Hypoxia inhibits cellular senescence to restore the therapeutic potential of old human endothelial progenitor cells via the hypoxia-inducible factor-1α-TWIST-p21 axis.
OBJECTIVE
Endothelial progenitor cells (EPCs) can significantly improve tissue repair by providing regeneration potential within injured cardiovascular tissue; however, it is challenging to obtain a sufficient amount of functional EPCs from aged patients for autologous stem cell therapy. To overcome this issue, we aimed to establish adequate ex vivo expansion protocols and identify repair modulators of cellular senescence. The senescence repair circuit of hypoxia-preconditioned senescent EPCs (hyp-old EPCs) was examined in an effort to enhance their regenerative potential.


RESULTS
Long-term culturing of EPCs in normoxic conditions markedly induced the expression of p21, whereas siRNA targeting of p21 in old EPCs significantly enhanced the proliferation potential of cells. Hyp-old EPCs displayed increased hypoxia-inducible factor-1α and TWIST expression. siRNA inhibition of TWIST, a target molecule of the hypoxia-inducible factor-1α, markedly upregulated the expression of p21 in hyp-old EPCs by reprogramming cell-cycle regulatory proteins. In a hindlimb model of ischemia, the transplantation of hyp-old EPCs enhanced the blood flow ratio and capillary density, improved cellular proliferation and cell survival at ischemic sites, and augmented the secretion of pivotal tissue angiogenic cytokines. It has been previously demonstrated that the restoration of old EPCs from a senescent state by hypoxia preconditioning is tightly mediated by the downregulation of p21 via the hypoxia-inducible factor-1α-TWIST axis.


CONCLUSIONS
This study introduces ex vivo expansion protocols involving hypoxic preconditioning that are suitable for efficiently expanding old EPCs without senescence through modulation of the hypoxia-induced hypoxia-inducible factor-1α-TWIST-p21 axis. In addition, the expanded cells are shown to be useful for therapeutic vasculogenesis.
From the Laboratory for Vascular Medicine and Stem Cell Biology, Medical Research Institute, Department of Physiology, School of Medicine, Pusan National University, Yangsan, Korea (S.H.L., J.H.L., S.Y.Y., S.M.K.); Immunoregulatory Therapeutics Group in Brain Busan 21 Project (S.H.L., J.H.L., S.Y.Y., S.M.K.), Department of Medical Biolotechnology (S.H.L.), Soonchunhyang University College of Medicine, Seoul, Korea; National Research Laboratory for Cardiovascular Stem Cell Niche, Innovative Research Institute for Cell Therapy (J.H., H.S.K.), Cardiovascular Center and Department of Internal Medicine, Seoul National University Hospital, and Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Korea (H.S.K.).
Sang Hun
SH
Lee
Jun Hee
JH
Lee
So Young
SY
Yoo
Jin
J
Hur
Hyo-Soo
HS
Kim
Sang Mo
SM
Kwon
D042501:Angiogenic Proteins / Q000378:metabolism; D000818:Animals; D002453:Cell Cycle; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism* / Q000637:transplantation*; D006614:Hindlimb; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology / Q000601:surgery*; D051379:Mice; D018482:Muscle, Skeletal / Q000098:blood supply*; D018919:Neovascularization, Physiologic; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020127:Recovery of Function; D012039:Regional Blood Flow; D015398:Signal Transduction; D013234:Stem Cells / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism*
D042501:Angiogenic Proteins; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1
aging; anoxia; progenitor cells; tissues; transplantation