The Interstitium of the Human Arterial Wall Contains Very Large Amounts of Extracellular Superoxide Dismutase

1. 2. 3. 4. Pontus Strlin, Kurt Karlsson, Bengt O. Johansson, Stefan L. Marklund

+ Author Affiliations 1. From the Department of Clinical Chemistry (P.S., K.K., S.L.M.) and the Department of Anatomy (B.O.J.), Ume University Hospital, Sweden. 1. Correspondence to Stefan L. Marklund, Department of Clinical Chemistry, Ume University Hospital, S-901 85 Ume, Sweden.

Next Section

Abstract
Abstract The levels of the secreted, interstitially located extracellular superoxide dismutase (EC-SOD), the cytosolic copper-and-zinccontaining SOD (CuZn-SOD), and the mitochondrial manganese-containing SOD (Mn-SOD) were measured in the walls of human coronary arteries, proximal thoracic aortas, and saphenous veins. The blood vessel walls, particularly the arteries, were found to contain exceptionally large amounts of EC-SOD, whereas the levels of CuZn-SOD and Mn-SOD were relatively low compared with other tissues. Analysis of EC-SOD by immunohistochemistry indicates an even distribution in the vessel wall, including large amounts in the arterial intima. Arterial smooth muscle cells were found to secrete large amounts of EC-SOD and likely are the principal source of the enzyme in the vascular wall. The EC-SOD concentration in the human arterial wall extracellular space is high enough to efficiently suppress the putative pathological effects of the superoxide radical, such as oxidation of LDL and reaction with nitric oxide to form the deleterious peroxynitrite. The levels of EC-SOD in the aortic wall are found to vary widely among species and were on average 6440 U/g in humans, 4340 U/g in the cow, 2660 U/g in the pig, 160 U/g in the dog, 770 U/g in the cat, 2390 U/g in the rabbit, 90 U/g in the rat, and 3400 U/g in the mouse. There were only moderate differences in the amounts of CuZn-SOD and Mn-SOD. This wide variation in EC-SOD content suggests that the susceptibility to pathologies induced by superoxide radicals in the vascular wall interstitium should vary widely among species. Key Words:

superoxide nitric oxide

oxygen free radicals atherosclerosis LDL Received March 10, 1995. Accepted August 25, 1995.

The effects of the superoxide radical and derived products on blood vessel functions have attracted increasing recent attention. Direct damaging effects on the endothelium1 and other vessel components, involvement in the oxidation of LDL2 3 4 5 and hence potential involvement in atherosclerosis, noxious interactions with NO,6 7 8 9 10 and direct effects on vessel tonus11 have all been reported. However, despite the potential importance of the superoxide radical, little is known about the endogenous protection of the extracellular space and cells of the vascular wall provided by SODs. To assess the role of SODs in blood vessel homeostasis we undertook the present study, in which we determined in human coronary artery, aorta, and saphenous vein the contents of the three SOD isoenzymes: the secreted EC-SOD,12 the cytosolic CuZn-SOD,13 and the mitochondrial matrix Mn-SOD.14 We find comparatively little CuZn-SOD and Mn-SOD but exceptionally large amounts of EC-SOD. For comparison, we also measured the SOD isoenzymes in the aortas of a number of other mammalian species and found considerable interspecies variation in EC-SOD content. Previous SectionNext Section

Methods
Extraction of the Vessel Wall
Macroscopically normal pieces (0.5 to 1.5 g) of human LAD, proximal thoracic aorta, and saphenous vein were cut out at autopsy within 48 hours after death. Thoracic aortas from the other mammals were collected within a few hours after death. The pieces were kept at 80C before analysis. For extraction, frozen pieces were pulverized in a Braun Microdismembrator II (B Brown Biotech Inc), and the frozen powder was added to 10 vol of 50 mmol/L potassium phosphate, pH 7.4, with 0.3 mol/L KBr and a set of antiproteolytic agents (0.5 mmol/L PMSF, 3 mmol/L DTPA , 90 mg/L aprotinin, 10 mg/L pepstatin, 10 mg/L chymostatin, and 10 mg/L leupeptin). The homogenates were then sonicated and finally extracted for 30 minutes at 4C. The extracts were centrifuged (20 000g for 15 minutes). Unless analyzed immediately, the supernatants were stored at 80C.

