A ti ti f I f b Vi f ti i M E d th li l C llActivation of Infammasomes by Visfatin in Mouse Endothelial CellsActivation of Infammasomes by Visfatin in Mouse Endothelial CellsActivation of Infammasomes by Visfatin in Mouse Endothelial CellsActivation of Infammasomes by Visfatin in Mouse Endothelial CellsyMi Xi Zh h W J j H Ch Zh Ni j Li d Pi l LiMin Xia Zhengchao Wang Junjun Hu Chun Zhang Ningjun Li and Pin lan LiMin Xia, Zhengchao Wang, Junjun Hu, Chun Zhang, Ningjun Li and Pin-lan LiMin Xia, Zhengchao Wang, Junjun Hu, Chun Zhang, Ningjun Li and Pin lan Li

D t t f Ph l d T i l M di l C ll f Vi i i C Vi i i C lth U i it Ri h d VA 23298Department of Pharmacology and Toxicology Medical College of Virginia Campus Virginia Commonwealth University Richmond VA 23298Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298p gy gy, g g p , g y, ,

ABSTRACTABSTRACT METHODSMETHODS RESULTSRESULTSABSTRACTABSTRACT METHODSMETHODS RESULTSRESULTSABSTRACTABSTRACT METHODSMETHODS RESULTSRESULTS

Inflammasomes have been reported to serve as intracellular Culture of MECs Mouse endothelial cells (MECs) were P f I fl i MEC NALP3 I fl C l F ti I hibiti f Vi f ti I d d I flInflammasomes have been reported to serve as intracellular Culture of MECs. Mouse endothelial cells (MECs) were Presence of Inflammasomes in MECs NALP3 Inflammasome Complex Formation Inhibition of Visfatin-Induced Inflammasomehi i iti ti d ti th i t i ti obtained from the American Type Culture Collection (ATCC)

Presence of Inflammasomes in MECs NALP3 Inflammasome Complex Formation Inhibition of Visfatin Induced Inflammasome machinery initiating and promoting the innate immune reaction obtained from the American Type Culture Collection (ATCC) I d d b Vi f ti C l F tiac e y t at g a d p o ot g t e ate u e eact o yp ( )

d i b t d d t d i D lb ’ difi d E l ’ Induced by Visfatin Complex Formationand inflammatory response to various danger signals The present and incubated and propagated in Dulbecco’s modified Eagle’s Induced by Visfatin Complex Formationand inflammatory response to various danger signals. The present p p g g

di (DMEM) i i 18 M di bi by p g g p

d h h i d h i i f i fl i f h medium (DMEM) containing 18 mM sodium bicarbonate, AAstudy hypothesized that activation of inflammasomes is one of the medium (DMEM) containing 18 mM sodium bicarbonate,Al 488 Al 555 Al 488 Al 555

AAlex488- Alex555-A

Alex488- Alex555-study hypothesized that activation of inflammasomes is one of the 4500 mg/L glucose 1 mM sodium pyruvate 1500 mg/L Alex488- Alex555- Alex488- Alex555-NALP3 ASC Overlay NALP3 Caspase-1 Overlay

i t t h i di ti d th li l i fl t4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L NALP3 ASC Overlay NALP3 Caspase-1 Overlay

important mechanisms mediating endothelial inflammatory sodium bicarbonate and 10% fetal Bovine serum (FBS) with Ctrlp g y sodium bicarbonate, and 10% fetal Bovine serum (FBS) with CtrlCtrlresponse to adipokines in obesity To test this hypothesis we first 4 mM L glutamine at 37°C in 5% CO atmosphereresponse to adipokines in obesity. To test this hypothesis, we first 4 mM L-glutamine at 37°C in 5% CO2 atmosphere. Neg NALP-3

h i d h i f 3 j i flg 2 p g

characterized the expression of 3 major inflammasome 2hrcharacterized the expression of 3 major inflammasomeI h i (ICC) Th MEC fi d i 4% Vehi

2hr

components in mouse endothelial cells (ECs) including cryopyrin Immunocytochemistry (ICC). The MECs were fixed in 4% Vehicomponents in mouse endothelial cells (ECs) including cryopyrin Immunocytochemistry (ICC). The MECs were fixed in 4%

rp ( ) g y pyparaformaldehyde and blocked with 1% BSA for 30 min Cells 4h4hr(also called NALP3) apoptosis-associated speck-like protein paraformaldehyde and blocked with 1% BSA for 30 min. Cells

in or 4tin mL 4hr(also called NALP3), apoptosis-associated speck-like protein permeabilization was performed by PBS with 0 05% Tween fa

tiL

fo

Scramfat /m

(ASC) d 1 B RT PCR d i t h i tpermeabilization was performed by PBS with 0.05% Tween- is

fm

L Scramisf

μg(ASC), and caspase-1. By RT-PCR and immunocytochemistry, we 20 (PBST) washing 3 × 5 min The cells were incubated in V g/mVi 2μ(ASC), and caspase 1. By RT PCR and immunocytochemistry, we 20 (PBST) washing 3 × 5 min. The cells were incubated in ASC Caspase-1 2μ

g8hrfound that these 3 molecules were abundantly expressed in mouse

( ) gi tib d i it bl dil ti f 1 h t

ASC Caspase 1 2

found that these 3 molecules were abundantly expressed in mouse primary antibody in suitable dilutions for 1 hour at room ASC y p p y yTh ll h d i PBST b ff f 3 10 siRNA

ECs Fluorescent confocal microscopy showed that these temperature. The cells were washed in PBST buffer for 3 × 10 Fi 1 ICC d i f i fl i MECsiRNA

16hrECs. Fluorescent confocal microscopy showed that these temperature. The cells were washed in PBST buffer for 3 10 Figure 1. ICC detection of inflammasomes components in MECs. 16hr

l l ld b d t t d i th t l f EC b t min and incubated with the secondary antibody for 30 minFigure 1. ICC detection of inflammasomes components in MECs.

molecules could be detected in the cytoplasm of mouse ECs, but min and incubated with the secondary antibody for 30 min. In permeabilized cells the representative images of ICC analysis Bo ecu es cou d be de ec ed e cy op s o ouse Cs, buThe cells incubated with Streptavindin HRP for 30 min was

In permeabilized cells, the representative images of ICC analysis Bthey were not colocalized under control condition However The cells incubated with Streptavindin-HRP for 30 min was showed that NALP3 mostly was distributed in the cytoplasm 0 8Bthey were not colocalized under control condition. However, followed after PBST washing for 3×10 min Diaminoben

showed that NALP3 mostly was distributed in the cytoplasm, 0.8NALP3 vs ASC

B0 8

yh h ll i b d i h di ki i f i f

followed after PBST washing for 3×10 min. Diaminoben- while ASC and Caspase 1 were not only detected in the cytoplasmNALP3 vs. ASCNALP3 vs Caspase 1

0.8 NALP3 vs Asc

when these cells were incubated with adipokine visfatin even forg

idi (DAB b ) d h Slidwhile ASC and Caspase-1 were not only detected in the cytoplasm,

* * #0 6NALP3 vs. Caspase-1NALP3 vs Caspase-1when these cells were incubated with adipokine, visfatin, even for zidine (DAB-brown) was used as chromogen. Slides were

p y y pb t d l l

*#

#0.60 6 *

t h th l l t d t f NALP3( ) gi d i h h li d d

but even more around nucleolus. #0.6*

*two hours, these molecules were aggregated to form a NALP3 counterstained with hematoxylin and mounted. CC 0 4C *

#, gg g counterstained with hematoxylin and mounted.

PC 0.4CC

0.4 ##

#inflammasome indicating the assembling and activation of this

PPC 0.4*

##inflammasome, indicating the assembling and activation of this

0 2i fl hi C l i i f 1 RNA extraction and real time RT PCR Total RNA was Eff t f Vi f ti RNA E i f

0.20.2inflammatory machinery. Colorimetric measurement of caspase-1 RNA extraction and real time RT-PCR. Total RNA was Effect of Visfatin on mRNA Expression ofinflammatory machinery. Colorimetric measurement of caspase 1

isolated from MECs by using TRIzol reagent (GIBCO LifeEffect of Visfatin on mRNA Expression of

0activity demonstrated that visfatin significantly increased isolated from MECs by using TRIzol reagent (GIBCO, Life I fl C t i MEC

00activity demonstrated that visfatin significantly increased y g g ( ,T h l i C l b d CA) di t th t l Inflammasome Components in MECs Ctrl Vehicle Scram ASC siRNACtrl 2h 4h 8h 16hy g y Technologies, Carlsbad, CA) according to the protocol Inflammasome Components in MECs Ctrl 2h 4h 8h 16h

caspase-1 activity by about 2 4 folds These results suggest thatg , , ) g p

d ib d b th f t Th RNA l l f t t VisfatinVisfatin (2μg/mL)caspase-1 activity by about 2.4 folds. These results suggest that described by the manufacturer. The mRNA levels for target AVisfatin

