coronary artery disease , as an important manifestation of atherosclerosis , is one of the most frequent causes of death and disability in the western world . it has been proposed that inflammation is the driving force in atherosclerosis [ 2 , 3 ] , and strong evidence supports the central role of proinflammatory cytokines , such as interleukin-1 ( il-1 ) and interleukin-6 ( il-6 ) in these pathologies . human coronary artery endothelial cells ( hcaec ) have previously been shown to have a strikingly greater responsiveness than human umbilical vein endothelial cells ( huvec ) . the endothelium is considered a dynamic organ with secretory , metabolic , immunologic roles , in addition to its regulatory function of nutrient transport . endothelial cells from diverse environments are heterogenous with respect to their surface phenotype , mrna expression , and levels of il-6 and procoagulant proteins , such as tissue factor ( tf ) . within the past decades , the majority of endothelial proinflammatory / coagulation studies have been performed on huvec , derived from a vascular bed not present in the adult , making these cells an inappropriate model of endothelial inflammation and coagulation [ 5 , 6 ] . tf is a pivotal factor found associated with endothelial cells within atherosclerotic plaques   that can transform the endothelial cell membrane from an anticoagulant to a procoagulant surface following vascular injury . unique sensitivity of hcaec to tumor necrosis factor  ( tnf)-stimulated expression of adhesion molecules ( as compared to human aortic endothelial cells )   and greater susceptibility of hcaec to inflammatory stimuli , ( as compared to huvec and human dermal microvascular endothelial cells ( hmvec ) )   have been reported . endothelial cells contribute to homeostasis and vasodilation , also by releasing prostacyclin ( pgi2 ) known to inhibit platelet aggregation and deposition . however , no reports as far as we know , have addressed the comparative influence of il-1 on pgi2 release , tf activity , or il-6 protein release from hcaec versus other endothelial cell types . previous epidemiological studies have suggested that black and green tea consumption may have beneficial effects on endothelial function and is associated with a decreased risk of cardiovascular events [ 1013 ] . black tea inhibited the proliferation of smooth muscle cells involved in the development and progression of atherosclerosis   and mechanisms for the beneficial effects of tea including vasculoprotective , antioxidative , antithrombogenic , anti - inflammatory , and lipid - lowering properties of tea flavonoids have been reported [ 11 , 1518 ] . animal studies confirmed that black tea extract ( bte ) has anti - inflammatory activity , however , its effects on different types of endothelial cells is still not clear . the polyphenolic stilbene resveratrol ( rsv ) is found in grape skins , red wine , and peanuts [ 19 , 20 ] . trans -rsv has been shown to inhibit tf expression in vascular cells and to act anti - inflammatory [ 2123 ] . however , it is still largely unknown how bte and/or rsv affect il-1-stimulated inflammatory and coagulation pathways in hcaec , as a novel model system , compared to huvec or other endothelial cell types . so , our focus has been to elucidate the mechanisms that could modulate il-1 proinflammatory / procoagulant responses in hcaec . the specific objectives of this study were to understand the influence of potential anti - inflammatory plant extracts , such as bte and rsv , on il-1-induced primary hcaec and to compare their il-6 , tf , and prostacyclin responses with other types of endothelial cells . lyophilized human il-1 ( invitrogen - carlsbad , california , usa ) , resveratrol and extract from black tea ( sigma - saint louis , missouri , usa ) were reconstituted according to manufacturer 's instructions to stock concentration and stored until usage at 20c or 80c . the black tea extract used in the current report is composed of more than 80% theaflavins ( theaflavin and theaflavin gallates ) . the final concentration of il-1 was 1000  pg / ml , resveratrol 40  mol / l , and the final concentration of black tea extract was 40  g / ml unless otherwise stated . huvec , hcaec , and hmvec were purchased from cambrex bioscience ( walkesville , maryland , usa ) . the cells were plated onto 12 or 6 well plates or 75  cm flasks ( tpp , trasadigen , switzerland ) at 37c in a humidified atmosphere at 5% co2 . hcaec and hmvec were grown in egm-2 m medium ( cambrex bioscience , walkesville , maryland , usa ) containing 5% fetal bovine serum ; for huvec we used egm medium containing 2% fetal bovine serum ( cambrex bioscience , walkesville , maryland , usa ) . for experiments , subconfluent cell cultures were used between 4 and 6 passages in serum - free medium with addition of stimulatory and/or modulatory substances for 24 hours , unless otherwise indicated . prior to experiments il-6 from cell culture supernatants was measured and analyzed by human il-6 elisa from biosource international ( camarillo , california , usa ) according to manufacturer 's instructions . enzyme immunoassay ( eia ) 6-keto pgf1 ( cayman diagnostica , michigan , usa ) was used to measure the concentration of nonenzymatically converted metabolite of prostacyclin or pgi2 in cell culture supernatants with competitive eia according to the instructions of the manufacturer . 6-keto pgf1 is a stable product of pgi2 . before rt - pcr , total rna from endothelial cell cultures was isolated using total rna isolation system ( promega , usa ) following manufacturer 's instructions . the purity and amount of rna were determined by measuring the od at a ratio of 260 to 280  nm . 