a number of diseases , including most forms of cardiovascular disease , diabetes , cancer , rheumatoid arthritis , and aging , are associated with endothelial dysfunction [ 13 ] . endothelial dysfunction is also a characteristic of bacterial sepsis [ 4 , 5 ] . to reduce or prevent the high incidence of morbidity and mortality associated with vascular complications , it is essential to understand further the mechanism by which inflammation regulates endothelial function . systemic inflammation causes an upregulation of a wide number of factors that lead to severe injury of vascular endothelial cells [ 610 ] . these include growth factors ( basic fibroblast growth factor ( bfgf or fgf-2 ) , vascular endothelial growth factor ( vegf ) ) ; proinflammatory cytokines ( il-1 , il-6 ) and chemokines ( il-8 ) ; extracellular matrix ( ecm ) degrading enzymes ( plasminogen activator inhibitor-1 ( pai-1 ) , matrix metalloproteinases ( mmps ) ) ; cell adhesion molecules ( intercellular adhesion molecule ( icam-1 ) , vascular cell adhesion protein ( vcam)-1 , p - selectin ) ; and the cyclooxygenase ( cox)-prostaglandin system . collectively , they participate in the inflammatory response and contribute to a vasoconstrictive and prothrombotic state . there is now increasing evidence that a number of transcription factors , including the sirtuin ( sirt ) family and nuclear factor b ( nf-b ) , regulate multiple genes whose products are putatively involved in the regulation of endothelial cell function . the sirtuin family consists of seven enzymes ( sirt1sirt7 ) that share a conserved core catalytic domain but differ in their cellular localisation and tissue distribution . to date , most of the studies have focussed on sirt1 . numerous genes involved in vascular growth , maturation , and remodelling are dysregulated in sirt1 deficient endothelial cells [ 1215 ] . additionally , administration of resveratrol , a sirt1 activator , improves endothelial dysfunction [ 1619 ] and reduces vascular inflammation in mice , two central pathophysiological processes involved in the initiation and progression of cardiovascular disease . importantly , sirt1 mediates these effects by interacting and inhibiting the proinflammatory transcription factor nf-b [ 1620 ] . a recent study in mice has suggested a role for sirt6 in inflammation ; however , the role of sirt6 in endothelial function is not known . in this study , a human umbilical vein endothelial cell ( huvec ) line will be used to determine the effect of the endotoxin lipopolysaccharide ( lps ) on sirt6 expression . in addition , the effect of sirt6 inhibition on mediators of vascular inflammation will be determined . specifically , the effect of sirt6 sirna on the expression of proinflammatory cytokines ( il-1 and il-6 ) , cox - prostaglandin system ( cox-2 and subsequent prostaglandin release ) , ecm remodelling enzymes ( mmp-2 , mmp-9 , pai-1 ) , adhesion molecules ( icam-1 , vcam-1 , p - selectin ) and angiogenic growth factors ( vegf and fgf-2 ) will be assessed . the migration and adhesion properties of sirt6 deficient huvecs will also be assessed . in order to elucidate if sirt6 mediates its effects via nf-b , luciferase assays will be used to determine the effect of sirt6 overexpression on nf-b activity . huvecs ( huv - ec - c ) were obtained from atcc ( manassas , va , usa ) . cells were incubated in 25  cm flask in dmem / f12 enriched with 10% fcs , 100  u / ml penicillin g , and 100  g / ml streptomycin until they reached 80% confluence . the cells were the treated with dmem / f-12 containing 2% heat - inactivated fcs in the absence or presence of 10  g / ml lps . after 48  h incubation , cells were collected and assessed for sirt6 expression by western blotting as detailed below . huvecs were incubated in dmem / f12 enriched with 10% fcs , 100  u / ml penicillin g , and 100  g / ml streptomycin . cells were transfected using transit - siquest reagent according to manufacturer guidelines ( mirus bio , madison , wi , usa ) . cells were transfected with either 50  nm sirt6 or nonspecific sirna from ambion ( austin , texas ) in dmem ( containing 100  u / ml penicillin g , 100  g / ml streptomycin ) for 72  h. the cells were then treated with either 10  g / ml lps ( in dmem / f-12 containing 2% heat - inactivated fcs ) for an additional 24  h. cells were collected and stored at 80c until assayed for mrna expression by qrt - pcr and protein expression by western blotting as detailed below . media were collected and stored at 80c until assayed for cytokine and prostaglandin release by elisa as detailed below . huvecs were transfected using lipofectamine 2000 as described by the manufacturer ( life technologies , carlsbad , ca ) . cells were cotransfected with 1  g reporter ( either pnf-b - luc or negative control ; qiagen ) and 1  g plasmid ( empty vector or sirt6 vector ; origene ) for 72  h. the cells were then treated with dmem / f-12 containing 2% heat - inactivated fcs in the absence or presence of 10  g / ml lps for 24  h. the media were collected and assayed for il-6 , il-8 , pge2 , and pgf2 as detailed above . the cells were harvested in lysis buffer and luminescence activity was measured using the dual - glo luciferase assay kit ( promega ) as instructed . the release of il-6 and il-8 was performed by sandwich elisa according to the manufacturer 's instructions ( invitrogen , carlsbad , ca ) . the release of pge2 and pgf2 into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer 's specifications ( kookaburra kits from sapphire bioscience , nsw , australia ) . assessment of protein expression was analysed by western blotting as previously described , with some minor modifications . two g protein was loaded into 10% mini - protean tgx gels ( bio - rad laboratories , hercules , ca ) . blots were incubated with 0.5  g / ml rabbit polyclonal anti - sirt6 ( s4197 , sigma , st . louis , mo , usa ) diluted in blocking buffer ( 5% skim milk / tbs - t ( 0.05% ) ) for 24  h at 4c . gels were stripped and reprobed with mouse monoclonal anti--actin ( a5316 ) for 24  h at 4c . total rna was extracted using tri - reagent according to manufacturer 's instructions ( sigma - aldrich , saint louis missouri ) . rna concentrations were quantified using a spectrophotometer ( smart spec , bio - rad ) . rna concentration and purity were measured using a nanodrop nd1000 spectrophotometer ( thermo scientific , pittsburgh , pa ) . rna was converted to cdna using the superscript vilo cdna synthesis kit ( invitrogen , carlsbad , california , usa ) according to the manufacturer 's instructions . the cdna was diluted tenfold , and 0.8  l of cdna was used to perform rt - pcr using sensimix plus sybr green ( quantace , alexandria , nsw , australia ) and 100  nm of prevalidated and designed primers ( quantitect primer assays , qiagen , germantown , maryland , usa ) . rna without reverse transcriptase during cdna synthesis as well as pcr reactions using water instead of template showed no amplification . average gene ct values were normalised to the average gapdh ct values of the same cdna sample . fold differences were determined using the comparative ct method , and the results are expressed as a ratio of the average of the control group . to assess the migration / invasion ability of sirt6 deficient cells , a gelatin invasion assay was used . briefly , transwell inserts ( 8  m pore size ) were coated on the underside with 0.1% gelatin . huvecs transfected with ns or sirt6 sirna were prepared as detailed above and 5  10 cells were allowed to migrate across inserts for 5 hours at 37c . unmigrated cells from the upper chamber were removed , and migrated cells in the lower side of the membrane were fixed , stained , and counted . the average number of migrated cells in ten randomly chosen fields of view per insert was taken to quantify the extent of migration . huvecs transfected with ns or sirt6 sirna were prepared as detailed above and 2  10 cells in 100  l per well were added to gelatin precoated 96-well plates and allowed to adhere for 1.5  h at 37c . unbound cells were washed , 10  l of mtt ( 5  mg / ml stock in pbs ) was added per well , and the incubation continued for 4  h. after washing , the bound dye was lysed with 200  l dmso and absorbance read at 570  nm . polymorphonuclear leukocyte ( pmn ) cells , isolated from whole blood , were a gift from dr . katherine woods ( ludwig institute for cancer research , austin hospital , heidelberg , victoria , australia ) . for these experiments , 48-well plates were used and huvecs transfected with ns or sirt6 sirna were prepared as detailed above . after 3 washings with serum - free medium , 1  10  pmn cells per ml were added to each well ( final 2.5  10  pmn cells per well ) , and incubated at 37c for an additional 60  min . the number of adhered pmns was evaluated by a colorimetric assay using mtt assay as detailed above . statistical analyses were performed using a commercially available statistical software package ( statgraphics plus version 3.1 , statistical graphics corp . , rockville , maryland , usa ) . two sample comparisons were analysed by a paired sample comparison . for all other comparisons , analysis was performed using a one - way anova ( using tukey hsd correction to discriminate among the means ) ; homogeneity of data was assessed by bartlett 's test , and when significant , data were logarithmically transformed before further