Hypoxic preconditioning-induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb.

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell-based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning-induced autophagy on survival of the engrafted EPCs. Autophagy of CD34(+) VEGFR-2(+) EPCs isolated from rat bone marrow increased after treatment with 1% O(2) . The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3-methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O(2) for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler-based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.