Abstract 1047: Role of Angiopoietin-2 in Neovascularization of the Chronically Ischemic Hindlimb of Transgenic Mice: Effect on Mobilization of Bone Marrow Progenitor Cells
Angiopoietin-1 (Ang1) activates Tie2 to promote endothelial cell (EC) quiescence and sequestration of bone marrow (BM) stem cells, and inhibits vascular inflammation. In contrast, Ang2 is a context-dependent antagonist of the Tie2 receptor mediating EC activation and angiogenesis, and also acts as an autocrine regulator of the EC inflammatory response.
Hypothesis: Ang2 is more effective than Ang1 in stimulating neovascularization in the mouse ischemic hindlimb, in part, due to its effects on mobilization of BM-derived endothelial progenitor cells (EPCs) and upregulation of homing signals, such as monocyte chemoattractant protein-1 (MCP1).
Methods: Binary transgenic (BT) mice were generated with liver-specific (Lap1) conditional overexpression of human Ang1 or Ang2 driven by a doxycycline (Dox) responsive promoter, providing an efficient system for Ang delivery into the circulation. Non-BT littermates served as controls. Femoral artery ligation was performed on day 0, together with Dox withdrawal to induce transgene expression. Limb perfusion, assessed with laser doppler imaging, is expressed as a ratio of ischemic (I) to non-ischemic (NI) leg. Effects of Ang1 or Ang2 overexpression on BM-derived circulating EPCs (%c-kit+/VEGFR2+ cells) was quantified by flow cytometry on day 7. MCP1 expression in mouse liver and cultured human microvascular ECs treated with Ang1 or Ang2 (0–250ng/ml, n=3) was determined by qRT-PCR.
Results: Plasma ELISA confirmed induction of transgene overexpression (>20-fold). Ang2 (n=5) but not Ang1 (n=5), increased ischemic limb perfusion (0.79±0.12 vs. 0.52±0.03, p<0.01) compared to controls (0.45±0.03, p<0.005, n=6). Ang2 increased circulating EPCs (1.60±0.47%, n=5) compared to Ang1 (0.13±0.08, n=4; (p<0.05)) or controls (0.48±0.22, n=6). Liver MCP1 expression was enhanced only in Ang2 BT mice (n=4). ECs treated with Ang1 or Ang2 resulted in upregulation (p<0.05) of MCP1 at 6hr that was sustained (24hr) only by Ang2.
Conclusions: Ang2 was superior to Ang1 in enhancing ischemic limb perfusion in transgenic mice. Ang2 may augment neovascularization by novel mechanisms involving stimulation of proinflammatory cytokine expression and mobilization of BM-derived EPCs.