Abstract 5802: Ischemia-induced Angiogenesis is Impaired in Aminopeptidase-A deficient Mice with Decreased Stability of HIF-1α
Background: Aminopeptidase A (APA) is a membrane-bound metalloprotease degreading bioactive peptidase by cleaving from N-terminal amino acids with several functions in angiogenesis. To assess whether APA regulates ischemia-induced angiogenesis, we explored the mechanism by which APA affects angiogenesis under hypoxia conditions and evaluated the process of perfusion recovery in the hindlimb ischemia model of APA knock-out mice (APAKO).
Methods and Results: Western blotting analysis and immunostaining of endothelial cells (ECs) from mouse aorta revealed that the accumulation HIF-1α protein and nuclear translocation in response to hypoxic challenge (1%O2, 12h) were blunted in APAKO (HIF-1α/β-Actin ratio, C57BL/6J (WT) vs APAKO: 0.987±0.069 vs 0.467±0.063, mean±SE, p=0.018). Although the levels of E3 ubiquitin ligase Siah2 and prolyl-hydroxylase 3 (PHD3), both upstream regulators of HIF-1α, did not alter under hypoxia (1%O2, 6h), proteasome inhibitor MG-132 restored the decreased accumulation of HIF-1α in ECs from APAKO similar to WT. Tube formation assay under hypoxia showed ECs of APAKO formed significantly less endothelial network (Tube length, mm/field, WT vs APAKO: 35.2±3.4 vs 27.6±5.0, p=0.024), and this was partly reversed by VEGF (50ng/mL) treatment (31.2±4.8, p=0.048), but not by soluble APA peptide supplementation (26.6±4.3, p=NS). APAKO and WT mice were subjected to unilateral hindlimb ischemia. Laser Doppler perfusion imaging demonstrated decreased blood flow recovery in APAKO, (Ischemic/Non ischemic limb, WT vs APAKO: 0.80±0.07 vs 0.70±0.04, at 4 weeks, p=0.046). Tissue capillary density at 2 weeks was significantly decreased in APAKO (ECs/Muscle Ratio, WT vs APAKO: 1.57±0.15 vs 1.25±0.12; p=0.039). FACS analysis of endothelial progenitor cells revealed no significant difference between in APAKO and WT mice, suggesting that vasculogenesis was minimally involved in the reparative process from ischemia in APAKO mice.
Conclusion: Ischemia-induced angiogenesis is impaired in APAKO mice due to decreased stability of HIF-1α with upregulation of proteasomal degradation under hypoxic conditions and subsequent shortage of growth factor secretion. APA would be a functional target of ischemia-induced angiogenesis.