SOD Activity Analysis

SOD enzymatic activity was determined by the direct spectrophotometric method, employing KO215 as modified.16 To distinguish between the cyanide-sensitive isoenzymes CuZn-SOD and EC-SOD and the resistant Mn-SOD, 3 mmol/L cyanide was used. One unit in the assay is defined as the activity that brings about a decay of O2 concentration at a rate of 0.1 s1 in 3 mL of buffer. It corresponds to 8.3 ng human CuZn-SOD, 6.3 ng bovine CuZn-SOD, 8.6 ng human EC-SOD, and 65 ng bovine Mn-SOD. The KO2 assay is carried out at pH 9.5 and at a relatively high superoxide concentration. In comparison, the xanthine oxidasecytochrome

C SOD assay13 is carried out under more physiological conditions, ie, neutral pH and low superoxide concentration. One unit in the KO2 assay corresponds to about 0.024 units CuZnSOD and EC-SOD and 0.24 units Mn-SOD in the xanthine oxidase assay. The KO2 assay is thus 10 times more sensitive for CuZn-SOD and EC-SOD activity than for Mn-SOD activity. The results presented in the tables were not corrected for this effect.

Specific Analysis of EC-SOD

EC-SOD in human blood vessel wall extracts was determined by ELISA.17 There is no crossreactivity with human CuZn-SOD. For conversion of results to activity units, 8.6 ng per unit was assumed.18 For the specific analysis of EC-SOD in vessel extracts from other species, chromatography on Con ASepharose (Pharmacia Biotech) was used. Unlike CuZn-SOD and Mn-SOD, the glycoprotein EC-SOD binds to the lectin concanavalin A. The procedure has been described previously,19 the only difference being that the extraction buffer described above was used as a solvent in all steps. The yield of EC-SOD in the procedure was tested with human blood vessel extracts. Seventy-five percent of the applied EC-SOD was found to be recovered as determined by ELISA, and all EC-SOD results for the other mammals were corrected accordingly. The CuZn-SOD activity of the extracts was then calculated as total cyanidesensitive SOD activity minus (corrected) EC-SOD activity.

Protein and DNA Analysis

For protein analysis, Coomassie brilliant blue G-250 was employed,20 standardized with human serum albumin. DNA concentration was determined with fluorimetry as a complex with bisbenzimidazole (Hoechst 33258)21 using calf thymus DNA as a standard.

Effect of Storage on SOD Activity

Since the human samples were collected from bodies stored under refrigeration up to 48 hours, the effects of storage on the various analyzed factors were determined. Small pieces (around 300 mg) of cow aorta, prepared under aseptic conditions, were stored in 1.5-mL capped tubes in a refrigerator at 5C. Tubes were transferred to 80C at 1.5 hours, 12 hours, 24 hours, and daily up to 6 days after death. Three 1.5-hour samples and two from the other storage times were then homogenized and analyzed as described above for the animal samples. There were no systematic increases or decreases in the activities of the SOD isoenzymes or the contents of protein or DNA in the daily samples over the 6-day period. The means of the parameters in the two pieces stored at 5C for 6 days deviated at most 13% from the means of the pieces stored for only 1.5 hours.

Immunohistochemistry
Vessels for immunostaining (LAD, thoracic aorta, and saphenous vein) were obtained at autopsy within 48 hours of death or immediately during vessel surgery. Cryostat sections were fixed for 45 minutes in a 1% paraformaldehyde solution. An avidinbiotinhorseradish peroxidase system (Dakopatts) was used for immunostaining. Anti-EC-SOD antibodies, raised against recombinant human protein18 in goat and rabbit and purified by adsorption/desorption on EC-SOD immobilized on CNBr-activated Sepharose, were used at

concentrations of 0.7 to 8.6 mg/L. As negative controls, primary antibodies were substituted with nonimmunized goat/rabbit IgG (2.4 to 11.6 mg/L).

