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

Yuliang Feng, Wei Huang, Mashhood Wani, Xiyong Yu, Muhammad Ashraf, Yuliang Feng, Wei Huang, Mashhood Wani, Xiyong Yu, Muhammad Ashraf

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. miR-22 is upregulated in Exo…
Figure 1. miR-22 is upregulated in ExoIPC.
A. Electron microscopy was used to image the structure of an exosome, which was approximately 100 = 100 nm. B. Histogram showing exosome size distribution. X values: Vesicle diameter (nm); Y value: frequency of vesicles. C. Equal amounts of proteins from exosomes and culture medium were analyzed by western blotting for the exosome-enriched protein, CD63. D. RNA analysis by bioanalyzer showed no ribosomal RNA 28 s and 18 s presence. E. microRNA microarray data showing upregulation of miRs in ExoIPC as compared to Exonon-IPC. ExoIPC exosomes from MSCs following ischemic preconditioning. Exonon-IPC: exosomes from MSCs without ischemic preconditioning. FC: fold change (ExoIPC vs. Exonon-IPC). F. qPCR was performed to confirm the enrichment of miR-22 in exosomeIPC as compared to exosomenon-IPC. cel-miR-39 was used as invariant control. (*) denotes P<0.05 for significant difference (n = 3). ExoIPC exosomes from MSCs following ischemic preconditioning.
Figure 2. miR-22 transfers from MSCs to…
Figure 2. 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).
Figure 3. Time-lapse imaging for the transfer…
Figure 3. Time-lapse imaging for the transfer of exosomal miR-22.
A. Flow chart of time-lapse confocal imaging experiment. B. Live cell imaging of MSCs co-transfected with lentivirus overexpressing fusion protein CD63-RFP (red) and miR-22 labeled with fluorescein (green).
Figure 4. miR-22 targets Mecp2.
Figure 4. miR-22 targets Mecp2.
A. Target scan showed that Mecp2 is predicted target of miR-22 with 4 potential binding sites on its 3′UTR. B. Western blot was performed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM) in MSCs. C. Luciferase activity was employed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM). (*) denotes P<0.05 vs. control for significant difference (n = 3). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). D. Western blot showing upregulation of Mecp2 in infarcted hearts. E. Western blot showing successful knockdown of Mecp2 in infarcted hearts with siRNA (si-Mecp2) F. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by si-Mecp2. (*) denotes P<0.05 vs. control for significant difference (n = 3 for each group). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). G. Western blot showing downregulation of Mecp2 in infarcted hearts by miR-22 mimic. H. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by miR-22 mimic. (*) denotes P<0.05 vs. control for significant difference. (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3 for each group).
Figure 5. Exo IPC ameliorated cardiac fibrosis.
Figure 5. ExoIPC ameliorated cardiac fibrosis.
A. Masson trichrome staining to determine the infarct size in various groups. B. Quantification of infarct size in various groups. (*) denotes P<0.05 vs. Scr for significant difference. (#) denotes P<0.05 vs. si-Mecp2, miR-22 mimic, Exonon-IPC, ExoIPC+miR-22 inhi for significant difference (n = 8). (∥) denotes P<0.01 vs. Exonon-IPC for significant difference (n = 8 for each group).

References

    1. Kim HW, Haider HK, Jiang S, Ashraf M (2009) Ischemic preconditioning augments survival of stem cells via miR-210 expression by targeting caspase-8-associated protein 2. J Biol Chem 284: 33161–33168.
    1. Leroux L, Descamps B, Tojais NF, Seguy B, Oses P, et al. (2010) Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4-dependent pathway. Mol Ther 18: 1545–1552.
    1. Ranganath SH, Levy O, Inamdar MS, Karp JM (2012) Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease. Cell Stem Cell 10: 244–258.
    1. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, et al. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9: 654–659.
    1. Buccini S, Haider KH, Ahmed RP, Jiang S, Ashraf M (2012) Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol 107: 301.
    1. Roccaro AM, Sacco A, Maiso P, Azab AK, Tai YT, et al. (2013) BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. J Clin Invest 123: 1542–1555.
    1. Kroh EM, Parkin RK, Mitchell PS, Tewari M (2010) Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods 50: 298–301.
    1. Lu G, Haider HK, Jiang S, Ashraf M (2009) Sca-1+ stem cell survival and engraftment in the infarcted heart: dual role for preconditioning-induced connexin-43. Circulation 119: 2587–2596.
    1. Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3: 3–22.
    1. Gnecchi M, Danieli P, Cervio E (2012) Mesenchymal stem cell therapy for heart disease. Vascul Pharmacol 57: 48–55.
    1. Pasha Z, Wang Y, Shiekh R, Zhang D, Zhao T, et al. (2008) Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium. Cardiovasc Res 77 1: 134–42.
    1. Uemura R, Xu M, Ahmad N, Ashraf M (2006) Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling. Circ Res 98: 1414–1421.
    1. Gnecchi M, He H, Liang OD, Melo LG, Morello F, et al. (2005) Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med 11: 367–368.
    1. Hergenreider E, Heydt S, Treguer K, Boettger T, Horrevoets AJ, et al. (2012) Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat Cell Biol 14: 249–256.
    1. Mittelbrunn M, Gutierrez-Vazquez C, Villarroya-Beltri C, Gonzalez S, Sanchez-Cabo F, et al. (2011) Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat Commun 2: 282.
    1. Huang ZP, Chen J, Seok HY, Zhang Z, Kataoka M, et al. (2013) MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress. Circ Res 112: 1234–1243.
    1. Gurha P, Abreu-Goodger C, Wang T, Ramirez MO, Drumond AL, et al. (2012) Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction. Circulation 125: 2751–2761.
    1. Katare R, Riu F, Mitchell K, Gubernator M, Campagnolo P, et al. (2011) Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Circ Res 109: 894–906.
    1. Alvarez-Saavedra M, Carrasco L, Sura-Trueba S, Demarchi AV, Walz K, et al. (2010) Elevated expression of MeCP2 in cardiac and skeletal tissues is detrimental for normal development. Hum Mol Genet 19: 2177–2190.

Source: PubMed

Sponsorit ja yhteistyökumppanit

Sairaudet

Huumeiden interventiot

3
Tilaa