Abstract We0096: FLRT2 silencing enhances endothelial cell retention on synthetic vascular graft materials under fluid shear stress in vitro.

Coronary heart disease is a leading cause of death, and surgical intervention with coronary artery bypass grafting (CABG) is often required to treat severe disease.Currently, autologous vessel grafts are the only option for CABG procedures. 8-25% of autologous grafts fail within 1 year, hence synthetic vascular grafts are needed to address this limitation. Synthetic vascular grafts less than 6 mm in diameter (size needed for CABG) have unacceptable patency rates. A promising strategy to improve patency is to endothelialize synthetic vascular grafts prior to implantation, but previous attempts have met with limited success due to detachment of cells upon exposure to fluid shear stress. We have previously shown that endothelial cells (ECs) which resist detachment from vascular grafts under fluid shear stress had significantly reduced fibronectin leucine-rich transmembrane protein 2 ( FLRT2 ) expression. We therefore hypothesize that FLRT2 gene silencing along with scaffold modifications will result in enhanced endothelial cell retention on synthetic vascular grafts upon exposure to fluid shear stress. We successfully downregulated FLRT2 protein expression using a silencing RNA (siRNA) approach and proceeded to seed FLRT2 silenced cells onto a commercially available Goretex graft material and subjected them to fluid shear stress of 15 dyn/cm 2 for 15 min. We observed an 88.9%±4.5% cell retention of FLRT2 silenced ECs on Goretex graft material in comparison to 44.5%±3.5% cell retention with non-specifically targeted scramble siRNA ECs post fluid shear stress (p<0.05). In addition, we performed western blots comparing both vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1(ICAM-1) levels to assess endothelial cell activation and found that silencing of FLRT2 decreased VCAM-1 expression by 49.2%±3.5% and ICAM-1 expression by 57.1%±3.4% relative to control, suggestive of decreased endothelial cell activation(p<0.01). Furthermore, we tested cell retention on fibronectin (a very well-studied ECM adhesive protein) and its integrin binding domain RGD peptide (Arg-Gly-Asp) under fluid shear stress and observed that there is a significantly improved retention of si-FLRT2 cells on RGD peptide (61.8%±11.7%) in comparison to fibronectin (21.1%±2.5%) (p<0.05). In conclusion, FLRT2 gene silencing enhanced EC retention on commercially available synthetic grafts and therefore is a attractive strategy for improving patency rates in CABG applications.