T.J. GUZIK, R. OLSZANECKI, J. SADOWSKI, B. KAPELAK, P. RUDZIÑSKI, A. JOPEK, A. KAWCZYNSKA, N. RYSZAWA, J. LOSTER, J. JAWIEN,

M. CZESNIKIEWICZ-GUZIK, K.M. CHANNON, R. KORBUT

SUPEROXIDE DISMUTASE ACTIVITY AND EXPRESSION IN HUMANVENOUS AND ARTERIAL BYPASS GRAFT VESSELS.

Department of Pharmacology, Department of Internal Medicine and Department ofCardiovascular and Transplant Surgery; Jagiellonian University School of Medicine, Cracow,

Poland and Department of Cardiovascular Medicine, University of Oxford, UK

Venous bypass grafts are more prone to accelerated atherosclerosis than arterialgrafts, which is partly related to increased oxidative stress and diminished nitricoxide bioavailability. In veins superoxide production is dependent primarily on nox2NAD(P)H oxidase expression, while in arteries nox4 appears to play an importantrole. This may in part explain differences in susceptibility to graft failure. Net levelsof oxidative stress are however determined in parallel by the production as well asby degradation of free radicals (eg. by superoxide dimutases, catalases, thioredoxinsetc). The differences in superoxide dismutase (SOD) expression and activity inhuman bypass conduit vessels remain unclear. Accordingly, we aimed to compareSOD activity and protein levels as well as its functional effects on superoxideproduction in segments of human internal mammary arteries (IMA) and saphenousveins (HSV) from patients undergoing bypass graft surgery (n=24). SOD activitywas assessed by inhibition of pyrogallol autoxidation, Cu-Zn SOD and Mn SODprotein levels were studied by immunoblotting. Basal superoxide release wasdetected by lucigenin (5µM) enhanced chemiluminescence. Total SOD activity didnot differ significantly between HSV and IMA. Similarly, no difference wasobserved in SOD activity in the presence of KCN (Mn-SOD). Human bypass conduitvessels show amounts of Cu-Zn SOD or Mn-SOD protein levels. In both HSV andIMA segments superoxide production was more than doubled in the presence ofSOD inhibitor - DETC. Conclusions: These studies suggest that the differences inoxidative stress between human arteries and veins are unlikely to be caused by SODactivity. However SOD plays and important role in amelioration of oxidative stressin both types of vessels.

K e y w o r d s : endothelium, oxidant stress, reactive oxygen species; superoxide dimutase;bypass grafts.

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2005, 56, 2, 313�323

www.jpp.krakow.pl

INTRODUCTION

Oxidative stress plays important roles in vascular physiology and pathology(1-3). Previous studies have shown that superoxide anion is produced byoxidases present in human vascular cells. These include NAD(P)H oxidases,xanthine oxidase and eNOS (4). However, net levels of oxidative stress aredetermined in parallel by production and biodegradation of free radicals (5) aswell as by genetic susceptibility (6). The major anti-oxidant systems in thevasculature include superoxide dismutases, catalase, glutathione peroxidaseand recently more and more appreciated thioredoxin (7). While all of the aboveenzymes play important roles in the modulation of oxidative stress, the primaryrole in the metabolism of superoxide anion radical is exerted by superoxidedismutases (5). There are 3 major forms of superoxide dismutases. MnSOD-present in mitochondria; CuZn SOD in cytoplasm, and extracellular SOD (ECSOD) secreted by vascular cells into the extracellular matrix (5). They catalysethe reaction of superoxide dismutation to hydrogen peroxide, which thenundergoes further reactions and exerts numerous biological functions. Thisprocess leads not only to the removal of superoxide from the vascular wall, butalso generates an important regulatory molecule - hydrogen peroxide (5).Therefore, the expression and activity of superoxide dimutases may be criticalin the regulation of vascular function and may be critical for cellular defensefrom injury and ischemia reperfusion (8, 9). The reaction of superoxide (.O2

-)with NO is however approximately three times faster than the elimination ofsuperoxide by superoxide dismutases, which could question the role ofsuperoxide dimutases in protecting nitric oxide bioavailability (4). Animalmodels have however clearly indicated that the inhibition of superoxidedimutases by DETC leads to a very significant attenuation of endotheliumdependent NO-mediated vasorelaxations (10). Similar findings have beenreported in Cu-Zn SOD deficient mice (11). All these data show, that in spite ofmuch slower rate of the reaction between SOD and superoxide, superoxidedismutases (including intracellular Cu-Zn SOD) are important in the regulationof NO bioavailability. Differences in SOD activity can also have impact ontherapeutic approaches, as gene therapy using SOD as well as therapeutic useof SOD mimetics have been recently proposed to decrease levels of oxidativestress and improve endothelial dysfunction and diminish the development ofatherosclerosis (12).

