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Focal Adhesion Kinase Mediates The Frequency And Depth Of Vascular Smooth Muscle Invasion In A Novel Three-Dimensional Assay

Luke P Brewster¹, Eric M Brey², Mark Liwanag³, Allen M Samarel¹, Howard P Greisler.^1
1Loyola University Medical Center, Maywood, IL; ²Illinois Institute of Technology, Maywood, IL; ³Millikan University, Decatur, IL.

OBJECTIVES: Vascular interventions denude the endothelium that stimulate vascular smooth muscle cells (VSMC), in response to growth factors and matrix changes, to invade in 3-D the extracellular matrix, migrate to and proliferate in the vessel intima, and contributing to myointimal hyperplasia. Focal adhesion kinase (FAK) is an intracellular non-kinase that mediates growth factor and integrin signaling important to cellular invasion, migration, and proliferation. In order to test whether FAK signaling is critical to VSMC invasion, migration, and proliferation in 3-D, VSMC and ECs were infected with a dominant negative inhibitor of FAK, FAK-related non-kinase (FRNK), and placed in our 3-D coculture assay.

METHODS: A 3-D model was developed by suspending VSMC and ECs (3:1 ratio) into a pellet, which was embedded in fibrin hydrogel and placed in growth media. Digital images captured the distance traveled from the pellet into the surrounding matrix (length of invasion) as well as the frequency of cells invading the matrix (Figure). FAK signaling was inhibited by infecting VSMCs with an adenovirus (Adv) expressing GFP-tagged-FRNK (GFP-FRNK) prior to cell suspension. Adv-GFP was used to control for nonspecific effects of infection. Western blot analysis with quantitative densitometry determined protein expression levels, and confocal microscopy was utilized to localize GFP-FRNK intracellularly.

RESULTS: Both the length (Figure) and frequency VSMC invasion was significantly decreased in the GFP-FRNK treatment group (A) compared to the GFP-infected (B) and uninfected control group up to 3 weeks (P<.02). Quantification of alpha-smooth muscle cell actin was not different between the treatment and control groups (P>.3). However, GFP-FRNK infection did significantly decrease FAK autophosphorylation (pFAK397) (23%+/-1% compared to 100%+/-4%; P<.002) without affecting FAK expression (P=.6) by displacing FAK from focal adhesion complexes intracellularly.

CONCLUSIONS: GFP-FRNK overexpression inhibits the invasiveness and length of migration in VSMCs in a 3-D matrix. The mechanism of action appears to be through the disruption of FAK signaling rather than preserving or promoting a contractile phenotype. FAK may be a novel therapeutic target for the prevention of VSMCs' contribution to myointimal hyperplasia.