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Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.

Feng Y, Huang W, Wani M, Yu X, Ashraf M - PLoS ONE (2014)

Bottom Line: Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2).In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis.Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Provincial Cardiovascular Institute, Southern Medical University, Guangzhou, China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Mesenchymal stem cells (MSCs) have potential application for the treatment of ischemic heart diseases. Besides differentiation properties, MSCs protect ischemic cardiomyocytes by secretion of paracrine factors. In this study, we found exosomes enriched with miR-22 were secreted by MSCs following ischemic preconditioning (Exo(IPC)) and mobilized to cardiomyocytes where they reduced their apoptosis due to ischemia. Interestingly, by time-lapse imaging, we for the first time captured the dynamic shedding of miR-22 loaded exosomes from cytosol to extracellular space. Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2). In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis. Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

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miR-22 transfers from MSCs to cardiomyocytes.A. A diagrammatic representation of experimental design for visualizing the transfer of miR-22 from MSCs into cardiomyocytes in a co-culture system. B. Fluorescent microscopy showed the existence of miR-22 (Green) in cardiomyocytes after 24 hr co-culture with MSCs. C. qPCR analysis showed the significant upregulation of miR-22 in cardiomyocytes co-cultured with MSCsmiR-22 as compared to MSCsNC (MSCsmiR-22; MSCs transfected with miR-22 mimic; MSCsNC transfected with negative control of microRNA mimic). (*) denotes P<0.05 for significant difference (n = 3). D. TUNEL assay showed reduced apoptosis in cardiomyocytes (co-cultured with MSCsmiR-22) as compared to control. (*) denotes P<0.05 for significant difference (n = 3).
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pone-0088685-g002: miR-22 transfers from MSCs to cardiomyocytes.A. A diagrammatic representation of experimental design for visualizing the transfer of miR-22 from MSCs into cardiomyocytes in a co-culture system. B. Fluorescent microscopy showed the existence of miR-22 (Green) in cardiomyocytes after 24 hr co-culture with MSCs. C. qPCR analysis showed the significant upregulation of miR-22 in cardiomyocytes co-cultured with MSCsmiR-22 as compared to MSCsNC (MSCsmiR-22; MSCs transfected with miR-22 mimic; MSCsNC transfected with negative control of microRNA mimic). (*) denotes P<0.05 for significant difference (n = 3). D. TUNEL assay showed reduced apoptosis in cardiomyocytes (co-cultured with MSCsmiR-22) as compared to control. (*) denotes P<0.05 for significant difference (n = 3).

Mentions: To determine whether miR-22 secreted from MSCs can transfer to cardiomyocytes in a non-contact manner, first, we transfected MSCs with a miR-22 mimic that was labeled with green fluorescein (MSCmiR-22) and then washed the MSCs with RNAse containing DMEM to degrade the residual miR-22 mimic on the MSCs. Figure S1 has shown RNase was effective. Then we employed a co-culture system of MSCs with neonatal cardiomyocytes in which the cells were separated by a membrane of 0.3 µm pore size to prevent direct cell contact or transfer of larger vesicles (Fig. 2A). Fluorescent imaging showed that after co-culture for 24 h, the green miR-22 was mainly concentrated in cardiomyocytes (Fig. 2B). In parallel, real-time PCR demonstrated that the expression of miR-22 was dramatically upregulated in cardiomyocytes co-cultured with MSCs. Moreover, these cardiomyocytes were more resistant to ischemic stress (Fig. 2C). These experiments demonstrate that miR-22 can mobilize from MSCs to cardiomyocytes.


Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.

Feng Y, Huang W, Wani M, Yu X, Ashraf M - PLoS ONE (2014)

miR-22 transfers from MSCs to cardiomyocytes.A. A diagrammatic representation of experimental design for visualizing the transfer of miR-22 from MSCs into cardiomyocytes in a co-culture system. B. Fluorescent microscopy showed the existence of miR-22 (Green) in cardiomyocytes after 24 hr co-culture with MSCs. C. qPCR analysis showed the significant upregulation of miR-22 in cardiomyocytes co-cultured with MSCsmiR-22 as compared to MSCsNC (MSCsmiR-22; MSCs transfected with miR-22 mimic; MSCsNC transfected with negative control of microRNA mimic). (*) denotes P<0.05 for significant difference (n = 3). D. TUNEL assay showed reduced apoptosis in cardiomyocytes (co-cultured with MSCsmiR-22) as compared to control. (*) denotes P<0.05 for significant difference (n = 3).
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pone-0088685-g002: miR-22 transfers from MSCs to cardiomyocytes.A. A diagrammatic representation of experimental design for visualizing the transfer of miR-22 from MSCs into cardiomyocytes in a co-culture system. B. Fluorescent microscopy showed the existence of miR-22 (Green) in cardiomyocytes after 24 hr co-culture with MSCs. C. qPCR analysis showed the significant upregulation of miR-22 in cardiomyocytes co-cultured with MSCsmiR-22 as compared to MSCsNC (MSCsmiR-22; MSCs transfected with miR-22 mimic; MSCsNC transfected with negative control of microRNA mimic). (*) denotes P<0.05 for significant difference (n = 3). D. TUNEL assay showed reduced apoptosis in cardiomyocytes (co-cultured with MSCsmiR-22) as compared to control. (*) denotes P<0.05 for significant difference (n = 3).
Mentions: To determine whether miR-22 secreted from MSCs can transfer to cardiomyocytes in a non-contact manner, first, we transfected MSCs with a miR-22 mimic that was labeled with green fluorescein (MSCmiR-22) and then washed the MSCs with RNAse containing DMEM to degrade the residual miR-22 mimic on the MSCs. Figure S1 has shown RNase was effective. Then we employed a co-culture system of MSCs with neonatal cardiomyocytes in which the cells were separated by a membrane of 0.3 µm pore size to prevent direct cell contact or transfer of larger vesicles (Fig. 2A). Fluorescent imaging showed that after co-culture for 24 h, the green miR-22 was mainly concentrated in cardiomyocytes (Fig. 2B). In parallel, real-time PCR demonstrated that the expression of miR-22 was dramatically upregulated in cardiomyocytes co-cultured with MSCs. Moreover, these cardiomyocytes were more resistant to ischemic stress (Fig. 2C). These experiments demonstrate that miR-22 can mobilize from MSCs to cardiomyocytes.

Bottom Line: Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2).In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis.Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Provincial Cardiovascular Institute, Southern Medical University, Guangzhou, China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Mesenchymal stem cells (MSCs) have potential application for the treatment of ischemic heart diseases. Besides differentiation properties, MSCs protect ischemic cardiomyocytes by secretion of paracrine factors. In this study, we found exosomes enriched with miR-22 were secreted by MSCs following ischemic preconditioning (Exo(IPC)) and mobilized to cardiomyocytes where they reduced their apoptosis due to ischemia. Interestingly, by time-lapse imaging, we for the first time captured the dynamic shedding of miR-22 loaded exosomes from cytosol to extracellular space. Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2). In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis. Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

Show MeSH
Related in: MedlinePlus