Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 754735 6 pageshttpdxdoiorg1011552013754735
Research ArticleOncogene- and Oxidative Stress-InducedCellular Senescence Shows Distinct Expression Patterns ofProinflammatory Cytokines in Vascular Endothelial Cells
Etsu Suzuki1 Masao Takahashi2 Shigeyoshi Oba2 and Hiroaki Nishimatsu3
1 Institute of Medical Science St Marianna University School of Medicine 2-16-1 Sugao Miyamae-ku Kawasaki 216-8512 Japan2The Department of Internal Medicine Faculty of Medicine University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-8655 Japan3The Department of Urology Faculty of Medicine University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-8655 Japan
Correspondence should be addressed to Etsu Suzuki esuzuki-tkyuminacjp
Received 11 August 2013 Accepted 11 September 2013
Academic Editors K Abdelmohsen and M Kakoki
Copyright copy 2013 Etsu Suzuki 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
Senescent cells are metabolically active and produce a variety of proinflammatory cytokines It was previously reported thatatherosclerotic plaques contain senescent cells suggesting that senescence may contribute to the progression of atherosclerosis Inthis study we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H
2O2) or an adenovirus
that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines Both H2O2treatment
and AdRas12V infection induced senescence in VECs as assessed by senescence-associated 120573-Gal activity and the expression ofproteins such as p53 and p21CIP1 In addition both treatments induced the expression of a variety of cytokines including interleukin-1120573 (IL-1120573) and nerve growth factor (NGF) AdRas12V infection induced IL-1120573 expression more significantly than H
2O2treatment
whereas both treatments induced comparablemRNAand protein expression levels ofNGFThese results suggest that senescent cellsexpress different patterns of proinflammatory cytokines depending on the trigger that induced senescence It is therefore possiblethat senescent cells can differentially induce inflammation in the surrounding tissues depending on the cause of senescence
1 Introduction
It is well established that primary cultured cells that havebeen explanted from tissues do not proliferate indefinitelyand eventually exit the cell cycle Hayflick observed thisphenomenon and hypothesized that the finite lifecycle ofthese cells may be an expression of aging or senescence at thecellular level [1] Cellular senescence therefore represents astable and long-term cell cycle arrest although cells remainviable and metabolically active Senescent cells produce avariety of cytokines and chemokines which may explainwhy senescent cells cause inflammation in the surroundingtissues [2] Although telomere shortening is a major cause ofcellular senescence (known as replicative cellular senescence)[3] other stimuli such as the activation of oncogenes andoxidative stress can also cause cellular senescence (termedpremature cellular senescence) [4 5] When cells are dam-aged they withdraw from the cell cycle and try to repair
the damage by activating the p53-p21CIP1 and retinoblastomaprotein (pRb)-p16INK4A pathways The activation of thesepathways is therefore an important factor during cellularsenescence [2 6]
Senescent cells exhibit a significantly alteredmorphologyThey become large flat and multinucleatedThey sometimesbecome spindle-shaped depending on the senescence triggerAlthough there is no single marker by which senescent cellsare identified senescence-associated (SA)-120573-Galactosidase(120573-Gal) activity is observed in senescent cells [7] Senescentcells also express p53 the hypophosphorylated (active) formof pRb and cyclin-dependent kinase (cdk) inhibitors suchas p16INK4A and p21CIP1 [2 6] because senescent cells havewithdrawn from the cell cycle
It has recently been shown that human atheroscleroticplaques contain SA-120573-Gal positive vascular endothelial cells(VECs) and vascular smooth muscle cells (VSMCs) that
2 The Scientific World Journal
exhibit the morphological features of senescence [8 9] It hasalso been reported that interleukin-1120573 (IL-1120573) is expressed insenescent cells located in human atherosclerotic lesions [8]suggesting that senescent cells may promote inflammationin the lesions Although several different pathways suchas telomere shortening oncogene activation and oxidativestress induce a common phenotype of senescence it remainsunclear whether senescent cells show the same patterns ofcytokines expression regardless of the trigger A distinctpattern of cytokine productionmay be stimulated dependingupon the nature of inducer thereby causing different patternsof inflammation in surrounding tissues
In this study we used a constitutively active mutant ofRas to induce oncogene-induced senescence and hydrogenperoxide (H
2O2) to induce oxidative stress-induced senes-
cence in human umbilical vein endothelial cells (HUVECs)We examined whether these two pathways induce differentpatterns of cytokines expression
2 Materials and Methods
21 Reagents Anti-p21CIP1 anti-NF-120581B p65 and anti-120573-actin antibodies were purchased from Santa-Cruz Biotech-nology Inc (Santa-Cruz CA) and anti-p53 antibody wasobtained from Abcam (Tokyo Japan) Anti-phospho-NF-120581B p65 (Ser536) antibody which recognizes the catalyticallyactive form of the p65 subunit of NF-120581B was obtained fromCell Signaling Technology Inc (Danvers MA) BAY11-7082was purchased from Sigma-Aldrich (St Louis MO)
22 Cell Culture HUVECs were purchased from Sanko Jun-yaku Co Ltd (Tokyo Japan) and cultured in HuMedia-EG2(Kurabo Osaka Japan) To induce senescence by treatmentwith H
2O2 HUVECs were incubated with 100 120583molL H
2O2
for 1 hr After washing with phosphate-buffered saline (PBS)HUVECs were cultured in HuMedia-EG2 for 7 days
23 Adenoviral Infection Infection with a replication-defective adenovirus that expresses a constitutively activemutant of mouse Ras (AdRas12V) was reported previously[10] A recombinant adenovirus that expresses greenfluorescence protein (AdGFP) was obtained from QuantumBiotechnologies (Montreal Canada) HUVECs were infectedwith these adenoviruses at a multiplicity of infection (MOI)of 20 and then were cultured in HuMedia-EG2 for 7 days toinduce senescence
24 SA-120573-Gal Assay SA-120573-Gal stainingwas performed usinga cellular senescence detection kit (Cell Biolabs Inc SanDiego CA) according to the manufacturerrsquos protocol
25 RNA Extraction and Real Time PCR Analysis Total RNAwas extracted using TRIzol reagent (Gibco-BRL RockvilleMD) according to themanufacturerrsquos instructions Total RNAwas reverse transcribed using a ReverTra Ace qPCR RTKit (TOYOBO Osaka Japan) The expression of human IL-1120573 interleukin-6 (IL-6) nerve growth factor (NGF) andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
