P290: Shear-induced detachment of endothelial cells is mediated by extrinsic apoptosis

Abstract

Introduction: Human endothelial cells (ECs) fail to remain adherent to prosthetic biomaterials in the vasculature. We have previously demonstrated that shear stress induces apoptosis of ECs adherent to Dacron, causing detachment. However, it is unclear through what pathway mechanical loading is inducing cell death. In this study, we test the hypothesis that shear-induced apoptosis is mediated through the extrinsic rather than the intrinsic pathway of apoptosis. Methods: Human aortic ECs were established on Dacron membranes and incubated in the presence or absence of inhibitors of the extrinsic [Z-IETD-FMK (25μM), a specific caspase-8 inhibitor] or intrinsic [Z-LEHD-FMK (25μM), a caspase-9 inhibitor] pathways of apoptosis. Membranes were inserted into a parallel plate bioreactor and exposed to 0 or 30 dynes/cm2 of shear stress for 24 hours. Following flow, cells were stained with ethidium bromide and hoechst 33258. Through image analysis, EC retention was calculated as a percentage of the cells remaining relative to the no flow controls. Percentage of remaining ECs that were apoptotic was identified by characteristic nuclear morphology. Results: Exposure to 30 dynes/cm2 of shear stress induced EC apoptosis and detachment. Inhibiting the extrinsic pathway of apoptosis with the caspase-8 inhibitor significantly decreased apoptosis and improved EC retention. However, inhibiting caspase-9, specific to the intrinsic pathway of apoptosis was ineffective at preventing cell detachment and apoptosis. Conclusion: These data demonstrate that shear stress-induced detachment of ECs seeded on Dacron is mediated through the extrinsic pathway of apoptosis. These findings implicate shear induced Fas activation or integrin disengagement as the initiating event leading to apoptosis of ECs. Future work will focus on elucidating further the mechanism of this extrinsic mediated apoptosis, which will guide tissue engineering approaches in the development of endothelialized prosthetic vascular grafts.