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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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Role of macrophages in AdIGF-I-MSCs induced pro-regenerative mechanisms. PCNA (a), Brca2 (b), and Gadd45α (c) in vivo mRNA expression levels were statistically compared with/without macrophages depletion. d Representative microphotographs of PCNA immunostained sections showing changes in proliferation in liver hepatocytes at 14 days after treatments. e Statistical comparisons of number of PCNA+ cells among groups. ANOVA Tukey’s post-test, **p < 0.01 and ***p < 0.001 vs. saline; ττp < 0.01 and τττp < 0.001 vs. saline + LipClod; σp < 0.05; σσσp < 0.001 and σσσσp < 0.0001 vs. AdIGF-I-MSCs + LipClod. n.s nonsignificant
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Fig8: Role of macrophages in AdIGF-I-MSCs induced pro-regenerative mechanisms. PCNA (a), Brca2 (b), and Gadd45α (c) in vivo mRNA expression levels were statistically compared with/without macrophages depletion. d Representative microphotographs of PCNA immunostained sections showing changes in proliferation in liver hepatocytes at 14 days after treatments. e Statistical comparisons of number of PCNA+ cells among groups. ANOVA Tukey’s post-test, **p < 0.01 and ***p < 0.001 vs. saline; ττp < 0.01 and τττp < 0.001 vs. saline + LipClod; σp < 0.05; σσσp < 0.001 and σσσσp < 0.0001 vs. AdIGF-I-MSCs + LipClod. n.s nonsignificant

Mentions: We then asked whether the depletion of macrophages could abrogate the pro-regenerative and DNA repair effects of AdIGF-I-MSCs treatment. For this, mRNA levels of PCNA, Brca2 and Gadd45α were analyzed by qPCR, 2 weeks after AdIGF-I-MSCs or vehicle administration. The effect of AdIGF-I-MSCs on hepatocytes proliferation and protection was lost when animals were depleted from macrophages, as suggested by changes in the expression of all markers analyzed (Fig. 8a-c). Numbers of PCNA+ hepatocytes were also reduced to control vehicle levels thus confirming an essential role of macrophages in AdIGF-I-MSCs-induced proliferation (Fig. 8d, e). In the case of PCNA and Brca2, control vehicle group depleted from macrophages (saline + Clod) showed a very strong pro-regenerative effect; nevertheless, PCNA and Brca2 expression levels were significantly reduced when compared to the saline + Clod control when AdIGF-I-MSCs were applied to macrophages-depleted animals and reached levels which were similar to saline (Fig. 8a,b,d,e). From these results we can conclude that the pro-regenerative mechanisms triggered by AdIGF-I-MSCs are likely dependent on macrophages through hepatocytes protection from DNA damage/DNA repair mechanisms.Fig. 8


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
Role of macrophages in AdIGF-I-MSCs induced pro-regenerative mechanisms. PCNA (a), Brca2 (b), and Gadd45α (c) in vivo mRNA expression levels were statistically compared with/without macrophages depletion. d Representative microphotographs of PCNA immunostained sections showing changes in proliferation in liver hepatocytes at 14 days after treatments. e Statistical comparisons of number of PCNA+ cells among groups. ANOVA Tukey’s post-test, **p < 0.01 and ***p < 0.001 vs. saline; ττp < 0.01 and τττp < 0.001 vs. saline + LipClod; σp < 0.05; σσσp < 0.001 and σσσσp < 0.0001 vs. AdIGF-I-MSCs + LipClod. n.s nonsignificant
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Fig8: Role of macrophages in AdIGF-I-MSCs induced pro-regenerative mechanisms. PCNA (a), Brca2 (b), and Gadd45α (c) in vivo mRNA expression levels were statistically compared with/without macrophages depletion. d Representative microphotographs of PCNA immunostained sections showing changes in proliferation in liver hepatocytes at 14 days after treatments. e Statistical comparisons of number of PCNA+ cells among groups. ANOVA Tukey’s post-test, **p < 0.01 and ***p < 0.001 vs. saline; ττp < 0.01 and τττp < 0.001 vs. saline + LipClod; σp < 0.05; σσσp < 0.001 and σσσσp < 0.0001 vs. AdIGF-I-MSCs + LipClod. n.s nonsignificant
Mentions: We then asked whether the depletion of macrophages could abrogate the pro-regenerative and DNA repair effects of AdIGF-I-MSCs treatment. For this, mRNA levels of PCNA, Brca2 and Gadd45α were analyzed by qPCR, 2 weeks after AdIGF-I-MSCs or vehicle administration. The effect of AdIGF-I-MSCs on hepatocytes proliferation and protection was lost when animals were depleted from macrophages, as suggested by changes in the expression of all markers analyzed (Fig. 8a-c). Numbers of PCNA+ hepatocytes were also reduced to control vehicle levels thus confirming an essential role of macrophages in AdIGF-I-MSCs-induced proliferation (Fig. 8d, e). In the case of PCNA and Brca2, control vehicle group depleted from macrophages (saline + Clod) showed a very strong pro-regenerative effect; nevertheless, PCNA and Brca2 expression levels were significantly reduced when compared to the saline + Clod control when AdIGF-I-MSCs were applied to macrophages-depleted animals and reached levels which were similar to saline (Fig. 8a,b,d,e). From these results we can conclude that the pro-regenerative mechanisms triggered by AdIGF-I-MSCs are likely dependent on macrophages through hepatocytes protection from DNA damage/DNA repair mechanisms.Fig. 8

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