removing the infusion pumps, ASCs harvested from subcutaneous adipose tissue 15

of green fluorescent protein (GFP)-transgenic rats and cultured as previously described [23] 16

were injected (3 × 107 cells/rat) into the peritoneal cavity on day 22 and sections were 17

harvested on days 29 and 35 (Tx group). Peritoneal samples were immediately fixed in 18

20% formalin solution, embedded in paraffin, and stained with Masson’s trichrome. 19

6

1

Histological analysis 2

As previously described [24], the maximum SMC thickness (μm) was measured in each 3

section. Five sections were randomly selected using a KS400 Imaging System (Kortron 4

Elektronik GmbH, Eching, Germany) [25]. The average peritoneal thickness was 5

determined for each specimen. 6

7

Immunohistochemical analysis 8

Immunohistochemical analysis was performed as previously described [21]. The mouse 9

anti-rat alpha-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, UK) was used 10

as primary antibody. The negative control was confirmed by incubation without primary or 11

secondary antibodies and exhibited no positive cells. The number of α-SMA positive cells 12

in the peritoneum were counted in 10 random regions in each tissue. The average number 13

was then calculated (the number of positive cells/SMC mm2) using the KS400 Imaging 14

System. 15

16

Double-immunofluorescence 17

Immunofluorescence were performed as previously described [21]. Sections were 18

incubated through overnight with following antibodies: FITC-conjugated goat anti-GFP 19

7

antibody (Abcam, Tokyo, Japan), mouse anti-VEGF antibody (Abcam), mouse anti-CD31 1

antibody (Abcam), rabbit anti-platelet-derived growth factor-BB (PDGF-BB) antibody 2

(Abcam), rabbit anti-PDGF-Rβ antibody (Abcam), and rabbit anti-α-SMA antibody 3

(Abcam). After incubation, sections were mounted in diluted Alexa 555 (Molecular Probes, 4

Inc., Eugene, OR, USA). Negative controls omitted primary antibodies. In all fluorescent 5

images, cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). 6

7

Real-time polymerase chain reaction (PCR) 8

RNA extraction and real-time PCR analysis were performed as previously described [21]. 9

For quantification, samples were normalized against glyceraldehyde-3-phosphate 10

dehydrogenase (GAPDH) mRNA. PCR primers are shown in Table 1. 11

12

Statistical analysis 13

Results are expressed as means ± standard deviations (SDs). Group results were compared 14

using one-way analysis of variance. Statistical analysis was performed using Graph Pad 15

Prism version 5.0 software (Graph Pad Software, Inc., San Diego, CA, USA). (p) < 0.05 16

was considered statistically significant. 17

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Results 1

Effects of ASCs transplantation on PF and collagen mRNA expression 2

Serial morphological changes are shown in Figure 1A. SMC thickness on day 35 in the Tx 3

group significantly decreased (Figure 1B). Type 3 collagen mRNA levels on day 29 in the 4

Tx group were lower than those in the CG group (Figure 1C), however those on day 35 5

were not significantly different between groups (data not shown). ASCs were widely 6

distributed in the peritoneum on day 25, and decreased on days 27 and 29 and disappeared 7

on day 35 (Figure 1D). 8

9

Effects of ASCs transplantation on EMT and inflammation 10

Since ASCs were almost disappeared on day 35 as above, we compared the manners of 11

ASCs on peritoneal repair between days 22 and 29. Snail mRNA levels on day 29 in the Tx 12

group were significantly lower than those in the CG group, however that of TGF-β were 13

comparable to those in the CG group (Figure 2A). α-SMA positive cells were widely 14

distributed in the peritoneum on day 22, and the distribution of α-SMA positive cells in 15

both CG and Tx groups are shown in Figure 2B. ASCs suppressed α-SMA mRNA 16

expression and the number of α-SMA-positive cells in the Tx group on day 29 (Figure 2C). 17

The FITC-positive transplanted cells (in green), which migrated into the peritoneal 18

interstitium, did not express α-SMA (in red) (Figure 2D). IL-1β, TNF-α, and MCP-1 19

