research article silence of nlrp3 suppresses atherosclerosis...

Research ArticleSilence of NLRP3 Suppresses Atherosclerosis andStabilizes Plaques in Apolipoprotein E-Deficient Mice

Fei Zheng1 Shanshan Xing2 Zushun Gong1 Wei Mu1 and Qichong Xing1

1 Department of Cardiology Qianfoshan Hospital Shandong University 16766 Jingshi RoadJinan 250014 China

2 Shandong University of Traditional Chinese Medicine Jinan 250355 China

Correspondence should be addressed to Qichong Xing xingqichong126com

Received 10 March 2014 Accepted 20 May 2014 Published 4 June 2014

Academic Editor Tania Silvia Frode

Copyright copy 2014 Fei Zheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives The role of the NLRP3 inflammasome in atherosclerosis remains controversial The aim of this study was to determinewhether inhibition ofNLRP3 signaling by lentivirus-mediatedRNA interference could reduce atherosclerosis and stabilizes plaquesWe 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 4weeks Lentivirus transfer was repeated in 2 weeks Four weeks after the first lentivirus injection we evaluated the effects of NLRP3gene silencing on plaque composition and stability and on cholesterol efflux and collagen metabolism by histopathologic analysesand real-time PCR Results Gene silence of NLRP3 prevented plaques progression and inhibited inductions of proinflammatorycytokines Moreover this RNA interference reduced plaque content of macrophages and lipid and increased plaque content ofsmooth muscle cells and collagen leading to the stabilizing of atherosclerotic plaques Conclusions NLRP3 inflammasomes mayplay a vital role in atherosclerosis and lentivirus-mediated NLRP3 silencing would be a new strategy to inhibit plaques progressionand to reduce local inflammation

1 Introduction

Atherosclerosis is a complicated inflammatory progress char-acterized by lipid deposition leukocyte infiltration andvascular smooth muscle cell proliferation in the vascularwalls [1 2] Because inflammation in the atheroscleroticprocess mostly occurs in the absence of microbial infectionit is considered to be sterile inflammation [3] Growingevidences suggest that some types of sterile inflammationare mediated by the inflammasome a large multiproteincomplex in the cytosol and a component of the innateimmune system [4 5] The nod-like receptor family pyrindomain containing 3 (NLRP3) inflammasome the bestcharacterized inflammasome to date contains the nod-likereceptor scaffold protein NLRP3 associated with the adapterprotein apoptosis-associated speck-like protein containinga CARD (ASC) which recruits caspase-1 and induces its

activation Caspase-1 induces secretion of interleukin-1120573 (IL-1120573) and interleukin-18 (IL-18) which plays a vital role inpromoting the development of lipid plaques and destabiliz-ing the plaques [6 7] These findings suggest the involve-ment of the NLRP3 inflammasome in the development ofatherosclerosis

Recent studies have shown that cholesterol crystal acti-vate the NLRP3 inflammasome and release of IL-1120573 whichindicates that the NLRP3 inflammasome may have a role inthe development of atherosclerosis [8 9] However anotherstudy of double-mutant mice did not find any differencesin atherosclerosis progression with or without the NLRP3inflammasome [10] The reason for this discrepancy isunclear The role of the NLRP3 inflammasome in atheroscle-rosis remains controversial

Small interfering RNAs (siRNAs) have proven effectivein silencing target genes and lentivirus can efficiently deliver

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 507208 8 pageshttpdxdoiorg1011552014507208

2 Mediators of Inflammation

siRNA [11] In the present study we constructed lentiviralvectors to knock down NLRP3 to evaluate the role of theNLRP3 inflammasome in atherosclerosis in apolipoprotein(Apo) E-deficient mice which can develop severe hyperc-holesterolemia and spontaneous atherosclerosis [12]

2 Materials and Methods

21 Lentivirus Vectors for NLRP3 RNAi Three differentNLRP3-specific target sequences were chosen using theNLRP3 reference sequence (Gene Bank Accession numberNC 000077) According to the structure of a pGCSIL-GFP viral vector (Genechem gene Shanghai China)double-stranded DNA were synthesized and inserted intoa linearized vector The positive clones were identifiedas lentiviral vectors and pGCSILNLRP3-1 (sequence 51015840-GGUGAAAUGUACUUAAAUC-31015840) induced the highestlevels of downregulation So pGCSILNLRP3-1 vector andviral packaging system were cotransfected into 293 cells toreplicate competent lentivirus The mock vector (sequence51015840-GUGCACAUGAGUGAGAUUU-31015840) was also packagedand used as a negative control which has no significanthomology to mouse gene sequences

