Extracellular vesicles of ETV2 transfected fibroblasts stimulate endothelial cells and improve neovascularization in a murine model of hindlimb ischemia

Abstract

Ischemia are common conditions related to lack of blood supply to tissues. Depending on the ischemic sites, ischemia can cause different diseases, such as hindlimb ischemia, heart infarction and stroke. This study aims to evaluate how extracellular vesicles (EVs) derived from ETV2 transfected fibroblasts affect endothelial cell proliferation and neovascularization in a murine model of hindlimb ischemia. Human fibroblasts were isolated and cultured under standard conditions and expanded to the 3th passage before use in experiments. Human fibroblasts were transduced with a viral vector containing the ETV2 gene. Transduced cells were selected by puromycin treatment. These cells were further cultured for collection of EVs, which were isolated from culture supernatant. Following co-culture with endothelial cells, EVs were evaluated for their effect on endothelial cell proliferation and were directly injected into ischemic tissues of a murine model of hindlimb ischemia. The results showed that EVs could induce endothelial cell proliferation in vitro and improved neovascularization in a murine model of hindlimb ischemia. Our results suggest that EVs derived from ETV2-transfected fibroblasts can be promising non-cellular products for the regeneration of blood vessels.

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Abbreviations

ADSCs:

Adipose derived stem cells

ALI:

Acute hindlimb ischemia

EPCs:

Endothelial progenitor cells

ETV-2:

ETS variant 2

EVs:

Extracellular vesicles

HUVECs:

Human umbilical vein endothelial cells

MCECs:

Mouse cardiac endothelial cells

MSCs:

Mesenchymal stem cells

PBMCs:

Peripheral blood mononuclear cells

PBS:

Phosphate buffer saline

VEGFR2:

Vascular endothelial growth factor receptor 2

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Acknowledgements

This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106-YS.06-2013.37.

Funding

Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106-YS.06-2013.37.

Authors’ contributions

PVP was responsible for suggesting the idea for this study, creating the experiment design, vector preparation, data analysis, writing the Discussion, preparing the figures, and revising the manuscript. VBN, TTTD, HTNL, LTP were responsible for performing murine model of hindlimb ischemia, evaluation of recovery of damaged limbs after EVs injection. OTH and MTHT were responsible for performing the essays of fibroblast culture, RT-PCR analysis, flow cytometry analysis, and writing the Methods. OTKN and NKP were were responsible for EVs isolation, characterization; performing the HUVEC cultures, Matrigel assays, and writing the Introduction. All authors read and approved the manuscript.

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Correspondence to Phuc Van Pham.

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Van Pham, P., Vu, N.B., Dao, T.TT. et al. Extracellular vesicles of ETV2 transfected fibroblasts stimulate endothelial cells and improve neovascularization in a murine model of hindlimb ischemia. Cytotechnology 69, 801–814 (2017). https://doi.org/10.1007/s10616-017-0095-2

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Keywords

  • Extracellular vesicles
  • EVs
  • ETV2
  • Fibroblasts
  • Ischemia
  • Hindlimb ischemia