Limits...
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.

Show MeSH

Related in: MedlinePlus

miR-221 transferred from MSC to CM and reduced PUMA expression in CM.Panel A: In situ hybridization staining of miR-221 in CM co-cultured with MSCmiR-221 for 48 hours. miR-221 is shown as a purple-blue signal (red circles) which was observed in both GFP positive MSC (green arrow) and GFP negative CM (red arrow). Panel B: Western blot of PUMA and corresponding semi-quantitative data in CM co-cultured with various MSC in a dual-chamber system.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756018&req=5

pone-0073304-g006: miR-221 transferred from MSC to CM and reduced PUMA expression in CM.Panel A: In situ hybridization staining of miR-221 in CM co-cultured with MSCmiR-221 for 48 hours. miR-221 is shown as a purple-blue signal (red circles) which was observed in both GFP positive MSC (green arrow) and GFP negative CM (red arrow). Panel B: Western blot of PUMA and corresponding semi-quantitative data in CM co-cultured with various MSC in a dual-chamber system.

Mentions: The transfer of bioactive molecules between MSC and CM was tracked using the red fluorescence of PKH26. MSC expressed green fluorescence following transfection with either lenti-miR-221 or lenti-miR-NC. MSC were pre-labeled with PKH26 and co-cultured with CM. PKH26 was not only visible in GFP positive MSC, but also in some CM after 4 days of co-culture with MSC (Figure 5A, red arrows). PKH26 appeared around nuclei of some GFP negative CM in cells fixed with 4% PFA and counterstained with DAPI (Figure 5B, red arrows). To directly detect the translocation of miRs from MSC to CM, ISH was performed after CM were co-cultured with MSCmiR-221 for 48 hours. miR-221 location was detected as a purple-blue signal by incubating cell cultures with NBT/BCIP. miR-221 was observed in GFP positive MSC (green arrow) and in GFP negative cells (red arrow) (Figure 6A).


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)

miR-221 transferred from MSC to CM and reduced PUMA expression in CM.Panel A: In situ hybridization staining of miR-221 in CM co-cultured with MSCmiR-221 for 48 hours. miR-221 is shown as a purple-blue signal (red circles) which was observed in both GFP positive MSC (green arrow) and GFP negative CM (red arrow). Panel B: Western blot of PUMA and corresponding semi-quantitative data in CM co-cultured with various MSC in a dual-chamber system.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073304-g006: miR-221 transferred from MSC to CM and reduced PUMA expression in CM.Panel A: In situ hybridization staining of miR-221 in CM co-cultured with MSCmiR-221 for 48 hours. miR-221 is shown as a purple-blue signal (red circles) which was observed in both GFP positive MSC (green arrow) and GFP negative CM (red arrow). Panel B: Western blot of PUMA and corresponding semi-quantitative data in CM co-cultured with various MSC in a dual-chamber system.
Mentions: The transfer of bioactive molecules between MSC and CM was tracked using the red fluorescence of PKH26. MSC expressed green fluorescence following transfection with either lenti-miR-221 or lenti-miR-NC. MSC were pre-labeled with PKH26 and co-cultured with CM. PKH26 was not only visible in GFP positive MSC, but also in some CM after 4 days of co-culture with MSC (Figure 5A, red arrows). PKH26 appeared around nuclei of some GFP negative CM in cells fixed with 4% PFA and counterstained with DAPI (Figure 5B, red arrows). To directly detect the translocation of miRs from MSC to CM, ISH was performed after CM were co-cultured with MSCmiR-221 for 48 hours. miR-221 location was detected as a purple-blue signal by incubating cell cultures with NBT/BCIP. miR-221 was observed in GFP positive MSC (green arrow) and in GFP negative cells (red arrow) (Figure 6A).

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