22 Animal Protocol The animal experimental protocol wasreviewed and approved by the Animal Care Committee ofShandong University (Jinan China) A total of 40 male ApoE-deficient mice aged 8 weeks were purchased from theBeijing University Animal Research Center (Beijing China)and were housed on a 12-hr light12-hr dark cycle in plentyof food and water All mice were fed a high-fat diet (025cholesterol and 15 cocoa butter) After 4 weeks mice weredivided into control group and NLRP3 interfering (NLRP3i)group which were injected via tail vein with mock viralsuspension (175 times 108 Tfu 20 uL) and NLRP3i viral suspen-sion (175 times 108 Tfu 20 uL) Lentivirus transfer was repeatedin 2 weeks Four weeks after the first lentivirus injectionmice were anesthetized and perfused with phosphate buffersaline (PBS) for 2min with needle inserted into the leftventricle The aortic root vessels were removed and fixedin 4 paraformaldehyde overnight and then embedded inoptimal cutting temperature (OCT) compound The rest ofaortas were collected for protein extraction and PCR

23 Serum Lipid Level Measurement Total triglycerides andcholesterol were detected with an automated enzymatictechnique (Boehringer Mannheim Diagnostics) [13] High-density lipoprotein (HDL) and low-density lipoprotein (LDL)levels were determined with an automated chemically modi-fied technique (RocheModular DPP System Roche Switzer-land)

24 Caspase-1 Activity Measurement and ELISA Caspase-1activity in protein extracted from aortas in the control andNLRP3i groups was measured using the Caspase-1 Colori-metric Assay Kit (BioVision Mountain View CA) accordingto the manufacturerrsquos instructions Absorbance reading was

taken at 410 nm using a microplate reader IL-1120573 and IL-18 were analysed using IL-1120573- and IL-18-specific ELISA kitsfrom RampD Systems

25 Histological Analysis OCT compound-embedded aorticroot vessels were cross-sectioned into pieces 6 120583m thick at50 120583m intervals Sections were stained with hematoxylinand eosin (HampE) for histological analysis The plaque wassubdivided into a fibrous cap and a necrotic core onthe basis of picrosirius red staining for collagen and oilred O staining for lipids respectively Corresponding sec-tions on separate slides were immunostained with mon-oclonal antibodies Macrophages and smooth muscle cells(SMCs) were immunostained with monocytemacrophage-specific antibody (MOMA)-2 (diluted 1 25 Abcam Cam-bridge MA) and anti-120572-actin antibodies (diluted 1 1000Sigma-Aldrich) respectively The sections were incubatedwith the appropriate HRP-conjugated secondary antibodies(diluted 1 100 Zhongshan) followed by incubationwith 331015840-diaminobenzidine and counterstaining with hematoxylinSections reacted with nonimmune IgG secondary antibodyonly and no primary and secondary antibodies were used asnegative controls

An automated image analysis system (Image-Pro Plus60 Media Cybernetics Silver Spring MD) was used forquantitative measurements The positive-staining area ofmacrophages SMCs lipids and collagen was quantified bycomputer-assisted color-gated measurement and the ratioof positive-staining area to intimal area was calculated Thevulnerability index was calculated by the following formulapositive-staining area of (macrophages + lipid)positive-staining area of (120572-SMCs + collagen) [14]

26 Quantitative Real-Time PCR Analysis After the aor-tas were collected total RNA was extracted using Trizolreagent (Invitrogen) in accordance with the manufacturerrsquosinstructions The mRNA levels were quantified by the use ofSYBR green technologyThe house-keeping gene 120573-actin wasquantified as an internal RNA control The primer sequencesfor all studied genes are listed in Table 1The 2minusΔΔCT methodfor comparing relative expression results was applied [15]

27 Data Analysis Data were analyzed using SPSS 180 (SPSSInc Chicago IL USA) Quantitative variables are presentedas mean plusmn SD A 2-tailed Studentrsquos 119905-test was used in thecomparison of continuous data 119875 lt 005 was consideredstatistically significant

3 Results

31 Safety and Efficiency of Lentiviral Transfection All micewere apparently healthy and no mice were lost before theday of sacrifice A systemic lentivirus delivery has proven anefficient approach to transferring gene into arterial walls [16]In order to evaluate the efficacy of lentivirus-mediated genesilencing in vivo the levels of NLRP3 mRNAs expressionthe activity of caspase-1 and the protein level of IL-1120573 andIL-18 (downstream targets of NLRP3 inflammasome) were

Mediators of Inflammation 3

Table 1 Primer sequences for real-time PCR

Gene Forward sequence Reverse sequenceNLRP3 51015840-ATGCCAGGAAGACAGCATTG-31015840 51015840-TCATCGAAGCCGTCCATGAG-31015840

