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 . 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-1 ( il-1 ) and interleukin-18 ( il-18 ) , which plays a vital role in promoting the development of lipid plaques and destabilizing the plaques [ 6 , 7 ] . recent studies have shown that cholesterol crystal activate the nlrp3 inflammasome and release of il-1 , 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 . small interfering rnas ( sirnas ) have proven effective in silencing target genes and lentivirus can efficiently deliver sirna . 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 . 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 ) , 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 . the animal experimental protocol was reviewed and approved by the animal care committee of shandong university ( jinan , china ) . a total of 40 male apo e - deficient mice aged 8 weeks were purchased from the beijing university animal research center ( beijing , china ) and were housed on a 12-hr light/12-hr dark cycle in plenty of food and water . all mice were fed a high - fat diet ( 0.25% cholesterol and 15% cocoa butter ) . after 4 weeks , mice were divided into control group and nlrp3 interfering ( nlrp3i ) group , which were injected via tail vein with mock viral suspension ( 1.75  10  tfu , 20  ul ) and nlrp3i viral suspension ( 1.75  10  tfu , 20  ul ) . four weeks after the first lentivirus injection , mice were anesthetized and perfused with phosphate buffer saline ( pbs ) for 2  min with needle inserted into the left ventricle . the aortic root vessels were removed and fixed in 4% paraformaldehyde overnight and then embedded in optimal cutting temperature ( oct ) compound . total triglycerides and cholesterol were detected with an automated enzymatic technique ( boehringer mannheim diagnostics ) . high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) levels were determined with an automated chemically modified technique ( roche modular dpp system , roche , switzerland ) . caspase-1 activity in protein extracted from aortas in the control and nlrp3i groups was measured using the caspase-1 colorimetric assay kit ( biovision , mountain view , ca ) according to the manufacturer 's instructions . absorbance reading was taken at 410  nm using a microplate reader . il-1 and il-18 were analysed using il-1- and il-18-specific elisa kits from r&d systems . oct compound - embedded aortic root vessels were cross - sectioned into pieces 6  m thick at 50  m intervals . the plaque was subdivided into a fibrous cap and a necrotic core on the basis of picrosirius red staining for collagen and oil red o staining for lipids , respectively . macrophages and smooth muscle cells ( smcs ) were immunostained with monocyte / macrophage - specific antibody ( moma)-2 ( diluted 1  :  25 ; abcam , cambridge , ma ) and anti--actin antibodies ( diluted 1  :  1000 ; sigma - aldrich ) , respectively . the sections were incubated with the appropriate hrp - conjugated secondary antibodies ( diluted 1  :  100 ; zhongshan ) , followed by incubation with 3,3-diaminobenzidine and counterstaining with hematoxylin . sections reacted with nonimmune igg , secondary antibody only , and no primary and secondary antibodies were used as negative controls . an automated image analysis system ( image - pro plus 6.0 ; media cybernetics , silver spring , md ) was used for quantitative measurements . the positive - staining area of macrophages , smcs , lipids , and collagen was quantified by computer - assisted color - gated measurement , and the ratio of positive - staining area to intimal area was calculated . the vulnerability index was calculated by the following formula : positive - staining area of ( macrophages + lipid)/positive - staining area of ( -smcs + collagen ) . after the aortas were collected , total rna was extracted using trizol reagent ( invitrogen ) in accordance with the manufacturer 's instructions . a 2-tailed student 's t - test was used in the comparison of continuous data . all mice were apparently healthy , and no mice were lost before the day of sacrifice . a systemic lentivirus delivery has proven an efficient approach to transferring gene into arterial walls . in order to evaluate the efficacy of lentivirus - mediated gene silencing in vivo , the levels of nlrp3 mrnas expression , the activity of caspase-1 , and the protein level of il-1 and il-18 ( downstream targets of nlrp3 inflammasome ) were measured . compared with the control group , the mrna levels of nlrp3 were significantly downregulated , the caspase-1 activity was strongly decreased , and the content of il-1 and il-18 was also decreased after silencing nlrp3 ( figure 1 , p < 0.05 ) . mice transfected with control lentivirus or nlrp3 lentivirus differed in histological and immunohistochemical staining of aortic root vessels ( figure 2 ) . in the atherosclerotic lesion area , smcs content was higher by 39% and collagen content was 46% higher with nlrp3 lentivirus than control transfection . the number of macrophages in the atherosclerotic lesion area , as determined by moma-2 , was significantly lower by nearly 22% and lipid content was 33% lower with nlrp3 lentivirus than control transfection . through calculation , likewise , serum total cholesterol , triglycerides , ldl , and hdl in the nlrp3i group did not differ significantly from those in the control group ( table 2 ) , suggesting that the effect of gene transfer on atherosclerosis was independent of serum lipid levels . in order to evaluate the effect of nlrp3 interfering on macrophage lipid efflux , we measured the mrna expression of atp - binding cassette ( abc ) transporters abca1 and abcg1 , also known as the cholesterol efflux regulatory protein . in comparison with control , mrna of abca1 and abcg1 increased significantly in aortas of apoe - deficient mice treated with nlrp3 silencing ( p < 0.05 ) ( figure 3 ) . collagens are main components of fibrous cap in plaques , and prolyl-4-hydroxylases 1 ( p4h1 ) are rate limiting