Silence of NLRP3 Suppresses Atherosclerosis and Stabilizes Plaques in Apolipoprotein E-Deficient Mice
Silence of NLRP3 Suppresses Atherosclerosis and Stabilizes Plaques in Apolipoprotein E-Deficient Mice
Fei Zheng,1 Shanshan Xing,2 Zushun Gong,1 Wei Mu,1 and Qichong Xing1
1Department of Cardiology, Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan 250014 , China
2Shandong University of Traditional Chinese Medicine, Jinan 250355, China
Received 10 March 2014; Accepted 20 May 2014; Published 4 June 2014
Academic Editor: Tânia Silvia Fröde
Copyright © 2014 Fei Zheng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Objectives. The role of the NLRP3 inflammasome in atherosclerosis remains controversial. The aim of this study was to determine whether inhibition of NLRP3 signaling by lentivirus-mediated RNA interference could reduce atherosclerosis and stabilizes plaques. We also tried to explore the mechanisms of the impact of NLRP3 inflammasome on atherosclerosis. Methods. Apolipoprotein E-deficient mice aged 8 weeks were fed a high-fat diet and were injected with NLRP3 interfering or mock viral suspension after 4 weeks. Lentivirus transfer was repeated in 2 weeks. Four weeks after the first lentivirus injection, we evaluated the effects of NLRP3 gene silencing on plaque composition and stability and on cholesterol efflux and collagen metabolism, by histopathologic analyses and real-time PCR. Results. Gene silence of NLRP3 prevented plaques progression and inhibited inductions of proinflammatory cytokines. Moreover, this RNA interference reduced plaque content of macrophages and lipid, and increased plaque content of smooth muscle cells and collagen, leading to the stabilizing of atherosclerotic plaques. Conclusions. NLRP3 inflammasomes may play a vital role in atherosclerosis, and lentivirus-mediated NLRP3 silencing would be a new strategy to inhibit plaques progression and to reduce local inflammation.
1. Introduction
Atherosclerosis is a complicated inflammatory progress characterized by lipid deposition, leukocyte infiltration, and vascular smooth muscle cell proliferation in the vascular walls [1, 2]. Because inflammation in the atherosclerotic process mostly occurs in the absence of microbial infection, it is considered to be sterile inflammation [3]. Growing evidences suggest that some types of sterile inflammation are mediated by the inflammasome, a large multiprotein complex in the cytosol and a component of the innate immune system [4, 5]. The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, the best characterized inflammasome to date, contains the nod-like receptor scaffold protein NLRP3 associated with the adapter protein, apoptosis-associated speck-like protein containing a CARD (ASC), which recruits caspase-1 and induces its activation. Caspase-1 induces secretion of interleukin-1β (IL-1β) and interleukin-18 (IL-18), which plays a vital role in promoting the development of lipid plaques and destabilizing the plaques [6, 7]. These findings suggest the involvement of the NLRP3 inflammasome in the development of atherosclerosis.
Recent studies have shown that cholesterol crystal activate the NLRP3 inflammasome and release of IL-1β, which indicates that the NLRP3 inflammasome may have a role in the development of atherosclerosis [8, 9]. However, another study of double-mutant mice did not find any differences in atherosclerosis progression with or without the NLRP3 inflammasome [10]. The reason for this discrepancy is unclear. The role of the NLRP3 inflammasome in atherosclerosis remains controversial.
Small interfering RNAs (siRNAs) have proven effective in silencing target genes and lentivirus can efficiently deliver siRNA [11]. In the present study, we constructed lentiviral vectors to knock down NLRP3 to evaluate the role of the NLRP3 inflammasome in atherosclerosis in apolipoprotein (Apo) E-deficient mice, which can develop severe hypercholesterolemia and spontaneous atherosclerosis [12].
2. Materials and Methods2.1. Lentivirus Vectors for NLRP3 RNAi
Three different NLRP3-specific target sequences were chosen using the NLRP3 reference sequence (Gene Bank Accession number NC_000077). According to the structure of a pGCSIL-GFP viral vector (Genechem gene, Shanghai, China), double-stranded DNA were synthesized and inserted into a linearized vector. The positive clones were identified as lentiviral vectors and pGCSIL/NLRP3-1 (sequence: 5′-GGUGAAAUGUACUUAAAUC-3′) induced the highest levels of downregulation. So pGCSIL/NLRP3-1 vector and viral packaging system were cotransfected into 293 cells to replicate competent lentivirus. The mock vector (sequence: 5′-GUGCACAUGAGUGAGAUUU-3′) was also packaged and used as a negative control, which has no significant homology to mouse gene sequences.
2.2. Animal Protocol
The animal experimental protocol was reviewed and approved by (...truncated)