ABCA1 51015840-GCGGACCTCCTGTGGTGTT-31015840 51015840-CAAGAATCTCCGGGCTTTAGG-31015840

ABCG1 51015840-AAGGCCTACTACCTGGCAAAGA-31015840 51015840-GCAGTAGGCCACAGGGAACA-31015840

P4H1205721 51015840-CCCTAAGGCAACTGGATGAA-31015840 51015840-CCCATTTGTTGCCAACTAG-31015840

MMP-2 51015840-ACCCAGATGTGGCCAACTAC-31015840 51015840-TCATTTTAAGGCCCGAGCAA-31015840

MMP-9 51015840-CCTGGAACTCACACGACATCTTC-31015840 51015840-TGGAAACTCACACGCCAGAA-31015840

120573-actin 51015840-TCATCACTATTGGCAACGAGC-31015840 51015840-AACAGTCCGCCTAGAAGCAC-31015840

Table 2 Body weight and serum lipid levels in control group andNLRP3i group

End point Control(119899 = 20)

NLRP3i(119899 = 20) 119875 value

Body weight g 288 plusmn 41 291 plusmn 38 NSTotal cholesterol mmolL 207 plusmn 087 201 plusmn 064 NSTriglycerides mgdL 352 plusmn 324 335 plusmn 24 NSLDL mgdL 615 plusmn 148 589 plusmn 073 NSHDL mgdL 1035 plusmn 175 1154 plusmn 179 NSNS indicates not significant

measured Compared with the control group the mRNA lev-els of NLRP3 were significantly downregulated the caspase-1activity was strongly decreased and the content of IL-1120573 andIL-18 was also decreased after silencing NLRP3 (Figure 1 119875 lt005)

32 Effects of NLRP3i on Plaque Composition and StabilityMice transfected with control lentivirus or NLRP3 lentivirusdiffered in histological and immunohistochemical stainingof aortic root vessels (Figure 2) In the atherosclerotic lesionarea SMCs content was higher by 39 and collagen contentwas 46 higher with NLRP3 lentivirus than control trans-fection The number of macrophages in the atheroscleroticlesion area as determined by MOMA-2 was significantlylower by nearly 22 and lipid content was 33 lowerwith NLRP3 lentivirus than control transfection Throughcalculation the vulnerability index was significantly lowerwith NLRP3 lentivirus than control transfection

33 Effects of NLRP3 Silencing on Body Weight and LipidsNLRP3 silencing resulted in no significant differences incomparison with controls in body weight Likewise serumtotal cholesterol triglycerides LDL and HDL in the NLRP3igroup did not differ significantly from those in the controlgroup (Table 2) suggesting that the effect of gene transfer onatherosclerosis was independent of serum lipid levels

34 Effects of NLRP3i on Lipid Efflux In order to evaluatethe effect of NLRP3 interfering on macrophage lipid effluxwe measured the mRNA expression of ATP-binding cassette(ABC) transporters ABCA1 and ABCG1 also known as thecholesterol efflux regulatory protein In comparisonwith con-trol mRNA of ABCA1 and ABCG1 increased significantly in

aortas of ApoE-deficient mice treated with NLRP3 silencing(119875 lt 005) (Figure 3)

35 Effects of NLRP3 Silencing on Collagen Synthesis andDegradation Collagens are main components of fibrous capin plaques and prolyl-4-hydroxylases 1205721 (P4H1205721) are ratelimiting for collagen synthesis On the contrary matrixmetalloproteinase-2 (MMP-2) andmatrixmetalloproteinase-9 (MMP-9) contribute to the breakdown of the collagenOur data showed that the mRNA expression of P4H1205721 wasmarkedly higher in the NRLP3i group than in the controlgroup (119875 lt 005) and the mRNA expression of MMP-2 andMMP-9 was significantly lower in the treatment group thanthe control group (119875 lt 005) (Figure 4) These two effects ledto the increasing of the collagen and thickness of the fibrouscap

4 Discussion

In the present study we applied lentivirus-mediated RNAinterfering methods to efficiently knock down NLRP3 geneand investigated its role in the pathogenesis of plaque vulner-ability in ApoE-deficientmice Our results demonstrated thatmRNA expression of NLRP3 was remarkably attenuated bytransfected lentiviral siRNAs Plaques of the NLRP3i groupshowed a higher content of collagen and SMCs lower contentof lipid and macrophages and lower vulnerability index thandid in the control group

