Abstract 5109: LKB1 in Vascular Endothelial Cells is Essential for Revascularization in a Mouse Ischemic Hind-limb Model
Recent reports have indicated that LKB1 is a regulator of AMP-activated protein kinase (AMPK) signaling in many tissues including liver, skeletal muscle and cardiac myocytes. In vascular endothelial cells, AMPK signaling is essential for cell function and angiogenic responses to tissue ischemia. Homozygous LKB1 deficiency in mice leads to embryonic lethality associated with abnormal vascular development. Here, we investigated whether heterozygous LKB1 deficiency in endothelial cells has a role in regulating revascularization to tissue ischemia in hind limb ischemic mouse model. We also explored the role of AMPK signaling downstream of LKB1 in vascular endothelial cells. Homozygous vascular endothelial cell deletion of LKB1, using the conditional loxP gene regulation system crossed with Tie2-Cre transgenic mice, led to an embryonic lethal phenotype. Thus, we produced endothelial specific reduced LKB1 model, Lkb1flox/+;Tie2Tg/+ (heterozygous-KO) mice. LKB1 expression level was decreased to half in the isolated endothelial cells from heterozygous-KO mice compared with those from Lkb1flox/+;Tie2+/+ (control) mice, but mice were normal in viability, body weight and vascular density. Revascularization of hind limb following ischemic surgery was significantly impaired in heterozygous-KO mice compared with control mice as evaluated by laser Doppler Flow method and capillary density analyses at day 14 and 28 after surgery. In cultured endothelial cells, reduction of LKB1, using both siRNA and adenovirus expressing non-functional kinase dead LKB1 protein, attenuated differentiation into network structures that was accompanied by diminished phosphorylation levels of AMPK (Thr-172) and acetyl-CoA carboxylase (ACC) (Ser-79). Conversely, LKB1 over-expression by adenovirus (Ad-LKB1) augmented differentiation into network structure with elevated phosphorylation levels of AMPK, ACC and eNOS (Ser-1177). The augmented network structure induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. Taken together, our observations suggest that LKB1-AMPK axis in endothelial cells plays a pivotal role in the revascularization response to tissue ischemia.