Insulin growth factor 2 contributes to adipose stem cell-derived exosome mediated angiogenesis against hind-limb ischemia injury

Critical limb ischemia is a severe stage when hypoperfusion occurs in patients with lower extremity arteriosclerotic obliterans. Exosomes have emerged as a key therapeutic agent for lower extremity ischemia. However, how these exosomes take effects on ischemic limb is still unknown. Insulin-like growth factor II (IGF2) is a key protein responsible for guiding angiogenesis. Mouse ischemic limb was injected with phosphate buffer saline (PBS), exosomes from murine adipose-derived stem cells (ADSC-Exo) and IGF2-specific knockout of ADSC-Exo (IGF2^−/−-Exo), respectively, then blood flow was detected at 21 days after injection. The level of dynamic limb movement and impairment were assessed by ambulatory impairment and tissue damage scores. Laser Doppler imaging was performed to measure the blood flow of the ischemic limbs. Molecular biology experiment is performed to investigate the mechanism of ADSC-Exo mediated limb protection against ischemia injury. Compared with the untreated group, ADSC-Exo injection significantly promoted blood perfusion of ischemic hind limbs, while knockout of IGF2 weakened the therapeutic effect of the ADSC-Exo. The fluorescence intensity of CD31⁺ in the IGF2^−/−-Exo-treated group was lower than that in the ADSC-Exo group. Tubular structures of endothelial cells in ADSC-Exo-treated group were significantly more than those in PBS-treated group, while endothelial cells in IGF2^−/−-Exo-treated group formed fewer tubular structures than those in ADSC-Exo-treated group. The protein levels of p-PI3K, p-AKT and p-eNOS in endothelial cells were upregulated by ADSC-Exo whereas IGF2-knockout impaired this promotion to activation states. Our study demonstrated that IGF2-expressing ADSC-Exo exerted a potent pro-proliferative and angiogenic effects against limb ischemia injury associated with the PI3K-Akt-eNOS pathway.