P288: Patterns of kinase activation induced by injury in the murine femoral artery

Abstract

Background: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. Cell signaling in vascular smooth muscle cells (VSMCs) remains a potential molecular target to modulate the development of intimal hyperplasia. Transgenic murine strains allow for a detailed examination of the role of individual molecules on biological outcomes. Purpose: The aim of this study is to define a baseline pattern of both histological changes and kinase activation in a murine model of intimal hyperplasia. Methods: The murine femoral wire injury model was employed in which a micro wire is passed through a branch of the femoral and used to denude the common femoral artery. Specimens were perfusion-fixed and sections were stained with H&E and Movat’s stain such that morphometry could be performed using an ImagePro system. Additional specimens of femoral artery were also harvested and snap frozen for western blotting to allow for the study of MAPK activation. Contrallateral vessels were used as controls. Results: The injured femoral arteries develop intimal hyperplasia, which is maximal at 28 days and does not change substantially between day 28 and day 56. Sham operated vessels did not produced such a response. Using PCNA as a marker of cell proliferation, we identified a peak in VSMC proliferation at 3-5 days after injury. There was a time dependent increase in kinase activity immediately after injury. MEK1/2 activation peaks at 10 mins after injury and is followed by a peak in ERK1/2 activation at 20 mins. The stress kinases p38MAPK and JNK peak between 10 and 20 mins. Activation of akt is later at 45 and 120 mins and activation of p70S6K occurs at 180 mins after injury. Conclusions: These data demonstrate that femoral wire injury in the mouse induces a consistent model of intimal hyperplasia that is associated with a time dependent increase in signaling kinase activity. Accurate characterization of cell signaling is a necessary step in the development of molecular therapeutics.