Abstract 3647: Identification of the Pro-Angiogenic Capacity of Neurotrophin 3
Neurotrophin 3 (NT-3) is a member of the neurotrophin family, a class of growth factors characterized as critical regulators of neural survival and growth. NT-3 was shown to be involved in mammalian pre-natal heart development. We hypothesize that the cardiovascular actions of NT-3 extend to the adult life. The present study, that was developed in a mouse limb ischemia model, is the first to document the pro-angiogenic activity of NT-3. Anaesthetized CD1 mice underwent left femoral artery occlusion and NT-3 was over-expressed in the ischemic adductor by local injection of an adenoviral vector carrying the rat NT-3 gene (Ad.NT-3, 10^8 PFU). Control mice received Ad.Null. Blood flow (BF) recovery to the ischemic foot was measured at 7d and 14d from ischemia by laser Doppler flowmetry. Limb muscles were then harvested to analyse capillary and arteriole density and expression of trkC (receptor of NT-3) in capillary endothelial cells (EC). To assess whether NT-3 stimulates EC proliferation, additional groups of mice received BrdU (25 mg/kg BW, IP, daily from surgery) and were sacrificed after 3d. EC proliferation was determined by staining for both incorporated BrdU and proliferating cell nuclear antigen (PCNA). Using different mice, levels of phosphorylated trkC, Akt (Ser473) and eNOS (Ser1177) were analyzed in ischemic muscles by western blot, whereas VEGF-A content was measured by real time PCR and ELISA. We found that capillary EC express trkC, thus suggesting possible direct NT-3 actions on these cells. Ad.NT-3 improved BF to the ischemic foot at both 7d (72±3% of perfusion recovery vs. 64±2% in Ad.Null, P<0.05) and 14d post-ischemia (82±2% vs. 68±4% in Ad.Null, P<0.01), increased capillary (1150±60 vs. 635±88 capillaries/mm2 in Ad.Null, P<0.01) and arteriole (12.9±0.4 vs. 8.7±1 arterioles/ mm2 in Ad.Null, P<0.05) density in ischemic muscles and enhanced EC proliferation (BrdU-positive EC/mm2: 17.4±2.7 vs. 6.6±2.7 in Ad.Null, P< 0.05; PCNA-positive EC/mm2: 5.4±0.6 vs. 1.7±0.7 in Ad.Null, P<0.01). Moreover, Ad.NT-3 stimulated trkC, Akt and eNOS phosphorylation (P<0.05 vs. Ad.Null for all comparisons), without changing VEGF-A expression. Taken together, our results provide evidence that NT-3 promotes therapeutic angiogenesis in ischemic limbs.