Thrombospondin-2 Gene Silencing in Human Aortic Smooth Muscle Cells Improves Cell Attachment

Abstract

Despite decades of research, anastomotic intimal hyperplasia remains a major cause of delayed prosthetic arterial graft failure. Previously, we reported profound upregulation of thrombospondin-2 (TSP-2) mRNA in neointimal smooth muscle cells after prosthetic arterial bypass graft placement. TSP-2 is an antiangiogenic matricellular protein with specific functions yet unknown. In this study, we hypothesized that inhibition of TSP-2 in human aortic smooth muscle cells (HAoSMCs) would reduce cell proliferation and migration in vitro, providing a therapeutic target to mitigate intimal hyperplasia. HAoSMCs were transfected with TSP-2 small interfering ribonucleic acid (siRNA) using a commercial transfection reagent. Gene silencing was evaluated using semiquantitative real-time polymerase chain reaction. ELISA was used to measure TSP-2 protein levels in cell culture supernatants. Cell migration and proliferation were assessed using scratch wound assays and alamar blue assays, respectively. Attachment assays were performed to assess the effect of TSP-2 silencing on HAoSMC adhesion to fibronectin. TSP-2 siRNA achieved consistent target gene silencing at 48 hours post-transfection in HAoSMCs. This single transfection allowed suppression of TSP-2 protein expression for more than 30 days. TSP-2 gene silencing did not affect HAoSMC migration or proliferation. MMP-2 levels were also unaffected by changes in TSP-2 protein levels. However, HAoSMC attachment to fibronectin improved significantly in cells treated with TSP-2 siRNA. siRNA-mediated TSP-2 silencing of human aortic HAoSMCs improved cell attachment but had no effect on cell migration or proliferation. The effect on cell attachment was unrelated to changes in MMP activity.