RNA interference is a powerful technique for selectivelysilencing mRNA for a wide range of proteins [17] and usingsiRNA is alsomore efficient than other gene-specific targetingapproaches [18] Furthermore gene silencing strategies usingsiRNA have been applied to treat or prevent several diseases[19 20] The major limitation for synthetic siRNAs andplasmid-based siRNAs as a delivering approach is theirtransient expression and poor transfection efficiency in vivoIn contrast the lentiviral vector-mediated gene deliverysystem applied in this study has proven effective and longlasting in silencing gene function in primary mammaliancells stem cells and transgenic mice [21] In the presentstudy the efficacy of this method was confirmed not onlyby our observation that NLRP3 mRNA expression levels ofthe treatment group were remarkably downregulated butalso by observation that the caspase-1 activity was stronglyreduced and the content of downstream targets of NLRP3


lowast

Control NLRP3i

35

30

25

20

15

10

05

00

Fold

indu

ctio

nNLRP3

(a)

120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

Caspase-1 activity

Control NLRP3i

lowast

(b)

lowast

Control NLRP3i

50

40

30

20

10

0

IL-1120573

(pg

mg

prot

ein)

IL-1120573

(c)

lowast

Control NLRP3i

350

300

250

200

150

100

50

0

IL-18

(pg

mg

prot

ein)

IL-18

(d)

Figure 1 Efficiency of lentivirus transfection (a) NLRP3 interference downregulated NLRP3 mRNA expression by 643 compared withthe control via real-time PCR analyses (b) Caspase-1 activity in protein extracted from aortas was reduced by 56 after NLRP3 silencingcompared with the control (c and d) Silence of NLRP3 decreased the downstream proinflammatory factors IL-1120573 and IL-18 using ELISAlowast

119875 lt 005 versus control Values are mean plusmn SD

inflammasomes IL-1120573 and IL-18 was decreased after silenc-ing NLRP3 In addition the fact that no adverse effects wereobserved in our mice during the experiment demonstratedthat lentivirus transfection was safe in these animals Thuswe regard that lentiviral vectors expressing siRNA provide anefficient specific and safe tool to knock down NLPR3 genes

A strong link between inflammation and atherosclerosisis becoming increasingly evident [22] A number of recentlandmark studies have implicated the activation of theNLRP3inflammasome in the progress of atherosclerosis Choles-terol crystals generated in vitro activate caspase-1 and thecleavage of both IL-1120573 and IL-18 in human peripheral bloodmononuclear cells andmousemacrophages and both dependon NLRP3 and ASC [8 9] In line with the in vitro datahypercholesterolemic mice deficient in the receptor for low-density lipoprotein (LDL) which are reconstituted with bonemarrow from Nlrp3minusminus Pycar119889minusminus or Il1119887minusminus mice developmany fewer atherosclerotic plaques than those reconstituted

with wild-type bone marrow [8] However the critical roleof the NLRP3 inflammasome in atherosclerosis has beenchallenged by another study of double-mutant mice gener-ated by crossing Apo119890minusminus mice with Nlrp3minusminus Pycar119889minusminus orCaspase1minusminusmice [10]That study did not find any differencesin atherosclerosis progression with or without the NLRP3inflammasome To clarify these differences we designed thisstudy applying RNA interference Our results demonstratedthat silence of NLRP3 suppressed atherosclerosis and stabi-lized atherosclerotic plaques as the NLRP3i group of miceconsistently exhibited a lower content of macrophages andlipids a higher content of collagen and a thicker fibrous capin atherosclerotic plaques than the control group of miceAll these morphological changes led to a decreased plaquevulnerability index in the NLRP3i group compared to thecontrol group By incorporating the cumulative effects ofprotective and destructive factors plaque vulnerability indexhas been recognized as a histological marker for vulnerable


Con

trol

NLR

P3i

HE Macrophage Lipid SMC Collagen

(a)

Control NLRP3i

lowast20

15

10

5

0

Mea

n ca

p th

ickn

ess (120583

m)

(b)

lowast

Control NLRP3i

40

30

20

10

0

Mac

roph

age (

)

(c)

lowast

Control NLRP3iLi

pid

()

40

30

20

10

0

(d)

lowast

Control NLRP3i

14

12

10

8

6

4

2

0

SMC

()

(e)

lowast

Control NLRP3i

35

30

25

20

15

10

5

0

Col

lage

n (

)

(f)

lowast

Control NLRP3i

30

25

20

15

10

05

00

Vuln

erab

ility

inde

x

(g)

Figure 2 Cross sections of aortic root vessels from the control and gene interference groups were stained with HampE vascular smoothmusclecells and collagen were immunostained with 120572-actin and Sirius red respectively and macrophages and lipids were stained with MOMA-2and oil red O respectively (a) The mean cap thickness was increased after the NLRP3 silencing compared with the control (b) Number ofmacrophages in the atherosclerotic lesion area was significantly lower by nearly 22 and lipid content was 33 lower with NLRP3 lentivirusthan control transfection (c and d) SMCs content was higher by 39 and collagen content was 46higher withNLRP3 lentivirus than controltransfection (e and f) The vulnerability index was significantly lower in NLRP3i group than control (g) lowast119875 lt 005 versus control Values aremean plusmn SD Scale bar is 100 120583m

plaques These findings suggested that NLRP3 was critical inthe progression of atherosclerosis

