inflammation is common for patients with cardiovascular diseases and is considered to be a sign or atherogenic response . reactive oxygen species ( ros ) such as superoxide anions , hydrogen peroxide , peroxynitrite , and hydroxyl radicals plays an important role in inflammation , leading to endothelial oxidative damage and dysfunction of the cardiovascular system . proinflammatory cytokines , for example , tumor necrosis factor - alpha ( tnf ) , are able to stimulate ros liberation . superfluous ros production not only causes endothelial dysfunction   but also stimulates signaling transduction pathways implicated in augmented gene expression of inflammation - related cytokine . in cardiovascular cells , the normal intracellular ros levels rely on the proper balance between ros formation and antioxidant defense systems . sophisticated interactions between leukocytes and the endothelium are involved in inflammatory reaction . the endothelial cell ( ec ) surface is relatively smooth and nonadhesive . in cardiovascular diseases , the interactions between the ecs and components of the blood are altered by adhesion molecules , for example , icam-1 and vcam-1 . the endothelial cells are activated , leading to overexpression of adhesion molecules at inflammation sites . additionally , substantial evidence demonstrates an augmented expression of vcam-1 in inflammatory animal models and human atherosclerotic plaques . its key function is to degrade heme to iron , carbon monoxide ( co ) , and biliverdin [ 10 , 11 ] . biliverdin , via the cytosolic enzyme biliverdin reductase , is converted to bilirubin , which possesses antioxidant characteristics . in addition , ho-1-derived co is involved in vasoregulation and signal transduction [ 13 , 14 ] . some signaling molecules , for instance , 5 adenosine monophosphate - activated protein kinase ( ampk ) , mitogen - activated protein kinases ( mapk ) , and pi3k / akt , as well as transcriptional factors including activator protein 1 ( ap-1 ) and nf - e2-related factor-2 ( nrf2 ) , regulate ho-1 gene expression [ 1518 ] . clopidogrel ( inn ) is an oral antiplatelet drug hindering blood clots and has been discovered to possess several biological properties such as anti - inflammation , antioxidation , and antiatherosclerosis [ 1921 ] . anti - inflammation and antioxidation can be used as therapeutic strategies to prevent or treat cardiovascular diseases . the present study was carried out to elucidate the role of ampk and ho-1 in the inn inhibition of tnf-stimulated expression of vcam-1 and the underlying mechanisms implicated . primary human aortic endothelial cells ( haecs ) and endothelial growth factors were purchased from cambrex bioscience ( rockland , me ) . medium 199 , gentamycin , fungizone , glutamine , collagenase , gelatin , trypsin / edta , penicillin , and streptomycin were purchased from sigma - aldrich ( st . louis , mo ) . fetal bovine serum ( fbs ) was purchased from hyclone ( logan , ut , usa ) . hl-60 cells were cultured in roswell park memorial institute ( rpmi ) 1640 plus l - glutamine and 25  mm hepes supplemented with 15% fbs . human tnf , dmso , hepes , sodium bicarbonate , compound c , and all other chemicals were from sigma . clopidogrel ( inn ) ( plavix , sanofi - aventis , bn.f-33565 , france ) stock solution ( 10  mm ) was prepared by dissolving it in dmso . trizol rna extraction reagent and 2,7-dichlorofluorescin diacetate ( h2dcf - da ) were purchased from invitrogen ( carlsbad , ca , usa ) . antibodies reacting with ampk and phosphor - ampk ( thr172 ) were obtained from cell signaling technology ( danvers , ma ) ; antibodies reacting with -actin and nrf2 were obtained from santa cruz biotechnology ( santa cruz , ca ) ; anti - ho-1 and vcam-1 antibodies were obtained from abcam ( cambridge , ma , usa ) . cell culture haecs were cultured in medium 199 added with 20  mm hepes , 2  mm glutamine , 20% fbs , 100  mu / l penicillin , and 100  mg / l streptomycin at 37c . preincubation with inhibitors was implemented in hepes - hsa buffer ( 10  mm hepes , 10  mm glucose , 1.5  mm cacl2 , 145  mm nacl , 5  mm kcl , 1  mm mgso4 , and 0.25% hsa ) . equivalent amounts of protein samples were separated by sds - page and then transferred to nitrocellulose membrane followed by immunoblotting with the primary antibodies including phosphor - ampk - thr172 , ampk , ho-1 , nrf2 , parp , and -actin . total rna was extracted by using a qiashredder column and rneasy kit ( qiagen ) and frozen at 80c for further rt - pcr analysis . primer sequences were as follows : -actin ( atgtttgagaccttcaacac , cacgtcacacttcatgatgg ) , vcam-1 ( cgtcttggtcagcccttcct , acattcatatactcccgcatccttc ) , and ho-1 ( gggtgatagaagaggccaaga , agctcctgcaactcctcaaa ) . cdna ( 1  l ) was amplified in pcr solution ( 25  l ) in a mini opticontm real - time pcr detection system . cycle parameters were set as 95c for 15  min , 40 cycles of 95c for 15  sec , 58c for 1  min , and 72c for 1  min . the ho-1 activity in ec lysates was calculated as picomoles bilirubin produced per hour per milligram of total protein ( pmol br h-1 mg-1 ) and the data were expressed as fold ho-1 activity compared to control cells . subcellular fractionation was performed with a subcellular protein fractionation kit ( thermo fisher scientific inc . , expression of parp was utilized as loading controls for the purity of the nuclear extracts . two distinct sequences targeting human nrf2 and ho-1 mrna were selected and obtained from sigma ( st . the shrna sequences were as follows : nrf2 shrna # 1 , 5-gctcctactgtgatgtgaaat-3 , shrna # 2 , 5-ccggcatttcactaaacacaa-3 ; ho-1 shrna # 1 , 5-gctgagttcatgaggaacttt-3 , shrna # 2 , 5-gctgagttcatgaggaacttt-3 ; shluc shrna , 5-caaatcacagaatcgtcgtat-3. shluc was used as a vector control . intracellular ros state was determined with the cell - permeant 2,7-dichlorodihydrofluorescein diacetate ( h2dcfda ) as previously described . concisely , haecs were grown to 50% confluence followed by serum starvation in medium 199 supplemented with 0.5% ( v / v ) fbs for another 24  h. the ecs were maintained in serum - free medium without phenol red for 15  min preceding being exposed to tnf. ecs were incubated with h2dcfda ( 10  m ) for 10  min and instantly observed under a confocal microscope ( leica tcs sp2 ) . the homogenate was prepared in the potassium phosphate buffer ( 20  mm , ph 7.0 ) and centrifuged at 10,000  g for 20  min at 4c . cell lysates ( 100  l ) were incubated with 5% tca ( 150  l ) and centrifuged at 5000  g for 10  min at 4c . the cell lysate was incubated with 0.4  m tris buffer and 0.01  m dtnb . after incubation at room temperature for 5  min , the intracellular gsh production was measured with a microplate reader at 412  nm ( model 680 , bio - rad ) . data were analyzed with one - way analysis of variance and fisher 's protected least significant difference test . to determine whether inn decreases tnf-stimulated ros formation , we pretreated ecs with 10  mol / l inn for 24  h and then treated ecs with 1  ng / ml tnf for another 20  min . as illustrated in figure 1 , tnf promoted ros liberation at 20  min , and pretreatment with 10  mol / l inn dramatically repressed this ros liberation . n - acetylcysteine ( nac ) is a potent antioxidant and used as a positive control . since ho-1 has a potent cytoprotective effect against cellular oxidative stress [ 28 , 29 ] , we sought to investigate the effect of inn on ho-1 expression . haecs were incubated with 10  m inn for various time periods . as illustrated in figures 2(a ) , 2(b ) , and 2(c ) , inn treatment time - dependently promoted expression of ho-1 at mrna and protein levels as well as its activity . furthermore , the role of ho-1 in the suppression of hl-60 adhesion by inn was determined with ho-1 shrna knockdown study . ecs were transfected with shho-1 , followed by incubation with 10  m inn for 24  h and then ecs were stimulated with tnf for another 6  h. knockdown of ho-1 was confirmed by western blot ( figure 2(d ) ) . as indicated in figure 2(e ) , shho-1 lessened the inn suppression of hl-60 adhesion . together , our data suggest that ho-1 plays an imperative role in the suppression of tnf-stimulated hl-60 adhesion by inn . previous studies   suggest that ampk modulates the antioxidant status of cardiovascular ecs by upregulating expression of genes implicated in antioxidant defense , for example , manganese superoxide dismutase , catalase , and thioredoxin . to determine the signaling pathways implicated in the inn - mediated ho-1 induction ecs were incubated with inn ( 10  m ) for different time periods . as illustrated in figure 3(a ) , ampk was activated by inn . however , inn did not change ampk protein expression . to further substantiate the role of the camkk/ampk pathway , their specific inhibitors , sto-609 and compound c , were used . as shown in figures 3(b ) , 3(c ) , and 3(d ) , sto-609 and compound c attenuated inn - triggered ho-1 expression and activity . as indicated in figure 3(e ) , the inn - stimulated elevation of cellular gsh content was repressed by sto-609 and compound c. our findings demonstrate that inn induces ho-1 expression and increases gsh synthesis through the camkk/ampk pathway . to illuminate the downstream signaling pathway of camkk/ampk for the stimulation of ho-1 by inn ecs were incubated with 10  m inn for the indicated time . as indicated in figure 4(a ) , nrf2 nuclear translocation was augmented as early as 8  h and continued until 24  h after inn treatment . furthermore , nrf2 nuclear translocation was repressed in ecs pretreated with the ampk inhibitor compound c for 1  h before being challenged with 10  m inn . our results suggest that the camkk/ampk pathway is implicated in inn - stimulated nrf2 nuclear translocation . to further determine the role of nrf2 in inn - stimulated ho-1 expression , ecs were transfected with shnrf2 and shluc . as illustrated in figures 4(b ) and 4(c ) , knockdown of nrf2 eliminated inn - induced ho-1 expression . together , our results demonstrate that nrf2 is a transcriptional factor responsible for inn - stimulated ho-1 expression . as indicated in figure 5 , tnf significantly increased expression of vcam-1 and hl-60 cell adhesion . inn treatment dramatically suppressed tnf-induced vcam-1 expression at mrna and protein levels as well as hl-60 cell adhesion . to further identify the role of ampk and nrf2 in the tnf-stimulated vcam-1 expression , we then used compound c to inhibit ampk and shnrf2 to knock down nrf2 expression . as illustrated in figure 5 , compound c and shnrf2 abolished tnf-triggered vcam-1 expression and hl-60 cell adhesion . together , these results suggest that ampk and nrf2 mediate inn inhibition of tnf-stimulated vcam-1 expression and monocyte adhesion in haec . improvement of intracellular antioxidant ability is thought to diminish the risk of oxidative stress - induced diseases . clopidogrel ( inn ) is an oral antiplatelet drug used to impede blood clots in coronary artery disease ( cad ) . previous studies have shown that inn has anti - inflammation and antioxidation activities [ 1921 ] . recent work has demonstrated that inn augments nitric oxide ( no ) and prostacyclin production in endothelial cells . in addition , inn impedes cd40 ligand both in vitro and in vivo , stimulating ho-1 expression . these findings suggest that inn can preserve endothelial function via a mechanism independent of its antiplatelet activity . suggest that inn protects against diabetes - induced vascular damage and reduces circulating endothelial cells ( cecs ) in type-2 diabetes patients . the effect of inn was to improve vascular function , protect against oxidative stress , and inhibit apoptosis in patients with type-2 diabetes . this involved an increase in the expression of both phosphorylated akt and ampk . in the present study , we revealed that inn repressed tnf-stimulated ros liberation , vcam-1 expression , and cell adhesion and that this effect was related to the increase in ho-1 expression and gsh content via the camkk/ampk / nrf2 pathway . tnf is a powerful proinflammatory cytokine secreted by various innate immune cells , predominantly activated macrophages , as well as neutrophils , mast cells , and eosinophils cells [ 3539 ] . in macrophages , the plasma levels of tnf are augmented in some pathologies , such as cancer , atherosclerosis , rheumatoid arthritis , and preeclampsia . several studies have revealed that tnf , via activation of nfb , induces expression of proinflammatory cytokines and adhesion molecules , for example , il-6 and vcam-1 [ 41 , 42 ] . in the current study , we revealed that tnf increased ros production , vcam-1 expression , and cell adhesion . increasing evidence supports that it protects against many chronic diseases such as cardiovascular diseases , hypertension , diabetes mellitus , and neurological disorders . using systemic manipulation of either ho-1 expression or activity , some studies suggested that ho-1 plays a vital role in atherosclerosis initiation and development . similarly , rabbits treated with snpp , an ho-1 inhibitor , exhibit a significant lipid deposits in abdominal aortic plaques . taken together , these studies substantiated the anti - inflammatory or antiatherosclerotic activity of ho-1 . the early step in atherogenesis is endothelial dysfunction resulting in several compensatory responses that change the vascular homeostasis . proinflammatory stimuli such as a diet rich in saturated fat , obesity , hypercholesterolemia , and hyperglycemia cause the expression of adhesion molecules , for example , vascular cell adhesion molecule-1 ( vcam-1 ) and p - selectin in endothelium , and these molecules facilitate the attachment of monocytes and lymphocyte [ 48 , 49 ] . in addition , turbulent flow resulting from an unfavorable serum lipid profile probably leads to overexpression of adhesion molecules in endothelial cells triggering atherosclerosis . overexpression of vcam-1 enhances recruitment of monocytes to endothelial injury locations ; succeeding liberation of monocyte chemoattractant protein-1 ( mcp-1 ) by leukocytes amplifies the inflammatory cascade through recruiting other leukocytes , stimulating leukocytes in the media , and initiating recruitment and proliferation of smooth muscle cells . here , we demonstrate that suppression of vcam-1 expression by inn reduced hl-60 cell adhesion to tnf-stimulated haecs . nevertheless , this inhibition was eliminated by ampk inhibitor compound c , shho-1 , and shnrf2 ( figures 2(d ) , 5(c ) , and 5(f ) ) . these data demonstrate the contribution of ampk , nrf2 , and ho-1 to the suppression of hl-60 cell adhesion by inn . nrf2 is an imperative transcriptional factor implicated in cellular anti - inflammatory action and related to the induction of ho-1 and glutathione s - transferase [ 52 , 53 ] . nrf2 interacts with its cytosolic inhibitor keap1 under basal circumstances . in response to stress , nrf2 is released from keap1 and successively translocates to the nucleus , activating its target gene transcription via are . in the current study , inn augmented nrf2 nuclear translocation , and silencing nrf2 abolished the inn induction of ho-1 . these results indicate that this transcriptional factor is indispensable for inn - induced ho-1 expression . nrf2 activation is controlled by several kinases , including jnk , p38 , erk , and pi3k / akt . in the current study , we demonstrated that inn activates camkk and ampk . by using specific inhibitors of camkk and ampk , we revealed that the camkk/ampk pathway is implicated in the inn - triggered ho-1 induction . in summary , we have revealed that inn hinders tnf-induced ros formation , expression of vcam-1 , and hl-60 cell adhesion by upregulating ho-1 gene expression and elevating gsh levels through the camkk/ampk / nrf2 pathway ( figure 6 ) . the antioxidant and anti - inflammatory characteristic of inn is thought to protect against oxidative stress - induced diseases including arthrosclerosis .
clopidogrel ( inn ) , an oral antiplatelet drug , has been revealed to have a number of biological properties , for instance , anti - inflammation and antioxidation . 
 oxidative stress plays an imperative role in inflammation , diabetes mellitus , atherosclerosis , and cancer . in the present study , human aortic endothelial cells ( haecs ) 
 were employed to explore the anti - inflammatory activity of inn . 
 inn reduced tnf-induced reactive oxygen species ( ros ) generation and time - dependently prompted the expression and activity of heme oxygenase 1 ( ho-1 ) . 
 cellular glutathione ( gsh ) levels were augmented by inn . 
 shho-1 blocked the inn suppression of tnf-induced hl-60 cell adhesion . 
 the camkk/ampk pathway and nrf2 transcriptional factor were implicated in the induction of ho-1 by inn . additionally , tnf dramatically augmented vcam-1 expression at protein and mrna levels . 
 inn treatment strikingly repressed tnf-induced expression of vcam-1 and hl-60 cell adhesion . 
 compound c , an ampk inhibitor , and shnrf2 abolished tnf-induced expression of vcam-1 and hl-60 cell adhesion . 
 our data suggest that inn diminishes tnf-stimulated vcam-1 expression at least in part via ho-1 induction , which is camkk/ampk pathway - dependent .