Enhanced Stabilization of Atherosclerotic Plaques in Apolipoprotein E-Knockout Mice by Combinatorial Toll-like Receptor-1 and-2 Gene Silencing

Because both Toll-like receptor-1 (TLR1) and TLR2 are expressed in atherosclerotic plaques, we hypothesized that TLR1 and TLR2 may play different roles in the formation of vulnerable plaques and that combinatorial knockdown of TLR1 and TLR2 genes may enhance the effects of isolated knockdown of the TLR1 or TLR2 gene on plaque stabilization. Lentiviruses carrying small interfering RNAs of TLR1 or TLR2 were constructed, which knocked down mRNA and protein expression of TLR1 or TLR2 significantly in vitro. One hundred and forty apolipoprotein E-deficient (apoE(-/-)) mice were randomly allocated to control, mock, TLR1 interference (TLR1i), TLR2 interference (TLR2i), and TLR1+2 interference (TLR1+2i) subgroups and a constrictive collar was placed around the carotid artery of these mice to induce plaque formation. TLR1i and TLR2i viral suspension was transfected into the carotid plaques separately in the TLR1i and TLR2i subgroups or together in the TLR1+2i subgroup. Four weeks after lentivirus transfection, expression of both TLR1 and TLR2 in the carotid plaques was remarkably attenuated. Plaques of the TLR1i subgroup showed lower macrophage content and interleukin (IL)-6 expression and a thicker fibrous cap compared with the control or mock subgroups. Plaques of the TLR2i subgroup showed a higher content of collagen and lower content of lipid and macrophages, a thicker fibrous cap, lower vulnerability index, and lower mRNA expression of IL-6 and monocyte chemoattractant protein-1 than the TLR1i subgroup. In the TLR1+2i subgroup, the macrophage and smooth muscle cell content, and the vulnerability index, were ameliorated as compared with those in the TLR2i subgroup. Lentivirus-mediated RNA interference can be used to efficiently knock down TLR1 and TLR2 genes in carotid plaques of apoE(-/-) mice. Although isolated knockdown of TLR1 or TLR2 is effective in attenuating plaque vulnerability, combinatorial interference with TLR1 and TLR2 exhibits enhanced improvement of plaque stability, and thus provides a useful approach to the stabilization of vulnerable plaques.