Overexpression of miR-126 promotes the differentiation of mesenchymal stem cells toward endothelial cells via activation of PI3K/Akt and MAPK/ERK pathways and release of paracrine factors

Feng Huang 1 , Zhen-fei Fang 1 , Xin-qun Hu 1 , Liang Tang 1 , Sheng-hua Zhou 1 , and Jian-ping Huang 2
  • 1 Department of Cardiology, Second Xiangya Hospital of Central South University, Changsha 410011, Hunan, China
  • 2 Alibaba Business College, Hangzhou Normal University, Hangzhou 310036, Zhejiang, China
Feng Huang, Zhen-fei Fang, Xin-qun Hu, Liang Tang, Sheng-hua Zhou and Jian-ping Huang

Abstract

The endothelial cell (EC)-specific miRNA, miR-126, is known to promote angiogenesis in response to angiogenic factors by repressing negative regulators of signal transduction pathways; however, whether miR-126 might regulate the differentiation of stem cells toward endothelial lineage remains unknown. To answer this question, in this study mesenchymal stem cells (MSCs) harvested from C57BL/6 mouse bone marrow were transfected with miR-126 (MSCmiR-126) using recombinant lentiviral vectors. Results showed the para-secretion and the expression levels of phosphorylated PI3K p85, Akt, p38, ERK1 protein in the MSCmiR-126 group were dramatically increased when compared with the control group. With half culture medium refreshed every 3 days, a small number of 6-day-cultured MSCmiR-126 differentiated into endothelial-like cells and most of 9-day-cultured MSCmiR-126 formed a cobblestone-like structure. These differentiated cells evidently expressed EC-specific makers and possessed mature ECs function, while inhibition of paracrine factors suppressed the MSC-EC differentiation. Strikingly, the increased secretion of MSCmiR-126 and their endothelial-differentiated potential were deprived by using a PI3K or MEK chemical inhibitor. Our results suggest that overexpression of miR-126 agumenting the endothelial differentiation of MSCs might in part be attributable to the activation of PI3K/Akt and MAPK/ERK pathways and an increased release of paracrine factors.

  • Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.

    • Crossref
  • Chen, J.J. and Zhou, S.H. (2011). Mesenchymal stem cells overexpressing MiR-126 enhance ischemic angiogenesis via the AKT/ERK-related pathway. Cardiol. J. 18, 675–681.

    • Crossref
  • Dimmeler, S., Aicher, A., Vasa, M., Mildner-Rihm, C., Adler, K., Tiemann, M., Rutten, H., Fichtlscherer, S., Martin, H., and Zeiher, A.M. (2001). HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J. Clin. Invest. 108, 391–397.

    • Crossref
  • Evseenko, D., Zhu, Y., Schenke-Layland, K., Kuo, J., Latour, B., Ge, S., Scholes, J., Dravid, G., Li, X., MacLellan, W.R., et al. (2010). Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells. Proc. Natl. Acad. Sci. USA 107, 13742–13747.

    • Crossref
  • Fish, J.E., Santoro, M.M., Morton, S.U., Yu, S., Yeh, R.F., Wythe, J.D., Ivey, K.N., Bruneau, B.G., Stainier, D.Y., and Srivastava, D. (2008). miR-126 regulates angiogenic signaling and vascular integrity. Dev. Cell 15, 272–284.

    • Crossref
  • Gnecchi, M., He, H., Noiseux, N., Liang, O.D., Zhang, L., Morello, F., Mu, H., Melo, L.G., Pratt, R.E., Ingwall, J.S., et al. (2006). Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 20, 661–669.

    • Crossref
    • PubMed
  • Gnecchi, M., Zhang, Z., Ni, A., and Dzau, V.J. (2008). Paracrine mechanisms in adult stem cell signaling and therapy. Circ. Res. 103, 1204–1219.

    • Crossref
  • Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H., and Kim, V.N. (2004). The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.

    • PubMed
  • He, L. and Hannon, G.J. (2004). MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531.

    • Crossref
    • PubMed
  • Ivey, K.N., Muth, A., Arnold, J., King, F.W., Yeh, R.F., Fish, J.E., Hsiao, E.C., Schwartz, R.J., Conklin, B.R., Bernstein, H.S., et al. (2008). MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2, 219–229.

    • Crossref
    • PubMed
  • Kane, N.M., Meloni, M., Spencer, H.L., Craig, M.A., Strehl, R., Milligan, G., Houslay, M.D., Mountford, J.C., Emanueli, C., and Baker, A.H. (2010). Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 30, 1389–1397.