9

mRNA levels on day 29 in the Tx group were significantly lower than those in the CG 1

group (Figure 2E). 2

3

Effects of ASCs transplantation on angiogenesis 4

VEGF and PDGF-BB mRNA levels on day 29 in the Tx group were significantly higher 5

than those in the CG group (Figure 3A and B), however that of HIF-1α was significantly 6

decreased (Figure 3C). Immunofluorescence showed that part of ASCs expressed VEGF on 7

day 29 (Figure 3E). In addition, a part of the transplanted ASCs that formed lumens also 8

expressed PDGF-BB and PDGF-Rβ (Figure 3F and G). Transplanted ASCs on day 29 also 9

expressed CD31, which is a marker of ECs (Figure 3H). The average vascular density on 10

days 29 in the Tx group tended to decrease compared to that in the CG group, but the 11

differences were not statistically significant (Figure 3D). 12

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Discussion 1

To the best of our knowledge, this is the first study to show that intraperitoneal ASCs 2

transplantation facilitated peritoneal repair in rats with CG-induced PF. 3

SMC thickness on day 35 was significantly reduced after ASCs injection. Transplanted 4

ASCs appeared in the peritoneum until day 29. Since the transplanted ASCs may conduct 5

to facilitate tissue repair transiently, we focus the manners at early stage of transplantation. 6

ASCs modified mRNA expression of fibrosis-related factors on day 29. And ASCs 7

suppressed Snail and α-SMA mRNA levels, but transplanted ASCs did not indicate α-SMA. 8

Since TGF-β levels in the Tx group were comparable to those in the CG group, EMT may 9

be regulated by TNF-α and HIF-1α in our study. When we examined the effects of ASCs on 10

inflammation and angiogenesis, ASCs remarkably suppressed TNF-α levels. Moreover, 11

VEGF and PDGF-BB levels significantly increased, and some ASCs secreted VEGF and 12

PDGF-BB and differentiated into vascular cells, resulting in decreased HIF-1α levels and 13

vascular density. 14

Recent studies suggested that one of the functions of MSCs was to act as guardians against 15

excessive inflammatory responses. Prockop et al. [8] demonstrated that activated MSCs 16

induced by pro-inflammatory factors modulated a negative feedback loop of a generic 17

pathway of TNF-α–regulated inflammation. In our study, ASCs significantly suppressed 18

IL-1β, TNF-α, and MCP-1 mRNA levels during the early repair phase. 19

11

Ueno et al. [14] reported that BMSCs ameliorated PF by suppressing inflammation and 1

inhibiting TGF-β1 signaling. In our study, mRNA levels of Snail, which is a transcriptional 2

repressor of E-cadherin and an inducer of EMT, were significantly inhibited independent of 3

TGF-β levels. Regulation of several EMT-related factors was altered by activation of the 4

TGF-β pathway, such as glycogen synthase kinase-3β, mitogen-activated protein kinase, 5

and Smad-related proteins, which were also examined in the present study, but we found no 6

significant difference in expression levels between the CG and Tx groups (data not shown). 7

Previous reports have demonstrated that several signals emerging from the extracellular 8

microenvironment are necessary for EMT induction [4, 26]. TGF-β as well as TNF-α and 9

hypoxia converge on nuclear factor kappa-B activation, resulting in Snail upregulation. 10

According to our results, Snail mRNA suppression was probably because of the inhibition 11

of HIF-1α and TNF-α expression. Actually, EMT suppression in the present study was 12

demonstrated by confirming the reduction not only in Snail expression but also in the 13

α-SMA expressions. Also, our results suggested that the transplanted ASCs, which 14

migrated into peritoneal interstitium, did not transform into fibroblasts. 15

It has been established that angiogenesis is induced by tissue injury and hypoxia. Pinelopi 16

et al. [27] demonstrated that pharmacological inhibition of HIFs before acute kidney injury 17

induced by occlusion of renal arteries ameliorated renal fibrosis. In our study, VEGF and 18

PDGF-BB levels were significantly higher, whereas HIF-1α levels were lower in the Tx 19

12

group. PDGF-BB/PDGF-Rβ signaling plays an important role in vascular maturation and 1

has been well documented by genetic studies [28, 29]. We observed a belt-like distribution 2

of PDGF-BB on day 29 in the Tx group. Because PDGF stimulates its own expression by 3

resident cells through a paracrine positive feedback effect and released PDGF binds to 4

surrounding extracellular matrix, PDGF expression appeared band-like. Thus, amplification 5

of both VEGF and PDGF signaling at the early repair stage improved tissue ischemia 6

through a substantial microvessel network. 7

Moreover, a previous report suggested that ASCs had the potential to differentiate into ECs 8

and pericytes [30]. Gehmert et al. [3] reported that PDGF-BB exerted potent biological 9

effects on mural cells and that ASCs expressed PDGF-Rβ as a receptor for PDGF-BB. 10

