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

View Article: PubMed Central - PubMed

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.


Related in: MedlinePlus

Regulation of cell cycle-related genes by AdIGF-I-MSCs treatment. a Screening analyses of mRNA expression profile differences in between fibrotic liver samples after saline (control) or AdIGF-I-MSCs treatments, using a functionally specific qPCR array. Punctated lines represent the thresholds; genes with scores above them were considered as differentially regulated (induced: to the right; repressed: to the left) by AdIGF-I-MSCs. b-g Changes in the most differentially regulated genes were validated by qPCR, and comparisons against AdGFP-MSCs condition were added. ANOVA Tukey’s post-test, **p < 0.01 and ****p < 0.0001 vs. saline; σp < 0.05; σσp < 0.01; σσσp < 0.001 and σσσσp < 0.0001 vs. AdGFP-MSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5120504&req=5

Fig7: Regulation of cell cycle-related genes by AdIGF-I-MSCs treatment. a Screening analyses of mRNA expression profile differences in between fibrotic liver samples after saline (control) or AdIGF-I-MSCs treatments, using a functionally specific qPCR array. Punctated lines represent the thresholds; genes with scores above them were considered as differentially regulated (induced: to the right; repressed: to the left) by AdIGF-I-MSCs. b-g Changes in the most differentially regulated genes were validated by qPCR, and comparisons against AdGFP-MSCs condition were added. ANOVA Tukey’s post-test, **p < 0.01 and ****p < 0.0001 vs. saline; σp < 0.05; σσp < 0.01; σσσp < 0.001 and σσσσp < 0.0001 vs. AdGFP-MSCs

Mentions: We have previously shown that AdIGF-I-MSCs treatment significantly enhanced hepatocyte proliferation, with a peak within 24 hours after cellular application [13]. With the aim of uncovering molecular mechanisms driving the pro-regenerative effect of MSCs, by using PCR array analyses for cell cycle markers, we evaluated the gene expression profile of fibrotic liver pooled samples obtained from AdIGF-I-MSCs and vehicle-treated animals at 1 day after their in vivo application. AdIGF-I-MSCs treatment resulted in the upregulation of seven genes and downregulation of four genes (out of 90 genes; >2 fold change; Fig. 7a). Many of the upregulated genes are involved in DNA repair, such as Breast cancer 2 (Brca2), Myeloblastosis oncogene (Myb), Abelson murine leukemia viral oncogene homolog 1 (Abl1) and G protein-coupled receptor 132 (Gpr132) (fold change: 14; 4.7; 4.3 and 2, respectively; vs. vehicle). Likewise, some of them and others are known to positively regulate cell cycle progression, such as Cyclin A1 (Ccna1), Efr transcription factor 2 (E2f2), Retinoblastoma-like protein 2 (Rbl2) and G protein-coupled receptor 132 (Gpr132) (fold change: 4; 2.4; 2 and 2, respectively). Interestingly, some upregulated genes, such as Myb, Rbl2 and Cyclin-dependent kinase inhibitor 2A (CDKN2A; fold change: 2), code for proteins able to act as tumor suppressors. Among downregulated genes, growth arrest and DNA-damage-inducible 45α (Gadd45α) had the highest score (fold change: 2.9).Fig. 7


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
Regulation of cell cycle-related genes by AdIGF-I-MSCs treatment. a Screening analyses of mRNA expression profile differences in between fibrotic liver samples after saline (control) or AdIGF-I-MSCs treatments, using a functionally specific qPCR array. Punctated lines represent the thresholds; genes with scores above them were considered as differentially regulated (induced: to the right; repressed: to the left) by AdIGF-I-MSCs. b-g Changes in the most differentially regulated genes were validated by qPCR, and comparisons against AdGFP-MSCs condition were added. ANOVA Tukey’s post-test, **p < 0.01 and ****p < 0.0001 vs. saline; σp < 0.05; σσp < 0.01; σσσp < 0.001 and σσσσp < 0.0001 vs. AdGFP-MSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120504&req=5

Fig7: Regulation of cell cycle-related genes by AdIGF-I-MSCs treatment. a Screening analyses of mRNA expression profile differences in between fibrotic liver samples after saline (control) or AdIGF-I-MSCs treatments, using a functionally specific qPCR array. Punctated lines represent the thresholds; genes with scores above them were considered as differentially regulated (induced: to the right; repressed: to the left) by AdIGF-I-MSCs. b-g Changes in the most differentially regulated genes were validated by qPCR, and comparisons against AdGFP-MSCs condition were added. ANOVA Tukey’s post-test, **p < 0.01 and ****p < 0.0001 vs. saline; σp < 0.05; σσp < 0.01; σσσp < 0.001 and σσσσp < 0.0001 vs. AdGFP-MSCs
Mentions: We have previously shown that AdIGF-I-MSCs treatment significantly enhanced hepatocyte proliferation, with a peak within 24 hours after cellular application [13]. With the aim of uncovering molecular mechanisms driving the pro-regenerative effect of MSCs, by using PCR array analyses for cell cycle markers, we evaluated the gene expression profile of fibrotic liver pooled samples obtained from AdIGF-I-MSCs and vehicle-treated animals at 1 day after their in vivo application. AdIGF-I-MSCs treatment resulted in the upregulation of seven genes and downregulation of four genes (out of 90 genes; >2 fold change; Fig. 7a). Many of the upregulated genes are involved in DNA repair, such as Breast cancer 2 (Brca2), Myeloblastosis oncogene (Myb), Abelson murine leukemia viral oncogene homolog 1 (Abl1) and G protein-coupled receptor 132 (Gpr132) (fold change: 14; 4.7; 4.3 and 2, respectively; vs. vehicle). Likewise, some of them and others are known to positively regulate cell cycle progression, such as Cyclin A1 (Ccna1), Efr transcription factor 2 (E2f2), Retinoblastoma-like protein 2 (Rbl2) and G protein-coupled receptor 132 (Gpr132) (fold change: 4; 2.4; 2 and 2, respectively). Interestingly, some upregulated genes, such as Myb, Rbl2 and Cyclin-dependent kinase inhibitor 2A (CDKN2A; fold change: 2), code for proteins able to act as tumor suppressors. Among downregulated genes, growth arrest and DNA-damage-inducible 45α (Gadd45α) had the highest score (fold change: 2.9).Fig. 7

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