Our previous work has revealed several differences in the mechanisms ofsuperoxide production between human arteries and veins (13, 14). Thesefindings were described using bypass graft conduit vessels as model systems.The differences between studied vessels are important for understanding ofvascular pathology, but also because these vessels are widely used for surgicaltreatment of coronary artery disease (15). Oxidative stress appears to be morepronounced in human saphenous veins than in mammary arteries, which could

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partially explain better prognosis in arterial revascularisations (13, 14).However, the contribution of oxidative stress to this process is unknown.Limited number of studies have looked at the effects of superoxide dismutaseinhibition on oxidative stress parameters in human blood vessels (16, 17).Those studies have found no significant differences in the increases ofsuperoxide production following SOD inhibition in human arteries and veins.These studies were inconclusive as one found greater SOD activity in arterieswhile another reported lack of difference. However there was an importantdrawback in both studies, which used 250 µM-500 µM concentrations oflucigenin that can artificially generate superoxide by redox cycling in thepresence of tissues and therefore change the potential differences in superoxidedimutase activity in studied vessels.

Accordingly, we aimed to directly measure superoxide dimutases activity inhuman arterial and venous bypass conduit vessels; compare their proteinexpression and functional effects of SOD inhibition on superoxide production.

MATERIALS AND METHODS

Patients and Blood Vessels

Excess segments of human saphenous vein (HSV) and internal mammary artery (IMA) wereobtained from a total of 27 patients undergoing coronary artery bypass graft surgery. We andothers have used this approach to study 'paired' artery and vein specimens, from the same patient,in several previous studies (16-18). Vessels were harvested using a no-touch technique, beforesurgical distension. The segment was immediately transferred to ice cold Krebs-Hensleit buffer,delicately flushed and carefully dissected to remove excess adventitial tissue, using microsurgicalinstruments. All vessels were collected before topical administration of drugs such as papaverine.The integrity of the endothelium after harvesting was checked by immunohistochemistry for theendothelial cell marker CD31, in preliminary studies. Patient characteristics and risk factorprofile was typical for patients undergoing bypass graft surgery. All patients were treated withHMG-CoA reductase inhibitor.

Collection of tissue specimens was approved by the Local Research Ethics Committees andinformed consent was obtained.

Superoxide Dismutase activity

Superoxide dismutase activity was measured by inhibition of pyrogallol autooxidation (19).Frozen vessel segments (-70°C) were ground in liquid nitrogen and suspended in 50 mM Tris buffer,pH 8.2. After centrifugation at 10000 g for 5 minutes, equal volumes of supernatants were mixedwith 0.9 ml 50 mM Tris buffer pH 8.2 containing 1 mM DTPA, 10 µM catalase and 200 µMpyrogallol. The change of absorbance at 420 nm was measured every 5 sec for 5 minutes in 23°Cusing Beckman DU 640 BV spectrophotometer equipped with kinetic software package. The SODactivity was expressed as units/ mg protein, one unit determined as the amount of enzyme thatinhibited the oxidation of pyrogallol by 50%. In parallel experiments, the activity of MnSOD wasestimated after inhibition of CuZnSOD in samples by the addition of 5 mM KCN.

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Western blotting

Portions of vascular homogenate were prepared in a homogenisation buffer (HEPES; 20mM;EDTA 0.01mM; protease inhibitors (Roche); PMSF 1mM; Triton X100 - 2% and SDS 0.5%).Protein content was measured using Bio-Rad system and samples were equalized for proteincontent. 20 µg protein portions of vascular homogenate were separated by SDS-PAGE (120mV)and transferred to nitrocellulose membranes for 4 hours at 4°C using 200 mAmp in a transferbuffer (25 mMTris; 190 mM Glycine; 20% methanol). MnSOD and Cu/Zn SOD were detectedusing anti-human SOD sheep polyclonal antibodies (Calbiochem, San Diego, CA). Membraneswere incubated for 1 hour at room temperature in 1:500 final dilution of primary antibodies.Anti alpha actin antibody (mAb; Sigma, Germany) (1:2500) was used to check for equal proteinloading and data were expressed per α-actin band. Goat horseradish peroxidase (HRP)conjugated anti-sheep IgG was used as secondary antibody to detect SODs (Calbiochem; SanDiego, CA). Bands were detected using chemiluminescence substrate and developed onphotographic membrane.

Vascular superoxide production

Superoxide production was measured by lucigenin-enhanced chemiluminescence (LGCL),using previously described and validated methods (6, 20). Briefly, intact vessel segments wereequilibrated in Krebs-HEPES gassed with 95%O2/5%CO2 for 30 minutes at 37°C. Lucigenin-enhanced chemiluminescence from intact vessels was measured in buffer (2 ml) containing lowconcentration lucigenin (5 µM). In order to address the role of SOD in modulating superoxiderelease in human saphenous veins and internal mammary arteries, superoxide release was measuredin the presence of SOD inhibitor - diethyldithiocarbamate - DETC (100 µM). Superoxideproduction was expressed as relative light units (RLU)/second/mg vessel dry weight.

Statistical analysis

Results are expressed as means ± SEM with n equal to the number of patients. Statisticalcomparisons between the two groups were made using Students t-test for independent or dependentsamples. p values <0.05 were considered statistically significant.

RESULTS

Superoxide dismutase activity in human arteries and veins

Since the superoxide dismutase (SOD) enzymes regulate superoxide levelsin the vascular wall, we next aimed to investigate whether differences in SODprotein levels and activities could account for differences in the superoxide-mediated effects observed in venous and arterial conduit vessels (Fig.1).Experiments comparing Cu/ZnSOD and MnSOD protein levels using WesternBlotting in HSV and IMA revealed no differences between these two conduitvessels (Fig.5 A and B). Similarly, both total SOD activity and MnSODactivity, measured in vascular homogenates using inhibition of pyrogallolauto-oxidation, was similar in both saphenous veins and mammary arteries(Fig.5 C).

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317 S

OD

act

ivity

(U

/mg

prot

ein)

0

5

10

Total SOD MnSOD

HSV IMA HSV IMA

NS

NS

Figure 1.

Fig.1. Superoxide dismutase(SOD) activity in human veinsand arteries. Total SOD activitywas assessed in vascularhomogenates in human saphenousvein (HSV) and internalmammary artery (IMA) conduitvessels. Spectrophotometricdetermination of the inhibition ofpyrogallol autooxidation (200µM) was used. Parallelexperiments were conducted inthe presence of KCN (5 mM) toinhibit CuZn SOD. Results werecalculated in units of activity permg of total protein. Data areexpressed as mean±SEM. NS -lack of significant differencebetween HSV and IMA.

HSV IMA

MnSOD

Cu/ZnSOD

α-actin

0

0.2

0.4

0.6

0.8

Cu/Zn SOD MnSOD

SO

D b

and

dens

ity/α

-act

in(A

U/A

U)

HSV IMA HSV IMA

NS

NS

Figure 2.

A.

B.

Fig.2. Superoxide dismutaseprotein levels and activity invenous and arterial bypassconduits Panel A. Westernblots showing MnSOD andCuZnSOD proteins in humansaphenous vein (HSV) andinternal mammary artery(IMA) conduit vessels. α-actin band shows equalloading of protein in eachsample. Panel B.Densitometric analysis ofSOD bands in HSV, IMA(mean±SEM; paired n=6),expressed in relation todensity of actin bands.

SOD protein levels in human venous and arterial conduit vessels

Since the superoxide dismutase (SOD) protein levels may have importanteffects on SOD activity in the vascular wall, we next aimed to investigate thedifferences in SOD protein levels between venous and arterial conduit vessels(Fig.2). Experiments comparing Cu/ZnSOD and MnSOD protein levels usingWestern Blotting in HSV and IMA revealed no differences between these twoconduit vessels (Fig.5 A and B).

Effects of SOD inhibition on vascular superoxide release.

In order to study the functional effect of superoxide dimutase activity inhuman vessels we measured vascular superoxide production by LGCL in thepresence and absence of SOD inhibitor DETC (100 µmol/L). This approachallowed not only to compare the functional effects of vascular SOD in humanbypass conduits, but was also an indirect indice of SOD activity measured inintact vessels as opposed to vascular homogenates used by majority of availablemethods of SOD activity determination.

Vessels were preincubated with DETC for 30 minutes prior to assay. DETCincreased ·O2

- release in both HSV and in IMA (Fig.3). The degree of increase ofsuperoxide release in response to DETC was similar in HSV and IMA, which wasin line with SOD activity measurements.

DISCUSSION

Vascular oxidative stress is determined by both production and degradation offree radicals in the vascular wall. While significant amount of data is available

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0

10

20

30

40

50

native +DETC 100uM

HSV IMA HSV IMA

NS

NS*

*

Figure 3.

Sup

erox

ide

prod

uctio

n (

RLU

/sec

/mg

dw)

Fig.3. Effects of SOD inhibitionby DETC (100 µM) on vascularsuperoxide release from HSV andIMA (n=8). Superoxide releasewas determined in intact vascularrings by lucigenin enhancedchemiluminescence (5 µM) in theabsence (native) and in thepresence of SOD inhibitor -DETC (100 µM). Data wereexpressed as relative light unitsper second per mg of dry weightof the vascular ring. Bars indicatemean±SEM. * p<0.05 vs native;NS - lack of significant differencebetween HSV and IMA.

about the activity and expression of oxidases in the vascular wall of human bypassconduit vessels, relatively little is known about the anti-oxidant enzyme systemsand their role in the regulation of oxidative stress in human veins and arteries.

In the present study, we did not find any significant differences in the activityor expression of superoxide dismutases in the vascular wall of human saphenousveins and internal mammary arteries. This suggests, that the differences in SODactivity are unlikely to be a primary cause for the variable characteristics ofoxidative stress in HSV and IMA. Our studies, however, show that in both humansaphenous veins and internal mammary arteries, in patients with generalizedatherosclerosis, SOD is important in limiting oxidative stress, as its inhibition byDETC caused significant increases in superoxide release from vascular rings.This is in line with studies showing the importance of superoxide dismutaseactivity in the regulation of NO-dependent endothelial function in both animalmodels and in humans. In animal models inhibition of SOD by DETC leads to asignificant impairment of NO bioavailability (10), and trangenic overexpressionof Cu-Zn SOD prevents the development of endothelial dysfunction caused bylipopolysaccharide (21). Landmesser et al. have nicely shown that in patientswith coronary artery disease (CAD), vascular EC-SOD activity is substantiallyreduced. The close relation between endothelium-bound EC-SOD activity andendothelial function suggests that reduced SOD activity contributes to endothelialdysfunction in patients with CAD (22). In our study all vessels were obtainedfrom patients with advanced atherosclerosis undergoing bypass graft surgery.SOD activity could be diminished in this group of patients and the differences inSOD activity could be observed in earlier stages of atherosclerosis or inphysiologic conditions. Similarly, activity and expression of superoxidedismutases may differ in younger and elderly individuals (23). Indeed, in younghypercholesterolemic subjects, SOD activity is increased as a defense mechanismto counteract impairment of endothelial function, as the result of increasedformation of oxygen free radicals (22). Moreover, although no significantdifferences in the expression of Cu-Zn SOD or Mn SOD between human conduitartery and vein was found, we cannot exclude a difference in extracellularsuperoxide dimutase (EC-SOD) protein levels (24), which we were unable tostudy. Indeed, Landmesser et al. have found that in human coronary arteries frompatients with coronary artery disease EC-SOD was increased along with totalSOD activity, while Cu-Zn SOD or Mn SOD was not different (22). It is,however, important to note, that both inhibition of pyrogallol autooxidation byvascular homogenates and effects of DETC on superoxide release were notdifferent, indicating that total activity of SOD (which includes EC-SOD) is notdifferent in both types of human vessels.

Some previous studies compared the activity of SOD in human saphenousveins and internal mammary arteries. They have, however, obtained contrastingresults. Schmalfuss et al. has found that SOD activity along with the generationof superoxide anion (measured by very high lucigenin concentrations) were all