Cell Culture
The arterial smooth muscle cell lines were initiated from pieces of human uterine artery as described by Fager et al.22 Their identity as smooth muscle cells was assessed by morphological appearance and by their expression of smooth muscle -actin23 (Boehringer Mannheim GmbH), and analyzed by flow cytometry. The cells were maintained in Waymouth MB 752/1 medium containing 15% fetal calf serum, 105 U/L benzylpenicillin, 100 mg/L streptomycin, 2 mmol/L glutamine, and 1 mmol/L sodium pyruvate and were studied between the fifth and eighth passages. Previous SectionNext Section

Results
Human Blood Vessel Walls
Human blood vessel walls, particularly the arteries, were found to contain exceptionally large amounts of EC-SOD, 10-fold more than average human tissues (Table 1). The levels of CuZn-SOD and Mn-SOD, on the other hand, were lower than in most other tissues. While in most tissues EC-SOD accounts for only a small percentage of the total SOD activity, the fraction in the arterial wall approaches 50%.

Localization of EC-SOD by Immunohistochemistry

The distribution of EC-SOD in human aorta, LAD, and saphenous veins was studied by means of immunohistochemistry in samples from several individuals. The staining of all the samples was essentially identical. A result with a thoracic aorta specimen is presented as an example (see the Figure). EC-SOD is apparently evenly distributed throughout the wall, with significant amounts observed in all layers.

View larger version:

In this page

In a new window Download as PowerPoint Slide

Figure 1. Immunostaining of EC-SOD in nondiseased thoracic aorta from a 40-year-old man, collected 48 hours postmortem. The antiEC-SOD antibody used (1.4 mg/L) (A) was raised against the recombinant enzyme in rabbit. Nonimmunized rabbit IgG was used as a negative control (2.4 mg/L) (B). e indicates endothelium; i, intima; m, media; and a, adventitia. The upper pair of arrows mark the internal elastic lamina; the lower pair, the border between media and adventitia. Bar=200 m.

SOD Isoenzymes in Aorta From Various Mammalian Species

We have previously found that the EC-SOD content of tissues shows a considerable interspecies variation, whereas the contents of CuZn-SOD and Mn-SOD vary to a lesser extent.24 To investigate whether these differences also occur in blood vessels, we measured the levels of the SOD isoenzymes in the aortas from seven additional mammalian species (Table 2). We find a remarkable variation in the EC-SOD content, whereas the differences in CuZn-SOD activity are relatively small. The Mn-SOD activity displays an intermediate variation among the species.

Sources of EC-SOD in the Vascular Wall

Although the intracellular isoenzymes CuZn-SOD and Mn-SOD occur in all nucleated cell types, EC-SOD is expressed by only a few.25 Expression by fibroblasts and glia cells has been demonstrated before, whereas other cell lines, eg, endothelial cells (n=6), lacked expression.25 All investigated suspension-growing cell lines of hematological origin likewise lacked expression. To further explore potential sources of EC-SOD in the vascular wall, expression by arterial smooth muscle cells was examined (Table 3). The smooth muscle cell lines all secreted EC-SOD in large amounts, similar to those previously found for fibroblast cell lines. Since smooth muscle cells constitute the major portion of the cells occurring in the vascular wall, they should be the principal source of the EC-SOD existing in this location.

Table 3. Expression of EC-SOD by Cultured Human Smooth Muscle Cells Previous SectionNext Section

Discussion
The major finding of the present study is the exceptionally high EC-SOD content in the extracellular space of the human arterial wall. The CuZn-SOD and Mn-SOD contents are relatively low. The cell densities of the vascular walls are low, however, and a comparison with other tissues on the basis of U/mg DNA26 indicates that the average content per cell of these intracellular isoenzymes is normal or slightly high. The present study, however, provides no information on the distribution of these intracellular isoenzymes among the various cell types in the vascular wall. Since the superoxide anion radical crosses membranes poorly,27 the SOD isoenzymes exert their protective functions in their distinct compartments. For these reasons, we focus this discussion on EC-SOD and events in the vascular wall extracellular space.

