Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors

Juliana Maíra Freitas Vieira; Laura Nicoleti Zamproni; Camila H C Wendt; Kildare Rocha de Miranda; Rafael Soares Lindoso; Sang Won Han

doi:10.1371/journal.pone.0272962

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors

PLoS One. 2022 Aug 16;17(8):e0272962. doi: 10.1371/journal.pone.0272962. eCollection 2022.

Authors

Juliana Maíra Freitas Vieira¹, Laura Nicoleti Zamproni², Camila H C Wendt^{3

4}, Kildare Rocha de Miranda^{3

4}, Rafael Soares Lindoso^{3

5}, Sang Won Han¹

Affiliations

¹ Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil.
² Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil.
³ Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
⁴ National Center for Structural Biology and Bioimaging/CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
⁵ National Institute of Science and Technology for Regenerative Medicine-REGENERA, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Abstract

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Angiogenesis Inducing Agents / metabolism
Animals
Extracellular Vesicles* / genetics
Extracellular Vesicles* / metabolism
Human Umbilical Vein Endothelial Cells / metabolism
Humans
Mesenchymal Stem Cells*
Mice
MicroRNAs* / genetics
MicroRNAs* / metabolism

Substances

Angiogenesis Inducing Agents
MIRN210 microRNA, human
MIRN296 microRNA, human
MicroRNAs

Grants and funding

JMFV received a scholarship from FAPESP (2018/21072-3). This work was supported by FAPESP (2015/20206-8) and CNPQ (303646/2019-5).