examined by real time PCR using SYBR Green (ThunderbirdSYBR qPCR Mix TOYOBO Japan) The following primerswere used
Real time PCR was performed using an ABI PRISM 7000sequence detection system (Applied Biosystems Foster CityCA) To confirm that no significant quantities of primerdimers were formed dissociation curves were analyzed
26 Protein Extraction and Western Blot Analysis Pro-teins were extracted from HUVECs in a cell lysis buffer(50mmolL Tris-HCl (pH 80) 150mmolL NaCl 1 NP-40) containing 2 120583gmL aprotinin 2120583gmL leupeptin and1mmolL phenylmethylsulfonyl fluoride Western blot anal-ysis was performed as previously described [11]
27 Enzyme-Linked Immunosorbent Assay Human IL-1120573 andNGF in culture media were measured with enzyme-linkedimmunosorbent assay (ELISA) kits (Abcam Tokyo Japan)according to the manufacturerrsquos instructions
28 Statistical Analyses All values are expressed as the meanplusmn SEM Statistical analyses were performed using analysisof variance followed by the Student-Neumann-Keuls testDifferences with a 119875 value of lt005 were considered to bestatistically significant
3 Results and Discussion
31 Oncogene Activation and Oxidative Stress Induce Cel-lular Senescence in HUVECs We first examined whetheroncogene activation and oxidative stress induced cellularsenescence in HUVECs by SA-120573-Gal staining (Figure 1)HUVECs pretreated with H
2O2or infected with AdRas12V
were positively stained in contrast to noninfected control cells(data not shown) or those infected with AdGFP suggest-ing that both treatments induced senescence in HUVECsBecause there were no significant differences in SA-120573-Galstaining between noninfected HUVECs and AdGFP-infectedHUVECs we used AdGFP-infected HUVECs as the neg-ative control throughout this study Interestingly although
The Scientific World Journal 3
AdGFP
H2O2 AdRasG12V
Figure 1 SA-120573-Gal staining of HUVECs HUVECs were infected with AdRas12V for 1 week to induce senescence or with AdGFP as thenegative control Senescence was also induced in HUVECs treated with 100120583molL H
2O2for 1 hr Arrows indicate large and round-shaped
senescent cells and arrowheads indicate flat or spindle-shaped senescent cells
both H2O2treatment and AdRas12V infection induced SA-
120573-Gal positive cells morphology of the cells was appar-ently different While cells treated with H
2O2were large
and round-shaped those infected with AdRas12V becamespindle-shaped or flat This suggests that the senescent cellsmay have different functions depending on the triggerthat induced senescence We also performed western blotanalysis to confirm that these treatments induced senescence(Figure 2) The expression of p53 in HUVECs treated withH2O2or infected with AdRas12V was significantly higher
than that in control cells suggesting that the DNA-damageresponse had been activated Both treatments also stimulatedthe expression of the cdk inhibitor p21CIP1 suggesting thatthe cells had withdrawn from the cell cycle It has beenreported that NF-120581B is activated in senescent cells whichhelps maintain the senescent phenotype and stimulates theproduction of cytokines [12 13] We therefore examinedwhether NF-120581B was activated in our systemThe p65 subunitof NF-120581B is reportedly phosphorylated at Serine-536 whenNF-120581B is activated Both H
2O2treatment and AdRas12V
infection stimulated the phosphorylation of p65 at Serine-536 suggesting that both treatments activated NF-120581B
32 Cytokine Production in Senescent Cells It is well estab-lished that senescent cells undergo massive changes in gene
AdGFP H2O2AdRas12V
p53
p21CIP1
P-p65NF120581B
p65NF120581B
120573-Actin
Figure 2 Western blot analysis of proteins extracted fromHUVECs HUVECs were infected with AdRas12V or treated withH2O2 Proteins were extracted from HUVECs 1 week after the
treatment and analyzed by western blot analysis
4 The Scientific World Journal
0
10
20
30IL
-1120573
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
lowastlowast
lowastdagger
(a)
0
1
2
IL-6
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
(b)
0
5
10
NG
FG
APD
H fo
ld in
duct
ion
H2O2AdGFP AdRas12V
lowast
lowast
(c)
Figure 3 Real time PCR analysis of the expression of proinflammatory cytokines in HUVECs HUVECs were infected with AdGFP orAdRas12V or treated with 100120583molL H
2O2 After 1 week total RNA was extracted for real time PCR analysis The expression of IL1-120573 IL-6
and NGF was normalized to GAPDH lowast and lowastlowast 119875 lt 005 and 119875 lt 001 respectively versus AdGFP infection dagger 119875 lt 001 versus AdRas12Vinfection (119899 = 6 per group)
expression and actively produce a variety of proinflammatorycytokines and immunemodulatorsThese changes have beenreferred to as the senescence-associated secretory phenotype[14] We used real time PCR to examine whether H
2O2
treatment and AdRas12V infection induced similar patternsof cytokine production (Figure 3) Although both treatmentssignificantly stimulated IL-1120573 expression compared withAdGFP infection the levels induced by AdRas12V weresignificantly higher than those induced by H
2O2 IL-6 is a
well-known cytokine that senescent cells produce [15 16]However expression of IL-6 in AdRas12V-infected or H
2O2-
treated HUVECs was similar to that in AdGFP-infectedHUVECs The expression of NGF in cells treated with H
2O2
or infected with AdRas12V was significantly higher than thatin AdGFP infection and the expression in the two treatmentgroups was similar In addition we assessed the expressionof several other cytokines including tumor necrosis factor-120572 and vascular endothelial growth factor-A Although bothtreatments significantly induced the expression of thesecytokines the extent (up to 2-fold induction) was smallerthan that observed in NGF expression (data not shown)We further examined the expression of IL-1120573 and NGF atthe protein level by ELISA (Figure 4(a)) H
2O2treatment
and AdRas12V infection increased the accumulation of IL-1120573 in the culture medium in a time-dependent mannerAdRas12V infection increased the accumulation of IL-1120573 in
the culture mediummore significantly than H2O2treatment
H2O2treatment and AdRas12V infection also increased the
accumulation of NGF in a time-dependent fashion andboth treatments had a similar effect To study the role ofNF-120581B in cytokine production we examined the effects ofBAY11-7082 an I120581B120572 inhibitor on IL-1120573 andNGF production(Figure 4(b)) Pretreatment with BAY11-7082 significantlyinhibited the secretion of IL-1120573 and NGF induced by H
2O2
treatment and AdRas12V infectionWe found that senescent HUVECs produced a variety
