Ultrasound molecular imaging of the angiogenic response induced by triple-point mutations of hypoxia-inducible factor-1.ALPHA. in aged mice with hind-limb ischemia

Abstract

Objective To evaluate the angiogenic response of the HIF-1α with triple-point mutations in aged mice with hind-limb ischemia by ultrasound molecular imaging of αv-integrin expression. Methods Twenty four aged mice under unilateral artery ligation were equally divided into 2 test groups and adenovirus (Ad) vector particles containing the gene for LacZ (Ad-LacZ) or HIF-1a 402/564/803(triple-point mutant, Ad-TM) were injected intramuscularly into the ischemic hind limb.Serial contrast-enhanced ultrasound perfusion imaging of the proximal adductor muscles in the ischemic and contralateral non-ischemic hind limbs was performed (n=6 per group at each time). Ultrasound molecular imaging of the endothelial αv-integrin expression was carried out in mice(n=6 per group) on day 7 after surgery at 8 minutes after intravenous injection of either αv-integrin targeting microbubbles or isotype control microbubbles in random with 30 min interval and the video intensity (VI) was measured.Following ultrasound imaging, the hind-limb was harvested for histological examination of αv-integrin expression. Results Immediately after femoral artery ligation, blood flow (as a percentage of that in the non-ischemia hind limb) was reduced to 15% in the 2 test groups (P>0.05). Although blood flow gradually recovered over time, recovery was persistently better in the Ad-TM group as compared with the Ad-LacZ group. On postoperative day 28, the blood flow ratio was significantly increased in the Ad-TM group (65.6±8.0)% as compared with the Ad-LacZ group (30.7±4.5)% (P<0.01). Minimal ultrasound signals were obtained with both MBαv [VI, (5.2±1.5)U] and MBiso [VI, (5.8±2.2)U] in the non-ischemic hind-limb.Progressive signal enhancement was noted in the ischemic hind limbs with MBαv in the order of the Ad-lacZ group [VI, (15.9±2.8)U], Ad-TM group [VI, (30.8±5.1)U], whereas no significant change of signals was noted in the Ad-TM group[VI, (7.9±1.6)U] and Ad-LacZ group[VI, (8.1±2.0)U] with MBiso. No αv-integrin expression was observed in the non-ischemic hind-limb, a marked endothelial αv-integrin expression in ischemic hind-limb was confirmed by immunofluorescence, especially in the Ad-TM group. Conclusions The angiogenic response is enhanced in the aged mice with hind-limb ischemia by using HIF-1α with triple-point mutations. Key words: Molecular probes; Ultrasound molecular imaging; Hind-limb ischemia; Angiogenesis; αv-integrin