1  g of total rna were transcribed into dna by reverse transcription system ( promega , usa ) and pcr was performed for -actin , il-6 , and tf ( table 1 ) . tissue factor activity was measured following the actichrome tf ( american diagnostica , stamford , connecticut , usa ) procedure . after cell treatment in 12 well plates , the medium was removed and 150  l cells were then scraped and frozen at 80c for 15 minutes and then thawed at 37c . cell lysates were then mixed with factor viia and factor x in 96 well plates . following a 15-minute incubation at 37c ,   spectrozyme fxa was added for 20 minutes and the reaction was stopped with glacial acetic acid . all experiments were repeated three times and the mrna expression studies were shown as 1 representative gel of two performed . means were compared between the treated and control groups using student 's t - test . in order to determine the inflammatory / coagulation responses of primary human endothelial cells , in particular hcaec , stimulated with different doses of il-1 ,   we measured released il-6 protein ( figure 1 ) . semiconfluent primary huvec , hcaec , and hmvec were incubated in 6-well plates with increasing doses of il-1 ( from 0 to 2000  pg / ml ) for 24 hours in serum - free media . released protein levels of il-6 were measured in supernatants by elisa , and in parallel , mrna expressions of -actin , il-6 , and tf were determined using rt - pcr . a comparison of released il-6 protein levels in the endothelial cell types showed highest levels coming from hcaec ( approximately 2-fold higher than from huvec ) and the lowest from hmvec ( figure 1 ) . the first rapidly increased il-6 protein levels were seen at an il-1 concentration of 500  pg / ml in hcaec and huvec , in comparison to hmvec where responses were consistently low . the mrna expression results show that il-6 , as well as tf mrna expression dose dependently increased with il-1 in all cell types ( data not shown ) . to investigate the effects of modulatory molecules on il-1-stimulated endothelial cells , we measured the il-6 protein levels in the presence or absence of bte and rsv . both bte and rsv significantly inhibited il-6 protein levels of hcaec ( figure 2 ) . il-1-induced tf activity showed the highest response in hcaec and was inhibited by bte and rsv in all three endothelial cell types ( figure 3(a ) ) . the il-1-induced mrna expression of il-6 and tf in all endothelial cell types is down - regulated with bte and rsv ( figure 3(b ) ) . to investigate the effects of il-1 on homeostasis of endothelial cells , released prostacyclin pgi2 was measured as 6-keto pgf1 in cell supernatants ( figure 4 ) .   to determine whether the responses were dose dependent , hcaec , huvec , and hmvec were incubated with increasing concentrations of il-1 ( figure 4(a ) ) . the highest responses were shown in huvec , which were around 3 - 4 fold higher than in hcaec . contrary to il-6 levels and tf activity , bte addition did not cause inhibition of il-1-stimulated prostacyclin release ( figure 4(b ) , left panel ) . however , rsv abrogated il-1-induced 6-keto pgf1 levels in all three cell types ( figure 4(b ) , right panel ) . in this report , hcaec were used as a model cell system , shown to be highly responsive to il-1-stimulated il-6 released protein levels , tf activity , and inhibition of these by bte and rsv . studies on hcaec reported in literature have looked at different stimulating molecules , such as il-1 and tnf ,   activated protein c , lipopolysaccharide [ 2528 ] , and their effects on different cytokine and chemokine expression , indicating unique cell type patterns and/or levels of expression . previously indicated that hcaec display greater susceptibility to inflammation and potential atherogenesis than huvec or hmvec . this is in accord with il-1-stimulated hcaec data indicated in this report . among the most accessible natural substances used worldwide are black tea , green tea , and wine , which have been suggested to be important modulators of cardiovascular disease ( cvd ) . both black and green tea have shown acute beneficial effects on aortic stiffness and wave reflections , as reported by vlachopoulos et al . . since aortic stiffness and wave reflections are markers of cvd and prognostic factors of cardiovascular risk , they are important parameters to be studied in healthy individuals one possible explanation for the lack of cardiovascular protection in some of the studies is the difference in infusion time , stirring , leaf size , and measurements in plasma / whole blood changes , although different brands and addition of milk did not show significant variances . however , flavonoids were reported to bind to protein , which causes variations in bioavailability and also results in changed biological activity especially in the case of binding to specific receptors and/or enzymes [ 31 , 32 ] . however , overall , the evidence suggests that individuals with the highest flavonoid intake have modestly reduced risks for cvd ( as reviewed in vita ja , 2005 ) . for tea , this conclusion is supported by a meta - analysis with 10 cohort studies and seven case - control studies included , which suggested an overall reduction in cvd risk of around 11% with consumption of 3 cups of tea per day . a meta - analysis was also performed recently on the association between green or black tea consumption and the risk of stroke . data from 9 studies