of proinflammatory cytokines and that the production wasmediated at least in part by the NF-120581B-dependent pathwayThus theNF-120581B-dependent pathway appears to play a pivotalrole in the production of cytokines in senescent VECs Wealso found that although H
2O2treatment and AdRas12V
infection significantly induced IL-1120573 expression AdRas12Vinfection had a more significant effect than H
2O2treatment
In contrast both treatments had a comparable effect onNGF expression IL-1120573 is a potent proinflammatory cytokinethat stimulates the expression of adhesion molecules onendothelial cells [17] and is frequently expressed in humanatherosclerotic plaques [8] In addition IL-1120573 deficiencydecreases the severity of atherosclerosis in apolipoprotein E(apoE) knock-out mice [18] IL-1120573 therefore potently inducesvascular inflammationNGF is amember of the neurotrophinfamily that stimulates not only nerve growth and survival
The Scientific World Journal 5
0 1 3 6(hr) (hr)
0
1
2
3
4
5
6
0 1 3 60
05
1
15
lowastlowastdagger
lowastlowast
lowastlowast
lowastlowast
dagger
lowastlowast
lowast
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(a)
0
1
2
3
4
5
6
0
05
1
15
AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2 AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2
Plt 005
Plt 005
Plt 001Plt 001
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(b)
Figure 4 The expression of IL-1120573 and NGF proteins in HUVECs treated with AdRas12V or H2O2 (a) Time course of the accumulation of
IL-1120573 and NGF in the culture media HUVECs were infected with AdGFP (closed squares) or AdRas12V (closed circles) or treated with H2O2
(open circles) After 1 week HUVECs were washed with PBS and medium was replaced with serum free Dulbeccorsquos modified Eagle medium(DMEM) HUVECs were incubated for the indicated periods and culture media were collected for ELISA lowast and lowastlowast 119875 lt 005 and 119875 lt 001respectively versus AdGFP infection at each time point dagger 119875 lt 005 versus H
2O2treatment at each time point (119899 = 6 per group) (b) The
effect of BAY11-7082 on cytokine production Experiments were performed as in (a) In addition some HUVECs were preincubated with10120583molL of BAY11-7082 (Bay) for 2 hrs and then washed with PBS The medium was replaced with serum free DMEM HUVECs were thenincubated for 6 hrs in the presence or absence of Bay (119899 = 6 per group)
but also mast cell accumulation in various organs [19]NGF can also promote angiogenesis [20] Although the roleof NGF in atherosclerosis has not been fully elucidatedevidence suggests that it plays a role in the progression ofatherosclerosis The expression of NGF was increased ininjured arteries after arterial balloon injury [21] NGF has apotential to stimulate the migration of VSMCs [21 22] Theproduction of IL-1120573 and NGF by senescent cells thereforehas the potential to stimulate vascular inflammation BecauseAdRas12V infection induced higher levels of IL-1120573 expressionthan H
2O2treatment senescent cells may differentially stim-
ulate inflammation in the surrounding tissues depending onthe senescence trigger However future studies are requiredto expand these observations
4 Conclusions
Oxidative stress and oncogene activation induced senescencein VECs and both stimuli promoted the production of
proinflammatory cytokines However oncogene activationstimulated production of IL-1120573more significantly than oxida-tive stress These data suggest that senescent cells may havedifferent proinflammatory actions on surrounding tissuesdepending on the senescence trigger
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] L Hayflick ldquoThe limited in vitro lifetime of human diploid cellstrainsrdquo Experimental Cell Research vol 37 no 3 pp 614ndash6361965
[2] J Campisi ldquoSenescent cells tumor suppression and organismalaging good citizens badneighborsrdquoCell vol 120 no 4 pp 513ndash522 2005
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
2 The Scientific World Journal
exhibit the morphological features of senescence [8 9] It hasalso been reported that interleukin-1120573 (IL-1120573) is expressed insenescent cells located in human atherosclerotic lesions [8]suggesting that senescent cells may promote inflammationin the lesions Although several different pathways suchas telomere shortening oncogene activation and oxidativestress induce a common phenotype of senescence it remainsunclear whether senescent cells show the same patterns ofcytokines expression regardless of the trigger A distinctpattern of cytokine productionmay be stimulated dependingupon the nature of inducer thereby causing different patternsof inflammation in surrounding tissues
In this study we used a constitutively active mutant ofRas to induce oncogene-induced senescence and hydrogenperoxide (H
2O2) to induce oxidative stress-induced senes-
cence in human umbilical vein endothelial cells (HUVECs)We examined whether these two pathways induce differentpatterns of cytokines expression
2 Materials and Methods
21 Reagents Anti-p21CIP1 anti-NF-120581B p65 and anti-120573-actin antibodies were purchased from Santa-Cruz Biotech-nology Inc (Santa-Cruz CA) and anti-p53 antibody wasobtained from Abcam (Tokyo Japan) Anti-phospho-NF-120581B p65 (Ser536) antibody which recognizes the catalyticallyactive form of the p65 subunit of NF-120581B was obtained fromCell Signaling Technology Inc (Danvers MA) BAY11-7082was purchased from Sigma-Aldrich (St Louis MO)
22 Cell Culture HUVECs were purchased from Sanko Jun-yaku Co Ltd (Tokyo Japan) and cultured in HuMedia-EG2(Kurabo Osaka Japan) To induce senescence by treatmentwith H
2O2 HUVECs were incubated with 100 120583molL H
2O2
for 1 hr After washing with phosphate-buffered saline (PBS)HUVECs were cultured in HuMedia-EG2 for 7 days
23 Adenoviral Infection Infection with a replication-defective adenovirus that expresses a constitutively activemutant of mouse Ras (AdRas12V) was reported previously[10] A recombinant adenovirus that expresses greenfluorescence protein (AdGFP) was obtained from QuantumBiotechnologies (Montreal Canada) HUVECs were infectedwith these adenoviruses at a multiplicity of infection (MOI)of 20 and then were cultured in HuMedia-EG2 for 7 days toinduce senescence
24 SA-120573-Gal Assay SA-120573-Gal stainingwas performed usinga cellular senescence detection kit (Cell Biolabs Inc SanDiego CA) according to the manufacturerrsquos protocol
25 RNA Extraction and Real Time PCR Analysis Total RNAwas extracted using TRIzol reagent (Gibco-BRL RockvilleMD) according to themanufacturerrsquos instructions Total RNAwas reverse transcribed using a ReverTra Ace qPCR RTKit (TOYOBO Osaka Japan) The expression of human IL-1120573 interleukin-6 (IL-6) nerve growth factor (NGF) andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