The present (Table 3) and previous25 analyses of EC-SOD expression by human cell lines indicate that the principal source of the enzyme in the vascular wall is the smooth muscle cells. EC-SOD shows high affinity for heparin and some related sulfated glycosaminoglycans28 29 and exists, in tissues, reversibly anchored primarily to heparan sulfate proteoglycans in the interstitial matrix and on cell surfaces.30 31 After secretion, the EC-SOD should slowly diffuse in the vascular wall and distribute itself according to amount and affinity of the glycosaminoglycans occurring in the various microenvironments. This process seems to result in a relatively even distribution of the enzyme to the various layers of the vessel wall (Figure). Assuming a distribution volume of 30% in the arterial wall, an average EC-SOD activity of 15 000 to 20 000 U/mL in the extracellular space can be estimated. CuZn-SOD, which is commercially available and has been widely used in similar studies, has an equivalent activity equal to 110 mg/L CuZn-SOD. The very high EC-SOD activity in the arterial interstitium, including the intima, could be important for the suppression of various pathological processes. LDL oxidation has been suggested to be a primary step in atherogenesis.32 In several in vitro models of LDL oxidation, addition of CuZn-SOD has been shown to be protective,2 3 4 5 6 suggesting a potential involvement of the superoxide radical in the process in vivo. The high EC-SOD activity in the human arterial intima should effectively suppress any direct involvement of the superoxide radical in LDL oxidation. NO produced by the endothelium is a major physiological vasodilator.33 34 It also reduces the adhesion of platelets35 and leukocytes36 to the vascular wall and by terminating lipid peroxidation may protect LDL from oxidation.37 38 Nitric oxide synthase can also be induced in phagocytic cells and smooth muscle cells.39 NO reacts with superoxide at an essentially diffusion-limited rate to form the very toxic compound peroxynitrite.6 7 Peroxynitrite, which is in itself an oxidizing agent, may nitrate proteins8 and induce LDL oxidation10 and may decompose to other strongly oxidizing species.9 Thus, the interaction between superoxide and NO reduces the physiological and protective effects of NO and leads to the formation of toxic compounds. The extensive nitration of protein tyrosines found in human atherosclerotic lesions shows that significant amounts of peroxynitrite may be formed under pathological conditions in vivo.40 With maximal stimulation of the endothelium of isolated rabbit aorta, NO concentrations of 0.85 mol/L have been measured in the wall,41 although in vivo the concentrations may be lower due to scavenging of NO by hemoglobin.42 With the high rate constant for peroxynitrite formation, 6.7109 Lmol1s1,7 it can be calculated that on the order of 19 000 U/mL (EC)SOD is needed to compete equally with 0.85 mol/L NO for superoxide radicals. Thus, the high EC-SOD activity in the human arterial wall interstitium should be sufficient to compete with basal concentrations of NO but may allow significant formation of peroxynitrite under maximal agonist stimulation of the endothelium and when NO synthase is induced in other cell types. In several pathological situations the rate of superoxide formation is also increased in the arterial wall43 44 45 46 47 and may equal or exceed the rate of NO formation, as indicated by the effects of administration of large amounts of SOD on NO-induced responses. Thus, the injection of a heparan sulfatebinding SOD derivative reduced the blood pressure in spontaneously hypertensive rats,43 addition of SOD enhanced acetylcholine-induced relaxation of aortas from diabetic rats,44 and in vivo administration of liposome-encapsulated CuZn-SOD enhanced the response to acetylcholine of isolated aortic rings derived from cholesterol-fed rabbits.45 All these studies were performed on species with less arterial wall EC-SOD than is found in humans, and the results emphasize the potential protective role of a high EC-SOD level of the arterial wall interstitium.

We have previously found considerable differences among mammalian species in organ ECSOD contents.24 Here we describe large differences in the levels in the aortic wall. One similarity among the species is that in all cases except rats and mice the arterial wall contains by far the largest amounts of EC-SOD of all organs analyzed, suggesting a particularly important role for the enzyme in this tissue. The rat species deviates from all other mammals in that the organ EC-SOD content is very low, but the plasma content is relatively high.24 Unlike the EC-SOD of other species, rat EC-SOD is dimeric and has a low affinity for heparin and does not bind to heparan sulfate under physiological conditions.48 In the mouse, the lungs and the aorta contain equally large amounts of EC-SOD. The wide interspecies differences indicate that the pathophysiological effects of superoxide radicals in the vascular wall should differ among species. In support of this suggestion we have shown that preincubation of rabbit aortic rings with recombinant human EC-SOD results in significant protection of NO-mediated relaxation against superoxide formed by pyrogallol autoxidation.49 The preincubation of the rabbit aortic rings resulted in a rise in (EC)SOD activity of about 5000 U/g wet weight, ie, to reach the levels of the human aorta. Thus, the difference in EC-SOD levels among species is an important factor to consider when extrapolating experimental findings in animal models to the situation in humans. In summary, the very high EC-SOD activity of the human arterial wall interstitium and the large differences among mammalian species deserve attention in all studies on vascular wall pathology potentially involving oxidative stress. Previous SectionNext Section

Selected Abbreviations and Acronyms

CuZn-SOD = copper-and-zinccontaining SOD EC-SOD = extracellular superoxide dismutase LAD = left anterior descending coronary artery Mn-SOD = manganese-containing SOD NO = nitric oxide SOD = superoxide dismutase Previous SectionNext Section

Acknowledgments
This study was supported by the Swedish Natural Science Research Council, grant 9204. The authors would like to thank Agneta berg, Eva Bern, and Els-Marie hman for their skillful technical assistance. Previous Section

References
1.

Mehta JL, Lawson DL, Nichols WW. Attenuated coronary relaxation after reperfusion: effects of superoxide dismutase and TxA2 inhibitor U 63557A. Am J Physiol. 1989;257:H1240-H1246. Abstract/FREE Full Text 2. Steinbrecher UP, Zhang H, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Radic Biol Med. 1990;9:155-168. Medline 3. Heinecke JW, Baker L, Rosen H, Chait A. Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. J Clin Invest. 1986;77:757-761. 4. Kawamura M, Heinecke JW, Chait A. Pathophysiological concentrations of glucose promote oxidative modification of low density lipoprotein by a superoxide-dependent pathway. J Clin Invest. 1994;94:771-778. 5. Heinecke JW, Kawamura M, Suzuki L, Chait A. Oxidation of low density lipoprotein by thiols: superoxide-dependent and -independent mechanisms. J Lipid Res. 1993;34:2051-2061. Abstract 6. Beckman JS, Beckman T, Chen J, Marshall PA, Freeman B. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620-1624. Abstract/FREE Full Text 7. Huie TE, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun.. 1993;18:195-199. Medline 8.

Ischiropoulos H, Zhu L, Chen J, Tsai M, Martin JC, Smith CD, Beckman JS. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys. 1992;298:431-437. CrossRefMedline 9. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, Beckman JS. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol. 1992;5:834-842. CrossRefMedline 10. Darley-Usmar VM, Hogg N, OLeary VJ, Wilson MT, Moncada S. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic Res Commun.. 1992;17:9-20. Medline 11. Katusic ZS, Vanhoutte PM. Superoxide anion is an endothelium-derived contracting factor. Am J Physiol. 1989;257:H33-H37. Abstract/FREE Full Text 12. Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A. 1982;79:7634-7638. Abstract/FREE Full Text 13. McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969;244:6049-6055. Abstract/FREE Full Text 14. Weisiger RA, Fridovich I. Mitochondrial superoxide dismutase: site of synthesis and intramitochondrial localization. J Biol Chem. 1973;248:4793-4796. Abstract/FREE Full Text

15. Marklund SL. Spectrophotometric study of spontaneous disproportionation of superoxide anion radical and sensitive direct assay for superoxide dismutase. J Biol Chem. 1976;251:7504-7507. FREE Full Text 16. Marklund SL. Direct assay of superoxide dismutase with potassium superoxide. In: Greenwald RA, ed. Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC Press Inc; 1985:249-255. 17. Karlsson K, Marklund SL. Plasma clearance of human extracellular superoxide dismutase C in rabbits. J Clin Invest. 1988;82:762-766. 18. Tibell L, Hjalmarsson K, Edlund T, Skogman G, Engstrm , Marklund SL. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci U S A. 1987;84:66346638. Abstract/FREE Full Text 19. Marklund SL. Extracellular superoxide dismutase in human tissues and human cell lines. J Clin Invest. 1984;74:1398-1403. 20. Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. CrossRefMedline 21. Labarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem. 1980;102:344-352. CrossRefMedline 22.

