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Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells

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ABSTRACT

Background: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution.

Methods: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed.

Results: One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes.

Conclusions: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0424-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


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Increased growth factors levels and MMP2 activity in hepatic macrophages after MSCs treatment. Quantitative analyses of IGF-I and HGF mRNA expression levels in macrophages, on in vivo (a) or in vitro (b) experiments, after saline/DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) pretreatments. c IGF-I protein levels in hMø supernatants on in vitro experiments. Macrophages obtained from fibrotic livers were preincubated with conditioned media on in vitro studies. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline (in vivo) or macrophages/DMEM (in vitro); σp < 0.05, σσp < 0.01 and σσσp < 0.001 vs macrophages/AdGFP-MSCs conditions. d MMP-2 activity in hMø supernatants after in vitro incubation evaluated by zymography. One representative zymogram is shown. Band intensity of three independent experiments was detected by densitometric evaluation and plotted as MMP-2 relative activity of DMEM/macrophages condition. Dunn’s multiple comparisons test, *p < 0.05 and **p < 0.01 macrophages/DMEM
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Fig4: Increased growth factors levels and MMP2 activity in hepatic macrophages after MSCs treatment. Quantitative analyses of IGF-I and HGF mRNA expression levels in macrophages, on in vivo (a) or in vitro (b) experiments, after saline/DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) pretreatments. c IGF-I protein levels in hMø supernatants on in vitro experiments. Macrophages obtained from fibrotic livers were preincubated with conditioned media on in vitro studies. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline (in vivo) or macrophages/DMEM (in vitro); σp < 0.05, σσp < 0.01 and σσσp < 0.001 vs macrophages/AdGFP-MSCs conditions. d MMP-2 activity in hMø supernatants after in vitro incubation evaluated by zymography. One representative zymogram is shown. Band intensity of three independent experiments was detected by densitometric evaluation and plotted as MMP-2 relative activity of DMEM/macrophages condition. Dunn’s multiple comparisons test, *p < 0.05 and **p < 0.01 macrophages/DMEM

Mentions: Considering that macrophages with restorative profile would likely express growth factors, such as IGF-I [19] or HGF, we analyzed their expression levels in hMø at 1 day after MSCs treatments. As shown in Fig. 4a, an upregulation in IGF-I mRNA expression was found in hMø isolated from AdGFP-MSC-treated mice when compared with vehicle, and expression levels of this gene were further increased in the AdIGF-I-MSCs group. Interestingly, the latter was the only condition able to significantly induce HGF expression (Fig. 4a). This result is of significant importance since hepatocytes are known to express receptors for HGF but not for IGF-I [20].Fig. 4


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
Increased growth factors levels and MMP2 activity in hepatic macrophages after MSCs treatment. Quantitative analyses of IGF-I and HGF mRNA expression levels in macrophages, on in vivo (a) or in vitro (b) experiments, after saline/DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) pretreatments. c IGF-I protein levels in hMø supernatants on in vitro experiments. Macrophages obtained from fibrotic livers were preincubated with conditioned media on in vitro studies. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline (in vivo) or macrophages/DMEM (in vitro); σp < 0.05, σσp < 0.01 and σσσp < 0.001 vs macrophages/AdGFP-MSCs conditions. d MMP-2 activity in hMø supernatants after in vitro incubation evaluated by zymography. One representative zymogram is shown. Band intensity of three independent experiments was detected by densitometric evaluation and plotted as MMP-2 relative activity of DMEM/macrophages condition. Dunn’s multiple comparisons test, *p < 0.05 and **p < 0.01 macrophages/DMEM
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Fig4: Increased growth factors levels and MMP2 activity in hepatic macrophages after MSCs treatment. Quantitative analyses of IGF-I and HGF mRNA expression levels in macrophages, on in vivo (a) or in vitro (b) experiments, after saline/DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) pretreatments. c IGF-I protein levels in hMø supernatants on in vitro experiments. Macrophages obtained from fibrotic livers were preincubated with conditioned media on in vitro studies. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline (in vivo) or macrophages/DMEM (in vitro); σp < 0.05, σσp < 0.01 and σσσp < 0.001 vs macrophages/AdGFP-MSCs conditions. d MMP-2 activity in hMø supernatants after in vitro incubation evaluated by zymography. One representative zymogram is shown. Band intensity of three independent experiments was detected by densitometric evaluation and plotted as MMP-2 relative activity of DMEM/macrophages condition. Dunn’s multiple comparisons test, *p < 0.05 and **p < 0.01 macrophages/DMEM
Mentions: Considering that macrophages with restorative profile would likely express growth factors, such as IGF-I [19] or HGF, we analyzed their expression levels in hMø at 1 day after MSCs treatments. As shown in Fig. 4a, an upregulation in IGF-I mRNA expression was found in hMø isolated from AdGFP-MSC-treated mice when compared with vehicle, and expression levels of this gene were further increased in the AdIGF-I-MSCs group. Interestingly, the latter was the only condition able to significantly induce HGF expression (Fig. 4a). This result is of significant importance since hepatocytes are known to express receptors for HGF but not for IGF-I [20].Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hM&oslash;) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hM&oslash; in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hM&oslash; behavior in the context of liver fibrosis resolution.

Methods: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8&nbsp;weeks). In vivo gene expression analyses, in vitro experiments using hM&oslash; isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of M&oslash; were performed.

Results: One day after treatment, hM&oslash; from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hM&oslash; from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hM&oslash;, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hM&oslash; depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hM&oslash; abrogated such effects on the expression of the most highly regulated genes.

Conclusions: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0424-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus