Inducible Nitric Oxide Synthase Deficiency Does Not Affectthe Susceptibility of Mice to Atherosclerosis but Increases

Collagen Content in LesionsXi-Lin Niu, MD, PhD; Xia Yang, MD; Kiyotaka Hoshiai, MS; Kazuo Tanaka, MD, PhD;

Sadaaki Sawamura; Yasuhiro Koga, MD, PhD; Hiroe Nakazawa, MD, PhD

Background—Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) inthe development of atherosclerosis is not established.

Methods and Results—We compared the susceptibility of iNOS knockout (iNOS2/2) and wild-type (iNOS1/1) mice to thedevelopment of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atheroscleroticlesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS1/1 2856733103 mm2,iNOS2/2 2936823103 mm2, n510). iNOS mRNA was detected in the lesions of iNOS1/1 but not iNOS2/2 mice throughRT-PCR. Immunohistochemically, iNOS1/1 mice showed iNOS staining in macrophages and medial smooth musclecells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS2/2 mice. Therewas no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesionsof the 2 strains. However, the lesions of iNOS1/1 mice showed a markedly decreased extracellular collagen contentcompared with those of iNOS2/2 mice

Conclusions—iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but theresulting lesions show decreased levels of extracellular collagen and may be more fragile.(Circulation. 2001;103:1115-1120.)

Key Words: mice, inbredn atherosclerosisn nitric oxide synthase

Chronic inhibition of NO production accelerates athero-sclerosis,1,2 whereas enhanced production of NO sup-

presses the development of atherosclerosis or even regressesthe lesions.3,4 These effects of NO in large part depend on itsability to terminate LDL oxidation,5 to inhibit the expressionof adhesion molecules and chemokines,6,7 and to attenuatesmooth muscle cell migration8 and are principally due to NOproduced by endothelial NO synthase (eNOS). Experimentswith eNOS knockout mice9,10 and eNOS gene transfer11

confirmed that eNOS is an important antiatherogenic player.However, the direct influence of inducible NOS (iNOS) onthe development of atherosclerosis is unknown, although theinduction of iNOS consistently occurs in atheroscleroticvessels of humans12,13 and experimental animals.14,15 Theinduction of iNOS may be a defense mechanism against thedisease. However, a high flux of NO from iNOS may favorformation of peroxynitrite, a powerful oxidant generated bythe reaction of NO and superoxide,16 and produce nitroty-rosine, which was shown to be present in human atheromaplaque.17 Thus, it is important to determine the overall impactof iNOS on atherogenesis, because this may have implica-

tions for preventive and therapeutic strategy. To examinedirectly the influence of iNOS on the development of athero-genesis and on the characteristics of the lesion, we comparedthe susceptibility of iNOS2/2 and iNOS1/1 mice to thedevelopment of atherosclerosis induced with an atherogenicdiet and analyzed the composition of the atheroscleroticlesions in the 2 strains.

Methods

Mice and Experimental ProtocolThe iNOS2/2 mice, with a mixed C57Bl/6J3129 SvEv geneticbackground,18 were obtained from Merck & Co, Inc. The iNOS1/1

mice were obtained by crossing 129 SvEv mice with C57 Bl/6J micetwice. The iNOS2/2 and iNOS1/1 strains have similar geneticbackgrounds of'75% C57Bl/6J and'25% 129/SvEv. The animalswere maintained in a pathogen-free barrier facility with a 12-hourlight/dark cycle and had free access to food and water. Fifteenage-matched (8-week-old) females of each strains were fed a dietthat contained 15% fat, 1% cholesterol, and 0.5% sodium cholate19

for 15 weeks. Six females of each strain (8 weeks old), fed normalchow for the same period of time, served as controls. The study wasapproved by the Animal Care Committee of Tokai University.

Received July 6, 2000; revision received August 29, 2000; accepted September 12, 2000.From the Departments of Physiology (X.-L.N., X.Y., K.H., H.N.) and Microbiology (K.T., S.S., Y.K.), School of Medicine, Tokai University, Isehara,

Kanagawa, Japan.Correspondence to Prof H. Nakazawa, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193 Japan. E-mail:

nakazawa@is.icc.u-tokai.ac.jp© 2001 American Heart Association, Inc.

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Measurement of Plasma Lipid LevelsMice were deprived of food for 16 hours, and blood was collectedvia the abdominal aorta into heparin-coated tubes with the animalsunder pentobarbital sodium–induced anesthesia. Plasma was ob-tained through centrifugation of the whole blood for 10 minutes at12 000gat 4°C. Concentrations of total cholesterol, HDL choles-terol, and triglycerides were measured enzymatically with aBoehringer-Mannheim Hitachi 717 analyzer.

Tissue Preparation and Histological AnalysisAfter the collection of blood, the heart and proximal aorta wereexcised and embedded in OCT compound (Tissue-Tek), frozen ondry ice, and stored at270°C until sectioning for histologicalanalysis. The OCT-embedded heart with aorta was serially sectionedfrom the middle of the ventricle to the root of the aorta into 5-mmslices. For the quantitative evaluation of atherosclerotic lesions,20 9sections were taken every 40mm, stained with oil red O, andcounterstained with hematoxylin. Remaining sections were frozen at270°C and used for other stainings. The lesion size was quantifiedthrough microscopic examination with a computer-assisted imageanalysis system (KS 300; Carl Zeiss). The average size of lesions inthe 9 sections was taken to represent the lesion size for each animal.Cellular density in the lesions was quantified by counting cell nucleiin the intima of the aortic lesions at 3 levels (sections 2, 4, and 6)with sections stained with oil red O and hematoxylin. Cellulardensity was expressed as the number of cells per intimal area(cells/mm2). The presence of collagen in the lesions was examined insections stained according to the standard Van Gieson method. Forevaluation of iNOS mRNA induction, the proximal aorta wassnap-frozen in liquid nitrogen and stored at280°C until use.

ImmunohistochemistrySerial cryostat sections were used to stain macrophages, smoothmuscle cells, iNOS, nitrotyrosine, and vascular cell adhesion mole-cule (VCAM)-1. The primary antibodies that we used were apolyclonal rabbit anti-mouse iNOS antibody (017-16001; Wako PureChemical Industries; diluted 1:1000), a monoclonal rat anti-mouseCD11b (Mac-1) antibody for macrophages (MAS 034p; HarlanSera-Laboratory Ltd; diluted 1:5), a polyclonal rabbit anti-nitrotyrosine antibody (06-248; Upstate Biotechnology; diluted

1:100), a monoclonal rat anti-mouse VCAM-1 antibody (RA140070; Antigenix America Inc; diluted 1:50), and a monoclonalmouse anti-human smooth muscle cella-actin antibody (IgG2a) (A2547; Sigma Chemical Co; diluted 1:400).21

Sections for immunohistochemical staining were fixed in acetoneat 220°C for 5 minutes. All subsequent incubations were performedat room temperature, except for incubation with primary antibodies.The endogenous peroxidase activity was neutralized with H2O2

(0.3% vol/vol) for 10 minutes. An avidin-biotin blocking kit (VectorLaboratories) was used to block nonspecific endogenous biotinstaining. After incubation with 4% (vol/vol) BSA (Sigma) diluted inPBS for 30 minutes, the sections were incubated with primaryantibodies overnight at 4°C. Then biotinylated goat anti-rabbit IgG(for iNOS and nitrotyrosine; BA-1000, Vector Laboratories), biotin-ylated rabbit anti-rat IgG (for macrophages; BA-4001, VectorLaboratories), or biotinylated goat anti-mouse IgG2a antibody (forsmooth muscle cells; GAM/IGG2A/BIO, Nordic ImmunologicalLaboratories BV) was applied at 5mg/mL and incubated for 45minutes. Subsequently, the sections were incubated withstreptavidin-peroxidase complex (Vector Laboratories) for 30 min-utes. The staining was visualized with 9-amino-3-ethylene-carbazole(Vector Laboratories) or diaminobenzidine (Vector Laboratories).Nonimmune rabbit or rat serum was used in place of the primaryantibody as a negative control.

Measurements of iNOS and mRNATo determine iNOS mRNA levels, the frozen aorta was homoge-nized in ISOGEN (Nippon Gene), total RNA was extracted, andRT-PCR was applied as previously described.22 Briefly, equalamounts of RNA were reverse-transcribed into cDNA. The RTproducts were amplified with the following primers for iNOS:59-CTTGCCCCTGGAAGTTTCTCTT-3 (upstream) and 39-CTTTGTTGTCCTTGGATGGTCG-59 (downstream), which af-forded a 519-bp fragment. As an internal control,b2-microglobulin(b2M) mRNA was estimated under the same cycling conditions with

Figure 1. iNOS expression in aorta. Effects of atherogenic dieton iNOS and b2-microglobulin (b2M) mRNAs were evaluatedwith RT-PCR in iNOS2/2 and iNOS1/1 mice.

Figure 2. Representative sections fromiNOS2/2 and iNOS1/1 mice fed athero-genic diet (oil red O and hematoxylin Hstainings). From this point, at level of 3complete valves with their attachmentsites, 5-mm sections were taken for next320 mm of aorta, and 9 sections takenevery 40 mm were used for evaluation oflesion size.

Plasma Lipid Levels in iNOS1/1 and iNOS2/2 Mice

Lipid, mg/dL

Normal Diet (n55) Atherogenic Diet (n510)

iNOS1/1 iNOS2/2 iNOS1/1 iNOS2/2

Total cholesterol 81.063.0 85.264.9 364.0661.8 406.5655.2

HDL cholesterol 55.563.4 51.063.7 128.5619.2 171.367.5

Triglyceride 5.061.3 6.063.0 3.560.3 4.260.9

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the primers 59-TGACCGGCTTGTATGCTATC-39(upstream) and59-CAGTGTGAGCCAGGATATAG-39(downstream), which gavea 223-bp fragment. The PCR products of iNOS andb2M were mixedand electrophoresed on a 1% agarose Tris-acetate EDTA gel, whichwas stained with ethidium bromide. The gel was photographed underultraviolet light.

Statistical AnalysisAll data are expressed as mean6SEM. To analyze differences inatherosclerotic lesion size between the 2 groups, Student’st test andMann-WhitneyU analysis were performed. To compare lipid mea-surements, 2-way ANOVA was performed. Probability values of,0.05 were considered significant.

Results

Plasma Lipid LevelsPlasma cholesterol was markedly elevated with the athero-genic diet in both iNOS2/2 and iNOS1/1 mice, but nosignificant difference was observed between the 2 groups(Table). Triglyceride levels were unaffected by the athero-genic diet, and there were no significant intergroup differ-ences. Basal levels of all lipids were comparable in the 2strains and remained in the same ranges after 15 weeks ofnormal chow (data not shown).

iNOS Expression in AortaAs shown in Figure 1, iNOS mRNA was not observed in theaorta of control mice of either strain. It also was not observedin iNOS2/2 mice fed the atherogenic diet, as expected.However, iNOS mRNA expression was clearly demonstratedin the aorta of iNOS1/1 mice fed the atherogenic diet.

Atherosclerotic Lesion FormationAll mice of both strains developed atherosclerotic lesionsalong the proximal aortic wall and at the valve cusps after the15-week atherogenic diet (Figure 2). Neither strain developedaortic atherosclerotic lesions when maintained on the normalchow for the same period of time. Individual lesions variedfrom simple fatty streaks to complex fibrous plaques, butthere was no consistent difference in morphological charac-teristics or sites of lesions between the 2 strains. The averagevalues of cumulative lesion size were similar in the 2 strains(iNOS1/1 2856733103 mm2, iNOS2/2 2936823103 mm2

[mean6SEM], n510) (Figure 3).

Collagen Content in LesionsAs shown in Figure 4, extracellular collagen content wasmarkedly decreased in the lesions of iNOS1/1 mice comparedwith that of comparable-sized lesions of iNOS2/2 mice. Thecellular densities in the lesions were equal in the 2 strains(iNOS1/1 38696315 cells/mm2, iNOS2/2: 35686248 cells/mm2 [mean6SEM], n510).

ImmunohistochemistryImmunocytochemical analyses showed that lesions consistedprimarily of macrophages (Figures 5A1, 5B1, and 5B5) withminimal smooth muscle cell involvement in both strains ofmice (data not shown). iNOS staining was extensive in thelesions of iNOS1/1 mice (Figures 5B2 and 5B6) but wasabsent in those of iNOS2/2 mice (Figure 5A2). Specifically,iNOS staining was seen in macrophages (identified in adja-cent sections by positive MAC-1 staining) distributedthroughout the intimal lesions. Staining for iNOS was alsoevident in medial smooth muscle cells beneath the intimallesions (Figure 5B2). iNOS was not detected in the aorta ofeither of these strains of mice maintained on normal chow(data not shown). Nitrotyrosine staining in the lesions of theiNOS1/1 mice showed a distribution similar to that of iNOSstainings but was more marked in the intimal area of lesions,where macrophages are the major component (Figures 5B3and 5B7). Nitrotyrosine staining was not present in sectionsof aorta from iNOS2/2 mice fed the atherogenic diet or insuch sections from both strains fed normal chow (data notshown). VCAM-1 staining was seen in the lesions of bothstrains, with no apparent difference in the intensity ordistribution between the 2 strains (Figures 5A4 and 5B4).

DiscussionThis is the first study in which iNOS knockout mice havebeen used to directly examine whether iNOS-mediated path-

Figure 3. Sizes of atherosclerotic lesions. Average sizes of ath-erosclerotic lesions were calculated from 9 sections in iNOS1/1

(n510) and iNOS2/2 (n510) mice fed atherogenic diet. Therewere no significant differences.

Figure 4. Collagen content detected withVan Gieson staining in aorta sectionsfrom iNOS2/2 and iNOS1/1 mice fedatherogenic diet. Interstitial collagen wasabundant in lesions of iNOS2/2 mice butscarce in lesions of iNOS1/1 mice. Mag-nification: 31000.

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ways promote or protect against development of atheroscle-rosis in vivo. iNOS was induced in the atherosclerotic lesionsonly in iNOS1/1 mice, and the sizes of atherosclerotic lesionswere similar in the iNOS2/2 and iNOS1/1 mice.

The involvement of iNOS in atherosclerosis has beenshown through a variety of observations, including thedetection of iNOS protein or mRNA in the atheroscleroticlesions of both humans12,13 and experimental animals.14 Ourresults confirm and extend previous reports of the inductionof iNOS in atherosclerotic vessels. iNOS may have paradox-ical effects on the development of atherosclerosis due to the

antiatherogenic character of NO5–8 and the proatherogenicproperty of NO-derived peroxynitrite.15,23,24 Our study pro-vides direct evidence that the overall impact of iNOS on theprogression of atherosclerosis is remarkably little. We knowof only 1 previous study in which iNOS knockout mice wereused to examine the influence of iNOS on vascular lesions.25

Using a transplant arteriosclerosis model in which an allo-genic heart was heterotropically transplanted into an iNOS2/2

or iNOS1/1 recipient, those authors concluded that iNOS hasan antiarteriosclerotic effect, based on the marked inhibitionof intimal hyperplasia of coronary arteries in hearts trans-

Figure 5. Immunohistochemical staining for macrophages, iNOS, nitrotyrosine, and VCAM-1. A, Sections were obtained from iNOS2/2

mice fed atherogenic diet. B, Sections were obtained from iNOS1/1 mice fed atherogenic diet. Magnification A1 to A3, A5, B1 to B3,and B8, 31000; B5 to B7, 32500; and A4 and B4, 3330. A1, B1, and B5, macrophage staining. A2, B2, and B6, iNOS staining. A3,B3, and B7, nitrotyrosine staining. A4 and B4, VCAM-1 staining. A5 and B8, negative control.

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planted into iNOS1/1 mice. Experiments with adenovirus-mediated iNOS gene transfer also demonstrated that iNOSinhibited intimal hyperplasia in allograft arteriosclerosis26

and balloon-injured arteries.27 These findings are not inconflict with our result, because the pathological processesthat underlie intimal hyperplasia are not the same as those inhyperlipidemic atherosclerosis. In intimal hyperplasia, immu-nological reaction is the initial trigger and neointimal smoothmuscle cell migration and proliferation are the major charac-teristics,25 whereas modified LDL accumulation and mono-cyte/macrophage recruitment play important roles in hyper-lipidemic atherosclerosis.28 As shown in Figures 5A1, 5B,and 5B5, macrophages are the predominant cells in the lesion,and the VCAM-1 induction, which is 1 of the earliest eventsin the development of atherosclerosis,28 was comparable iniNOS2/2 and iNOS1/1 mice. Thus, induced iNOS appears notto have affected the lesion size or adhesion molecule expres-sion. However, it may increase plaque instability, because amarked decrease in extracellular collagen content was ob-served in the lesions of iNOS1/1 mice compared with those ofiNOS2/2 mice. The collagen content in the lesion determinesthe biomechanical strength of the atherosclerotic lesion andits vulnerability to disruption.28–30 NO-induced apoptosis orinhibition of cell proliferation is unlikely to account for thedecreased collagen content, because lesion cellularity wassimilar in both strains. Other possible mechanisms are NO-induced inhibition of collagen formation31 and the activationof collagen-degrading metalloproteinase.3,29,30,32,33We haverecently shown that peroxynitrite is a novel activator ofprocollagenase,34 so the finding that iNOS was colocalizedwith nitrotyrosine, which is formed by peroxynitrite-inducednitration,17 in the lesions of iNOS1/1 mice suggests thatiNOS-catalyzed NO production may have enhanced per-oxynitrite formation and activated MMP. It remains to beexamined whether MMP induction or inhibition of collagenformation contributed to the reduced collagen content in ourexperiment. As a next step, it would be useful to confirm ourfindings by using mice crossed iNOS2/2 and either apoE orLDL receptor2/2 mice, which develop advanced atheroscle-rotic lesions that are more relevant to human disease than thecurrent model.35

In conclusion, iNOS appeared to have neither a proathero-genic nor an antiatherogenic influence in hyperlipidemia-induced atherosclerosis under our in vivo conditions, but itdoes decrease the collagen content of the lesion, which mayincrease plaque instability. This finding may offer a noveltherapeutic approach to the prevention of acute coronaryevents.

AcknowledgmentsThis work was supported by a grant from Tokyo BiochemicalResearch Foundation of Japan and a Grant-in-Aid for ScientificResearch (C) (11838018) from the Ministry of Education, Science,Sports and Culture.

References1. Cayatte AJ, Palacino JJ, Horten K, et al. Chronic inhibition of nitric oxide

production accelerates neointima formation and impairs endothelialfunction in hypercholesterolemic rabbits.Arterioscler Thromb. 1994;14:753–759.

2. Naruse K, Shimizu K, Muramatsu M, et al. Long-term inhibition of NOsynthesis promotes atherosclerosis in the hypercholesterolemic rabbitthoracic aorta: PGH2 does not contribute to impaired endothelium-dependent relaxation.Arterioscler Thromb. 1994;14:746–752.

3. Wang BY, Ho HK, Lin PS, et al. Regression of atherosclerosis: role ofnitric oxide and apoptosis.Circulation. 1999;99:1236–1241.

4. Candipan RC, Wang BY, Buitrago R, et al. Regression or progression:dependency on vascular nitric oxide.Arterioscler Thromb Vasc Biol.1996;16:44–50.

5. Hogg N, Kalyanaraman B. Nitric oxide and low-density lipoproteinoxidation.Free Radic Res. 1998;28:593–600.

6. Qian H, Neplioueva V, Shetty GA, et al. Nitric oxide synthase genetherapy rapidly reduces adhesion molecule expression and inflammatorycell infiltration in carotid arteries of cholesterol-fed rabbits.Circulation.1999;99:2979–2982.

7. De Caterina R, Libby P, Peng HB, et al. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endo-thelial expression of adhesion molecules and proinflammatory cytokines.J Clin Invest. 1995;96:60–68.

8. Sarkar R, Meinberg EG, Stanley JC, et al. Nitric oxide reversibly inhibitsthe migration of cultured vascular smooth muscle cells.Circ Res. 1996;78:225–230.

9. Moroi M, Zhang L, Yasuda T, et al. Interaction of genetic deficiency ofendothelial nitric oxide, gender, and pregnancy in vascular response toinjury in mice.J Clin Invest. 1998;101:1225–1232.

10. Rudic RD, Shesely EG, Maeda N, et al. Direct evidence for theimportance of endothelium-derived nitric oxide in vascular remodeling.J Clin Invest. 1998;101:731–736.

11. Von der Leyen HE, Gibbons GH, Morishita R, et al. Gene therapyinhibiting neointimal vascular lesion: in vivo transfer of endothelial cellnitric oxide synthase gene.Proc Natl Acad Sci U S A. 1995;92:1137–1141.

12. Buttery LD, Springall DR, Chester AH, et al. Inducible nitric oxidesynthase is present within human atherosclerotic lesions and promotes theformation and activity of peroxynitrite.Lab Invest. 1996;75:77–85.

13. Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multipleisoforms of nitric oxide synthase in normal and atherosclerotic vessels.Arterioscler Thromb Vasc Biol. 1997;17:2479–2488.

14. Alfon J, Guasch JF, Berrozpe M, et al. Nitric oxide synthase II (NOS II)gene expression correlates with atherosclerotic intimal thickening: pre-ventive effects of HMG-CoA reductase inhibitors.Atherosclerosis. 1999;145:325–331.

15. Luoma JS, Stralin P, Marklund SL, et al. Expression of extracellular SODand iNOS in macrophages and smooth muscle cells in human and rabbitatherosclerotic lesions: colocalization with epitopes characteristic ofoxidized LDL and peroxynitrite-modified proteins.Arterioscler ThrombVasc Biol. 1998;18:157–167.

16. Ischiropoulos H, Zhu L, Beckman JS. Peroxynitrite formation frommacrophage-derived nitric oxide.Arch Biochem Biophys. 1992;298:446–451.

17. Beckmann JS, Ye YZ, Anderson PG, et al. Extensive nitration of proteintyrosines in human atherosclerosis detected by immunohistochemistry.Biol Chem Hoppe Seyler. 1994;375:81–88.

18. MacMicking JD, Nathan C, Hom G, et al. Altered responses to bacterialinfection and endotoxic shock in mice lacking inducible nitric oxidesynthase.Cell. 1995;81:641–650.

19. Munday JS, Thompson KG, James KA, et al. Dietary antioxidants do notreduce fatty streak formation in the C57BL/6 mouse atherosclerosismodel.Arterioscler Thromb Vasc Biol. 1998;18:114–119.

20. Suzuki H, Kurihara Y, Takeya M, et al. A role for macrophage scavengerreceptors in atherosclerosis and susceptibility to infection.Nature. 1997;386:292–296.

21. Moulton KS, Heller E, Konerding MA, et al. Angiogenesis inhibitorsendostatin or TNP-470 reduce intimal neovascularization and plaquegrowth in apolipoprotein E– deficient mice.Circulation. 1999;99:1726–1732.

22. Singh I, Grams M, Wang WH, et al. Coordinate regulation of renalexpression of nitric oxide synthase, renin, and angiotensinogen mRNA bydietary salt.Am J Physiol. 1996;270:F1027–F1037.

23. Leeuwenburgh C, Hardy MM, Hazen SL, et al. Reactive nitrogen inter-mediates promote low density lipoprotein oxidation in human athero-sclerotic intima.J Biol Chem. 1997;272:1433–1436.

24. Graham A, Hogg N, Kalyanaraman B, et al. Peroxynitrite modification oflow-density lipoprotein leads to recognition by the macrophage scavengerreceptor.FEBS Lett. 1993;330:181–185.

Niu et al Influence of iNOS on Atherosclerosis Development 1119

by guest on June 27, 2018http://circ.ahajournals.org/

Dow

nloaded from

25. Koglin J, Glysing Jensen T, Mudgett JS, et al. Exacerbated transplantarteriosclerosis in inducible nitric oxide-deficient mice.Circulation.1998;97:2059–2065.

26. Shears LL, Kibbe MR, Murdock AD, et al. Efficient inhibition of intimalhyperplasia by adenovirus-mediated inducible nitric oxide synthase genetransfer to rats and pigs in vivo.J Am Coll Surg. 1998;187:295–306.

27. Shears LL, Kawaharada N, Tzeng E, et al. Inducible nitric oxide synthasesuppresses the development of allograft arteriosclerosis.J Clin Invest.1997;100:2035–4226.

28. Ross R. Atherosclerosis–an inflammatory disease.N Engl J Med. 1999;340:115–126.

29. Libby P. Molecular bases of the acute coronary syndromes.Circulation.1995;91:2844–2850.

30. Sukhova GK, Schonbeck U, Rabkin E, et al. Evidence for increasedcollagenolysis by interstitial collagenases-1 and -3 in vulnerable humanatheromatous plaques.Circulation. 1999;99:2503–2509.

31. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide-generating compoundsinhibit total protein and collagen synthesis in cultured vascular smoothmuscle cells.Circ Res. 1995;76:305–309.

32. Rajagopalan S, Meng XP, Ramasamy S, et al. Reactive oxygen speciesproduced by macrophage-derived foam cells regulate the activity ofvascular matrix metalloproteinases in vitro: implications for athero-sclerotic plaque stability.J Clin Invest. 1996;98:2572–2579.

33. Rekhter MD. Collagen synthesis in atherosclerosis: too much and notenough.Cardiovasc Res. 1999;41:376–384.

34. Okamoto T, Akaike T, Nagano T, et al. Activation of human neutrophilprocollagenase by nitrogen dioxide and peroxynitrite: a novel mechanismfor procollagenase activation involving nitric oxide.Arch BiochemBiophys. 1997;342:261–274.

35. Breslow JL. Mouse models of atherosclerosis.Science. 1996;272:685–688.

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and Hiroe NakazawaXi-Lin Niu, Xia Yang, Kiyotaka Hoshiai, Kazuo Tanaka, Sadaaki Sawamura, Yasuhiro Koga

Atherosclerosis but Increases Collagen Content in LesionsInducible Nitric Oxide Synthase Deficiency Does Not Affect the Susceptibility of Mice to

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