Atherosclerosis progresses through lipid accumulationwithin macrophages resulting in the formation of foam celland necrotic core Although cholesterol lowering is a wayto decrease lipid retention increasing lipid efflux from

macrophages mostly through ABCA1 and ABCG1 has beenregarded as another significant strategy to limit atherosclero-sis Overexpression of ABCA1 and ABCG1 limits atheroscle-rotic plaque formation while genetic deletion of these genespromotes foam cell formation and accelerates atherosclerosisin animal models [23ndash26] Our data showed that mRNA


ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)

Figure 3 ABCA1 and ABCG1 mRNA expression levels were determined by real-time PCR analyses Compared with control mRNA ofABCA1 and ABCG1 increased significantly in aortas of ApoE-deficient mice treated with NLRP3 silencing lowast119875 lt 005 versus control Valuesare mean plusmn SD

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)

Figure 4 P4H1205721 MMP-2 and MMP-9 mRNA levels in aortas of ApoE-deficient mice were determined with real-time PCR The mRNAexpression of P4H1205721 was markedly higher in the NRLP3i group than in the control group and the mRNA expression levels of MMP-2 andMMP-9 were significantly lower in the treatment group than the control group lowast119875 lt 005 versus control Values are mean plusmn SD


levels of ABCA1 and ABCG1 were increased after the treat-ment of NLRP3i suggesting that NLRP3 gene silencing couldbe a potentially therapeutic mean to increase macrophagecholesterol efflux for the prevention of atherosclerosis

Collagens are the main components of fibrous cap inatherosclerotic plaques and play a vital role in stabilizingplaques and preventing plaque rupture Prolyl-4-hydroxylase(P4H) is one of the key enzymes essential for the collagensynthesis P4H1205721 an isoenzyme of P4H is a rate-limitingenzyme that folds the procollagen polypeptide chains into thestable triple helical molecules which is necessary for collagenmaturation and secretion [27] In the present study weshowed that themRNAexpression of P4H1205721 was significantlyhigher and the mRNA expression of MMP-2 and MMP-9that contributed to collagen degradation was markedly lowerin the NRLP3 interfering group than in the control groupresulting in the thickness of fibrous cap and the stabilizingof atherosclerotic plaques

In conclusion the present study demonstrated thatlentivirus-mediated RNA interferencewas effective in knock-ing down NLRP3 genes in ApoE deficient mice resulting inreduced inflammatory cytokines and plaque content of lipidand macrophages increased plaque content of collagen andlowered plaque vulnerability index NLRP3 inflammasomesplay important roles in the pathogenesis of plaque vulner-ability Our findings raised the possibility that lentivirus-mediated gene silencing of NLRP3 is a potential therapeutictarget for inhibiting progression of atherosclerotic plaquesand subsequently preventing cardiovascular events

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgment

This study was supported by the Foundation for OutstandingYoung Scientist in Shandong Province (No 2009BSB01312)

References

[1] P Libby P M Ridker and A Maseri ldquoInflammation andatherosclerosisrdquo Circulation vol 105 no 9 pp 1135ndash1143 2002

[2] G K Hansson ldquoMechanisms of disease inflammation athero-sclerosis and coronary artery diseaserdquoTheNewEngland Journalof Medicine vol 352 no 16 pp 1626ndash1695 2005

[3] G Y Chen and G Nunez ldquoSterile inflammation sensing andreacting to damagerdquo Nature Reviews Immunology vol 10 no12 pp 826ndash837 2010

[4] BKDavisHWen and J P-Y Ting ldquoThe inflammasomeNLRsin immunity inflammation and associated diseasesrdquo AnnualReview of Immunology vol 29 pp 707ndash735 2011

[5] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[6] A Tedgui and Z Mallat ldquoCytokines in atherosclerosis patho-genic and regulatory pathwaysrdquo Physiological Reviews vol 86no 2 pp 515ndash581 2006

[7] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1szligdecreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003

[8] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 no 7293 pp 1357ndash1361 2010

[9] K Rajamaki J Lappalainen K Oorni et al ldquoCholesterol crys-tals activate the NLRP3 inflammasome in humanmacrophagesa novel link between cholesterol metabolism and inflamma-tionrdquo PLoS ONE vol 5 no 7 Article ID e11765 2010

[10] P Menu M Pellegrin J-F Aubert et al ldquoAtherosclerosis inApoE-deficient mice progresses independently of the NLRP3inflammasomerdquoCell Death andDisease vol 2 no 3 article e1372011