examined by real time PCR using SYBR Green (ThunderbirdSYBR qPCR Mix TOYOBO Japan) The following primerswere used
Real time PCR was performed using an ABI PRISM 7000sequence detection system (Applied Biosystems Foster CityCA) To confirm that no significant quantities of primerdimers were formed dissociation curves were analyzed
26 Protein Extraction and Western Blot Analysis Pro-teins were extracted from HUVECs in a cell lysis buffer(50mmolL Tris-HCl (pH 80) 150mmolL NaCl 1 NP-40) containing 2 120583gmL aprotinin 2120583gmL leupeptin and1mmolL phenylmethylsulfonyl fluoride Western blot anal-ysis was performed as previously described [11]
27 Enzyme-Linked Immunosorbent Assay Human IL-1120573 andNGF in culture media were measured with enzyme-linkedimmunosorbent assay (ELISA) kits (Abcam Tokyo Japan)according to the manufacturerrsquos instructions
28 Statistical Analyses All values are expressed as the meanplusmn SEM Statistical analyses were performed using analysisof variance followed by the Student-Neumann-Keuls testDifferences with a 119875 value of lt005 were considered to bestatistically significant
3 Results and Discussion
31 Oncogene Activation and Oxidative Stress Induce Cel-lular Senescence in HUVECs We first examined whetheroncogene activation and oxidative stress induced cellularsenescence in HUVECs by SA-120573-Gal staining (Figure 1)HUVECs pretreated with H
2O2or infected with AdRas12V
were positively stained in contrast to noninfected control cells(data not shown) or those infected with AdGFP suggest-ing that both treatments induced senescence in HUVECsBecause there were no significant differences in SA-120573-Galstaining between noninfected HUVECs and AdGFP-infectedHUVECs we used AdGFP-infected HUVECs as the neg-ative control throughout this study Interestingly although
The Scientific World Journal 3
AdGFP
H2O2 AdRasG12V
Figure 1 SA-120573-Gal staining of HUVECs HUVECs were infected with AdRas12V for 1 week to induce senescence or with AdGFP as thenegative control Senescence was also induced in HUVECs treated with 100120583molL H
2O2for 1 hr Arrows indicate large and round-shaped
senescent cells and arrowheads indicate flat or spindle-shaped senescent cells
both H2O2treatment and AdRas12V infection induced SA-
120573-Gal positive cells morphology of the cells was appar-ently different While cells treated with H
2O2were large
and round-shaped those infected with AdRas12V becamespindle-shaped or flat This suggests that the senescent cellsmay have different functions depending on the triggerthat induced senescence We also performed western blotanalysis to confirm that these treatments induced senescence(Figure 2) The expression of p53 in HUVECs treated withH2O2or infected with AdRas12V was significantly higher
than that in control cells suggesting that the DNA-damageresponse had been activated Both treatments also stimulatedthe expression of the cdk inhibitor p21CIP1 suggesting thatthe cells had withdrawn from the cell cycle It has beenreported that NF-120581B is activated in senescent cells whichhelps maintain the senescent phenotype and stimulates theproduction of cytokines [12 13] We therefore examinedwhether NF-120581B was activated in our systemThe p65 subunitof NF-120581B is reportedly phosphorylated at Serine-536 whenNF-120581B is activated Both H
2O2treatment and AdRas12V
infection stimulated the phosphorylation of p65 at Serine-536 suggesting that both treatments activated NF-120581B
32 Cytokine Production in Senescent Cells It is well estab-lished that senescent cells undergo massive changes in gene
AdGFP H2O2AdRas12V
p53
p21CIP1
P-p65NF120581B
p65NF120581B
120573-Actin
Figure 2 Western blot analysis of proteins extracted fromHUVECs HUVECs were infected with AdRas12V or treated withH2O2 Proteins were extracted from HUVECs 1 week after the
treatment and analyzed by western blot analysis
4 The Scientific World Journal
0
10
20
30IL
-1120573
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
lowastlowast
lowastdagger
(a)
0
1
2
IL-6
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
(b)
0
5
10
NG
FG
APD
H fo
ld in
duct
ion
H2O2AdGFP AdRas12V
lowast
lowast
(c)
Figure 3 Real time PCR analysis of the expression of proinflammatory cytokines in HUVECs HUVECs were infected with AdGFP orAdRas12V or treated with 100120583molL H
2O2 After 1 week total RNA was extracted for real time PCR analysis The expression of IL1-120573 IL-6
and NGF was normalized to GAPDH lowast and lowastlowast 119875 lt 005 and 119875 lt 001 respectively versus AdGFP infection dagger 119875 lt 001 versus AdRas12Vinfection (119899 = 6 per group)
expression and actively produce a variety of proinflammatorycytokines and immunemodulatorsThese changes have beenreferred to as the senescence-associated secretory phenotype[14] We used real time PCR to examine whether H
2O2
treatment and AdRas12V infection induced similar patternsof cytokine production (Figure 3) Although both treatmentssignificantly stimulated IL-1120573 expression compared withAdGFP infection the levels induced by AdRas12V weresignificantly higher than those induced by H
2O2 IL-6 is a
well-known cytokine that senescent cells produce [15 16]However expression of IL-6 in AdRas12V-infected or H
2O2-
treated HUVECs was similar to that in AdGFP-infectedHUVECs The expression of NGF in cells treated with H
2O2
or infected with AdRas12V was significantly higher than thatin AdGFP infection and the expression in the two treatmentgroups was similar In addition we assessed the expressionof several other cytokines including tumor necrosis factor-120572 and vascular endothelial growth factor-A Although bothtreatments significantly induced the expression of thesecytokines the extent (up to 2-fold induction) was smallerthan that observed in NGF expression (data not shown)We further examined the expression of IL-1120573 and NGF atthe protein level by ELISA (Figure 4(a)) H
2O2treatment
and AdRas12V infection increased the accumulation of IL-1120573 in the culture medium in a time-dependent mannerAdRas12V infection increased the accumulation of IL-1120573 in
the culture mediummore significantly than H2O2treatment
H2O2treatment and AdRas12V infection also increased the
accumulation of NGF in a time-dependent fashion andboth treatments had a similar effect To study the role ofNF-120581B in cytokine production we examined the effects ofBAY11-7082 an I120581B120572 inhibitor on IL-1120573 andNGF production(Figure 4(b)) Pretreatment with BAY11-7082 significantlyinhibited the secretion of IL-1120573 and NGF induced by H
2O2
treatment and AdRas12V infectionWe found that senescent HUVECs produced a variety
of proinflammatory cytokines and that the production wasmediated at least in part by the NF-120581B-dependent pathwayThus theNF-120581B-dependent pathway appears to play a pivotalrole in the production of cytokines in senescent VECs Wealso found that although H
2O2treatment and AdRas12V
infection significantly induced IL-1120573 expression AdRas12Vinfection had a more significant effect than H
2O2treatment
In contrast both treatments had a comparable effect onNGF expression IL-1120573 is a potent proinflammatory cytokinethat stimulates the expression of adhesion molecules onendothelial cells [17] and is frequently expressed in humanatherosclerotic plaques [8] In addition IL-1120573 deficiencydecreases the severity of atherosclerosis in apolipoprotein E(apoE) knock-out mice [18] IL-1120573 therefore potently inducesvascular inflammationNGF is amember of the neurotrophinfamily that stimulates not only nerve growth and survival
The Scientific World Journal 5
0 1 3 6(hr) (hr)
0
1
2
3
4
5
6
0 1 3 60
05
1
15
lowastlowastdagger
lowastlowast
lowastlowast
lowastlowast
dagger
lowastlowast
lowast
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(a)
0
1
2
3
4
5
6
0
05
1
15
AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2 AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2
Plt 005
Plt 005
Plt 001Plt 001
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(b)
Figure 4 The expression of IL-1120573 and NGF proteins in HUVECs treated with AdRas12V or H2O2 (a) Time course of the accumulation of
IL-1120573 and NGF in the culture media HUVECs were infected with AdGFP (closed squares) or AdRas12V (closed circles) or treated with H2O2
(open circles) After 1 week HUVECs were washed with PBS and medium was replaced with serum free Dulbeccorsquos modified Eagle medium(DMEM) HUVECs were incubated for the indicated periods and culture media were collected for ELISA lowast and lowastlowast 119875 lt 005 and 119875 lt 001respectively versus AdGFP infection at each time point dagger 119875 lt 005 versus H
2O2treatment at each time point (119899 = 6 per group) (b) The
effect of BAY11-7082 on cytokine production Experiments were performed as in (a) In addition some HUVECs were preincubated with10120583molL of BAY11-7082 (Bay) for 2 hrs and then washed with PBS The medium was replaced with serum free DMEM HUVECs were thenincubated for 6 hrs in the presence or absence of Bay (119899 = 6 per group)
but also mast cell accumulation in various organs [19]NGF can also promote angiogenesis [20] Although the roleof NGF in atherosclerosis has not been fully elucidatedevidence suggests that it plays a role in the progression ofatherosclerosis The expression of NGF was increased ininjured arteries after arterial balloon injury [21] NGF has apotential to stimulate the migration of VSMCs [21 22] Theproduction of IL-1120573 and NGF by senescent cells thereforehas the potential to stimulate vascular inflammation BecauseAdRas12V infection induced higher levels of IL-1120573 expressionthan H
2O2treatment senescent cells may differentially stim-
ulate inflammation in the surrounding tissues depending onthe senescence trigger However future studies are requiredto expand these observations
4 Conclusions
Oxidative stress and oncogene activation induced senescencein VECs and both stimuli promoted the production of
proinflammatory cytokines However oncogene activationstimulated production of IL-1120573more significantly than oxida-tive stress These data suggest that senescent cells may havedifferent proinflammatory actions on surrounding tissuesdepending on the senescence trigger
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] L Hayflick ldquoThe limited in vitro lifetime of human diploid cellstrainsrdquo Experimental Cell Research vol 37 no 3 pp 614ndash6361965
[2] J Campisi ldquoSenescent cells tumor suppression and organismalaging good citizens badneighborsrdquoCell vol 120 no 4 pp 513ndash522 2005
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
The Scientific World Journal 3
AdGFP
H2O2 AdRasG12V
Figure 1 SA-120573-Gal staining of HUVECs HUVECs were infected with AdRas12V for 1 week to induce senescence or with AdGFP as thenegative control Senescence was also induced in HUVECs treated with 100120583molL H
2O2for 1 hr Arrows indicate large and round-shaped
senescent cells and arrowheads indicate flat or spindle-shaped senescent cells
both H2O2treatment and AdRas12V infection induced SA-
120573-Gal positive cells morphology of the cells was appar-ently different While cells treated with H
2O2were large
and round-shaped those infected with AdRas12V becamespindle-shaped or flat This suggests that the senescent cellsmay have different functions depending on the triggerthat induced senescence We also performed western blotanalysis to confirm that these treatments induced senescence(Figure 2) The expression of p53 in HUVECs treated withH2O2or infected with AdRas12V was significantly higher
than that in control cells suggesting that the DNA-damageresponse had been activated Both treatments also stimulatedthe expression of the cdk inhibitor p21CIP1 suggesting thatthe cells had withdrawn from the cell cycle It has beenreported that NF-120581B is activated in senescent cells whichhelps maintain the senescent phenotype and stimulates theproduction of cytokines [12 13] We therefore examinedwhether NF-120581B was activated in our systemThe p65 subunitof NF-120581B is reportedly phosphorylated at Serine-536 whenNF-120581B is activated Both H
2O2treatment and AdRas12V
infection stimulated the phosphorylation of p65 at Serine-536 suggesting that both treatments activated NF-120581B
32 Cytokine Production in Senescent Cells It is well estab-lished that senescent cells undergo massive changes in gene
AdGFP H2O2AdRas12V
p53
p21CIP1
P-p65NF120581B
p65NF120581B
120573-Actin
Figure 2 Western blot analysis of proteins extracted fromHUVECs HUVECs were infected with AdRas12V or treated withH2O2 Proteins were extracted from HUVECs 1 week after the
treatment and analyzed by western blot analysis
4 The Scientific World Journal
0
10
20
30IL
-1120573
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
lowastlowast
lowastdagger
(a)
0
1
2
IL-6
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
(b)
0
5
10
NG
FG
APD
H fo
ld in
duct
ion
H2O2AdGFP AdRas12V
lowast
lowast
(c)
Figure 3 Real time PCR analysis of the expression of proinflammatory cytokines in HUVECs HUVECs were infected with AdGFP orAdRas12V or treated with 100120583molL H
2O2 After 1 week total RNA was extracted for real time PCR analysis The expression of IL1-120573 IL-6
and NGF was normalized to GAPDH lowast and lowastlowast 119875 lt 005 and 119875 lt 001 respectively versus AdGFP infection dagger 119875 lt 001 versus AdRas12Vinfection (119899 = 6 per group)