2μg/mL for 4hr( μg )

ti ti f i fl b f i iti tidescribed by the manufacturer. The mRNA levels for target A 2μg/mL for 4hr

activation of inflammasomes may be one of initiating Figure 3 Co-localization of ASC Caspase-1 and NALP3 in MECsgenes were analyzed by real-time quantitative RT-PCR using a Figure 4 Co localization of ASC Caspase 1 and NALP3 in1activation of inflammasomes may be one of initiating Figure 3. Co-localization of ASC, Caspase-1 and NALP3 in MECs.genes were analyzed by real time quantitative RT PCR using a Figure 4. Co-localization of ASC, Caspase-1 and NALP3 in

on 15mechanisms responsible for the action of adipokine visfatin to Using confocal microscopy we examined the aggregation ofBio Rad iCycler system (Bio Rad Hercules CA) The mRNA MECs after different stimulations: visfatin Scramble sRNA +si

omechanisms responsible for the action of adipokine-visfatin to Using confocal microscopy, we examined the aggregation ofBio-Rad iCycler system (Bio-Rad, Hercules, CA). The mRNA MECs after different stimulations: visfatin, Scramble sRNA +

3 ssp p g py gg gi fl t t f ti l f i fllevel was normalized to the 18S mRNA i f ti d ASC iRNA + i f ti (A) R t ti f lP

-3 re

induce endothelial dysfunction and inflammatory injury inflammasome components to form active complex of inflammasomes.level was normalized to the 18S mRNA. visfatin and ASC siRNA + visfatin. (A) Representative confocalLP xpinduce endothelial dysfunction and inflammatory injury p pP l A h t ti f l fl t i h

( ) pi i l A h d th t 4 h i f ti ti l tiA

L ex

( t d b NIH t HL057244 HL091464 d Panel A shows representative confocal fluorescent images, where we images in panel A showed that 4 hours visfatin stimulationNA

NA 10(supported by NIH grants HL057244, HL091464 and Panel A shows representative confocal fluorescent images, where we

b d di ib d d l l d klC f l I fl A l i MECimages in panel A showed that 4 hours visfatin stimulationi ifi l i d d i fl l f i dR

N

110( pp y g ,

observed ASC distributed around nucleolus area and NALP3 weaklyConfocal Immunofluorescence Analysis. MECs grown on significantly induced inflammasomes complex formation andmR 1

HL075316)observed ASC distributed around nucleolus area and NALP3 weaklyConfocal Immunofluorescence Analysis. MECs grown on

l li d i h diff i li i di dsignificantly induced inflammasomes complex formation andm 0HL075316). spread in the cytoplasm under normal condition Visfatin induced theirglass coverslips were treated with different stimuli as indicated they were attenuated by Silencing of ASC gene by ASC siRNACtrl 2h 4h 8h 16h spread in the cytoplasm under normal condition. Visfatin induced theirglass coverslips were treated with different stimuli as indicated they were attenuated by Silencing of ASC gene by ASC siRNACtrl 2h 4h 8h 16h

aggregated as shown by co localization of both inflammasomein different figures presented in this study The cells were transfection also block the action of visfatin Panel B summarizedaggregated as shown by co-localization of both inflammasomein different figures presented in this study. The cells were transfection also block the action of visfatin. Panel B summarizedVisfatin (2μg/mL) gg g ycomponents ( ello in the left panel) starting at 2 ho rs and reachingwashed and fixed in 4% paraformaldehyde and blocked with l li ti ffi i t d t h i l li ti l l f

( μg )B components (yellow in the left panel), starting at 2 hours and reachingwashed and fixed in 4% paraformaldehyde and blocked with co-localization coefficient data showing co-localization levels ofB p (y p ), g g

i d 4 h f i f ti i b ti A h th i htp y

1% BSA f 30 i Th ll bili d b PBSTg

NALP3 d ASC C 1 b f ft ASC1 8n maximum around 4 hours of visfatin incubation. As shown on the right1% BSA for 30 min. The cells were permeabilized by PBST, NALP3 and ASC or Caspase-1 before or after ASC gene1.8

on maximum around 4 hours of visfatin incubation. As shown on the rightl l li i b A 3 d C 1 h d i il

p y ,hi t i d PBS ith 0 2% T it hi b f

NALP3 and ASC or Caspase 1 before or after ASC geneil i h d

sio

BACKGROUNDBACKGROUND panel co-localization between NALP3 and Caspase-1 showed similarwashing twice and once PBS with 0.2% Triton washing before silencing Both NALP3 vs ASC and NALP3 vs Caspase-1 co-1 2esBACKGROUNDBACKGROUND panel, co localization between NALP3 and Caspase 1 showed similarwashing twice and once PBS with 0.2% Triton washing beforebl ki f l i li i d d i f i

