Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice

Su Yeon An, Yu Jin Jang, Hee Joung Lim, Jiyou Han, Jaehun Lee, Gyunggyu Lee, Ji Young Park, Seo Young Park, Ji Hyang Kim, Byung Rok Do, Choongseong Han, Hee Kyung Park, Ok Hee Kim, Myeong Jun Song, Say June Kim, Jong-Hoon Kim

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background & Aims Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. Methods Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor β 1 (TGFβ1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. Results In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFβ, and Smad proteins in the TGFβ signaling pathways. In HSC cell lines and primary HSCs, TGFβ1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFβ-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFβ type I receptor by binding to αvβ3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. Conclusions MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFβ signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

Original languageEnglish
Pages (from-to)1174-1186
Number of pages13
JournalGastroenterology
Volume152
Issue number5
DOIs
Publication statusPublished - 2017 Apr 1

Fingerprint

Umbilical Cord
Mesenchymal Stromal Cells
Epidermal Growth Factor
Liver Cirrhosis
Hepatic Stellate Cells
Liver
Transforming Growth Factors
Proteins
Injections
Extracellular Matrix
Smooth Muscle
Actins
Fibrosis
Smad Proteins
Thioacetamide
Cell Line
Extracellular Matrix Proteins
Metalloproteases
Human Milk
Korea

Keywords

  • Decorin
  • MMP
  • Mouse Model
  • Signal Transduction

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice. / An, Su Yeon; Jang, Yu Jin; Lim, Hee Joung; Han, Jiyou; Lee, Jaehun; Lee, Gyunggyu; Park, Ji Young; Park, Seo Young; Kim, Ji Hyang; Do, Byung Rok; Han, Choongseong; Park, Hee Kyung; Kim, Ok Hee; Song, Myeong Jun; Kim, Say June; Kim, Jong-Hoon.

In: Gastroenterology, Vol. 152, No. 5, 01.04.2017, p. 1174-1186.

Research output: Contribution to journalArticle

An, SY, Jang, YJ, Lim, HJ, Han, J, Lee, J, Lee, G, Park, JY, Park, SY, Kim, JH, Do, BR, Han, C, Park, HK, Kim, OH, Song, MJ, Kim, SJ & Kim, J-H 2017, 'Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice', Gastroenterology, vol. 152, no. 5, pp. 1174-1186. https://doi.org/10.1053/j.gastro.2016.12.003
An, Su Yeon ; Jang, Yu Jin ; Lim, Hee Joung ; Han, Jiyou ; Lee, Jaehun ; Lee, Gyunggyu ; Park, Ji Young ; Park, Seo Young ; Kim, Ji Hyang ; Do, Byung Rok ; Han, Choongseong ; Park, Hee Kyung ; Kim, Ok Hee ; Song, Myeong Jun ; Kim, Say June ; Kim, Jong-Hoon. / Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice. In: Gastroenterology. 2017 ; Vol. 152, No. 5. pp. 1174-1186.
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TY - JOUR

T1 - Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice

AU - An, Su Yeon

AU - Jang, Yu Jin

AU - Lim, Hee Joung

AU - Han, Jiyou

AU - Lee, Jaehun

AU - Lee, Gyunggyu

AU - Park, Ji Young

AU - Park, Seo Young

AU - Kim, Ji Hyang

AU - Do, Byung Rok

AU - Han, Choongseong

AU - Park, Hee Kyung

AU - Kim, Ok Hee

AU - Song, Myeong Jun

AU - Kim, Say June

AU - Kim, Jong-Hoon

PY - 2017/4/1

Y1 - 2017/4/1

N2 - Background & Aims Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. Methods Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor β 1 (TGFβ1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. Results In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFβ, and Smad proteins in the TGFβ signaling pathways. In HSC cell lines and primary HSCs, TGFβ1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFβ-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFβ type I receptor by binding to αvβ3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. Conclusions MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFβ signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

AB - Background & Aims Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. Methods Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor β 1 (TGFβ1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. Results In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFβ, and Smad proteins in the TGFβ signaling pathways. In HSC cell lines and primary HSCs, TGFβ1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFβ-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFβ type I receptor by binding to αvβ3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. Conclusions MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFβ signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

KW - Decorin

KW - MMP

KW - Mouse Model

KW - Signal Transduction

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