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. Approach and 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.