Abstract 348: Genetic Disruption of the PI3 Kinase γ Impairs Reparative Neovascularization of Ischemic Limb Muscles and Depresses the Functionality of Bone Marrow-Derived Progenitor Cells
The PI3 Kinase γ (PI3Kγ), which lays upstream to Akt, modulates the directed migration of leukocytes and the interaction between leukocytes and vascular cells. The present study aimed to test the role of PI3Kγ in reparative neovascularization of ischemic limb muscles. PI3Kγ knockout (KO) and wild type (WT) mice (n=10 per group) were submitted to unilateral limb ischemia. Blood flow (BF) recovery was measured by laser Doppler flowmetry and muscular capillarization by histology. The expression of PI3Kγ, total and phosho Erk1/2 (pErk1/2) and total and Ser473-phospho Akt (Akt) in muscles was analyzed by western blot. In addition, endothelial progenitor cells (EPC) were enriched from bone marrow mononuclear cells and then studied in vitro to determine the impact of PI3Kγ deletion on survival, migration toward SDF-1, and pAkt content and localization. BF recovery was delayed in KO (ischemic to contralateral ratio at 2 weeks: 0.64±0.05 vs. 0.86±0.04 in WT, P<0.01). In WT muscles, ischemia increased capillary density (1929±432 vs. 916±149 cap/mm2 in contralateral normoperfused muscles, P<0.01) and augmented the PI3Kγ mRNA content as well as the pErk1/2 and pAkt levels (P<0.05). In contrast, the ischemic muscles of KO showed impaired capillarization (557±155 vs. 777±183 cap/mm2 in contralateral, P=N.S.), abnormal leukocyte infiltration, increased myocyte apoptosis, and reduced phospho to total Akt ratio (P<0.05 vs. WT). Consistently, cultured KO EPC displayed reduced amount and decreased nuclear localization of pAkt under normoxia (1.3±0.2 vs. 5.3±1.0% of total cells in WT, P<0.05) or hypoxia (6.5±0.6 vs. 13.8±6.2%, P<0.05). The apoptosis marker, cleaved caspase-3, was present in a larger fraction of KO EPC under normoxia (22.8±3.1 vs. 12.2±1.0% of total cells in WT, P<0.05) or hypoxia (60.2±3.7 vs. 19.5±2.1%, P±0.05). Moreover, KO EPC showed impaired migratory capacity toward SDF-1 (2.9±0.4 vs.7.1±1.0% of total cells in WT, P<0.02). These findings demonstrate, for the first time, that the genetic disruption of PI3Kγ impairs post-natal angiogenesis and disturbs the functionality of bone marrow-derived EPC. Thus, PI3Kγ may represent a target for therapeutic manipulation of neovascularization.