Intra‐arterial adenoviral delivery of GFP‐FRNK inhibits FAK‐ and PYK2 signaling following balloon injury

Abstract

The focal adhesion kinase family of nonreceptor protein tyrosine kinases ‐ focal adhesion kinase (FAK) and proline‐rich tyrosine kinase‐2 (PYK2)‐play a central role in integrin‐dependent vascular smooth muscle (VSMC) signaling. FRNK (FAK‐Related Non‐Kinase) the autonomously expressed C‐terminus of FAK, inhibits both FAK and PYK2 signaling in cultured VSMC. To determine the role of FAK, PYK2 and FRNK in vascular remodeling, we subjected rats to carotid artery balloon injury and analyzed their expression by Western Blotting. FAK, FRNK and PYK2 were all upregulated (FAK=3.5±0.56; PYK2=6.0±2.2; FRNK=8.9±2.8‐fold) vs uninjured contralateral control arteries 2wk later. To analyze if early FRNK overexpression inhibits FAK and/or PYK2 dependent signaling, adenoviruses (Adv) expressing GFP or GFP‐FRNK were delivered to the arterial lumen at the time of injury. Tissue extracts 1wk later were analyzed with GFP antibody to detect transgene expression, and FAK and PYK2‐ dependent signaling with phosphospecific antibodies. GFP‐FRNK overexpression inhibited FAK‐Y397 phosphorylation, as well as paxillin phosphorylation at Y118. GFP‐FRNK also downregulated PYK2 expression and its phosphorylation at Y402. To analyze whether specific knockdown of FAK or PYK2 attenuates neointimal formation, 2 specific Adv‐shRNAs were designed to target either FAK or PYK2 and were successfully tested in cultured VSMC. We are now examining their use in vivo to ascertain which kinase is responsible for paxillin phosphorylation and downstream signaling. Supported by the Falk Medical Research Trust.