Fager G, Hansson GK, Ottosson P, Dahllf B, Bondjers G. Human arterial smooth muscle cells in culture: effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res. 1988;176:319-335. CrossRefMedline 23. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Biol Chem. 1986;103:2787-2796. 24. Marklund SL. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J. 1984;222:649-655. Medline 25. Marklund SL. Expression of extracellular superoxide dismutase by human cell lines. Biochem J. 1990;266:213-219. Medline 26. Sandstrm JS, Karlsson K, Edlund T, Marklund SL. Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues. Biochem J. 1993;294:853-857. 27. Winterbourn CC, Stern A. Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical. J Clin Invest. 1987;80:1486-1491. 28. Sandstrm J, Carlsson L, Marklund SL, Edlund T. The heparin-binding domain of extracellular superoxide dismutase, and formation of variants with reduced heparin affinity. J Biol Chem. 1992;267:18205-18209. Abstract/FREE Full Text 29. Karlsson K, Lindahl U, Marklund SL. Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans. Biochem J. 1988;256:29-33.

Medline 30. Karlsson K, Marklund SL. Binding of human extracellular superoxide dismutase C to cultured cell lines and to blood cells. Lab Invest. 1989;60:659-666. Medline 31. Karlsson K, Sandstrm J, Edlund A, Marklund SL. Turnover of extracellular superoxide dismutase in tissues. Lab Invest. 1994;70:705-710. Medline 32. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. Medline 33. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. CrossRefMedline 34. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987;84:9265-9269. Abstract/FREE Full Text 35. Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet. 1987;2:1057-1058. Medline 36. Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes P. Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol. 1993;265:H862-H867.

Abstract/FREE Full Text 37. Hogg N, Kalyanaraman B, Joseph J, Struck A, Parthasarathy S. Inhibition of lowdensity lipoprotein oxidation by nitric oxide: potential role in atherogenesis. FEBS Lett. 1993;334:170-174. CrossRefMedline 38. Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation: formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem. 1994;269:26066-26075. Abstract/FREE Full Text 39. Beasley D, Schwartz JH, Brenner BM. Interleukin 1 induces prolonged l-argininedependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest. 1991;87:602-608. 40. Beckman JS, Yao ZY, Anderson PG, Chen J, Accavitti MA, Tarpey MM, White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler. 1994;175:81-88. 41. Malinski T, Taha Z, Grunfeld S, Pattou S, Kapturczak M, Tomboulian P. Diffusion of nitric oxide in the aorta wall monitored in situ by porphyrinic microsensors. Biochem Biophys Res Commun.. 1993;193:1076-1082. CrossRefMedline 42. Lancaster JR. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc Natl Acad Sci U S A. 1994;91:8137-8148. Abstract/FREE Full Text 43.

Nakazono K, Watanabe N, Matsuno K, Sasaki J, Sato T, Inoue M. Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci U S A. 1991;88:10045-10048. Abstract/FREE Full Text 44. Hattori Y, Kawasaki H, Abe K, Kanno M. Superoxide dismutase recovers altered endothelium-dependent relaxation in diabetic rat aorta. Am J Physiol. 1991;261:H1086-H1094. Abstract/FREE Full Text 45. White RC, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:1044-1048. Abstract/FREE Full Text 46. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546-2551. 47. Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. J Clin Invest. 1995;95:187-194. 48. Karlsson K, Marklund SL. Extracellular superoxide dismutase in the vascular system of mammals. Biochem J. 1988;255:223-228. Medline 49. Abrahamsson T, Brandt U, Marklund SL, Sjquist PO. Vascular bound recombinant extracellular superoxide dismutase type C protects against the detrimental effects of superoxide radicals on endothelium-dependent