E. S. Lee1, K. Samadzadeh1, A. Afkhami1, A. Rona1, D. Hao2, A. Wang2 1Sacramento Veterans Affairs Medical Center,Research Service,Mather, CA, USA 2University Of California – Davis,UC Davis Medical Center,Sacramento, CA, USA
Introduction: Endothelial cell seeding has been shown to improve long term vascular graft patency and minimize intimal hyperplasia. Previous work has shown endothelial cells wash away after exposure to shear stress conditions. We hypothesize that the use of a novel ligand targeting αVβ3 can improve upon endothelial cell recruitment and adhesion under shear stress conditions. The purpose of this study is to compare the effects of shear stress in vitro on cell recruitment and adhesion between an αVβ3 Ligand and other conventional ligand substrates.
Methods: Channels in a BioFlux200 48 well plate were treated with Avidin (20 μg/mL) + Biotin (2 μM) and at various concentrations of the αVβ3 ligand or a fibronectin analog arginylglycylaspartic acid (RGD) (0.05 μM, 0.2 μM, 0.5 μM, 2.0 μM, and 5.0 μM) to determine optimal standard concentration. All channels were treated with phosphate-buffered saline (PBS), Avidin (20 μg/mL), RGD (2 μM) or the αVβ3 ligand (2 μM). Human umbilical vein endothelial cells (HUVECs) from commercial stock of low passage were suspended in media and seeded into channels at a concentration of 1×106 cells/mL under 2 dyn/cm2 shear stress for 2 hours at 37 C. Following cell seeding, the channels were fixed using 3.7% PFA and imaged using an Olympus IX81 fluorescent microscope to determine recruited cell count.
Results: Under shear stress, greater cell recruitment was seen from the αVβ3 ligand treatment (2.62×105 ± 1.02×105) when compared to RGD treatment (1.13 x105 ± 1.03×104) in static concentrations (Figure 1, p = 0.03). Cell adhesion and recruitment were directly correlated with increasing concentrations of both the αVβ3 ligand and RGD treatments. Cell recruitment between 2 μM and 5 μM αVβ3 ligand was not significantly different. In concentrations above 2 μM, greater adhesion overall was seen from the αVβ3 ligand, which influenced more cell-cell adhesion and clustering resulting in a vastly greater number of recruited HUVECs (4.16×105 vs. 2.57×106).
Conclusion: Targeting for the integrin αVβ3 increases endothelial cell recruitment and adhesion under shear stress conditions much greater than other accepted standard ligand substrates. Clinical application in targeting for the αVβ3 ligand may assist in improving endothelial cell seeding and thereby decreasing vascular bypass graft complications.
