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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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Related in: MedlinePlus

Morphology of cultured MSC and CM.Panel A: MSC obtained from rat bone marrow. Panel B: Quantitative real-time PCR of GATA-4 expression in MSC transduced with GATA-4 (MSCGATA-4) or empty vector (MSCNull). Panel C: Immunostaining of MSCGATA-4 and MSCNull. Panel D: CM obtained from rat neonatal ventricles. Panel E: CM were immunostained positive for α-actinin (green) and connexin 43 (red, white arrows). Myofibers were seen with clear Z-lines in sarcomeres. E1: DAPI; E2: α-actinin; E3: connexin 43; and E4: merged images of E1 to E3. §, p<0.05 vs MSCNull.
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pone-0073304-g001: Morphology of cultured MSC and CM.Panel A: MSC obtained from rat bone marrow. Panel B: Quantitative real-time PCR of GATA-4 expression in MSC transduced with GATA-4 (MSCGATA-4) or empty vector (MSCNull). Panel C: Immunostaining of MSCGATA-4 and MSCNull. Panel D: CM obtained from rat neonatal ventricles. Panel E: CM were immunostained positive for α-actinin (green) and connexin 43 (red, white arrows). Myofibers were seen with clear Z-lines in sarcomeres. E1: DAPI; E2: α-actinin; E3: connexin 43; and E4: merged images of E1 to E3. §, p<0.05 vs MSCNull.

Mentions: MSC obtained from femurs and tibias appeared as a morphologically heterogeneous population. These cells exhibited fibroblast-like morphology with irregularly shaped large euchromatic and lobulated nuclei as seen in Figure 1A. MSCGATA-4 exhibited a higher expression of GATA-4 measured using quantitative real-time PCR (Figure 1B). Both MSCGATA-4 and its empty vector control (MSCNull) expressed GFP, only MSCGATA-4 stained intensely for GATA-4 (Figure 1C). CM displayed a flattened shape with cell clusters (Figure 1D). CM were significantly smaller than MSC and had spontaneous beating. CM were positive for α-actinin and expressed the gap junction protein connexin 43 (Figure 1E). Clear Z-lines in sarcomeres were observed in CM myofibers.


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)

Morphology of cultured MSC and CM.Panel A: MSC obtained from rat bone marrow. Panel B: Quantitative real-time PCR of GATA-4 expression in MSC transduced with GATA-4 (MSCGATA-4) or empty vector (MSCNull). Panel C: Immunostaining of MSCGATA-4 and MSCNull. Panel D: CM obtained from rat neonatal ventricles. Panel E: CM were immunostained positive for α-actinin (green) and connexin 43 (red, white arrows). Myofibers were seen with clear Z-lines in sarcomeres. E1: DAPI; E2: α-actinin; E3: connexin 43; and E4: merged images of E1 to E3. §, p<0.05 vs MSCNull.
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Related In: Results  -  Collection

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pone-0073304-g001: Morphology of cultured MSC and CM.Panel A: MSC obtained from rat bone marrow. Panel B: Quantitative real-time PCR of GATA-4 expression in MSC transduced with GATA-4 (MSCGATA-4) or empty vector (MSCNull). Panel C: Immunostaining of MSCGATA-4 and MSCNull. Panel D: CM obtained from rat neonatal ventricles. Panel E: CM were immunostained positive for α-actinin (green) and connexin 43 (red, white arrows). Myofibers were seen with clear Z-lines in sarcomeres. E1: DAPI; E2: α-actinin; E3: connexin 43; and E4: merged images of E1 to E3. §, p<0.05 vs MSCNull.
Mentions: MSC obtained from femurs and tibias appeared as a morphologically heterogeneous population. These cells exhibited fibroblast-like morphology with irregularly shaped large euchromatic and lobulated nuclei as seen in Figure 1A. MSCGATA-4 exhibited a higher expression of GATA-4 measured using quantitative real-time PCR (Figure 1B). Both MSCGATA-4 and its empty vector control (MSCNull) expressed GFP, only MSCGATA-4 stained intensely for GATA-4 (Figure 1C). CM displayed a flattened shape with cell clusters (Figure 1D). CM were significantly smaller than MSC and had spontaneous beating. CM were positive for α-actinin and expressed the gap junction protein connexin 43 (Figure 1E). Clear Z-lines in sarcomeres were observed in CM myofibers.

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