since the breakthrough discovery of the obligatory role of the vascular endothelial cells by furchgott and zawadzki in 1980 , endothelial pathophysiology has been intensely investigated , and the term " endothelial dysfunction " has been referred to in the scientific literature over 50,000 times ( pubmed search , may 2012 ) .
it is well known that the endothelial dysfunction is associated with aging , hypertension , atherosclerosis , and numerous other physiological and pathophysiological processes .
nitric oxide ( no ) , prostacyclin , and endothelium - derived hyperpolarizing factor ( epoxyeicosatrienoic acid ) are the three major substances involved in the endothelium - dependent vasodilator pathways .
the mechanism of endothelial dysfunction is largely due to the reduced bioavailability of endothelium - derived no by oxidative stress .
accordingly , the hallmark of endothelial dysfunction is impaired endothelium - dependent vasodilation , which is mediated by no .
a free radical is defined as any species that contains one or more unpaired electrons .
reactive oxygen species ( ros ) is a collective term that includes both oxygen free radicals , such as superoxide ( o2 ) , hydroxyl ( oh ) , peroxyl ( roo ) , and hydroperoxyl ( ho2 ) radicals , and certain non - radical oxidizing agents , such as hydrogen peroxide ( h2o2 ) , hypochlorous acid ( hocl ) , and ozone ( o3 ) , that can be readily converted to free radicals .
no and peroxynitrite ( onoo ) are the most relevant reactive nitrogen species in the cardiovascular system .
many studies have demonstrated that angiotensin - converting enzyme ( ace ) inhibitors have induced many beneficial effects on the endothelial function in animal models and humans , have improved the endothelial function in hypertension , and have reduced the risk associated with cardiovascular disease .
there have been numerous attempts to decrease the major complications associated with cardiac surgery , and there is increasing evidence that ace inhibitors hold promise as protective cardiovascular agents for cardiac surgery patients .
the mechanisms of the protective effects of ace inhibitors involve multiple factors , including those relating to their antioxidant effects . in several experimental studies , only sulfhydryl ( sh)-containing ace inhibitors such as captopril and zofenopril exerted the endothelium protective effect , but in other studies , the non - sh - containing ace inhibitor also showed similar effects .
therefore , the present study was designed to evaluate the antioxidant effects of ace inhibitors with or without a sh group ( captopril and enalapril , respectively ) and to identify the involved mechanisms .
all of the animal procedures were approved by the animal care and use committee of hanyang university .
twenty - five male new zealand white rabbits ( koatech , pyeongtaek , korea ) weighing between 2.0 and 2.5 kg were anesthetized with 3% to 5% sevoflurane in 4 l / min of 100% oxygen in a restraint chamber .
the marginal ear vein was cannulated , and heparin ( 1,000 iu / kg ) was administered intravenously . after 10 minutes , the rabbit was sacrificed by exsanguination from the carotid artery .
the infrarenal abdominal aorta was carefully excised and placed in a petri dish filled with 4 krebs - henseleit solution of the following composition ( mm ) : nacl 120.0 , nahco3 25.0 , kcl 5.0 , nah2po4 1.4 , mgso4 1.2 , cacl2 2.5 , and glucose 11.0 .
the vessel was cleaned of fat and connective tissue with extreme caution not to damage the endothelium or smooth muscle .
the vessel rings were mounted between two l - shaped steel hooks in water - jacketed ( 370.5 ) organ baths filled with 5 ml of krebs - henseleit solution .
the medium was continuously gassed with a mixture of 95% of o2 and 5% of co2 throughout the experiment .
the lower hook was fixed to the organ chamber , and the upper hook to the force transducer ( tsd125c ; biopac systems inc . ,
the isometric tension was continuously recorded on a personal computer with a data acquisition system ( mp100 system , biopac systems inc . ) via a transducer amplifier ( da100c , biopac systems inc . ) .
all the vessels were first equilibrated for 90 minutes , and the resting tension was set to 2 g gradually . during the equilibration period , the chamber medium was exchanged every 15 minutes .
after the equilibration period , the endothelium integrity was assessed by precontracting the aortic ring with 10 m of norepinephrine ( ne ) and a subsequent relaxation with cumulative concentrations ( 310 , 10 , 310 , and 10 m ) of acetylcholine ( ach ) .
an aortic ring with an endothelial function ( relaxation ) greater than or equal to 80% was considered to have an intact endothelium , and was included in the experiment .
a constant current of 15 ma was applied for 35 seconds using two circular platinum wire electrodes ( each 7 mm in length ) located at the bottom of the organ baths .
the vessel ring was mounted over 1 cm apart from the electrodes to avoid direct electrical injury . at 15 minutes of equilibration after the pretest and medium exchange , various concentrations ( 10 , 310 , 10 , and 310 m ) of captopril or enalapril were added and incubated for 15 minutes .
after exposure to ros generated by electrolysis , the medium was exchanged again . the contraction and relaxation curves
changes of the aortic tone by ach were expressed as percentages , and they were used as the experimental values .
cu / zn superoxide dismutase ( sod ) and catalase , the two important antioxidant enzymes , were inhibited to investigate the mechanism of the antioxidant activity of captopril and enalapril .
diethyldithiocarbamate ( detca ) was used for cu / zn sod inhibition , and 3-amino-1,2,4-triazole ( 3at ) for catalase inhibition .
after the pretest and equilibration period ( 15 minutes ) , the vessels were pre - incubated with 0.5 mm of detca for 30 minutes or 50 mm of 3at for 60 minutes .
captopril ( 310 m ) or enalapril ( 310 m ) was added and incubated during the last 15 minutes of pre - incubation , and the vessel rings were then exposed to electrolysis - induced ros .
after refreshing the medium , ne - induced contraction and the series of ach applications were then repeated .
ne , ach , captopril , enalapril , detca , and 3at were purchased from sigma - aldrich , inc .
the responses to ach are expressed as the percentage of the ach - induced relaxation , in the same vessel , during the pretest .
when appropriate , the student t - test or one - way analysis of variance ( anova ) followed by a post test for linear trend and the dunnett test were performed .
all of the curve fittings and statistical analyses were performed using prism ver . 5.0 ( graphpad software inc . , san diego , ca , usa ) . a p - value less than 0.05
twenty - five male new zealand white rabbits ( koatech , pyeongtaek , korea ) weighing between 2.0 and 2.5 kg were anesthetized with 3% to 5% sevoflurane in 4 l / min of 100% oxygen in a restraint chamber .
the marginal ear vein was cannulated , and heparin ( 1,000 iu / kg ) was administered intravenously . after 10 minutes , the rabbit was sacrificed by exsanguination from the carotid artery .
the infrarenal abdominal aorta was carefully excised and placed in a petri dish filled with 4 krebs - henseleit solution of the following composition ( mm ) : nacl 120.0 , nahco3 25.0 , kcl 5.0 , nah2po4 1.4 , mgso4 1.2 , cacl2 2.5 , and glucose 11.0 .
the vessel was cleaned of fat and connective tissue with extreme caution not to damage the endothelium or smooth muscle .
the vessel rings were mounted between two l - shaped steel hooks in water - jacketed ( 370.5 ) organ baths filled with 5 ml of krebs - henseleit solution .
the medium was continuously gassed with a mixture of 95% of o2 and 5% of co2 throughout the experiment .
the lower hook was fixed to the organ chamber , and the upper hook to the force transducer ( tsd125c ; biopac systems inc . , goleta , ca , usa ) .
the isometric tension was continuously recorded on a personal computer with a data acquisition system ( mp100 system , biopac systems inc . ) via a transducer amplifier ( da100c , biopac systems inc . ) .
all the vessels were first equilibrated for 90 minutes , and the resting tension was set to 2 g gradually . during the equilibration period , the chamber medium was exchanged every 15 minutes .
after the equilibration period , the endothelium integrity was assessed by precontracting the aortic ring with 10 m of norepinephrine ( ne ) and a subsequent relaxation with cumulative concentrations ( 310 , 10 , 310 , and 10 m ) of acetylcholine ( ach ) . an aortic ring with an endothelial function ( relaxation ) greater than or equal to 80%
a constant current of 15 ma was applied for 35 seconds using two circular platinum wire electrodes ( each 7 mm in length ) located at the bottom of the organ baths .
the vessel ring was mounted over 1 cm apart from the electrodes to avoid direct electrical injury .
at 15 minutes of equilibration after the pretest and medium exchange , various concentrations ( 10 , 310 , 10 , and 310 m ) of captopril or enalapril were added and incubated for 15 minutes .