involving 4  378 strokes among 194  965 individuals were pooled . the authors conclude that although a randomized clinical trial would be necessary to confirm the effect , this meta - analysis suggests that regardless of their country of origin , individuals consuming 3 cups of tea per day had a 21% lower risk of ischemic stroke than those consuming   less than 1 cup per day . in a large sample ( 6  597 subjects ) of the elderly ( over 65 years ) , debette et al . reported for the first time that carotid plaques were less frequent with increasing tea consumption in women . usually 1  g of tea is used for 100  ml of infusion [ 30 , 32 ] . in the process of manufacturing , the black tea leaf catechins are allowed to oxidize into theaflavins which give the black tea its characteristic colour and taste . the black tea flavonoid content accounts for 20%30% catechins , 10% theaflavins and 50%60% thearubigins , and both catechins as well as theaflavins have been shown to act as cardio - protectants in cardiomyocytes . the present data in huvec , hcaec , and hmvec indicate that bte significantly abrogates il-1-induced il-6 protein released levels similarly to rsv ( figure 2 ) , while also decreasing both tf activity levels and expression ( figure 3 ) . however , bte does not seem to have any effects on il-1-induced pgi2 in any of the three cell types ( figure 4(b ) , left panel ) . these differences imply that cytokine - specific processes and differential signaling pathways might be involved . since black tea consumption has been previously associated with a decreased risk of cardiovascular events [ 10 , 11 ] , signaling events in response to bte are relevant . bovine aortic endothelial cells when exposed to bte showed enos activity mediated through p38 mapk and estrogen receptors leading to phosphatidylinositol 3-kinase / akt pathway and enos generation [ 39 , 40 ] and vasorelaxation of aortic rat rings . rsv - mediated cardio - protection is achieved through the preconditioning effect and thus achieves a number of cardio - protective functions ( reviewed in ) , such as effecting release and/or generation of inflammatory mediators and attenuation of various soluble intercellular cytokines . rsv functions in scavenging free radicals and inhibiting lipid peroxidation , up - regulation of inducible no synthase , vascular endothelial growth factor , kinase insert domain - containing receptor , and endothelial no synthase . dealcoholized wine ( 1 cup ) delivered in a single oral dose to healthy subjects less than 40 years old was found to increase endothelium - dependent vasodilation . the authors indicate that this adds support to the hypothesis that antioxidant properties of red wine , rather than ethanol , may protect against cardiovascular diseases , however more research is needed on subjects with coronary heart disease . usually the concentrations of rsv in cellular models of cvd protection are 0.1100  mol/ l , although some studies showed different effects in low / high doses in enhancing proliferation and inducing apoptosis . when rsv in humans is absorbed around 75% serum levels were independent from meals and its lipid content , but rsv is rapidly metabolized into glucuronides and sulfates , which stay in the blood for 9 hours . biologic activity of metabolites was not elucidated yet [ 20 , 42 ] , and it is suggested that prolonged administration could lead to increased concentrations [ 42 , 46 ] . it is necessary to apply caution when interpreting the literature data translating concentrations of rsv on potential cardiovascular effects . to our knowledge , this is the first report to show the effects of rsv on il-1-induced il-6 and tf responses in hcaec , which could serve in cardioprotective processes similar to the ones described previously [ 24 , 42 , 47 , 48 ] . mol / l ) in huvec   at comparable concentrations to the 40  m used in the present report , to reduce expression of adhesion molecules on stimulated human saphenus vein endothelial cells , to inhibit adhesion of activated platelets to collagen or fibrinogen   and lower icam , vcam [ 24 , 51 ] . rsv was also reported to enhance the inhibitory activity of pgi2 on platelet aggregation in low doses , as well as to inhibit tnf-induced nad(p)h oxidase and nf-b activation and inflammatory markers ( at 0.110 mol / l ) [ 24 , 42 , 51 ] , similarly to our results . in porcine coronary arteries , short term treatment with rsv significantly inhibited mapk activities , with reduced phosphorylation seen of erk1/2 , jnk-1 and p38 mapk [ 53 , 54 ] , and stat3 phosphorylation . the data shown in huvec ( figure 4 ) indicates a similar level of il-1-stimulated pgi2 ( measured using 6-keto pgf1 ) as shown by olszanecki et al . . all three cell types huvec , hcaec , and hmvec indicate a similar vasoregulatory role for il-1 ,   while rsv addition abrogated pgi2 levels ( figure 4(b ) , right panel ) . rsv has also been shown to decrease the expression of vasoconstrictor endothelin and increase enos in huvec , which might counterbalance pgi2 inhibition   and decrease nad(p)h oxidase activity . in conclusion , a growing body of evidence indicates that inflammation not only provides the baseline for future atherosclerotic events , but is a necessity for coronary plaque formation and coagulation leading to thrombosis . the unique responsiveness of hcaec could account for the greater susceptibility of coronary arteries to inflammation and atherogenesis leading to cardiovascular pathology and substances , such as bte and rsv , also influence these effects at the cellular level .