Evaluation of the morphological and cytoskeletal reorganization of retrovirally transduced human umbilical vein endothelial cells in response to steady, laminar flow

The role of the vascular endothelial cells in maintaining vascular wall integrity and a thrombus-free flow surface has been well documented. Extending this knowledge to the field of vascular grafting has the potential to improve the patency of synthetic vascular grafts, especially small inner diameter grafts. Research into seeding synthetic vascular grafts has shown promise in animal models, while limited human trials have been disappointingGenetic alteration of endothelial cells in order to enhance their fibrinolytic action has emerged as a plausible improvement for endothelial cell seeded grafts; however, preliminary in vitro and in vivo experiments have suggested that genetically altered endothelial cells may be impaired in their ability to adhere to synthetic graft surfaces. The cause of this decreased adherence may be a consequence of integrin alteration secondary to retroviral gene transductionThe aim of the present study was to examine the effect of this integrin loss after retroviral gene transduction on human umbilical vein endothelial cell cytoskeletal development following fluid imposed shear stress. Genetically modified endothelial cells used in this study were transduced with the LN retroviral vector, conferring neomycin resistance to the cells. Naive human umbilical vein endothelial cells served as controls. The cells were seeded onto fibronectin coated glass slides and subjected to steady laminar flow for 66 hours with 25 dynes/cm² shear stress. A recirculating flow system and a parallel plate chamber provided the desired flow properties while maintaining a viable cell environment and facilitating collection of data. Through video microscopy, the response of endothelial cells within randomly chosen fields were quantified by analyzing the digitized images at specified time points. The images were used to obtain two parameters related to endothelial cell morphology, the cell shape index and angle of orientation. Following the 66 hour flow period, the cells were immunofluorescently stained to allow inspection of the cytoskeletal protein, F-actin, and a focal adhesion protein, vinculin. The results of the experiment indicated a statistically significant difference in cell morphology between the initial and final time points when the same cell lines were compared as well as for transduced versus naive cell lines. Since the measures were different at the initial time point, the difference of the measures at the final time point may not be indicative of a difference in the ability of the cells to respond to the shear stress. Changes in shape index and angle followed a similar trend in both the naive and transduced endothelial cells for the duration of the experiment. Immunofluorescent staining of the cytoskeleton revealed no qualitative difference in the amount or pattern of the stress fibers. Vinculin staining was unsuccessful in allowing inspection of vinculin association with the actin fibers of the cytoskeleton.