Abstract 718: Local Lentiviral ShRNA Mediated Silencing of CCR2 Inhibits Intimal Hyperplasia in ApoE3Leiden Mice
Intimal hyperplasia and accelerated atherosclerosis in venous bypass grafts are major causes of graft failure, in which inflammatory responses to vascular injury and smooth muscle cell (SMC) migration and proliferation are key players. RNA interference is a promising strategy to counteract inflammation and SMC activation. To avoid non-specific effects of RNAi in vivo, local delivery of siRNAs is to be preferred at the site of injury. Here, we study the effect of local application of lentiviral short hairpin RNA (shRNA), targeted against CC-chemokine receptor 2 (CCR2), on intimal hyperplasia in a murine model for vein graft disease. In this model, a venous interposition is placed into the carotid artery of recipient hypercholesterolemic ApoE3Leiden mice to induce intimal hyperplasia with features of accelerated atherosclerosis in 4 weeks. CCR2 is abundantly expressed in both macrophages and vSMCs as determined by RT-PCR. Also, vascular CCR2 mRNA expression levels were significantly upregulated during lesion progression in the vein graft (10-fold; P < 0.05), while its counterligand CCL2/JE was upregulated in particular at 6 hours after surgery (P < 0.001). A lentiviral shRNA was designed targeting murine CCR2. This shCCR2 silenced CCR2 expression at the mRNA (> 80%, P < 0.001) and protein level (> 60%, P < 0.001) in vitro. Also, infection of vSMCs with shCCR but not control lentivirus completely abolished the JE induced vSMC migration (P < 0.05). To determine the effect of CCR2 silencing on intimal thickening, a pluronic gel containing lentiviral shCCR2 or control virus was applied around the vein graft at time of surgery. Morphometric analysis showed a significant reduction in total amount of intimal hyperplasia in the shCCR2 treated animals (controls: 0.42 ± 0.05 mm2, shCCR2: 0.26 ± 0.03 mm2, P = 0.007) at 4 weeks after surgery. No differences were observed in relative macrophage and smooth muscle cell content of the lesions. In conclusion, these data demonstrate that vascular CCR2 contributes to vein graft disease and that local application of shRNA against CCR2 to the vessel wall prevents intimal hyperplasia in a hypercholesterolemic ApoE3Leiden mice. Therefore, this local application of RNAi is a very promising therapeutic tool to prevent vein graft failure.