analysis . the first aim was to determine the effect of lps on sirt6 expression in huvecs . to do this , huvecs were incubated in the absence or presence of 10  g / ml lps for 48  h and protein expression assessed by western blotting ( n = 6 experiments ) . as shown in figure 1 , lps significantly decreased sirt6 protein expression , without affecting -actin expression . having shown that lps inhibits sirt6 expression in huvecs , the next aim was to determine the effect of sirt6 knockdown on endothelial function in the presence of lps . to do this , huvecs were transfected with and without sirt6 sirna and nonspecific ( ns ) sirna . after 72  h transfection , cells were incubated in the absence or presence of 10  g / ml lps for 24  h. the efficacy of transfection was analysed by qrt - pcr and western blotting . compared to ns sirna transfected huvecs , transfection with sirt6 sirna resulted in a significant decrease in sirt6 mrna ( figure 2(a ) ) and protein expression ( figure 2(b ) ) . there was , however , no effect of sirt6 sirna on -actin protein expression ( figure 2(b ) ) . treatment of huvecs with lps significantly increased the release ( figure 3(a ) ) and gene expression ( figure 3(b ) ) of il-1 , il-6 , and il-8 . in support with the hypothesis that sirt6 regulates endothelial cell inflammation , sirna knockdown of sirt6 is associated with significantly increased gene expression of the proinflammatory cytokine il-6 and the chemokine il-8 ( figure 3(a ) ) . to determine whether inhibition of sirt6 plays a role in the steady state of cytokine mrna expression , consistent with the release data , when compared to ns sirna transfected huvecs , sirt6 sirna induced il-6 and il-8 mrna expression ( figure 3(b ) ) , as well as increasing the mrna expression of il-1. the next aim was to determine the effect of sirt6 sirna on the cox - prostaglandin system , specifically cox-1 and cox-2 gene expression and subsequent pge2 and pgf2 release . lps significantly induced cox-2 mrna expression ( figure 4(a ) ) and release of pge2 and pgf2 ( figure 4(b ) ) . as shown in figure 4 , inhibition of sirt6 significantly increased cox-2 mrna expression and subsequent prostaglandin release . we next sought to determine the effect of sirt6 knockdown on enzymes involved in ecm degradation and ecm remodelling , namely pai-1 , mmp-2 , and mmp-9 . as shown in figure 5 , lps induced mmp-9 and pai-1 gene expression ; an effect that was even higher in sirt6 deficient huvecs . on the other hand , there was no effect of lps or sirt6 inhibition on mmp-2 gene expression . to investigate putative promigratory effects of sirt6 sirna , endothelial vcam-1 , icam-1 , and p - selectin mrna expression was quantified by qrt - pcr . lps significantly increased the gene expression of the cell adhesion molecule icam-1 ( figure 6(a ) ) . this was also associated with an increase in the secretion of icam-1 into the conditioned media ( figure 6(b ) ) . additionally , when compared to lps alone , inhibition of sirt6 significantly increased icam-1 gene expression and release ( figures 6(a ) , 6(b ) ) . there was , however , no effect of lps or lps plus sirt6 sirna on vcam-1 and p - selectin gene expression ( figure 6(a ) ) . to evaluate the role of sirt6 in the cell adhesion potential of huvecs , two different assays were performed . in the first assay , ns or sirt6 sirna transfected huvecs were plated in 1% gelatin - coated 96 well plates for 1.5  h. thereafter , unattached cells were washed and mtt colorimetric assay was used to determine the number of adherent cells . as depicted in figure 6(c ) , in ns sirna transfected cells , lps induced an increase in absorbance , indicating an increase in cell adhesion . the effect of sirt6 sirna transfected huvecs to bind to leukocytes was also assessed . in these experiments , leukocytes were added to confluent monolayers of huvecs transfected with ns or sirt6 sirna . as shown in figure 6(d ) , in ns sirna transfected huvecs , stimulation with lps induced a significant increase in the adhesion of leukocytes . when sirt6 was knocked out in these cells , although there was no effect of lps on the expression of the angiogenic growth factors vegf and fgf2 ( figure 7(a ) ) , in huvecs transfected with sirt6 sirna , vegf and fgf2 gene expression was significantly upregulated ( figure 7(b ) ) . to measure the migration potential of sirna - treated huvecs , an invasion chamber assay was performed . this assay measures the number of cells migrating through pores of a polycarbonate filter , simulating a basement membrane . 72  h after transfection , the number of cells on the underside of each membrane was counted and the mean calculated . the effect of sirt6 knockdown on huvec cell migration is depicted in figure 7(c ) . in ns sirna transfected cells , lps treatment increased the mean number of cells that migrated through the membrane . when compared to lps treated ns sirna transfected huvecs , the mean number of cells that migrated through the membrane was increased in sirt6 sirna transfected huvecs . the final aim of this study was to determine if sirt6 regulates proinflammatory factors in huvecs via the nf-b pathway . to assess this , the effect of sirt6 sirna on nf-b transcription was investigated first . as shown in figure 8(a ) , knockdown of sirt6 significantly increased nf-b p65 mrna expression . next , whether sirt6 regulates the transcriptional activity of nf-b by luciferase assays using nf-b - luc reporter was determined . the cells were also transfected with sirt6 expression vector or control vector pcdna3.1 . as shown in figure 8(b ) , transfection with nf-b luc reporter significantly increased luciferase activity . however , overexpression of sirt6 inhibited nf-b - mediated luciferase reporter expression in huvecs . the effect of nf-b overexpression with and without co - transfection with sirt6 in huvecs on a number of nf-b target genes was also evaluated . as shown in figure 9 , overexpression of nf-b significantly increased il-6 and pgf2 levels in the conditioned media . epidemiological , clinical , and animal studies demonstrate that bacterial infection can induce endothelial dysfunction cells [ 610 , 23 ] . for example , infection decreases sirt1 expression and activity [ 22 , 24 ] and activation of sirt1 exerts beneficial effects on endothelial cell dysfunction [ 18 , 25 ] . this is the first study to report that lps can also decrease sirt6 expression in huvecs . this led to the hypothesis that sirt6 may also play an important role in infection induced endothelial dysfunction . in order to test this theory , sirt6 was inhibited in huvecs using sirna , and then a number of factors known to play an important role in vascular disease were investigated . in sirt6 deficient cells , in the presence of lps , there was an increase in the expression of the proinflammatory cytokines il-1 and il-6 , the chemokine il-8 , ecm degrading enzymes mmp-9 and pai-1 , cox - prostaglandin system , and the angiogenic growth factors fgf-2 and vegf . the loss of sirt6 in huvec was associated with increased expression of the cell adhesion molecule icam-1 , as well as increased cell adhesion properties . in addition , loss of sirt6 increases the expression of the proinflammatory transcription factor nf-b , whereas overexpression of sirt6 decreases the transcriptional activity of nf-b as well as the expression of its target genes . thus , activation of sirt6 may be beneficial for many inflammatory diseases associated with endothelial dysfunction . endothelial cells themselves are a major source of proinflammatory cytokines , which are significantly enhanced during inflammation . these proinflammatory stimuli can induce a phenotypic change of the quiescent endothelium to an activated endothelium . this results in an increase or induction of the endothelial expression of adhesion molecules and chemokines ( chemoattractant cytokines ) , leading to leukocyte recruitment , adhesion , and infiltration into the subendothelium . activated endothelium also leads to an increase in cytokine expression and production that may act in an autocrine fashion to enhance endothelial cell activation further . in this study , lps induced the expression and release of the proinflammatory cytokines il-1 and il-6 , the chemokine il-8 ; this increase was even higher in sirt6 deficient huvecs . as expected , lps stimulation induced the adhesion molecule icam-1 as well as leukocyte cell adhesion to huvecs , and this increase was further augmented in sirt6 deficient cells . of note , there was no effect of lps or sirt6 inhibition on the expression of vcam-1 or p - selection . although there are no studies on sirt6 and adhesion molecules , these studies are in support of the few other studies demonstrating that sirt6 regulates inflammatory cytokines [ 26 , 27 ] . in the process of vascular inflammation , activated endothelial cells can also migrate to surrounding tissues to spur the formation of new blood vessels , or angiogenesis , which plays an important role in a number of pathologies such as atherosclerosis , diabetes , and tumour development [ 2830 ] . indeed , in experimental studies , lps has been shown to promote human endothelial cell migration . in this study , lps stimulation not only promoted huvec migration but the effect was intensified in sirt6 deficient huvecs . vegf and fgf-2 are two angiogenic growth factors whose activation leads to dysregulated blood vessel formation as seen in various chronic inflammatory conditions such as cancer and rheumatoid arthritis [ 28 , 29 ] . in this study its proinflammatory products have been implicated in the pathogenesis of several inflammatory diseases including diabetes and atherosclerosis . in addition , cox-2 can also contribute to the pathogenesis of endothelial dysfunction by disrupting vascular homeostasis . it also generates vasoconstrictor substances , including vasoconstrictor prostanoids that have proatherogenic effects [ 32 , 33 ] . this study demonstrates that in the presence of infection , sirt6 is a regulator of the cox - prostaglandin pathway in endothelial cells . specifically , the loss of sirt6 in huvecs resulted in a significant increase in cox-2 expression concomitant with an increase in pge2 and pgf2 release . mmps play a key role in the processes of remodelling and reorganisation of the vasculature , which is a key feature of endothelial dysfunction [ 34 , 35 ] . pai-1 , a member of the serine protease inhibitor family , is a key inhibitor of fibrinolysis ; impaired endothelial fibrinolytic potential contributes to the development of thrombotic attacks and atherosclerosis [ 36 , 37 ] . in addition , pai-1 is also involved degradation of the ecm [ 36 , 37 ] . in this study , this is the first study to demonstrate a role for sirt6 in the regulation of matrix proteins in endothelial cells ; however , a role for sirt1 has been reported . nf-b is a master regulatory component driving many inflammatory diseases ; it is thought to play an important role in endothelial cell function [ 1 , 39 , 40 ] . there is now much evidence to show that sirt1 exerts many of its protective effects in endothelial cells by inhibiting nf-b activity . in this study , additionally , using luciferase assays , nf-b transcriptional activity was significantly reduced in huvecs that also overexpressed sirt6 . this decrease was also associated with a decrease in the expression of nf-b target genes , namely , il-6 and pgf2. collectively , this suggests that in the presence of sirt6 , huvecs display hypoactive nf-b , leading to decreased transcription of proinflammatory genes . our data is in support of recent studies that report increased expression of nf-b and its target genes in a number of tissues of sirt6deficient mice . functional alterations of the endothelium are hallmark features of many inflammatory diseases , including diabetes , hypertension , atherosclerosis , rheumatoid arthritis , and aging [ 1 , 4244 ] . therefore , the inhibition of inflammatory mediators in activated endothelial cells might be of great benefit in the maintenance of endothelial homeostasis and the prevention of vascular diseases . this study highlights the relevance of sirt6 as an important player in inflammatory reactions of the human endothelium in the context of sepsis . there is now growing literature implicating gut bacteria on the development of insulin resistance , cardiovascular disease , and atherosclerosis . it is , therefore , tempting to speculate that activators of sirt6 might be an effective strategy for a number of inflammatory vascular diseases such as diabetes and atherosclerosis .
a prominent feature of inflammatory diseases is endothelial dysfunction . 
 factors associated with endothelial dysfunction include proinflammatory cytokines , adhesion molecules , and matrix degrading enzymes . at the transcriptional level , 
 they are regulated by the histone deacetylase sirtuin ( sirt ) 1 via its actions on the proinflammatory transcription factor nuclear factor-b ( nf-b ) . 
 the role of sirt6 , also a histone deacetylase , in regulating inflammation in endothelial cells is not known . 
 the aim of this study was to determine the effect of sirt6 knockdown on inflammatory markers in human umbilical vein endothelial cells ( huvecs ) in the presence of lipopolysaccharide ( lps ) . 
 lps decreased expression of sirt6 in huvecs . 
 knockdown of sirt6 increased the expression of proinflammatory cytokines ( il-1 , il-6 , il-8 ) , cox - prostaglandin system , ecm remodelling enzymes ( mmp-2 , mmp-9 and pai-1 ) , the adhesion molecule icam-1 , and proangiogenic growth factors vegf and fgf-2 ; cell migration ; cell adhesion to leukocytes . 
 loss of sirt6 increased the expression of nf-b , whereas overexpression of sirt6 was associated with decreased nf-b transcriptional activity . 
 taken together , these results demonstrate that the loss of sirt6 in endothelial cells is associated with upregulation of genes involved in inflammation , vascular remodelling , and angiogenesis . 
 sirt6 may be a potential pharmacological target for inflammatory vascular diseases .