expression and actively produce a variety of proinflammatorycytokines and immunemodulatorsThese changes have beenreferred to as the senescence-associated secretory phenotype[14] We used real time PCR to examine whether H
2O2
treatment and AdRas12V infection induced similar patternsof cytokine production (Figure 3) Although both treatmentssignificantly stimulated IL-1120573 expression compared withAdGFP infection the levels induced by AdRas12V weresignificantly higher than those induced by H
2O2 IL-6 is a
well-known cytokine that senescent cells produce [15 16]However expression of IL-6 in AdRas12V-infected or H
2O2-
treated HUVECs was similar to that in AdGFP-infectedHUVECs The expression of NGF in cells treated with H
2O2
or infected with AdRas12V was significantly higher than thatin AdGFP infection and the expression in the two treatmentgroups was similar In addition we assessed the expressionof several other cytokines including tumor necrosis factor-120572 and vascular endothelial growth factor-A Although bothtreatments significantly induced the expression of thesecytokines the extent (up to 2-fold induction) was smallerthan that observed in NGF expression (data not shown)We further examined the expression of IL-1120573 and NGF atthe protein level by ELISA (Figure 4(a)) H
2O2treatment
and AdRas12V infection increased the accumulation of IL-1120573 in the culture medium in a time-dependent mannerAdRas12V infection increased the accumulation of IL-1120573 in
the culture mediummore significantly than H2O2treatment
H2O2treatment and AdRas12V infection also increased the
accumulation of NGF in a time-dependent fashion andboth treatments had a similar effect To study the role ofNF-120581B in cytokine production we examined the effects ofBAY11-7082 an I120581B120572 inhibitor on IL-1120573 andNGF production(Figure 4(b)) Pretreatment with BAY11-7082 significantlyinhibited the secretion of IL-1120573 and NGF induced by H
2O2
treatment and AdRas12V infectionWe found that senescent HUVECs produced a variety
of proinflammatory cytokines and that the production wasmediated at least in part by the NF-120581B-dependent pathwayThus theNF-120581B-dependent pathway appears to play a pivotalrole in the production of cytokines in senescent VECs Wealso found that although H
2O2treatment and AdRas12V
infection significantly induced IL-1120573 expression AdRas12Vinfection had a more significant effect than H
2O2treatment
In contrast both treatments had a comparable effect onNGF expression IL-1120573 is a potent proinflammatory cytokinethat stimulates the expression of adhesion molecules onendothelial cells [17] and is frequently expressed in humanatherosclerotic plaques [8] In addition IL-1120573 deficiencydecreases the severity of atherosclerosis in apolipoprotein E(apoE) knock-out mice [18] IL-1120573 therefore potently inducesvascular inflammationNGF is amember of the neurotrophinfamily that stimulates not only nerve growth and survival
The Scientific World Journal 5
0 1 3 6(hr) (hr)
0
1
2
3
4
5
6
0 1 3 60
05
1
15
lowastlowastdagger
lowastlowast
lowastlowast
lowastlowast
dagger
lowastlowast
lowast
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(a)
0
1
2
3
4
5
6
0
05
1
15
AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2 AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2
Plt 005
Plt 005
Plt 001Plt 001
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(b)
Figure 4 The expression of IL-1120573 and NGF proteins in HUVECs treated with AdRas12V or H2O2 (a) Time course of the accumulation of
IL-1120573 and NGF in the culture media HUVECs were infected with AdGFP (closed squares) or AdRas12V (closed circles) or treated with H2O2
(open circles) After 1 week HUVECs were washed with PBS and medium was replaced with serum free Dulbeccorsquos modified Eagle medium(DMEM) HUVECs were incubated for the indicated periods and culture media were collected for ELISA lowast and lowastlowast 119875 lt 005 and 119875 lt 001respectively versus AdGFP infection at each time point dagger 119875 lt 005 versus H
2O2treatment at each time point (119899 = 6 per group) (b) The
effect of BAY11-7082 on cytokine production Experiments were performed as in (a) In addition some HUVECs were preincubated with10120583molL of BAY11-7082 (Bay) for 2 hrs and then washed with PBS The medium was replaced with serum free DMEM HUVECs were thenincubated for 6 hrs in the presence or absence of Bay (119899 = 6 per group)
but also mast cell accumulation in various organs [19]NGF can also promote angiogenesis [20] Although the roleof NGF in atherosclerosis has not been fully elucidatedevidence suggests that it plays a role in the progression ofatherosclerosis The expression of NGF was increased ininjured arteries after arterial balloon injury [21] NGF has apotential to stimulate the migration of VSMCs [21 22] Theproduction of IL-1120573 and NGF by senescent cells thereforehas the potential to stimulate vascular inflammation BecauseAdRas12V infection induced higher levels of IL-1120573 expressionthan H
2O2treatment senescent cells may differentially stim-
ulate inflammation in the surrounding tissues depending onthe senescence trigger However future studies are requiredto expand these observations
4 Conclusions
Oxidative stress and oncogene activation induced senescencein VECs and both stimuli promoted the production of
proinflammatory cytokines However oncogene activationstimulated production of IL-1120573more significantly than oxida-tive stress These data suggest that senescent cells may havedifferent proinflammatory actions on surrounding tissuesdepending on the senescence trigger
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] L Hayflick ldquoThe limited in vitro lifetime of human diploid cellstrainsrdquo Experimental Cell Research vol 37 no 3 pp 614ndash6361965
[2] J Campisi ldquoSenescent cells tumor suppression and organismalaging good citizens badneighborsrdquoCell vol 120 no 4 pp 513ndash522 2005
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
4 The Scientific World Journal
0
10
20
30IL
-1120573
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
lowastlowast
lowastdagger
(a)
0
1
2
IL-6
GA
PDH
fold
indu
ctio
n
H2O2AdGFP AdRas12V
(b)
0
5
10
NG
FG
APD
H fo
ld in
duct
ion
H2O2AdGFP AdRas12V
lowast
lowast
(c)
Figure 3 Real time PCR analysis of the expression of proinflammatory cytokines in HUVECs HUVECs were infected with AdGFP orAdRas12V or treated with 100120583molL H
2O2 After 1 week total RNA was extracted for real time PCR analysis The expression of IL1-120573 IL-6
and NGF was normalized to GAPDH lowast and lowastlowast 119875 lt 005 and 119875 lt 001 respectively versus AdGFP infection dagger 119875 lt 001 versus AdRas12Vinfection (119899 = 6 per group)
expression and actively produce a variety of proinflammatorycytokines and immunemodulatorsThese changes have beenreferred to as the senescence-associated secretory phenotype[14] We used real time PCR to examine whether H
