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Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.

Yu B, Gong M, Wang Y, Millard RW, Pasha Z, Yang Y, Ashraf M, Xu M - PLoS ONE (2013)

Bottom Line: MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA).Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC.Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Introduction: microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).

Methods and results: Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC(Null)). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC(GATA-4). MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.

Conclusions: Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

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The morphology and characterization of MSC derived MVs (MSC-MVs).Panel A: Morphological features of MSC-MVs under electron microscope. Panel B: Western blot results show that MSC-MVs highly expressed HSP70, CD63, and CD9 compared to MSC; Panel C: The expression of miR-221 in MSC-MVs, MSC, and CM analyzed using real-time PCR.
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pone-0073304-g007: The morphology and characterization of MSC derived MVs (MSC-MVs).Panel A: Morphological features of MSC-MVs under electron microscope. Panel B: Western blot results show that MSC-MVs highly expressed HSP70, CD63, and CD9 compared to MSC; Panel C: The expression of miR-221 in MSC-MVs, MSC, and CM analyzed using real-time PCR.

Mentions: MVs derived from MSC (MSC-MVs) exhibited a rounded morphology with a transparent center (Figure 7A). MSC-MVs highly expressed HSP70, CD63, and CD9, while the expression level of these proteins was lower in MSC (Figure 7B). Notably, the expression of miR-221 in MSC-MVs was significantly higher than that found in MSC (10.7-fold) or in CM (74.2-fold) (Figure 7C).


Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.

Yu B, Gong M, Wang Y, Millard RW, Pasha Z, Yang Y, Ashraf M, Xu M - PLoS ONE (2013)

The morphology and characterization of MSC derived MVs (MSC-MVs).Panel A: Morphological features of MSC-MVs under electron microscope. Panel B: Western blot results show that MSC-MVs highly expressed HSP70, CD63, and CD9 compared to MSC; Panel C: The expression of miR-221 in MSC-MVs, MSC, and CM analyzed using real-time PCR.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756018&req=5

pone-0073304-g007: The morphology and characterization of MSC derived MVs (MSC-MVs).Panel A: Morphological features of MSC-MVs under electron microscope. Panel B: Western blot results show that MSC-MVs highly expressed HSP70, CD63, and CD9 compared to MSC; Panel C: The expression of miR-221 in MSC-MVs, MSC, and CM analyzed using real-time PCR.
Mentions: MVs derived from MSC (MSC-MVs) exhibited a rounded morphology with a transparent center (Figure 7A). MSC-MVs highly expressed HSP70, CD63, and CD9, while the expression level of these proteins was lower in MSC (Figure 7B). Notably, the expression of miR-221 in MSC-MVs was significantly higher than that found in MSC (10.7-fold) or in CM (74.2-fold) (Figure 7C).

Bottom Line: MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA).Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC.Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Introduction: microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).

Methods and results: Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC(Null)). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC(GATA-4). MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.

Conclusions: Our results demonstrate that cardioprotection by MSC(GATA-4) may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

Show MeSH
Related in: MedlinePlus