[11] K V Morris and J J Rossi ldquoLentiviral-mediated delivery ofsiRNAs for antiviral therapyrdquo Gene Therapy vol 13 no 6 pp553ndash558 2006

[12] S Zadelaar R Kleemann L Verschuren et al ldquoMouse modelsfor atherosclerosis and pharmaceutical modifiersrdquo Arterioscle-rosis Thrombosis and Vascular Biology vol 27 no 8 pp 1706ndash1721 2007

[13] J R McNamara and E J Schaefer ldquoAutomated enzymatic stan-dardized lipid analyses for plasma and lipoprotein fractionsrdquoClinica Chimica Acta vol 166 no 1 pp 1ndash8 1987

[14] M Shiomi T Ito Y Hirouchi and M Enomoto ldquoFibromuscu-lar cap composition is important for the stability of establishedatherosclerotic plaques in mature WHHL rabbits treated withstatinsrdquo Atherosclerosis vol 157 no 1 pp 75ndash84 2001

[15] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[16] Z-H Wang Y-Y Shang S Zhang et al ldquoSilence of TRIB3suppresses atherosclerosis and stabilizes plaques in diabeticApoEminusminusLDL receptorminusminus micerdquo Diabetes vol 61 no 2 pp463ndash473 2012

[17] S M Elbashir J Harborth W Lendeckel A Yalcin K Weberand T Tuschl ldquoDuplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cellsrdquo Nature vol 411 no6836 pp 494ndash498 2001

[18] A W Tong Y-A Zhang and J Nemunaitis ldquoSmall interferingRNA for experimental cancer therapyrdquo Current Opinion inMolecular Therapeutics vol 7 no 2 pp 114ndash124 2005

[19] A de Fougerolles H-P Vornlocher J Maraganore and JLieberman ldquoInterfering with disease a progress report onsiRNA-based therapeuticsrdquoNature ReviewsDrugDiscovery vol6 no 6 pp 443ndash453 2007

[20] L Zender S Hutker C Liedtke et al ldquoCaspase 8 small inter-fering RNA prevents acute liver failure in micerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 13 pp 7797ndash7802 2003

[21] HMatta B Hozayev R Tomar P Chugh and PM ChaudharyldquoUse of lentiviral vectors for delivery of small interfering RNArdquoCancer Biology andTherapy vol 2 no 2 pp 206ndash210 2003

[22] G K Hansson and A Hermansson ldquoThe immune system inatherosclerosisrdquoNature Immunology vol 12 no 3 pp 204ndash2122011

[23] M Van Eck R R Singaraja D Ye et al ldquoMacrophageATP-binding cassette transporter A1 overexpression inhibitsatherosclerotic lesion progression in low-density lipoproteinreceptor knockout micerdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 26 no 4 pp 929ndash934 2006


[24] X Wang H L Collins M Ranalletta et al ldquoMacrophageABCA1 and ABCG1 but not SR-BI promote macrophagereverse cholesterol transport in vivordquo Journal of Clinical Investi-gation vol 117 no 8 pp 2216ndash2224 2007

[25] R Out M Hoekstra K Habets et al ldquoCombined deletionof macrophage ABCA1 and ABCG1 leads to massive lipidaccumulation in tissue macrophages and distinct atherosclero-sis at relatively low plasma cholesterol levelsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 28 no 2 pp 258ndash2642008

[26] L Yvan-Charvet M Ranalletta N Wang et al ldquoCombineddeficiency ofABCA1 andABCG1 promotes foamcell accumula-tion and accelerates atherosclerosis in micerdquo Journal of ClinicalInvestigation vol 117 no 12 pp 3900ndash3908 2007

[27] K L Gorres and R T Raines ldquoProlyl 4-hydroxylaserdquo CriticalReviews in Biochemistry and Molecular Biology vol 45 no 2pp 106ndash124 2010

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


siRNA [11] In the present study we constructed lentiviralvectors to knock down NLRP3 to evaluate the role of theNLRP3 inflammasome in atherosclerosis in apolipoprotein(Apo) E-deficient mice which can develop severe hyperc-holesterolemia and spontaneous atherosclerosis [12]

2 Materials and Methods

21 Lentivirus Vectors for NLRP3 RNAi Three differentNLRP3-specific target sequences were chosen using theNLRP3 reference sequence (Gene Bank Accession numberNC 000077) According to the structure of a pGCSIL-GFP viral vector (Genechem gene Shanghai China)double-stranded DNA were synthesized and inserted intoa linearized vector The positive clones were identifiedas lentiviral vectors and pGCSILNLRP3-1 (sequence 51015840-GGUGAAAUGUACUUAAAUC-31015840) induced the highestlevels of downregulation So pGCSILNLRP3-1 vector andviral packaging system were cotransfected into 293 cells toreplicate competent lentivirus The mock vector (sequence51015840-GUGCACAUGAGUGAGAUUU-31015840) was also packagedand used as a negative control which has no significanthomology to mouse gene sequences

