Background: It has been shown that human bone marrow-derived mesenchymal stem cells (BM-MSC) repair infarcted hearts mainly through the production and release of cytoprotective paracrine factors. However, donor age may negatively influence the paracrine properties of BM-MSC. Recently, MSC of fetal origin have been isolated from the amniotic membrane of human placenta (A-MSC). These cells showed promising results in experimental models of myocardial infarction and, due to their immunoprivileged phenotype, A-MSC may be used for heterologous transplantation. However, their cytoprotective properties have never been investigated. Our goal was to establish if A-MSC can mediate cytoprotection and which pathways are eventually involved. Methods: A-MSC were isolated from placenta donated by healthy women undergoing cesarian section. Conditioned medium (CM) was obtained by growing A-MSC for 36 hours in serum starvation. Rat neonatal cardiomyocytes (H9c2) were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or CM from A-MSC (A-MSC-CM). H9c2 viability was evaluated by MTS assay and the rate of apoptosis was quantified by TUNEL staining. Cleaved Caspase 3 was evaluated by colorimetric assay and Western blotting. SAPK/JNK, p38 MAPK and Akt activation was analyzed by Western blotting and the expression of standard pro- and anti-apoptotic genes were analyzed in H9c2 cells by RT-PCR. Finally we performed RT-PCR to compare the expression profile of several known cytoprotective factors in A-MSC vs BM-MSC. Results: The hypoxia/reoxygenation protocol reduced the H9c2 viability by 55% (p<0.001 vs basal conditions); the A-MSC-CM remarkably increased cell viability by 45% compared with CTRL-M (p<0.001). In the presence of CTRL-M, TUNEL staining documented apoptotic death in 35 ± 8% of H9c2 cells. The A-MSC-CM significantly reduced the number of TUNEL positive nuclei by 91% vs CTRL-M (p<0.001). The colorimetric assay documented that, in H9c2 fed with CRTL-M, the amount of cleaved Caspase 3 was significantly increased by 60% after hypoxia/reoxygenation (p<0.001) while A-MSC-CM prevented Caspase 3 cleveage (p= n.s. vs normoxia). Western blot analysis confirmed the reduction of Caspase 3 in the presence of A-MSC-CM. As for the pathways involved, after hypoxia/reoxigenation we observed a marked activation of SAPK/JNK and p38 MAPK that was strongly reduced by A-MSC-CM; Akt phosphorilation did not show significant variation. Furthermore, the A-MSC-CM increased the expression of the anti-apoptotic genes Bcl-2 and Stat3 and inhibited the transcription of TNF-α and FasL compared with CTRL-M. Finally, the RT-PCR analysis documented that the A-MSC express several known cytoprotective factors such as PDGF-β , BMP2, EPO, IGF-1, FGF2 and VEGF at significantly higher levels compared with BM-MSC. Conclusions: We demonstrated that A-MSC express high levels of several cytoprotective factors and that A-MSC-CM remarkably protects cardiac myocytes against hypoxia/reoxygenation damage. The cytoprotective effects exerted by the A-MSC-CM seem to be mediated through the inhibition of SAPK/JNK and p38 MAPK pro-apoptotic pathways and by a concomitant over-expression of anti-apoptotic genes Bcl-2 and Stat3 and by a reduction of pro-apoptotic factors TNF-α and FasL. A-MSC theraphy may represent a novel and powerful approach for cardioprotection in ischemic heart disease.

Soluble factors released by human mesenchymal stem cells of fetal origin lead to cardiomyocyte protection through the inhibition of pro-apoptotic signaling

GNECCHI, MASSIMILIANO
2010-01-01

Abstract

Background: It has been shown that human bone marrow-derived mesenchymal stem cells (BM-MSC) repair infarcted hearts mainly through the production and release of cytoprotective paracrine factors. However, donor age may negatively influence the paracrine properties of BM-MSC. Recently, MSC of fetal origin have been isolated from the amniotic membrane of human placenta (A-MSC). These cells showed promising results in experimental models of myocardial infarction and, due to their immunoprivileged phenotype, A-MSC may be used for heterologous transplantation. However, their cytoprotective properties have never been investigated. Our goal was to establish if A-MSC can mediate cytoprotection and which pathways are eventually involved. Methods: A-MSC were isolated from placenta donated by healthy women undergoing cesarian section. Conditioned medium (CM) was obtained by growing A-MSC for 36 hours in serum starvation. Rat neonatal cardiomyocytes (H9c2) were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or CM from A-MSC (A-MSC-CM). H9c2 viability was evaluated by MTS assay and the rate of apoptosis was quantified by TUNEL staining. Cleaved Caspase 3 was evaluated by colorimetric assay and Western blotting. SAPK/JNK, p38 MAPK and Akt activation was analyzed by Western blotting and the expression of standard pro- and anti-apoptotic genes were analyzed in H9c2 cells by RT-PCR. Finally we performed RT-PCR to compare the expression profile of several known cytoprotective factors in A-MSC vs BM-MSC. Results: The hypoxia/reoxygenation protocol reduced the H9c2 viability by 55% (p<0.001 vs basal conditions); the A-MSC-CM remarkably increased cell viability by 45% compared with CTRL-M (p<0.001). In the presence of CTRL-M, TUNEL staining documented apoptotic death in 35 ± 8% of H9c2 cells. The A-MSC-CM significantly reduced the number of TUNEL positive nuclei by 91% vs CTRL-M (p<0.001). The colorimetric assay documented that, in H9c2 fed with CRTL-M, the amount of cleaved Caspase 3 was significantly increased by 60% after hypoxia/reoxygenation (p<0.001) while A-MSC-CM prevented Caspase 3 cleveage (p= n.s. vs normoxia). Western blot analysis confirmed the reduction of Caspase 3 in the presence of A-MSC-CM. As for the pathways involved, after hypoxia/reoxigenation we observed a marked activation of SAPK/JNK and p38 MAPK that was strongly reduced by A-MSC-CM; Akt phosphorilation did not show significant variation. Furthermore, the A-MSC-CM increased the expression of the anti-apoptotic genes Bcl-2 and Stat3 and inhibited the transcription of TNF-α and FasL compared with CTRL-M. Finally, the RT-PCR analysis documented that the A-MSC express several known cytoprotective factors such as PDGF-β , BMP2, EPO, IGF-1, FGF2 and VEGF at significantly higher levels compared with BM-MSC. Conclusions: We demonstrated that A-MSC express high levels of several cytoprotective factors and that A-MSC-CM remarkably protects cardiac myocytes against hypoxia/reoxygenation damage. The cytoprotective effects exerted by the A-MSC-CM seem to be mediated through the inhibition of SAPK/JNK and p38 MAPK pro-apoptotic pathways and by a concomitant over-expression of anti-apoptotic genes Bcl-2 and Stat3 and by a reduction of pro-apoptotic factors TNF-α and FasL. A-MSC theraphy may represent a novel and powerful approach for cardioprotection in ischemic heart disease.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/220708
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