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greater in the IMA than in the HSV. Interestingly, Western blot analysis showedno differences in SOD protein expression in IMA and HSV (16). Berry et al., inturn, have found no differences in either activity or protein for SOD (16). Ourstudy brings some additional important insights. Firstly, we used lowconcentrations of lucigenin in the measurements of superoxide production.Secondly, we paid particular attention to using the same size (weight) vascularrings for both HSV or IMA, which was not discussed by previous studies. Ourresults of functional effect of SOD inhibition on superoxide release are inagreement with previous studies conducted using high concentrations oflucigenin, thus confirming the validity of the approach taken by Schmalfuss et al.(16) or Berry et al. (17). This may suggest that therapeutic approaches aimed onincreasing endogenous antioxidant systems may be very useful in limitingvascular oxidative stress in humans. This approach may be even more importantin the light of findings that exogenous antioxidant vitamins do not seem to fulfillthis aim. Some novel therapeutic approaches have been proposed recently andcould include L-tryptophan (melatonin precursor) which can be useful in re-establishing SOD activity in the model of inflammatory diseases (25). Otherpotential approaches could include pentoxifylline in inflammation (26), L-carnitine (27), ferulic acid (28) in alcoholic abusive disease, all of which havebeen shown to induce intrinsic anti-oxidant mechanisms. The mechanisms ofSOD activation and regulation of expression are however not fully understoodand require further investigation, as novel studies suggest neural component inthis process (29).

The determination of mechanisms of oxidative stress in human bypass conduitvessels have important practical implications. Indeed, it has been found thatoxidative stress plays an important role in the pathogenesis of intimal hyperplasiain experimental venous bypass conduits (30). Oxidative stress is critical inendothelial dysfunction (2, 3) as well as in the regulation of other factors involvedin atherogenesis - including platelet and coagulation regulation (31). The role ofsuperoxide dismutases in this process is not clear. In the present study we showthat although the differences in SOD activity can rather not account fordifferences in the susceptibility to develop graft disease between HSV and IMA,SODs play important limiting role in the oxidative stress pathology in both typesof vessels. In previous studies we found that vascular superoxide production hasimportant functional differences between venous and arterial conduits used inpatients undergoing coronary artery bypass grafting (4, 14, 18, 20). Specifically,we found that the NAD(P)H oxidase system is quantitatively and proportionatelya greater source of superoxide in veins, whereas xanthine oxidase appears toadditionally contribute to superoxide production in arteries. We found thatincreased vascular NAD(P)H oxidase activity is associated with increased proteinlevels of p22phox, p47phox and p67phox, and increased p22phox and nox2(gp91phox) mRNA expression (14). The NAD(P)H oxidase is predominantlynox2-based in saphenous veins, whereas a nox4-based oxidase appears

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proportionately more important in mammary arteries. The importance ofdifferential expression of NAD(P)H oxidase homologues has been shownrecently by several investigators (32, 33). Another important difference includesdifferential response to angiotensin II, which is not observed in veins (17).Despite these differences in the functional and molecular characteristics ofsuperoxide production between veins and arteries, several findings suggest thatoxidative stress and endothelial dysfunction are regulated by similar systemicfactors in human atherosclerosis. First, both superoxide production andendothelial dysfunction are significantly correlated in arteries and veins fromindividual patients (14). Second, the expression of both p22phox and nox4mRNA are strikingly correlated in arteries and veins (14). Finally, protein kinaseC signaling has a key role in regulating superoxide production in both veins andarteries (14). The present study adds to that, the superoxide dismutase activity isalso a common feature of oxidative stress regulation in veins and arteries. It isalso important to remember that systemic nature of oxidative stress may begreatly affected by both clinical risk factors for atherosclerosis includinghypertension, hypecholesterolemia or diabetes (18, 20) as well as by geneticfactors. The latter may include NAD(P)H oxidase subunit genetic polymorphisms(6), as well as polymorphisms within genes encoding SOD (34).

In conclusion, our study shows that although superoxide dismutase activityand expression do not seem to differ significantly between human conduit arteriesand veins, SOD does play a regulatory role in oxidative stress, therefore novelantioxidant approaches are necessary to enhance SOD activity, and improveendothelial function. Some natural products which may be useful in limitingoxidative stress include flavonoids (35) or grapefruit seeds extract (36), as thesehave been suggested to increase SOD activity in various tissues. However, betterunderstanding of the regulation of vascular antioxidants is necessary to introducebetter antioxidant therapeutic strategies for atherosclerosis and graft failure.

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R e c e i v e d : May 3, 2005A c c e p t e d : May 16, 2005

Author's address: Assoc. Prof. Tomasz J Guzik MD PhD MSc, Department of Pharmacology,Cracow, Poland, Tel/fax +48 12 6310440.E-mail: tguzik@well.ox.ac.uk

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