22 Animal Protocol The animal experimental protocol wasreviewed and approved by the Animal Care Committee ofShandong University (Jinan China) A total of 40 male ApoE-deficient mice aged 8 weeks were purchased from theBeijing University Animal Research Center (Beijing China)and were housed on a 12-hr light12-hr dark cycle in plentyof food and water All mice were fed a high-fat diet (025cholesterol and 15 cocoa butter) After 4 weeks mice weredivided into control group and NLRP3 interfering (NLRP3i)group which were injected via tail vein with mock viralsuspension (175 times 108 Tfu 20 uL) and NLRP3i viral suspen-sion (175 times 108 Tfu 20 uL) Lentivirus transfer was repeatedin 2 weeks Four weeks after the first lentivirus injectionmice were anesthetized and perfused with phosphate buffersaline (PBS) for 2min with needle inserted into the leftventricle The aortic root vessels were removed and fixedin 4 paraformaldehyde overnight and then embedded inoptimal cutting temperature (OCT) compound The rest ofaortas were collected for protein extraction and PCR

23 Serum Lipid Level Measurement Total triglycerides andcholesterol were detected with an automated enzymatictechnique (Boehringer Mannheim Diagnostics) [13] High-density lipoprotein (HDL) and low-density lipoprotein (LDL)levels were determined with an automated chemically modi-fied technique (RocheModular DPP System Roche Switzer-land)

24 Caspase-1 Activity Measurement and ELISA Caspase-1activity in protein extracted from aortas in the control andNLRP3i groups was measured using the Caspase-1 Colori-metric Assay Kit (BioVision Mountain View CA) accordingto the manufacturerrsquos instructions Absorbance reading was

taken at 410 nm using a microplate reader IL-1120573 and IL-18 were analysed using IL-1120573- and IL-18-specific ELISA kitsfrom RampD Systems

25 Histological Analysis OCT compound-embedded aorticroot vessels were cross-sectioned into pieces 6 120583m thick at50 120583m intervals Sections were stained with hematoxylinand eosin (HampE) for histological analysis The plaque wassubdivided into a fibrous cap and a necrotic core onthe basis of picrosirius red staining for collagen and oilred O staining for lipids respectively Corresponding sec-tions on separate slides were immunostained with mon-oclonal antibodies Macrophages and smooth muscle cells(SMCs) were immunostained with monocytemacrophage-specific antibody (MOMA)-2 (diluted 1 25 Abcam Cam-bridge MA) and anti-120572-actin antibodies (diluted 1 1000Sigma-Aldrich) respectively The sections were incubatedwith the appropriate HRP-conjugated secondary antibodies(diluted 1 100 Zhongshan) followed by incubationwith 331015840-diaminobenzidine and counterstaining with hematoxylinSections reacted with nonimmune IgG secondary antibodyonly and no primary and secondary antibodies were used asnegative controls

An automated image analysis system (Image-Pro Plus60 Media Cybernetics Silver Spring MD) was used forquantitative measurements The positive-staining area ofmacrophages SMCs lipids and collagen was quantified bycomputer-assisted color-gated measurement and the ratioof positive-staining area to intimal area was calculated Thevulnerability index was calculated by the following formulapositive-staining area of (macrophages + lipid)positive-staining area of (120572-SMCs + collagen) [14]

26 Quantitative Real-Time PCR Analysis After the aor-tas were collected total RNA was extracted using Trizolreagent (Invitrogen) in accordance with the manufacturerrsquosinstructions The mRNA levels were quantified by the use ofSYBR green technologyThe house-keeping gene 120573-actin wasquantified as an internal RNA control The primer sequencesfor all studied genes are listed in Table 1The 2minusΔΔCT methodfor comparing relative expression results was applied [15]

27 Data Analysis Data were analyzed using SPSS 180 (SPSSInc Chicago IL USA) Quantitative variables are presentedas mean plusmn SD A 2-tailed Studentrsquos 119905-test was used in thecomparison of continuous data 119875 lt 005 was consideredstatistically significant

3 Results

31 Safety and Efficiency of Lentiviral Transfection All micewere apparently healthy and no mice were lost before theday of sacrifice A systemic lentivirus delivery has proven anefficient approach to transferring gene into arterial walls [16]In order to evaluate the efficacy of lentivirus-mediated genesilencing in vivo the levels of NLRP3 mRNAs expressionthe activity of caspase-1 and the protein level of IL-1120573 andIL-18 (downstream targets of NLRP3 inflammasome) were












NLRP3i(119899 = 20) 119875 value








4 Discussion




lowast

Control NLRP3i

35

30

25

20

15

10

05

00

Fold

indu

ctio

nNLRP3

(a)