2O2
treatment and AdRas12V infection induced similar patternsof cytokine production (Figure 3) Although both treatmentssignificantly stimulated IL-1120573 expression compared withAdGFP infection the levels induced by AdRas12V weresignificantly higher than those induced by H
2O2 IL-6 is a
well-known cytokine that senescent cells produce [15 16]However expression of IL-6 in AdRas12V-infected or H
2O2-
treated HUVECs was similar to that in AdGFP-infectedHUVECs The expression of NGF in cells treated with H
2O2
or infected with AdRas12V was significantly higher than thatin AdGFP infection and the expression in the two treatmentgroups was similar In addition we assessed the expressionof several other cytokines including tumor necrosis factor-120572 and vascular endothelial growth factor-A Although bothtreatments significantly induced the expression of thesecytokines the extent (up to 2-fold induction) was smallerthan that observed in NGF expression (data not shown)We further examined the expression of IL-1120573 and NGF atthe protein level by ELISA (Figure 4(a)) H
2O2treatment
and AdRas12V infection increased the accumulation of IL-1120573 in the culture medium in a time-dependent mannerAdRas12V infection increased the accumulation of IL-1120573 in
the culture mediummore significantly than H2O2treatment
H2O2treatment and AdRas12V infection also increased the
accumulation of NGF in a time-dependent fashion andboth treatments had a similar effect To study the role ofNF-120581B in cytokine production we examined the effects ofBAY11-7082 an I120581B120572 inhibitor on IL-1120573 andNGF production(Figure 4(b)) Pretreatment with BAY11-7082 significantlyinhibited the secretion of IL-1120573 and NGF induced by H
2O2
treatment and AdRas12V infectionWe found that senescent HUVECs produced a variety
of proinflammatory cytokines and that the production wasmediated at least in part by the NF-120581B-dependent pathwayThus theNF-120581B-dependent pathway appears to play a pivotalrole in the production of cytokines in senescent VECs Wealso found that although H
2O2treatment and AdRas12V
infection significantly induced IL-1120573 expression AdRas12Vinfection had a more significant effect than H
2O2treatment
In contrast both treatments had a comparable effect onNGF expression IL-1120573 is a potent proinflammatory cytokinethat stimulates the expression of adhesion molecules onendothelial cells [17] and is frequently expressed in humanatherosclerotic plaques [8] In addition IL-1120573 deficiencydecreases the severity of atherosclerosis in apolipoprotein E(apoE) knock-out mice [18] IL-1120573 therefore potently inducesvascular inflammationNGF is amember of the neurotrophinfamily that stimulates not only nerve growth and survival
The Scientific World Journal 5
0 1 3 6(hr) (hr)
0
1
2
3
4
5
6
0 1 3 60
05
1
15
lowastlowastdagger
lowastlowast
lowastlowast
lowastlowast
dagger
lowastlowast
lowast
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(a)
0
1
2
3
4
5
6
0
05
1
15
AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2 AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2
Plt 005
Plt 005
Plt 001Plt 001
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(b)
Figure 4 The expression of IL-1120573 and NGF proteins in HUVECs treated with AdRas12V or H2O2 (a) Time course of the accumulation of
IL-1120573 and NGF in the culture media HUVECs were infected with AdGFP (closed squares) or AdRas12V (closed circles) or treated with H2O2
(open circles) After 1 week HUVECs were washed with PBS and medium was replaced with serum free Dulbeccorsquos modified Eagle medium(DMEM) HUVECs were incubated for the indicated periods and culture media were collected for ELISA lowast and lowastlowast 119875 lt 005 and 119875 lt 001respectively versus AdGFP infection at each time point dagger 119875 lt 005 versus H
2O2treatment at each time point (119899 = 6 per group) (b) The
effect of BAY11-7082 on cytokine production Experiments were performed as in (a) In addition some HUVECs were preincubated with10120583molL of BAY11-7082 (Bay) for 2 hrs and then washed with PBS The medium was replaced with serum free DMEM HUVECs were thenincubated for 6 hrs in the presence or absence of Bay (119899 = 6 per group)
but also mast cell accumulation in various organs [19]NGF can also promote angiogenesis [20] Although the roleof NGF in atherosclerosis has not been fully elucidatedevidence suggests that it plays a role in the progression ofatherosclerosis The expression of NGF was increased ininjured arteries after arterial balloon injury [21] NGF has apotential to stimulate the migration of VSMCs [21 22] Theproduction of IL-1120573 and NGF by senescent cells thereforehas the potential to stimulate vascular inflammation BecauseAdRas12V infection induced higher levels of IL-1120573 expressionthan H
2O2treatment senescent cells may differentially stim-
ulate inflammation in the surrounding tissues depending onthe senescence trigger However future studies are requiredto expand these observations
4 Conclusions
Oxidative stress and oncogene activation induced senescencein VECs and both stimuli promoted the production of
proinflammatory cytokines However oncogene activationstimulated production of IL-1120573more significantly than oxida-tive stress These data suggest that senescent cells may havedifferent proinflammatory actions on surrounding tissuesdepending on the senescence trigger
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] L Hayflick ldquoThe limited in vitro lifetime of human diploid cellstrainsrdquo Experimental Cell Research vol 37 no 3 pp 614ndash6361965
[2] J Campisi ldquoSenescent cells tumor suppression and organismalaging good citizens badneighborsrdquoCell vol 120 no 4 pp 513ndash522 2005
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
The Scientific World Journal 5
0 1 3 6(hr) (hr)
0
1
2
3
4
5
6
0 1 3 60
05
1
15
lowastlowastdagger
lowastlowast
lowastlowast
lowastlowast
dagger
lowastlowast
lowast
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(a)
0
1
2
3
4
5
6
0
05
1
15
AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2 AdRas12V AdRas12V+ BAY
H2O2 + BAYH2O2
Plt 005
Plt 005
Plt 001Plt 001
NG
F (p
g120583
g pr
otei
n)
IL-1120573
(pg120583
g pr
otei
n)
(b)
Figure 4 The expression of IL-1120573 and NGF proteins in HUVECs treated with AdRas12V or H2O2 (a) Time course of the accumulation of
IL-1120573 and NGF in the culture media HUVECs were infected with AdGFP (closed squares) or AdRas12V (closed circles) or treated with H2O2