for collagen synthesis . on the contrary , matrix metalloproteinase-2 ( mmp-2 ) and matrix metalloproteinase-9 ( mmp-9 ) contribute to the breakdown of the collagen . our data showed that the mrna expression of p4h1 was markedly higher in the nrlp3i group than in the control group ( p < 0.05 ) , and the mrna expression of mmp-2 and mmp-9 was significantly lower in the treatment group than the control group ( p < 0.05 ) ( figure 4 ) . these two effects led to the increasing of the collagen and thickness of the fibrous cap . in the present study , we applied lentivirus - mediated rna interfering methods to efficiently knock down nlrp3 gene and investigated its role in the pathogenesis of plaque vulnerability in apoe - deficient mice . our results demonstrated that mrna expression of nlrp3 was remarkably attenuated by transfected lentiviral sirnas . plaques of the nlrp3i group showed a higher content of collagen and smcs , lower content of lipid and macrophages , and lower vulnerability index than did in the control group . rna interference is a powerful technique for selectively silencing mrna for a wide range of proteins , and using sirna is also more efficient than other gene - specific targeting approaches . furthermore , gene silencing strategies using sirna have been applied to treat or prevent several diseases [ 19 , 20 ] . the major limitation for synthetic sirnas and plasmid - based sirnas as a delivering approach is their transient expression and poor transfection efficiency in vivo . in contrast , the lentiviral vector - mediated gene delivery system applied in this study has proven effective and long lasting in silencing gene function in primary mammalian cells , stem cells , and transgenic mice . in the present study , the efficacy of this method was confirmed not only by our observation that nlrp3 mrna expression levels of the treatment group were remarkably downregulated , but also by observation that the caspase-1 activity was strongly reduced and the content of downstream targets of nlrp3 inflammasomes , il-1 and il-18 , was decreased after silencing nlrp3 . in addition , the fact that no adverse effects were observed in our mice during the experiment demonstrated that lentivirus transfection was safe in these animals . thus , we regard that lentiviral vectors expressing sirna provide an efficient , specific , and safe tool to knock down nlpr3 genes . a number of recent landmark studies have implicated the activation of the nlrp3 inflammasome , in the progress of atherosclerosis . cholesterol crystals generated in vitro activate caspase-1 and the cleavage of both il-1 and il-18 in human peripheral blood mononuclear cells and mouse macrophages , and both depend on nlrp3 and asc [ 8 , 9 ] . in line with the in vitro data , hypercholesterolemic mice deficient in the receptor for low - density lipoprotein ( ldl ) which are reconstituted with bone marrow from nlrp3 , pycard , or il1b mice however , the critical role of the nlrp3 inflammasome in atherosclerosis has been challenged by another study of double - mutant mice generated by crossing apoe mice with nlrp3 , pycard , or caspase1 mice . that study did not find any differences in atherosclerosis progression with or without the nlrp3 inflammasome . to clarify these differences , we designed this study , applying rna interference . our results demonstrated that silence of nlrp3 suppressed atherosclerosis and stabilized atherosclerotic plaques , as the nlrp3i group of mice consistently exhibited a lower content of macrophages and lipids , a higher content of collagen , and a thicker fibrous cap in atherosclerotic plaques than the control group of mice . all these morphological changes led to a decreased plaque vulnerability index in the nlrp3i group compared to the control group . by incorporating the cumulative effects of protective and destructive factors , atherosclerosis progresses through lipid accumulation within macrophages , resulting in the formation of foam cell and necrotic core . although cholesterol lowering is a way to decrease lipid retention , increasing lipid efflux from macrophages , mostly through abca1 and abcg1 , has been regarded as another significant strategy to limit atherosclerosis . overexpression of abca1 and abcg1 limits atherosclerotic plaque formation , while genetic deletion of these genes promotes foam cell formation and accelerates atherosclerosis in animal models [ 2326 ] . our data showed that mrna levels of abca1 and abcg1 were increased after the treatment of nlrp3i , suggesting that nlrp3 gene silencing could be a potentially therapeutic mean to increase macrophage cholesterol efflux for the prevention of atherosclerosis . collagens are the main components of fibrous cap in atherosclerotic plaques and play a vital role in stabilizing plaques and preventing plaque rupture . prolyl-4-hydroxylase ( p4h ) is one of the key enzymes essential for the collagen synthesis . p4h1 , an isoenzyme of p4h , is a rate - limiting enzyme that folds the procollagen polypeptide chains into the stable triple helical molecules , which is necessary for collagen maturation and secretion . in the present study , we showed that the mrna expression of p4h1 was significantly higher , and the mrna expression of mmp-2 and mmp-9 that contributed to collagen degradation was markedly lower in the nrlp3 interfering group than in the control group , resulting in the thickness of fibrous cap and the stabilizing of atherosclerotic plaques . in conclusion , the present study demonstrated that lentivirus - mediated rna interference was effective in knocking down nlrp3 genes in apoe deficient mice , resulting in reduced inflammatory cytokines and plaque content of lipid and macrophages , increased plaque content of collagen , and lowered plaque vulnerability index . our findings raised the possibility that lentivirus - mediated gene silencing of nlrp3 is a potential therapeutic target for inhibiting progression of atherosclerotic plaques and subsequently preventing cardiovascular events .