120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

Caspase-1 activity

Control NLRP3i

lowast

(b)

lowast

Control NLRP3i

50

40

30

20

10

0

IL-1120573

(pg

mg

prot

ein)

IL-1120573

(c)

lowast

Control NLRP3i

350

300

250

200

150

100

50

0

IL-18

(pg

mg

prot

ein)

IL-18

(d)







Con

trol

NLR

P3i


(a)

Control NLRP3i

lowast20

15

10

5

0

Mea

n ca

p th

ickn

ess (120583

m)

(b)

lowast

Control NLRP3i

40

30

20

10

0

Mac

roph

age (

)

(c)

lowast

Control NLRP3iLi

pid

()

40

30

20

10

0

(d)

lowast

Control NLRP3i

14

12

10

8

6

4

2

0

SMC

()

(e)

lowast

Control NLRP3i

35

30

25

20

15

10

5

0

Col

lage

n (

)

(f)

lowast

Control NLRP3i

30

25

20

15

10

05

00

Vuln

erab

ility

inde

x

(g)






ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)


Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)








Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























NLRP3i(119899 = 20) 119875 value








4 Discussion




lowast

Control NLRP3i

35

30

25

20

15

10

05

00

Fold

indu

ctio

nNLRP3

(a)

120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

Caspase-1 activity

Control NLRP3i

lowast

(b)

lowast

Control NLRP3i

50

40

30

20

10

0

IL-1120573

(pg

mg

prot

ein)

IL-1120573

(c)

lowast

Control NLRP3i

350

300

250

200

150

100

50

0

IL-18

(pg

mg

prot

ein)

IL-18

(d)







Con

trol

NLR

P3i


(a)

Control NLRP3i

lowast20

15

10

5

0

Mea

n ca

p th

ickn

ess (120583

m)

(b)

lowast

Control NLRP3i

40

30

20

10

0

Mac

roph

age (

)

(c)

lowast

Control NLRP3iLi

pid

()

40

30

20

10

0

(d)

lowast

Control NLRP3i

14

12

10

8

6

4

2

0

SMC

()

(e)

lowast

Control NLRP3i

35

30

25

20

15

10

5

0

Col

lage

n (

)

(f)

lowast

Control NLRP3i

30

25

20

15

10

05

00

Vuln

erab

ility

inde

x

(g)






ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)


Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)








Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

Control NLRP3i

35

30

25

20

15

10

05

00

Fold

indu

ctio

nNLRP3

(a)

120

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

Caspase-1 activity

Control NLRP3i

lowast

(b)

lowast

Control NLRP3i

50

40

30

20

10

0

IL-1120573

(pg

mg

prot

ein)

IL-1120573

(c)

lowast

Control NLRP3i

350

300

250

200

150

100

50

0

IL-18

(pg

mg

prot

ein)

IL-18

(d)







Con

trol

NLR

P3i


(a)

Control NLRP3i

lowast20

15

10

5

0

Mea

n ca

p th

ickn

ess (120583

m)

(b)

lowast

Control NLRP3i

40

30

20

10

0

Mac

roph

age (

)

(c)

lowast

Control NLRP3iLi

pid

()

40

30

20

10

0

(d)

lowast

Control NLRP3i

14

12

10

8

6

4

2

0

SMC

()

(e)

lowast

Control NLRP3i

35

30

25

20

15

10

5

0

Col

lage

n (

)

(f)

lowast

Control NLRP3i

30

25

20

15

10

05

00

Vuln

erab

ility

inde

x

(g)






ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)


Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)








Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

NLR

P3i


(a)

Control NLRP3i

lowast20

15

10

5

0

Mea

n ca

p th

ickn

ess (120583

m)

(b)

lowast

Control NLRP3i

40

30

20

10

0

Mac

roph

age (

)

(c)

lowast

Control NLRP3iLi

pid

()

40

30

20

10

0

(d)

lowast

Control NLRP3i

14

12

10

8

6

4

2

0

SMC

()

(e)

lowast

Control NLRP3i

35

30

25

20

15

10

5

0

Col

lage

n (

)

(f)

lowast

Control NLRP3i

30

25

20

15

10

05

00

Vuln

erab

ility

inde

x

(g)






ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)


Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)








Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ABCA1

Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

(a)

ABCG1

Control NLRP3i

Relat

ive m

RNA

expr

essio

n

25

20

15

10

05

00

lowast

(b)


Control NLRP3i

lowast18

16

14

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

P4H1205721

(a)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-2

(b)

lowast

Control NLRP3i

12

10

08

06

04

02

00

Relat

ive m

RNA

expr

essio

n

MMP-9

(c)








Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgment


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article silence of nlrp3 suppresses atherosclerosis...

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