(open circles) After 1 week HUVECs were washed with PBS and medium was replaced with serum free Dulbeccorsquos modified Eagle medium(DMEM) HUVECs were incubated for the indicated periods and culture media were collected for ELISA lowast and lowastlowast 119875 lt 005 and 119875 lt 001respectively versus AdGFP infection at each time point dagger 119875 lt 005 versus H
2O2treatment at each time point (119899 = 6 per group) (b) The
effect of BAY11-7082 on cytokine production Experiments were performed as in (a) In addition some HUVECs were preincubated with10120583molL of BAY11-7082 (Bay) for 2 hrs and then washed with PBS The medium was replaced with serum free DMEM HUVECs were thenincubated for 6 hrs in the presence or absence of Bay (119899 = 6 per group)
but also mast cell accumulation in various organs [19]NGF can also promote angiogenesis [20] Although the roleof NGF in atherosclerosis has not been fully elucidatedevidence suggests that it plays a role in the progression ofatherosclerosis The expression of NGF was increased ininjured arteries after arterial balloon injury [21] NGF has apotential to stimulate the migration of VSMCs [21 22] Theproduction of IL-1120573 and NGF by senescent cells thereforehas the potential to stimulate vascular inflammation BecauseAdRas12V infection induced higher levels of IL-1120573 expressionthan H
2O2treatment senescent cells may differentially stim-
ulate inflammation in the surrounding tissues depending onthe senescence trigger However future studies are requiredto expand these observations
4 Conclusions
Oxidative stress and oncogene activation induced senescencein VECs and both stimuli promoted the production of
proinflammatory cytokines However oncogene activationstimulated production of IL-1120573more significantly than oxida-tive stress These data suggest that senescent cells may havedifferent proinflammatory actions on surrounding tissuesdepending on the senescence trigger
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] L Hayflick ldquoThe limited in vitro lifetime of human diploid cellstrainsrdquo Experimental Cell Research vol 37 no 3 pp 614ndash6361965
[2] J Campisi ldquoSenescent cells tumor suppression and organismalaging good citizens badneighborsrdquoCell vol 120 no 4 pp 513ndash522 2005
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
6 The Scientific World Journal
[3] C B Harley A B Futcher and C W Greider ldquoTelomeresshorten during ageing of human fibroblastsrdquo Nature vol 345no 6274 pp 458ndash460 1990
[4] Q Chen A Fischer J D Reagan L-J Yan and B N AmesldquoOxidative DNA damage and senescence of human diploidfibroblast cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 92 no 10 pp 4337ndash43411995
[5] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16(INK4a)rdquoCell vol88 no 5 pp 593ndash602 1997
[6] I Ben-Porath and R A Weinberg ldquoThe signals and path-ways activating cellular senescencerdquo International Journal ofBiochemistry and Cell Biology vol 37 no 5 pp 961ndash976 2005
[7] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995
[8] T Minamino T Yoshida K Tateno et al ldquoRas induces vascularsmooth muscle cell senescence and inflammation in humanatherosclerosisrdquo Circulation vol 108 no 18 pp 2264ndash22692003
[9] C Matthews I Gorenne S Scott et al ldquoVascular smoothmuscle cells undergo telomere-based senescence in humanatherosclerosis effects of telomerase and oxidative stressrdquoCirculation Research vol 99 no 2 pp 156ndash164 2006
[10] E Suzuki H Nishimatsu D Nagata et al ldquoConstitutiveactivation of proto-oncogen protein p21 induces cell cycle arrestin the G1 phase in contact-inhibited vascular endothelial cellsrdquoHypertension Research vol 25 no 5 pp 773ndash778 2002
[11] E Suzuki D Nagata M Yoshizumi et al ldquoReentry into the cellcycle of contact-inhibited vascular endothelial cells by a phos-phatase inhibitor Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinaserdquo Journal ofBiological Chemistry vol 275 no 5 pp 3637ndash3644 2000
[12] Y Chien C Scuoppo X Wang et al ldquoControl of the senes-cence-associated secretory phenotype by NF-120581B promotessenescence and enhances chemosensitivityrdquo Genes and Devel-opment vol 25 no 20 pp 2125ndash2136 2011
[13] J S Tilstra A R Robinson JWang et al ldquoNf-kappab inhibitiondelays DNA damage-induced senescence and aging in micerdquoJournal of Clinical Investigation vol 122 no 7 pp 2601ndash26122012
[14] J-P Coppe C K Patil F Rodier et al ldquoSenescence-associatedsecretory phenotypes reveal cell-nonautonomous functions ofoncogenic RAS and the p53 tumor suppressorrdquo PLoS Biologyvol 6 no 12 pp 2853ndash2868 2008
[15] J C Acosta A OrsquoLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008
[16] T Kuilman C Michaloglou L C W Vredeveld et alldquoOncogene-induced senescence relayed by an interleukin-dependent inflammatory networkrdquoCell vol 133 no 6 pp 1019ndash1031 2008
[17] H Baumann and J Gauldie ldquoThe acute phase responserdquoImmunology Today vol 15 no 2 pp 74ndash80 1994
[18] H Kirii T Niwa Y Yamada et al ldquoLack of interleukin-1120573decreases the severity of atherosclerosis in apoE-deficientmicerdquoArteriosclerosisThrombosis and Vascular Biology vol 23 no 4pp 656ndash660 2003
[19] L Aloe and R Levi-Montalcini ldquoMast cells increase in tissuesof neonatal rats injected with the nerve growth factorrdquo BrainResearch vol 133 no 2 pp 358ndash366 1977
[20] P Lazarovici C Marcinkiewicz and P I Lelkes ldquoCross talkbetween the cardiovascular and nervous systems neurotrophiceffects of vascular endothelial growth factor (VEGF) andangiogenic effects of nerve growth factor (NGF)-implicationsin drug developmentrdquo Current Pharmaceutical Design vol 12no 21 pp 2609ndash2622 2006
[21] M J Donovan R CMiranda R Kraemer et al ldquoNeurotrophinand neurotrophin receptors in vascular smooth muscle cellsregulation of expression in response to injuryrdquo AmericanJournal of Pathology vol 147 no 2 pp 309ndash324 1995
[22] R Kraemer H Nguyen K L March and B Hempstead ldquoNGFactivates similar intracellular signaling pathways in vascularsmooth muscle cells as PDGF-BB but elicits different biologicalresponsesrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 19 no 4 pp 1041ndash1050 1999
![Page 1: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/1.jpg)
![Page 2: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/2.jpg)
![Page 3: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/3.jpg)
![Page 4: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/4.jpg)
![Page 5: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/5.jpg)
![Page 6: Oncogene- and Oxidative Stress-Induced Cellular Senescence ... · 2 TheScientificWorldJournal exhibitthemorphologicalfeaturesofsenescence[8,9].Ithas alsobeenreportedthatinterleukin-1𝛽(IL-1𝛽)isexpressedin](https://reader030.documents.pub/reader030/viewer/2022041204/5d54559588c99339758b635a/html5/thumbnails/6.jpg)