silencing. Both NALP3 vs. ASC and NALP3 vs. Caspase 1 co1.2C

pr

eBACKGROUNDBACKGROUND pattern to NALP3-ASC The bar graph summarized the co-localizationblocking Confocal visualization and detection of antigens localization was attenuated by ASC gene silencing n=6 batchesSC

exp pattern to NALP3-ASC. The bar graph summarized the co-localizationblocking. Confocal visualization and detection of antigens localization was attenuated by ASC gene silencing. n=6 batches

0 6AS

A e

coefficient showing significant co localization of NALP3 and ASC aswere performed as we described in previous studies of cells * P<0 05 vs NALP3 with ASC control group; # P<0 050.6NA coefficient showing significant co-localization of NALP3 and ASC aswere performed as we described in previous studies of cells, * P<0.05 vs. NALP3 with ASC control group; # P<0.05R

N g gell as Caspase 1 after isfatin stim lation n 6 batches of cells *(Hypertension 2006;57(1)74 80 and Antioxid Redox Signal

g ps NALP3 ith Caspase 1 control gro p0m

R

well as Caspase-1 after visfatin stimulation. n=6 batches of cells, *(Hypertension 2006;57(1)74-80 and Antioxid Redox Signal vs. NALP3 with Caspase-1 control group.0m p ,P<0 05 NALP3 ith ASC t l # P<0 05 NALP3 ith

( yp ( ) g2008 9 1417 1426) Co locali ation as anal ed b Image

p g pCtrl 2h 4h 8h 16h P<0.05 vs. NALP3 with ASC control group; # P<0.05 vs. NALP3 with2008; 9, 1417–1426). Co-localization was analyzed by Image Ctrl 2h 4h 8h 16h P 0.05 vs. NALP3 with ASC control group; # P 0.05 vs. NALP3 with

C 1 l; , ) y y g

P Pl ft d th l li ti ffi i t Vi f ti (2 / L) Caspase-1 control groupPro Plus software, and the co-localization coefficient wasVi f ti tl d fi d di ki h b t dVisfatin (2μg/mL) Caspase 1 control group.Pro Plus software, and the co localization coefficient was

d b ’ l i ffi i ( CC)Visfatin, a recently defined adipokine, has been reported Crepresented by Pearson’s correlation coefficient (PCC)Visfatin, a recently defined adipokine, has been reported Crepresented by Pearson s correlation coefficient (PCC).increased in blood and tissues of obese or diabetic patients or Increased Caspase-1 Activity Induced by Visfatin2.8nincreased in blood and tissues of obese or diabetic patients or Increased Caspase-1 Activity Induced by Visfatin2.8

onp p y y2 11 ss

i

Caspase 1 activity Caspase 1 activity was measured byanimals It has been considered as a pro-inflammatory factor 2.1

e-1

res

Caspase-1 activity. Caspase-1 activity was measured byanimals. It has been considered, as a pro inflammatory factor,1 4se pr A Bp y p y y

quantifying the cleavage of Tyr Val Asp p nitroanilineth i l ti iti l k f d th li l i fl ti d 1.4pa ex

A*quantifying the cleavage of Tyr-Val-Asp p-nitroanilinethe circulating critical marker of endothelial inflammation and ap A

e

3 * *2.5Figure 5 Effect of visfatin on Caspase 1

q y g g y p p(YVAD NA) ith l i t i 1 kit

g0.7C N

A 3 *2.5Figure 5. Effect of visfatin on Caspase-1(YVADpNA) with a colorimetric caspase-1 assay kitfunction However the mechanism mediating the action of

0.7

mR

activation As shown in panel A visfatin(YVADpNA) with a colorimetric caspase 1 assay kit(Bi i i ) Th d d h l

function. However, the mechanism mediating the action of0m 2.4 * * 2activation. As shown in panel A, visfatin(Biovision) The measurement was accorded to the protocoli f i i k0 te

te p ,

ti l ti k dl i d C 1(Biovision). The measurement was accorded to the protocolvisfatin remains unknown Ctrl 2h 4h 8h 16h 1 8ni

tg) ni

tg) 1 5stimulation markedly increased Caspase-1described by the manufacturer

visfatin remains unknown. Ctrl 2h 4h 8h 16h 1.8U /m U /m 1.5y pi i i i d d b i

described by the manufacturer.Visfatin (2μg/mL) m

e te/

me te/

activity in a time-dependent manner, but it wasVisfatin (2μg/mL)1 2ym ni

t

ym nit

1activity in a time dependent manner, but it wasInflammasome is an intracellular molecular platform to

