Abstract 5675: Local Gene Silencing of TLR4 Expression in vivo Improves Vein Graft Patency in Mice

Abstract

Introduction: Patency of vein grafts is limited by vascular remodelling processes such as neointima formation and accelerated atherosclerosis. Toll Like Receptor 4 (TLR4) is associated with neointima formation and atherosclerosis. Hypothesis: We hypothesize that TLR4 can serve as a local therapeutic target against vein graft disease. Methods: In human saphenous veins (huSV) immunohistochemical staining for TLR4 was performed on cross-sections of either directly harvested huSV (n=6), in vitro perfused huSV (n=6) and coronary graft huSV derived at autopsy (n=5). Mouse venous bypass grafting was performed in Balb/c mice (n=8) and in TLR4 deficient (C3H/Hej) mice (n=7) using a venous interposition model in the carotid artery. Five lentiviral TLR4 shRNAs were created and validated for local TLR4 gene silencing by measurement of murine TLR4/MD2 expression by FACS analysis and the best vector was selected for in vivo analysis. To test the effect of local TLR4 deficiency on vein graft disease virus was administered in pluronic gel and lubricated around vein grafts after vein grafting in APOE3*Leiden mice fed a high cholesterolemic diet. Results: A marked induction of focal TLR4 expression was observed in HuSV, reperfused ex vivo with whole blood under pulsatile arterial pressure. Moreover, TLR4 was abundantly present in the neointima formed in autopsy grafts and preexisting intimal lesions in directly harvested huSV. Vein grafts in C3H/Hej mice had 48±12% less vessel wall thickening (p=0.04) than Balb/c controls, 28 days after surgery. Hypercholesterolemic APOE3*Leiden mice showed massive vein graft disease due to neointima formation and accelerated atherosclerosis after vein grafting. Local gene silencing of TLR4 led to a significant reduction of vessel wall thickening (38±10%, n=10, p=0.03) in the graft segment and superior graft patency (p=0.013) as compared to the control. In conclusion, the upregulation of TLR4 expression in huSV during remodelling, the reduced vein graft thickening in TLR deficient C3H/Hej mice and reduction of vessel wall thickening after local interfering in the TLR4 pathway indicate the potential of TLR4 as a local therapeutic target against vein graft disease to increase graft patency.