Toll-like receptors 4 induces expression of matrix metalloproteinase-9 in human aortic smooth muscle cells

Abstract

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. It was confirmed that the presence of functional TLR4 promotes a proinflammatory phenotype and proliferation of vascular smooth muscle cells (VSMCs). Here we tested whether designed TLR4 small interfering RNAs (TLR4 siRNAs) is capable of inducing TLR4 deficient and simultaneously regulating the expression of matrix metalloproteinase-9 (MMP-9) in human aortic smooth muscle cells (HASMCs). Human aortic smooth muscle cells were obtained from Cascade Biologics (Portland, USA). The siRNAs used in this study were chemically synthesized by Ambion, diluted in RNase free water at concentration of 2 μg/ml. The TLR4 siRNAs were complexed with Lipofectamine^TM2000 in transfection buffer. After 30 min incubation at room temperature, the complexes were added to the cells. Subsequent to 5 h incubation, cells were treated with 10 ng/ml LPS for 24 h. RT–PCR analysis was used to detect mRNA expression of GAPDH, TLR4 and MMP-9; Western blot analysis was used to examine GAPDH, TLR4 and MMP-9 protein expression. It was shown that all three designed TLR4 siRNAs inhibited the expression of TLR4 in HASMCs as compared to nontargeting siRNA. Notably, TLR4 siRNA-1 exhibited the strongest inhibition effect. Transfection of HASMCs with TLR4 siRNA-1 resulted in down-regulation of LPS-induced expression of MMP-9. It was concluded that TLR4 siRNA-transfected HASMCs were capable for regulating the expression of MMP-9, providing support for the rational design of siRNAs as atherosclerotic therapy.