Immunofluorescence showed that some PDGF-BB– and PDGF-Rβ–positive ASCs formed 11

lumens. Therefore, the facilitative effect of ASCs transplantation was not only the 12

stimulation of resident cells to produce angiogenesis-related factors, but also the induction 13

of ASCs themselves to differentiate into vascular cells that, in turn, secrete PDGF-BB and 14

express PDGF-Rβ. 15

The present study has two main limitations which should be addressed. First, we could not 16

determine peritoneal function. Second, although several factors contributing to activation 17

of the TGF-β pathway were measured, we found no significant differences on day 29. 18

Therefore, serial examinations to assess the effects of ASCs in earlier periods are needed. 19

13

In conclusion, we propose that ASCs transplantation may be an effective method for 1

treating PF by contributing to anti-EMT and anti-inflammation effects, and improving 2

hypoxia at an early phase of repair. 3

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5

Acknowledgments 6

This study was supported by Grants-in-Aid for science from the Ministry of Education, 7

Culture, Sport, Science, and Technology of Japan. We thank Terumi Shibata for her 8

excellent technical assistance. We also thank Takako Ikegami and Tomomi Ikeda (Division 9

of Molecular and Biochemical Research, Juntendo University Graduate School of 10

Medicine) for their excellent technical assistance. 11

12

Disclosures 13

The authors declare that they have no conflict of interest 14

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Legends for figures 1

Figure 1: Effects of ASCs transplantation on morphological change and collagen mRNA 2

expression 3

A: Serial morphological changes in the CG group and Tx group. Masson’s trichrome stain; 4

magnification: ×200. B: SMC thickness in the Tx group on day 35 was significantly 5

decreased compared to that in CG group. C: Type 3 collagen mRNA levels in the Tx group 6

significantly decreased compared to that in the CG group. D: ASCs (green) and 7

DAPI-stained (blue) nuclei were visualized in the peritoneum. ASCs were widely 8

distributed on day 25. ASC numbers decreased on days 27 and 29 and disappeared on day 9

35; magnification: ×60. I: Error bars represent standard deviations (SDs). *: significantly 10

different (p < 0.05). ns: no significance. 11

12

Figure 2: Effects of ASCs transplantation on EMT and inflammation 13

A: Snail mRNA levels in the Tx group were significantly decreased on day 29, although 14

TGF-β levels were not significantly different. B, C: α-SMA positive cell were widely 15

distributed in the peritoneum on day 22. α-SMA mRNA levels the number of 16

α-SMA–positive cells in the Tx group were significantly decreased on day 29; 17

magnification: ×200. D: We detected transplanted cells that migrated into the peritoneal 18

interstitium; however, they were not double-stained by GFP (green) and α-SMA antibody 19

20

(red); magnification: ×40. E: IL-1β, TNF-α, and MCP-1 mRNA levels on day 29 in the Tx 1

group were significantly lower than those in the CG group. Error bars represent SDs. *: 2

significantly different (p < 0.05). ns: no significance. 3

4

Figure 3: Effects of ASCs transplantation on angiogenesis 5

A, B: VEGF and PDGF-BB mRNA levels on day 29 in the Tx group were significantly 6

increased. C: HIF-1α levels on day 29 in the Tx group were significantly decrease. D: 7

Average vascular density at days 29 tended to decrease in the Tx group compared to that in 8

the CG group. E: Transplanted ASCs (green) expressed VEGF (red) on day 29 in the Tx 9

group; magnification: ×40. F–H: A part of the transplanted ASCs (green) that formed 10

lumens also expressed PDGF-BB (F, red; ×40), PDGF-Rβ (G, red; ×40) and CD31 (H, red; 11

×60). Error bars represent SDs. *: significantly different (p < 0.05). 12

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Table 1. The PCR primers were designed as follows: 1