    • Crossref
  • Kane, N.M., Howard, L., Descamps, B., Meloni, M., McClure, J., Lu, R., McCahill, A., Breen, C., Mackenzie, R.M., Delles, C., et al. (2012). Role of microRNAs 99b, 181a, and 181b in the differentiation of human embryonic stem cells to vascular endothelial cells. Stem Cells 30, 643–654.

    • Crossref
  • Kinnaird, T., Stabile, E., Burnett, M.S., Lee, C.W., Barr, S., Fuchs, S., and Epstein, S.E. (2004). Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ. Res. 94, 678–685.

    • Crossref
  • Krampera, M., Pasini, A., Rigo, A., Scupoli, M.T., Tecchio, C., Malpeli, G., Scarpa, A., Dazzi, F., Pizzolo, G., and Vinante, F. (2005). HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation. Blood 106, 59–66.

    • Crossref
  • Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.

    • Crossref
  • Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H., and Kim, V.N. (2004). MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060.

    • Crossref
    • PubMed
  • Li, H., Zuo, S., He, Z., Yang, Y., Pasha, Z., Wang, Y., and Xu, M. (2010). Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival. Am. J. Physiol. Heart Circ. Physiol. 299, H1772–1781.

    • Crossref
  • Lim, S.Y., Kim, Y.S., Ahn, Y., Jeong, M.H., Hong, M.H., Joo, S.Y., Nam, K.I., Cho, J.G., Kang, P.M., and Park, J.C. (2006). The effects of mesenchymal stem cells transduced with Akt in a porcine myocardial infarction model. Cardiovasc. Res. 70, 530–542.

    • Crossref
  • Ohtani, K. and Dimmeler, S. (2011). Control of cardiovascular differentiation by microRNAs. Basic Res. Cardiol. 106, 5–11.

    • Crossref
  • Small, E.M. and Olson, E.N. (2011). Pervasive roles of microRNAs in cardiovascular biology. Nature 469, 336–342.

    • Crossref
  • Takaya, T., Ono, K., Kawamura, T., Takanabe, R., Kaichi, S., Morimoto, T., Wada, H., Kita, T., Shimatsu, A., and Hasegawa, K. (2009). MicroRNA-1 and MicroRNA-133 in spontaneous myocardial differentiation of mouse embryonic stem cells. Circ. J. 73, 1492–1497.

    • Crossref
  • Tiscornia, G. and Izpisua Belmonte, J.C. (2010). MicroRNAs in embryonic stem cell function and fate. Genes Dev. 24, 2732–2741.

    • Crossref
    • PubMed
  • Urbich, C., Aicher, A., Heeschen, C., Dernbach, E., Hofmann, W.K., Zeiher, A.M., and Dimmeler, S. (2005). Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J. Mol. Cell. Cardiol. 39, 733–742.

    • Crossref
  • Urbich, C., Kuehbacher, A., and Dimmeler, S. (2008). Role of microRNAs in vascular diseases, inflammation, and angiogenesis. Cardiovasc. Res. 79, 581–588.

    • Crossref
  • Wang, S., Aurora, A.B., Johnson, B.A., Qi, X., McAnally, J., Hill, J.A., Richardson, J.A., Bassel-Duby, R., and Olson, E.N. (2008). The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev. Cell 15, 261–271.

    • Crossref
  • Wang, H., Cao, F., De, A., Cao, Y., Contag, C., Gambhir, S.S., Wu, J.C., and Chen, X. (2009). Trafficking mesenchymal stem cell engraftment and differentiation in tumor-bearing mice by bioluminescence imaging. Stem Cells 27, 1548–1558.

    • Crossref
    • PubMed
  • Wu, F., Yang, Z., and Li, G. (2009). Role of specific microRNAs for endothelial function and angiogenesis. Biochem. Biophys. Res. Commun. 386, 549–553.

    • Crossref
  • Xu, J., Liu, X., Jiang, Y., Chu, L., Hao, H., Liua, Z., Verfaillie, C., Zweier, J., Gupta, K., and Liu, Z. (2008). MAPK/ERK signalling mediates VEGF-induced bone marrow stem cell differentiation into endothelial cell. J. Cell. Mol. Med. 12, 2395–2406.

    • Crossref
  • Zou, J., Li, W.Q., Li, Q., Li, X.Q., Zhang, J.T., Liu, G.Q., Chen, J., Qiu, X.X., Tian, F.J., Wang, Z.Z., et al. (2011). Two functional microRNA-126s repress a novel target gene p21-activated kinase 1 to regulate vascular integrity in zebrafish. Circ. Res. 108, 201–209.

    • Crossref
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