Abstract 239: Hemeoxygenase-1 Promotes Functional Regeneration after Critical Hind-Limb Ischemia
Neovascularization is crucial for preservation of tissue function after critical ischemia. We postulated that hemeoxygenase-1 (HO-1), a stress- and ischemia-inducible enzyme, may be involved in neovascularization and functional recovery after hind-limb ischemia. Hind-limb ischemia was induced in 10 to 12 week-old, male FVB/N mice by complete ligation of the proximal femoral artery including all superficial and deep branches. The day before the procedure and every other day thereafter, mice were treated with the HO-1 inhibitor tin protoporphyrin-IX (SnPP, 25 mg/kg i.p.). Complete hind-limb ischemia was verified by laser Doppler imaging. Doppler-derived flow (ischemic vs. non-ischemic limb ratio) did not differ between placebo and SnPP-treated animals immediately after femoral artery ligation (placebo: 0.28±0.01 vs. SnPP: 0.27±0.02 units; n=10, mean±SEM, P=n.s.). Recovery of hind-limb perfusion, however, was delayed in SnPP-treated animals 3 days (placebo: 0.76±0.06 vs. SnPP: 0.42±0.07; P<0.01) and 7 days (placebo: 0.91±0.04 vs. SnPP: 0.65±0.02; P<0.01) after femoral artery ligation, which was associated with an enhanced amputation rate (amputation score: placebo: 1.2±0.1 vs. SnPP: 1.9±0.2; P<0.01). FACS analyses performed 7 days after femoral artery ligation, revealed a significant impairment of sca-1+/kdr+ endothelial progenitor cell (EPC) mobilization into the circulation during HO-1 inhibition (sham-OP/placebo: 100%, sham-OP/SnPP: 101±3%, hind-limb ischemia/placebo: 215±9%, hind-limb ischemia/SnPP: 120±9%, P<0.01). As shown by immunohistochemistry, capillary density (isolectin+ cells per myocyte) in the ischemic gastrocnemius muscle after 7 days was significantly lower in SnPP-treated mice (placebo: 1.5±0.1 vs. SnPP: 1.0±0.1; P<0.01). After transplantation of Tie2-promotor/lacZ-reporter transgenic bone-marrow into lethally-irradiated wild-type mice, and consecutive femoral artery ligation, less lacZ-positive, putative endothelial cells were detected in the ischemic muscle during HO-1 inhibition (placebo: 8.3<1.5 vs. SnPP: 5.4<1.1 cells/mm2; P<0.05). In conclusion, HO-1 promotes mobilization and homing of EPCs, neovascularization, and functional regeneration after critical hind-limb ischemia.