1.2nzy

(U nzy

(U

1

significant at 4 hours of visfatin treatment PanelStatistics Data are presented as means ± SE SignificantInflammasome is an intracellular molecular platform toFi 2 Eff t f i f ti RNA i f E

n (

En (

significant at 4 hours of visfatin treatment. PanelStatistics. Data are presented as means ± SE. Significantpt t ti d ti f i fl t i t l ki Figure 2. Effect of visfatin on mRNA expression of 0 6 0 5

B showed that ASC siRNA inhibited the increasep g

diff b t d ithi lti lpromote maturation and secretion of inflammatory interleukins Figure 2. Effect of visfatin on mRNA expression ofi fl i h h l i

0.6 0.5B showed that ASC siRNA inhibited the increasedifferences between and within multiple groups werepromote maturation and secretion of inflammatory interleukins

h l i f i i j NALP3 inflammasome components in MECs through real time RT-PCRin Caspase 1 activity induced by visfatin

d e e ces betwee a d w t u t p e g oups we ei d i ANOVA f d f ll d bas the early response to infection or injury NALP3 inflammasome components in MECs through real time RT PCR

0 0in Caspase-1 activity induced by visfatin.examined using ANOVA for repeated measures followed byas the early response to infection or injury. NALP3assay As shown in panel A short time treatment of visfatin didn’t 0

Ct l 2h 4h 8h 16h Ctrl Vehi Scrmb ASCp y yexamined using ANOVA for repeated measures, followed by

inflammasome as the major member consists of NALP3 assay. As shown in panel A, short time treatment of visfatin didn t Ctrl 2h 4h 8h 16h Ctrl Vehi Scrmb ASC iRNADuncan’s multiple-range test P<0 05 was consideredinflammasome, as the major member, consists of NALP3

affect the NALP3 mRNA expression but 16 hours treatment Visfatin (2μg/mL)siRNADuncan s multiple-range test. P<0.05 was considered

scaffold the ASC (PYCARD) adaptor and caspase 1 affect the NALP3 mRNA expression, but 16 hours treatment Visfatin (2μg/mL)Visfatinstatistically significantscaffold, the ASC (PYCARD) adaptor, and caspase-1. p

i ifi tl i d NALP3 RNA l l H ASCVisfatin

2 / L f 4hstatistically significant. significantly increased NALP3 mRNA level. However, ASC 2μg/mL for 4hrg y ,RNA i t k dl i fl d b i f ti CONCLUSIONCONCLUSIONTh t t d d i d t t t th h th i th t mRNA expression was not markedly influenced by visfatin CONCLUSIONCONCLUSIONThe present study was designed to test the hypothesis that mRNA expression was not markedly influenced by visfatin

h ( ) CONCLUSIONCONCLUSIONe p ese t study was des g ed to test t e ypot es s t ati i f i fl i f h i treatment even at 16 hours treatment (B) Moreover pro-caspase-1activation of inflammasomes is one of the important treatment even at 16 hours treatment (B). Moreover, pro caspase 1activation of inflammasomes is one of the important

mRNA expression had a similar profile to NALP3 when MECsmechanisms mediating endothelial inflammatory response to mRNA expression had a similar profile to NALP3 when MECs Adipokine visfatin upregulated NALP3 and Caspase 1 expression level at hours of treatment but it initiated NALP3 inflammasomemechanisms mediating endothelial inflammatory response towere treated by visfatin namely no effect occurred until 16 hours Adipokine visfatin upregulated NALP3 and Caspase-1 expression level at hours of treatment, but it initiated NALP3 inflammasome

adipokines in obesity were treated by visfatin, namely, no effect occurred until 16 hours p p g p pl i d h i i i l i h i i hi hadipokines in obesity. y y

stim lation hen pro caspase 1 mRNA e pression increased b complex formation around 2 hours of exposure of visfatin NALP3 inflammasomes formation promotes the activation of caspase-1 whichstimulation, when pro-caspase-1 mRNA expression increased by complex formation around 2 hours of exposure of visfatin. NALP3 inflammasomes formation promotes the activation of caspase 1, which, p p p yt f ld b tl t i ti f i fl t f t IL 1β lti i d th li l d f ti d i fl t i jtwo folds. subsequently triggers secretion of inflammatory factor IL-1β resulting in endothelial dysfunction and inflammatory injury.two folds. q y gg y β g y y j y