Early vessel destabilization mediated by Angiopoietin-2 and subsequent vessel maturation via Angiopoietin-1 induce functional neovasculature after ischemia.

We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and
partial antagonist of Angiopoietin-1 (Ang1), is required for early
vessel destabilization during postischemic angiogenesis, when
combined with vascular growth factors. In vitro, matrigel
co-cultures assessed endothelial-cell tube formation and pericyte
recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with
or without Ang2. In a murine hindlimb ischemia model,
adeno-associated virus (rAAV, 3×10(12) virusparticles) transduction
of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early
expression d0-3) was performed intramuscularly (d-14). Femoral
artery ligation was performed at d0, followed by laser doppler
perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and
d14 (late timepoint), histological analysis of capillary/muscle
fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R,
NG2) was performed. In vitro, VEGF-A, APLN and Ang1 induced ring
formation, but only APLN and Ang1 recruited pericytes. Ang2 did not
affect tube formation by APLN, but reduced pericyte recruitment
after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not
alter LDI-perfusion at d14, consistent with an impaired PC-R
despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with
or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1
improved perfusion at d14, when combined with rAAV.Ang2 (d0-3),
accompanied by an increased CMF-R and PC-R. The combination of
early vessel destabilization (Ang2 d0-3) and continuous Ang1
overexpression improves hindlimb perfusion, pointing to the
importance of early vessel destabilization and subsequent vessel
maturation for enhanced therapeutic neovascularization.