Abstract 614: ERK Signaling: the Pivotal Regulator of Arterial Differentiation
Background: We have previously observed defective arterial morphogenesis characterized by reduced size and branch number of smaller arteries and ongoing low-level ischemia in mice with homozygous disruption of synectin gene expression (Chittenden et al Dev Cell 2006). Endothelial cells derived from synectin knock out (KO) mice demonstrate reduced responsiveness to VEGF, especially in terms of activation of Erk-1/2. Since Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin KO mice and endothelial cells (ECs) would restore arterial formation.
Hypothesis: Erk-1/2 activation is required for arteriogenesis
Methods: In vitro studies were conducted with primary arterial ECs isolated from synectin KO mice. In vivo studies were carried out in 5–6 week old synectin KO mice.
Results: Since partial inhibition of PI3K activity has previously been shown to activate Erk-1/2 by inhibiting a direct negative feedback loop, we treated synectin−/−and +/+ arterial EC with a selective PI3K inhibitor GS4898. Immunofluorescence confocal microscopy and Western blot showed that synectin−/− arterial EC had significantly downregulated Erk1/2 phosphorylation in response to VEGF but it was restored to normal following GS4898 administration. GS4898 treated KO EC demonstrated improved migration and the ability to form vascular tubes in 3D collagen gels in response to VEGF to the level of wild type EC. Implantation of angiogenic bioreactors in mice showed increased angiogenic response following IP administration of GS4898 was as potent as VEGF and a combination of GS4898 and VEGF was especially potent.
Conclusions: Synectin knockout is associated with reduced VEGF-dependent activation of Erk-1/2 and reduced arteriogenesis. Restoration of Erk-1/2 activation by partial inhibition of PI3K-depedent Akt activation restores this phenotype. Thus, Erk signaling plays a key role in arteriogenesis and its manipulation may provide new means for therapeutic interventions.