after exposure to ros generated by electrolysis , the medium was exchanged again . the contraction and relaxation curves
changes of the aortic tone by ach were expressed as percentages , and they were used as the experimental values .
cu / zn superoxide dismutase ( sod ) and catalase , the two important antioxidant enzymes , were inhibited to investigate the mechanism of the antioxidant activity of captopril and enalapril .
diethyldithiocarbamate ( detca ) was used for cu / zn sod inhibition , and 3-amino-1,2,4-triazole ( 3at ) for catalase inhibition .
after the pretest and equilibration period ( 15 minutes ) , the vessels were pre - incubated with 0.5 mm of detca for 30 minutes or 50 mm of 3at for 60 minutes .
captopril ( 310 m ) or enalapril ( 310 m ) was added and incubated during the last 15 minutes of pre - incubation , and the vessel rings were then exposed to electrolysis - induced ros .
after refreshing the medium , ne - induced contraction and the series of ach applications were then repeated .
ne , ach , captopril , enalapril , detca , and 3at were purchased from sigma - aldrich , inc .
the responses to ach are expressed as the percentage of the ach - induced relaxation , in the same vessel , during the pretest .
when appropriate , the student t - test or one - way analysis of variance ( anova ) followed by a post test for linear trend and the dunnett test were performed .
all of the curve fittings and statistical analyses were performed using prism ver . 5.0 ( graphpad software inc . , san diego , ca , usa ) . a p - value less than 0.05
in a dose - dependent manner , the captopril - treated vessel rings preserved ach - induced endothelium - dependent relaxation against the ros attack ( fig .
1 ) . at the highest concentration ( 10 m ) of ach , the 10 , 310 , 10 , and 310 m captopril treated groups showed a relaxation rate of 1.9%1.2% , 81.2%1.9% , 86.0%2.1% , and 89.1%0.8% , respectively ( n=15 for each group ; anova and post test for linear trend ; both p<0.0001 ) . the treatment of enalapril showed a similar effect ( fig .
the 10 , 310 , 10 , and 310 m enalapril - treated groups had relaxation rates of 1.8%1.2% , 10.0%2.1% , 73.9%5.7% , and 89.1%0.8% , respectively ( n , 13 to 15 ; anova and post test for linear trend ; both p<0.0001 ) .
the relaxation rate of the detca+captopril group was significantly lower than the control ( captopril - only ) group after the ros attack ( 73.0%2.4% vs. 86.8%1.1% ; n=15 ; p<0.0001 ) ( fig .
detca exerted the same effect with enalapril ( 73.0%2.4% vs. 86.8%1.1% ; n=15 ; p<0.0001 ) ( fig .
, 3at pretreatment did not alter the relaxation rate of the captopril - treated group nor the enalapril - treated group ( fig .
in a dose - dependent manner , the captopril - treated vessel rings preserved ach - induced endothelium - dependent relaxation against the ros attack ( fig .
1 ) . at the highest concentration ( 10 m ) of ach , the 10 , 310 , 10 , and 310 m captopril treated groups showed a relaxation rate of 1.9%1.2% , 81.2%1.9% , 86.0%2.1% , and 89.1%0.8% , respectively ( n=15 for each group ; anova and post test for linear trend ; both p<0.0001 ) . the treatment of enalapril showed a similar effect ( fig .
the 10 , 310 , 10 , and 310 m enalapril - treated groups had relaxation rates of 1.8%1.2% , 10.0%2.1% , 73.9%5.7% , and 89.1%0.8% , respectively ( n , 13 to 15 ; anova and post test for linear trend ; both p<0.0001 ) .
the relaxation rate of the detca+captopril group was significantly lower than the control ( captopril - only ) group after the ros attack ( 73.0%2.4% vs. 86.8%1.1% ; n=15 ; p<0.0001 ) ( fig .
detca exerted the same effect with enalapril ( 73.0%2.4% vs. 86.8%1.1% ; n=15 ; p<0.0001 ) ( fig .
, 3at pretreatment did not alter the relaxation rate of the captopril - treated group nor the enalapril - treated group ( fig .
in the present study , both captopril and enalapril exerted an antioxidant effect against the electrolysis - induced ros in the rabbit aortic rings .
this antioxidant effect was partially reduced by detca pretreatment , but it was not affected by 3at pretreatment .
ach - induced vasorelaxation requires the integrity of the vascular endothelium , thus " endothelium - dependent " relaxation .
ach binds to the muscarinic receptor of the endothelial cell , and the intracellular calcium level increases , which leads the endothelial nitric oxide synthase ( enos ) to produce no . the produced no diffuses to the vascular smooth muscle cell , and it activates the soluble guanylyl cyclase , which eventually converts the guanosine triphosphate to cyclic guanosine monophosphate ( gmp ) inducing smooth muscle relaxation .
in addition , the cyclooxygenase ( cox ) pathway producing prostacyclin is also related to the endothelium - dependent vasorelaxation .
the endothelium is a major regulator of vascular homeostasis that maintains the balance between vasodilation and vasoconstriction , inhibition and promotion of the proliferation and migration of smooth muscle cells , prevention and stimulation of the adhesion and aggregation of platelets , as well as thrombogenesis and fibrinolysis . upsetting this balance leads to endothelial dysfunction and damage to the arterial wall .
these homeostatic effects are largely mediated by no , a pivotal endothelium - derived substance .
no is a diffusible substance with a half - life of a few seconds , and it is formed from l - arginine by oxidation of the guanidine - nitrogen terminal . vascular endothelial cells normally generate ros as " second messengers " that might regulate endothelial cell growth and proliferation , endothelial barrier function , vasorelaxation , and vascular remodeling .
ros production has been known to occur in the endothelium , but also within the media and adventitia , all of which may impair no signaling within vascular tissue to endothelium - dependent and endothelium - independent vasodilators .
superoxide anion can be generated by various enzymes such as nicotinamide adenine dinucleotide phosphate oxidase , xanthine oxidase , cox , no synthase , lipoxygenase , cytochrome p450 monooxygenases , and enzymes of the mitochondrial respiratory chain .
superoxide anion and transition metals such as iron and copper react with hydrogen peroxide to form highly reactive hydroxyl radicals through the fenton reaction ( fe+h2o2fe+oh+oh ) and the haber - weiss reaction ( o2+h2o2oh+oh+o2 ) .
no and superoxide anion rapidly interact to form peroxynitrite , a potent oxidant also capable of oxidizing sh groups and thioethers , as well as nitrating and hydroxylating aromatic groups , including tyrosine , tryptophan , and guanine . at high concentrations , peroxynitrite is cytotoxic and may cause oxidative damage to proteins , lipids , and dna .
oxidative stress , however , may cause endothelial dysfunction or cell death through various pathways .
superoxide anions reduce the bioavailability of no and directly inhibit its main target : soluble guanylyl cyclase .
the major target of ros is no , but the resultant formation of peroxynitrite also causes endothelial dysfunction by oxidation of the zn - thiolate complexes within enos and the essential cofactor tetrahydrobiopterin ( bh4 ) . as a result of the oxidation of bh4 , enos becomes " uncoupled " and produces ros rather than no .
in addition , peroxynitrite inhibits guanylyl cyclase , inactivates the prostacyclin synthase by tyrosine nitration , and further enhances oxidative stress by inhibiting sod .
hydroxyl radicals , another ros , can induce cell damage through the peroxidation of lipids and sh groups . on the other hand
, oxidative stress can also induce apoptosis of endothelial cells by activation of apoptosis signal - regulating kinase 1 and c - jun n - terminal kinase . in the present study
, we used the buffer solution electrolysis method to produce the oxidative stress to the vascular rings .
the ros generation system by electrolysis of a physiological salt solution has the advantage that a wide range of ros are produced without addition of enzymes or chemicals , which themselves may influence the experiment .
furthermore , the short duration of exposure to ros allows the measurement of responses to various drugs , without the continuous inactivation of no or the possible oxidation of drugs by ros .
this electrolysis system generates superoxide anions , hydroxyl radicals , hydrogen peroxide , singlet oxygen , and hypochlorite .
the endothelium - dependent vasorelaxation is almost completely abolished by exposure to ros . in the present study ,
endothelium - dependent vasorelaxation was severely compromised in the aortic rings with low concentrations of captopril or enalapril pretreatment .
the vasoconstrictor response to 1 adrenoceptor stimulation is impaired by exposure to ros . because we used ne for preconstriction of the vessels in the present experiment
, we applied a duration and intensity ( 35 seconds and direct current 15 ma , respectively ) of constant current electrolysis that compromised endothelium - dependent vasorelaxation but did not affect ne - induced vasoconstriction .
the predominant isoform cu / zn sod is inhibited by the cu chelator detca . in the present study ,
this represents that superoxide anion scavenging activity is related to the mechanism of the antioxidant effect of captopril and enalapril .
hydrogen peroxide is scavenged by catalase , and this enzyme is inhibited by 3at . in the present experiment , pretreatment with 3at did not affect the antioxidant effect of captopril and enalapril , suggesting that their endothelium protective effect is not associated with hydrogen peroxide scavenging action .
many researchers have reported that only sh - containing ace inhibitors possess an antioxidant effect [ 10,24 - 26 ] .
the sh moiety easily undergoes oxidation and disulfide exchange reactions and can be converted into disulfides through the interaction with free radicals . by this process , it can serve as a free radical scavenger , thereby protecting the endothelial function . on the other hand ,
some investigators have reported that non - sh - containing ace inhibitors also exerted an endothelium protective effect against free radical injury .
it was reported that ramiprilat , a non - sh - containing ace inhibitor , protects against free radical injury in isolated working rat hearts .
the investigators explained that ramiprilat inhibits free radical - induced damages mainly by stimulation of prostacyclin synthesis and/or release , because their data showed that the addition of indomethacin ( 5 mol / l ) completely abolished the protective effects of ramiprilat .
this interpretation seems confusing , however , because in a later study it was demonstrated that cox inhibitors , such as indomethacin , possess antioxidant properties . in another experiment concerning this issue by fujita et al .
, they found that quinaprilat , a non - sh - containing ace inhibitor , could ameliorate both apoptosis and necrosis through the up - regulation of constitutive enos via an increase of bradykinin , with the resulting increase of nitric oxide in the cultured human aortic endothelial cells .
the b2 receptor is constitutively expressed , but the b1 receptor is normally absent and is only induced under inflammatory conditions in humans .
rabbit blood vasculature , however , is rich in b1 receptor sites ; hence , this difference between species should be considered when interpreting the results .
one limitation of the present study is that the amount of ros generated by electrolysis could not be measured due to the lack of methodologies .
a device that can measure the real - time amount of ros in the vicinity of the electrodes would be beneficial .
this method may offer clues for elucidating the exact ros composition ratio in the electrolyzed buffer solution .
it remains to be investigated whether other ace inhibitors , with or without a sh group , also protect endothelium against ros .
further studies on prostacyclin inhibition without any interactions with ros may clarify the relevance of prostacyclin in the endothelium protective effects of ace inhibitors . performing these studies
would more clearly explain the exact mechanism of the known beneficial effects of ace inhibitors in cardiac surgery patients .
both captopril ( a sh - containing ace inhibitor ) and enalapril ( a non - sh - containing ace inhibitor ) protect endothelium against free radical injury in a dose - dependent manner in isolated rabbit abdominal aortas .
 backgroundreactive oxygen species ( ros ) are known to be related to cardiovascular diseases . 
 many studies have demonstrated that angiotensin - converting enzyme inhibitors have beneficial effects against ros . 
 we investigated the antioxidant effect of captopril and enalapril in nitric oxide mediated vascular endothelium - dependent relaxations.materials and methodsisolated rabbit abdominal aorta ring segments were exposed to ros by electrolysis of the organ bath medium ( krebs - henseleit solution ) after pretreatment with various concentrations ( range , 10 - 5 to 310 - 4 m ) of captopril and enalapril . before and after electrolysis , the endothelial function was measured by preconstricting the vessels with norepinephrine ( 10 - 6 m ) followed by the cumulative addition of acetylcholine ( range , 310 - 8 to 10 - 6 m ) . 
 the relevance of the superoxide anion and hydrogen peroxide scavenging effect of captopril and enalapril was investigated using additional pretreatments of diethyldithiocarbamate ( detca , 0.5 mm ) , an inhibitor of cu / zn superoxide dismutase , and 3-amino-1,2,4-triazole ( 3at , 50 mm ) , an inhibitor of catalase.resultsboth captopril and enalapril preserved vascular endothelium - dependent relaxation after exposure to ros in a dose - dependent manner ( p<0.0001 ) . 
 pretreatment with detca attenuated the antioxidant effect of captopril and enalapril ( p<0.0001 ) , but pretreatment with 3at did not have an effect.conclusionboth captopril and enalapril protect endothelium against ros in a dose - dependent fashion in isolated rabbit abdominal aortas . 
 this protective effect is related to superoxide anion scavenging . 