Gene Forward primer Reverse primer

GAPDH 5′-ACGTAAACGGCCACAAGTTC-3′ 5′-AAGTCGTGCTGCTTCATGTG-3′

Type 3

collagen 5′-CCACCCTGAACTCAAGAGCGGAGAA-3′ 5′-GGGACAGTCATGGGACTGGCATTT-3′

TNF-α 5′-TCGGTCCCAACAAGGAGGAGAAGT-3′ 5′-TGATCTGAGTGTGAGGGTCTGGGC-3′

IL-1β 5′-TGGAGAAGCTGTGGCAGCTACCTAT-3′ 5′-CACAGAGGACGGGCTCTTCTTCAA-3′

MCP-1 5′-AGGTCTCTGTCACGCTTCTGGG-3′ 5′-TAGCAGCAGGTGAGTGGGGCAT-3′

Snail 5′-ACAGCGAACTGGACACACACACA-3′ 5′-AGGGGAGTGGAATGGAACTGCTGA-3′

TGF-β 5′-ATGGTGGACCGCAACAACGCAAT-3′ 5′-CAGCTCTGCACGGGACAGCAAT-3′

HIF-1α 5′-ACACACAGAAATGGCCCAGTGAGAA-3′ 5′-GCACCTTCCACGTTGCTGACTTGAT-3′

α-SMA 5′-AGTCGCCATCAGGAACCTCGAGAA-3′ 5′-ACCAGCAAAGCCCGCCTTACA-3′

VEGF 5′-ACTGGACCCTGGCTTTACTGCTG-3′ 5′-TTCACCACTTCATGGGCTTTCTGCT-3′

PDGF-BB 5′-AGCGAGAGTGTGGGCAGGGTTA-3′ 5′-GCACCATTGGCCCGTCCGAATCA-3′

2

A: day 0 day 22 (CG) day 35 (CG)

Figure 1

day 35 (Tx)

B

D: day 25 day 27 day 29 day 35

C

day 29 (CG)

0

100

200

300

400

500

*

CG groupTx group

day0 day22 day35

SM

C (

m)

*

0

2

4

6

8

*

day22 day29

Ty

pe

3 c

olla

ge

n/G

AP

DH

*

Figure 2

B: CG group on day 22 CG group on day 29 Tx group on day 29

A

D

0

2

4

6

8

TG

F-

/GA

PD

H

day22 day29

ns

ns

0

5

10

15

20

Sn

ail/

GA

PD

H

day22 day29

*

CG groupTx group

ns

E

C

TN

F-

/GA

PD

H

0

5

10

15

20

day22 day29

**

MC

P-1

/GA

PD

H

0

5

10

15

20

day22 day29

*

*

IL-1

/GA

PD

H

0

10

20

30

*

day22 day29

*

0

1

2

3

4

5

*

day22 day29

-S

MA

/GA

PD

H

*

0

500

1000

1500

2000*

day22 day29

-S

MA

(+)

ce

lls/m

m2

*

E: DAPI VEGFGFP Merge

F: DAPI GFP PDGF-BB Merge

G: DAPI GFP PDGF-Rβ Merge

H: DAPI GFP CD31 Merge

Figure 3

A B C D

0

2

4

6

8

day22 day29

*

VE

GF

/GA

PD

H CG groupTx group

*

0

2

4

6

8

day22 day29

*

PD

GF

-BB

/GA

PD

H

*

0

10

20

30

40

HIF

-1

/GA

PD

H

day22 day29

**

0

100

200

300

400

Va

sc

ula

r d

en

sit

y/m

m2

ns

day22 day29

*

Gene Forward primer Reverse primer

GAPDH 5′-ACGTAAACGGCCACAAGTTC-3′ 5′-AAGTCGTGCTGCTTCATGTG-3′

Type1 collagen 5′-CTGCACGAGTCACACCGGAACTT-3′ 5′-TCCTGGTCTGGGGCACCAATGT-3′

Type3 collagen 5′-CCACCCTGAACTCAAGAGCGGAGAA-3′ 5′-GGGACAGTCATGGGACTGGCATTT-3′

TNF-α 5′-TCGGTCCCAACAAGGAGGAGAAGT-3′ 5′-TGATCTGAGTGTGAGGGTCTGGGC-3′

interleukin-1beta (IL-1β) 5′-TGGAGAAGCTGTGGCAGCTACCTAT-3′ 5′-CACAGAGGACGGGCTCTTCTTCAA-3′

Monocyte chemoteraticprotein-1 (MCP-1)

5′-AGGTCTCTGTCACGCTTCTGGG-3′ 5′-TAGCAGCAGGTGAGTGGGGCAT-3′

Snail 5′-ACAGCGAACTGGACACACACACA-3′ 5′-AGGGGAGTGGAATGGAACTGCTGA-3′

TGF-β 5′-ATGGTGGACCGCAACAACGCAAT-3′ 5′-CAGCTCTGCACGGGACAGCAAT-3′

HIF-1α 5′-ACACACAGAAATGGCCCAGTGAGAA-3′ 5′-GCACCTTCCACGTTGCTGACTTGAT-3′

α-SMA 5′-AGTCGCCATCAGGAACCTCGAGAA-3′ 5′-ACCAGCAAAGCCCGCCTTACA-3′

VEGF 5′-ACTGGACCCTGGCTTTACTGCTG-3′ 5′-TTCACCACTTCATGGGCTTTCTGCT-3′

PDGF-BB 5′-AGCGAGAGTGTGGGCAGGGTTA-3′ 5′-GCACCATTGGCCCGTCCGAATCA-3′

Table 1. The PCR primers were designed as follows: