Enhancement of vascular function by modulation of endogenous nitric oxide production or activity (US...

7/29/2019 Enhancement of vascular function by modulation of endogenous nitric oxide production or activity (US patent 589

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5,891,459Page 2OTHER PUBLICATIONS

Rock et al. Lrginyl Lysine and LrginylLrginine. . . Med. Sci.Res. 1990, vol. 18, pp. 165166.Andrews et al. , "Lowensity Lipoproteins inhibit endothe-lium ependent Relaxation in Rabbit Aorta", Nature (1987),327:23739.Bath et al. , "Nitric Oxide and Prostacyclin: Divergence ofInhibitory EIfects on Monocyte Chemotaxis and Adhesion toEndothelium in Vitro", Arterioscierosis and Thrombosis(1991),11:25460.Cooke, "Endothelial Dysfunction in Disease States", Cur-rent Opinion in Cardiology (1990), 5:63744.Drexler et al. , "Correction of Endothelial Dysfunction inCoronary Micocirculation of HypercholesterolaemicPatients", The Lancet (1991),338:15461550.Garg and Hassid, "Nitric Oxideenerating Vasodilators and8BromoCyclic Guanosine Monophosphate Inhibit Mitro-genesis and Proliferation of Cultured Rat Vascular SmoothMuscle Cells", J. Clin. Invest. (1989), 83:17741777.Girerd et al. , "LArginine Augments Endothelium Depen-dent Vasodilation in Cholesterol ed Rabbits", CirculationResearch (1990), 67:13011308.Heistad et al. , "Augmented Responses to VasoconstrictorStimuli in Hypercholesterolemic and Atherosclerotic Mon-keys", Circulation Research (1984), 54:71118.Kubes et al. , "Nitric Oxide: An Endogenous Modulator ofLeukocyte Adhesion", PNAS USA (1991),88:4651655.Kugiyama et al. , "Impairment of Endothelium DependentArterial Relaxation by Lysolecithin in Modified Lowen-sity Lipoproteins", Nature (1990), 344:16062.Kuo et al. , "Pathophysiological Consequences of Athero-sclerosis Extend Into the Coronary Microcirculation: Res-toration of Endothelium ependent Responses by LArgi-nine", Circulation Research (19920, 70:46576.Lefer et al. , "Role of Endothelium erived Relaxing Factoras a Cardioprotective Agent in Myocardial Ischemia", Basil,Karger (1990), 190197.Minor et al. , "Dietnduced Atherosclerosis Increases theRelease of Nitrogen Oxides from Rabbit Aorta", J. Clin.Invest. (1990), 86:2109116.

Mitchell et al. , "Native LDL Inhibits the Release of Endot-helial Derived Relaxing Factors by Reducing the Activity ofEndothelial Nitric Oxide Synthase", (Abstract), J.Vase. Res.(1992), 29:169.Pohl and Busse, "EDRF Increases Cyclic GMP in PlateletsDuring Passage Through the Coronary Vascular Bed", Cir-culation Research (1989), 65:17981803.Radomski et al. , "Comparative Pharmacology of Endothe-lium erived Relaxing Factor, Nitric Oxide and Prostacy-clin in Platelets", Br. J. Pharmacol. (1987), 92:181187.Ross, "The Pathogenesis of Atherosclerosis n Update",The New England Journal of Medicine (1986),314:48800.Rossitch, Jr. et al. , "LArginine Normalizes EndothelialFunction in Cerebral Vessels from HypercholesterolemicRabbits", J. Clin. Invest. , 87:12951299 (1991).Stamler et al. , "NAcetylcysteine Potentiates Platelet Inhi-bition by Endothelium erived Relaxing Factor", Circula-tion Research (1989), 65:78995.Tanner et al. , "Oxidized Low Density Lipoproteins InhibitRelaxations of Porcine Coronary Artieries: Role of Scaven-ger Receptor and Endotheiium erived Nitric Oxide", Cir-culation, 83:2109116 (1991).Tomita et al. , "Rapid and Reversible Inhibition by LowDensity Lipoprotein of the Endothelium ependent Relax-ation to Hemostatic Substances in Porcine Coronary Arter-ies", Circulation Research (1990), 66:187.Weidinger et al. , "Persistent Dysfunction of RegeneratedEndothelium After Balloon Angioplasty of Rabbit lliacArtery", Circulation (1990), 81:16671679.Yamamoto et al. , "Videomicroscopic Demonstration ofDefective Cholinergic Arteriolar Vasodilation in Atheroscle-rotic Rabbit", J. Clin. Invest. (1988), 81:1752758.Lankin, "Atherosclerosis as a Free Radical Pathology", Inst.Congr. Ser. Excerpta Med. (1992), 998:385.Zembowicz, "The Biological Role of LArginine/NitricOxide Pathway", Folia Med. Cracov. (1992), 33:103116.


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5,891,459ENHANCEMENT OF VASCULAR FUNCTIONBYMODULATION OF ENDOGENOUSNITRIC OXIDE PRODUCTION ORACTIVITY

CROSS-REFERENCE TO RELATEDAPPLICATIONSThis application is a continuation-in-part of applicationSer. No. 08/336, 159, filed Nov. 8, 1994, now abandoned,which is a continuation-in-part of Ser. No. 08/076, 312, filedJun. 11, 1993, now U.S. Pat. No. 5,428,070.

INTRODUCTIONThis invention was supported in part by the United StatesGovernment under Grant 1KO7HCO2660 (NHLBI). TheU.S. Government may have an interest in this application.1. Technical FieldThe field of this invention is the modulation of NO

activity, which finds application in maintaining and improv-ing vascular function and thereby preventing or improvingvascular degenerative diseases.2. BackgroundAtherosclerosis and vascular thrombosis are a major

cause of morbidity and mortality, leading to coronary arterydisease, myocardial infarction, and stroke. Atherosclerosisbegins with an alteration in the endothelium, which lines theblood vessels. The endothelial alteration results in adherenceof monocytes, which penetrate the endothelial lining andtake up residence in the subintimal space between theendothelium and the vascular smooth muscle of the bloodvessels. The monocytes absorb increasing amounts of cho-lesterol (largely in the form of oxidized or modified low-density lipoprotein) to form foam cells. Oxidized low-density lipoprotein (LDL) cholesterol alters theendothelium, and the underlying foam cells distort andeventually may even rupture through the endothelium.Platelets adhere to the area of endothelial disruption and

release a number of growth factors, including plateletderived growth factor (PDGF). PDGF, which is also releasedby foam cells and altered endothelial cells, stimulates migra-tion and proliferation of vascular smooth muscle cells intothe lesion. These smooth muscle cells release extracellularmatrix (collagen and elastin) and the lesion continues toexpand. Macrophages in the lesion elaborate proteases, andthe resulting cell damage creates a necrotic core filled withcellular debris and lipid. The lesion is then referred to as a"complex lesion. "Rupture of this lesion can lead to throm-bosis and occlusion of the blood vessel. In the case of acoronary artery, rupture of a complex lesion may precipitatea myocardial infarction, whereas in the case of a carotidartery, stroke may ensue.One of the treatments that cardiologists and other inter-

ventionalists employ to reopen a blood vessel which isnarrowed by plaque is balloon angioplasty (approximately300,000 coronary and 100,000 peripheral angioplasties areperformed annually). Although balloon angioplasty is suc-cessful in a high percentage of the cases in opening thevessel, it unfortunately denudes the endothelium and injuresthe vessel in the process. This damage causes the migrationand proliferation of vascular smooth muscle cells of theblood vessel into the area of injury to form a lesion, known

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In atherosclerosis, thrombosis and restenosis there is alsoa loss of normal vascular function, such that vessels tend toconstrict, rather than dilate. The excessive vasoconstrictionof the vessel causes further narrowing of the vessel lumen,limiting blood flow. This can cause symptoms such asangina (if a heart artery is involved), or transient cerebralischemia (i.e. a "small stroke", if a brain vessel is involved).This abnormal vascular function (excessive vasoconstrictionor inadequate vasodilation) occurs in other disease states aswell. Hypertension (high blood pressure) is caused by exces-sive vasoconstriction, as well as thickening, of the vesselwall, particularly in the smaller vessels of the circulation.This process may alfect the lung vessels as well causingpulmonary (lung) hypertension. Other disorders known to beassociated with excessive vasoconstriction, or inadequatevasodilation include transplant atherosclerosis, congestiveheart failure, toxemia of pregnancy, Raynaud'sphenomenon, Prinzmetal's angina (coronary vasospasm),cerebral vasospasm, hemolytic-uremia and impotence.Because of their great prevalence and seriousconsequences, it is critically important to find therapies

which can diminish the incidence of atherosclerosis, vascu-lar thrombosis, restenosis, and these other disorders charac-terized by abnormality of vascular function and structure.Ideally, such therapies would inhibit the pathological vas-cular processes associated with these disorders, therebyproviding prophylaxis, retarding the progression of thedegenerative process, and restoring normal vasodilation.As briefly summarized above, these pathological pro-cesses are extremely complex, involving a variety of dilfer-

ent cells which undergo changes in their character,composition, and activity, as well as in the nature of thefactors which they secrete and the receptors that are up- ordown-regulated. A substance released by the endothelium,"endothelium derived relaxing factor" (EDRF), may play animportant role in inhibiting these pathologic processes.EDRF is now known to be nitric oxide (NO) or a labilenitroso compound which liberates NO. (For purposes of thesubject invention, unless otherwise indicated, nitric oxide(NO) shall intend nitric oxide or the labile nitroso compoundwhich liberates NO. ) This substance relaxes vascularsmooth muscle, inhibits platelet aggregation, inhibits mito-genesis and proliferation of cultured vascular smoothmuscle, and leukocyte adherence. Because NO is the mostpotent endogenous vasodilator, and because it is largelyresponsible for exercise-induced vasodilation in the conduitarteries, enhancement of NO synthesis could also improveexercise capacity in normal individuals and those withvascular disease. NO may have other elfects, either direct orindirect, on the various cells associated with vascular wallsand degenerative diseases of the vessel.Relevant LiteratureGirerd et al. (1990) Circulation Research 67:1301308report that intravenous administration of L-arginine poten-

tiates endothelium-dependent relaxation in the hind limb ofcholesterol-fed rabbits. The authors conclude that synthesisof EDRF can be increased by L-arginine in hypercholester-olemia. Rossitch et al. (1991)J. Clin. Invst. 87:1295299report that in vitro administration of L-arginine to basilararteries of hypercholesterolemic rabbits reverses the impair-ment of endothelium-dependent vasodilation and reducesvasoconstriction. They conclude that the abnormal vascularresponses in hypercholesterolemic animals is due to a


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5,891,459endothelium-derived NO-dependent vasodilation in hyper-cholesterolemic patients.Cooke et al. , "Endothelial Dysfunction in Hypercholes-terolemia is Corrected by L-arginine, " Endothelial Mecha-nisms ofVasomotor Control, eds. Drexler, Zeiher, Bassenge,

and Just; Steinkopff Verlag Darmstadt, 1991,pp. 173181,review the results of the earlier references and suggest, "Ifthe result of these investigations may be extrapolated, exog-enous administration of L-arginine (i.e., in the form ofdietary supplements) might represent a therapeutic adjunctin the treatment and/or prevention of atherosclerosis".Cooke (1990) Cuvvent Opinion in Cavdiology 5:63744discusses the role of the endothelium in the atherosclerosisand restenosis, and the elfect that these disorders have onendothelial function.Cooke (1992)J. Clin. Invest. 90:11681172, describe theelfect of chronic administration of oral L-arginine in hyper-

cholesterolemic animals on atherosclerosis. This is the firstdemonstration that oral L-arginine supplements can improvethe release of NO from the vessel wall. The increase in NOrelease from the vessel wall was associated with a strikingreduction in atherosclerosis in hypercholesterolemic ani-mals. This is the first evidence to support the hypothesis thatincreasing NO production by the vessel wall inhibits thedevelopment of atherosclerosis.Cooke and Tsao (1992) Cuvvent Opinion in Cavdiology7:79904 describe the mechanism of the progression ofatherosclerosis and suggest that inhibition of nitric oxide

may disturb vascular homeostasis and contribute to athero-genesis.Cooke and Santosa (1993) In: Steroid Hormones andDysfunctional Bleeding, AAAS Press, review the activitiesof EDRF in a variety of roles and suggest that reversibilityof endothelial dysfunction may be affected by the stage ofatherosclerosis. They conclude that EDRF is a potentvasodilator, plays a key role in modulating conduit andresistance vessel tone, has important elfects on cell growthand interactions of circulatory blood cells with a vessel wall,and that disturbances of EDRF activity may initiate orcontribute to septic shock, hypertension, vasospasm, tox-emia and atherosclerosis.Fitzpatrick et al. , American Journal of Physiology 265(Heart Circ. Physiol. 34):H774H778, 1993 report that wine

and other grape products may have endothelium-dependentvasorelaxing activity in vitro.Wang et al. (1994) J. Am. Cell. Cardiol. 23:452458,report that oral administration of arginine prevents athero-

sclerosis in the coronary arteries of hypercholesterolemicrabbits.Drexler et al. (1994) Circulation 89:16151623 describe

the elfect of intravenous arginine upon coronary vasculartone. This was the first evidence that intravenous argininecould restore normal NO-dependent vasodilation in thecoronary arteries of patients with cardiac transplants, Tsao etal. (1994) Circulation 89:2176182 demonstrates that oraladministration of arginine to hypercholesterolemic rabbitsenhances the release of nitric oxide by the vessel wall, andinhibits monocytes from sticking to the vessel.Tsao et al. (1994)J. Avtevioscl. Thvomb. 14:1529533reveals that oral arginine administration to hypercholester-olemic rabbits inhibits platelet aggregation (blood clotting).Platelet aggregation plays an important role in causingvascular thrombosis in vascular degenerative disorders.

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4enhances vasodilation and inhibits thickening of the vesselwall after balloon angioplasty.Noruse et al. (1994)Avteviosclev. Thvomb. 14:746752,

report that oral administration of an antagonist of NOproduction accelerates atherogenesis in hypercholester-olemic rabbits.Cayette et al. (1994)Avteviosclev. Thvomb. 14:753759,also report that oral administration of an antagonist of NOproduction accelerates plaque development in hypercholes-terolemic rabbits.Other references which refer to activities attributed to NOor its precursor include: Pohl and Busse (1989) Circ. Res.65:17981803; Radomski et al. (1987) Br. J. Pharmacol.92:181187; Stamler et al. (1989) Circ. Res. 65:789795;

anti-platelet activity); Garg and Hassid (1989) J. Clin.Invest. 83:1774-1777;Weidinger et al. (1990) Circulation81:16671679; NO activity in relation to vascular smoothmuscle growth); Ross (1986)N. Engl. J.Med. 314:488500;Bath et al. (1991)Avteviosclev. Thvomb. 11:254260; Kubeset al. (1991)Proc. Natl. Acad. Sci. USA 89:63486352;Lefer et al. (1990) In: Endothelium-Derived ContractingFactors. Basel, S. Karger, pp. 190197; NO activity in rela-tion to leukocyte adhesion and migration); Heistad et al.(1984)Circ. Res. 43:711718;Rossitch et al. (1991)J. Clin.Invest. 87:12951299; Yamamoto et al. (1988) ibid81:17521758;Andrews et al. (1987)Nature 327:237239;Tomita et al. (1990) Circ. Res. 66:1827; Kugiyama et al.(1990)Nature 344:160162; Mitchell et al. (1992)J. Vase.Res. 29:169 (abst.); Minor et al. (1990) J. Clin. Invest.86:21092116; NO activity in relation tohypercholesterolemia); Tanner et al. (1991) Circulation83:20122020; Kuo et al. (1992) Circ. Res. 70:f465476;Drexler et al. (1991)Lancet 338:15461550; Schuschke etal. (1994) Int. J. of Micvocivcu: Clin. and Expev. 14(4):20411; Yao et al. (1992) Circulation 86:13021309;Higashi et al. (1995) Hypertension 25(4 Pt 2):898902;Kharitonov et al. (1995)Clin. Sci. 88(2):135139;Smulderset al. (1994)Cli n. Sci.87(1):3743; Bode-boger et al. (1994)Clin. Sci. 87(3):303310; Bode-Boger et al. (1994) Clin.Sci.; Randall et al. (1994) Clin. Sci. 87(1):5359; Dubois-Rande et al. (1992)J. Card. Phavm. 20 Suppl. 12:S2113;Otsuji et al. (1995)Am. Heart J.129(6):1094100;Nakan-ishi et al. (1992)Am. J. of Physio. 263(6 Pt 2):H16508;Kuo et al. (1992) Civc. Reseavch 70(3): 465476; Tanner et

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:983943; Davies et al. (1994)Surgery 116(3):557568; andRaij (1994)Kidney Institute 45:77581.

SUMMARY OF THE INVENTIONMethods are provided for improving vascular function

and structure, particularly modulating vascular relaxation,cellular adhesion, infiltration and proliferation by modulat-ing the level of nitric oxide or active precursor at a physi-ological site. The methods find use in preventing the deg-radation of vascular function, particularly as involved withthe occurrence of atherosclerosis, restenosis, thrombosis,hypertension, impotence, and other disorders characterizedby reduced or inadequate vasodilation. The enhancement ofendogenous nitric oxide or secondary messenger availability

45 al. (1991)Circulation 83(6):20122020; Meng et al. (1995)J. Am. Col. Card. 25(1):269275; Lefer and Ma (1993)Avtevioscl. and Thvomb. 13(6):771776; McNamara et al.(1993)Biochem. and Biophys. Res. Comm. 193(1):291296;Tarry and Makhoul (1994) Avtev. and Thvomb. 14(6)


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oxide also inhibits initiation and the progression ofatherosclerosis, restenosis, vascular hypertrophy or hyper-plasia and thrombosis. This is due to the fact that nitric oxideis not only a potent modulator, but can also inhibit plateletsand white blood cells from adhering to the vessel wall. Asa prophylaxis or treatment for vascular functiondeterioration, particularly in atherosclerotic susceptiblehosts, the agent is chronically administered at an elfectivedosage. For restenosis, the agent may be administered for alimited period since this pathological process generallyabates 3 months after the vascular injury (i.e. angioplastyor atherectomy). Oral administration of L-arginine, precur-sors to L-arginine, e.g. oligopeptides or polypeptides com-prising L-arginine, or proteins comprising high levels ofL-arginine, by i tself or in combination with L-lysine, par-ticularly further supplemented with GRAS substances whichenhance the elfectiveness of the active agents, as a dietarysupplement will increase NO elaboration and thereby dimin-ish the elfects of atherogenesis. Other techniques to enhanceNO or secondary messenger availability may be utilizedsuch as increasing the availability of NO synthase, forexample, as a result of enhanced expression of NO synthasein the vessel wall, particularly at the lesion site, release ofNO from the vessel wall or reduction of degradation of NOor the secondary messenger, cyclic guanosine monophos-phate ("cGMP").

BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 is a bar diagram of histomorphometric studies of

the elfect of L-arginine on atherosclerotic plaque in hyper-cholesterolemic animals. (See Ex. I)FIGS. 2A, 2B, and 2C are nephelometric scans of theelfect of L-arginine diet supplement on platelet reactivity asevidenced by platelet aggregation initiated by adenosinediphosphate. (See Ex. 2) A) aggregation of platelets fromhypercholesterolemic rabbit; B) reduced aggregation ofplatelets from hypercholesterolemic rabbit treated withL-arginine; C) antagonism of NO synthase by LNMMAreverses the beneficial elfect of L-arginine.FIG. 3 is a bar diagram comparing the elfect of L-arginine

diet supplement on cell binding to aortic endothelium ofhypercholesterolemic animals. (See Ex. 4)FIG. 4. Lesion surface area of thoracic aortae from allarginine treated hypercholesterolemic animals (ARG, weeks1423) is reduced in comparison to that of hypercholester-olemic animals receiving vehicle (CHOL, weeks 143).(See Ex. 5)FIG. 5. Macrophage accumulation in iliac arteries 4weeks following balloon injury. (Macrophage infiltrationinto the vessel wall initiates and accelerates plaqueformation). Data is expressed as a percent of the vessel thatcontain macrophages. Balloon injury in hypercholester-olemic rabbits (CHOL) results in a marked increase inarterial macrophage accumulation compared with injurediliac arteries from rabbits on normal chow (CONT). Mac-rophage accumulation in iliac arteries from hypercholester-olemic rabbits receiving L-arginine (ARG) is significantlyreduced compared to the CHOL group. (*;p&0.01,ARG v.CHOL). This study revealed that oral arginine treatmentmarkedly reduced the infiltration of monocytes/macrophages into the vessel wall, explaining in part theelfect of arginine to inhibit plaque formation. (See Ex. 6)

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Exposure of human aortic endothelial cells to oxLDLincreased the ex vivo binding ofmonocytes when comparedto Control. In comparison to cells not exposed to flow(static), previous exposure to flow inhibited the monocyteadhesion induced by oxLDL. These elfects of flow wereblocked by NO synthase inhibitors and mimicked by NOdonors (PAPA-NO) or cyclic GMP (cGMP). Bars representmean ~SEM. *p&0.05; **p&0.01. (See Ex. S)FIG. 7 is a bar diagram of morphometric measurements ofintimal lesion thickening two weeks after a balloon angio-plasty in animals treated with a plasmid construct containingthe gene for NO synthase (INJ+NOS) in comparison tocontrol vector (INJ+CV) or untreated injured vessels (INJ).(See Ex. 11)FIG. II is a histogram showing the elfect of local intralu-minal administration of arginine on restenosis. Hypercho-lesterolemic rabbits had balloon angioplasty of the iliacartery. Immediately thereafter some animals received aninfusion of arginine directly into the vessel by means of acatheter designed to apply high local concentrations ofarginine to the vessel. Two to four weeks later, vessels wereremoved from the animals, and examined microscopically.Thickening of the vessel wall (internal thickening or"restenosis") was reduced in the animals treated withintraluminal infusion of arginine (ARG) in comparison tothose treated with vehicle. (See Ex. 12)FIG. 9 is a set of dose-response curves showing the elfectof chronic lysine administration on endothelium dependentvasodilation in hypercholesterolemic rabbits. Chronic oraladministration of lysine (for ten weeks) improvedNO-mediated vasodilation; this improvement in NO activitywas also associated with a marked reduction in plaque area.Chronic administration of lysine was just as elfective asarginine in restoring vascular function and structure. (SeeEx. 14)FIG. 10 is a scatter-diagram illustrating the relationshipbetween the level of blood LDL-cholesterol and monocytebinding. Monocytes were isolated from the blood of humanswith normal or elevated cholesterol levels. The binding ofthese monocytes to endothelial cells in culture wasobserved. Monocytes from individuals with high cholesterollevels have a greater adhesiveness for endothelial cells. Thismonocyte-endothelial cell interaction in vivo is the first stepin the development of atherosclerotic plaque. (See Ex. 15)FIG. 11 is a bar diagram showing the adhesiveness ofmonocytes obtained from subjects with normal cholesterollevels (CONT) and those from hypercholesterolemic (HC)humans, before, during, and after treatment with arginine(the NO precursor). Prior to initiating arginine (Arg) orplacebo (plac) treatment, monocytes from hypercholester-olemic individuals have a greater tendency to bind toendothelial cells ex vivo (baseline). After 2 weeks of argi-nine treatment monocytes from these hypercholesterolemicindividuals have a significantly reduced adhesiveness andare no dilferent from those of the normal subjects. At thispoint arginine therapy was discontinued and there was awashout (4 weeks). At this time point, monocytes from thepatients previously treated with arginine now have increasedadhesiveness, olf of the arginine treatment. (See Ex. 15)FIG. 12 is a bar diagram which shows that monocytesfrom individuals with elevated cholesterol (CHOL) havegreater adhesiveness for endothelial cells. However, aftertreatment with sodium nitroprusside (CHOL +SNP), the


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5,891,459individuals with normal cholesterol levels (nc). Plateletaggregation ex vivo in response to adenosine diphosphate(ADP) is increased in hc individuals in comparison tonormal individuals. After 2 weeks of treatment with oralL-arginine, platelet aggregation is attenuated in the hyper-cholesterolemic individuals, while an even greater elfect ofthe treatment is seen at four weeks. (See Ex. 16)FIG. 14 is a bar graph showing increases in coronaryblood flow in response to intracoronary infusions of acetyl-choline (ACH) before and after intravenous infusion ofL-arginine (30 g), in patients with transplant atherosclerosis.Acetylcholine stimulates the release of NO from the vesselwall causing vasodilation and increased blood flow. There isimproved NO-dependent vasodilation after L-arginineadministration. (See Ex. 18)

DESCRIPTION OF SPECIFIC EMBODIMENTSIn accordance with the subject invention, vascular func-

tion is maintained or its deterioration inhibited or retardedby enhancing the level or activity of endogenous nitricoxide. By enhancing the level or activity of endogenousnitric oxide, common vascular degenerative diseases such asatherosclerosis, restenosis, hypertension, vasospasm,impotence, angina, and vascular thrombosis, can be treatedprophylactically and/or therapeutically. The enhanced levelor activity of nitric oxide (which is intended to include anyprecursor of nitric oxide which results in such enhancedlevel) can be achieved by modulating the activity, synthesisor concentration of any of the components associated withthe formation of nitric oxide in the nitric oxide syntheticpathway, or inhibiting the rate of degradation of nitric oxide,its precursors, or the secondary messengers associated withthe relaxation signal. In referring to the enhanced level oractivity, the term "elfect" will be used to encompass the twosituations. The enhanced elfect of nitric oxide may be aresult of oral or intravenous administration to the patient ofa precursor in the metabolic pathway to the production ofnitric oxide (such as L-arginine, L-lysine, polypeptidescomprising these amino acids, and the like), providing anenzyme in the metabolic pathway to NO, particularly NOsynthase, by introduction of the gene for NO synthase underconditions for integration of the gene into the endothelial orother cells and expression of the gene, or by directly addingan enzyme associated with the production of nitric oxide.The enhanced level of nitric oxide may also result fromadministration of an agent to protect the NO fromdegradation, such as an oxidant, reductant or superoxidedismutase. Alternatively, the agent may serve to enhance thebioavailability or elfectiveness of the primary active agent,such as L-arginine or L-lysine. The agent, individually or incombination, will be administered in a form of other than anatural food source, such as meat or plants as natural proteinsources, fruits or products derived therefrom.One approach is to employ L-arginine and/or L-lysine, as

individual amino acids, in combination, or as a precursor toL-arginine, e. g. a monomer or a polypeptide, as a dietarysupplement. The amino acid(s) are administered as anyphysiologically acceptable salt, such as the hydrochloridesalt, glutamate salt, etc. They can also be administered as apeptide (e.g., poly-L-arginine, poly-L-lysine, or combina-tions thereof) so as to increase plasma levels of the NOprecursor. Oligopeptides of particular interest include oli-gopeptides of from 2 to 30, usually 2 to 20, preferably 2 to

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tides can be modified by being ligated to other compounds,which can enhance absorption from the gut, provide forenhancement of NO synthesis or stability, e.g. reducingagents and antioxidants, and the like.Naturally occurring sources include protamine or other

naturally occurring L-arginine or -lysine containing protein,which is high in one or both of the indicated amino acids,e.g. greater than about 40%, preferably greater than about50%.The administration of L-arginine, other convenient NO

precursor, or other agent which enhances NO availability,would be in accordance with a predetermined regimen,which would be at least once weekly and over an extendedperiod of time, generally at least one month, more usually atleast three months, and as a chronic treatment, could last forone year or more, including the life of the host. The dosageadministered will depend upon the frequency of theadministration, the blood level desired, other concurrenttherapeutic treatments, the severity of the condition, whetherthe treatment is for prophylaxis or therapy, the age of thepatient, the natural level of NO in the patient, and the like.Desirably, the amount of L-arginine and/or L-lysine (Rand/or K) or biologically equivalent compound which isused would generally provide a plasma level in the range ofabout 0.15 to 30 mM. The oral administration of R and/or Kcan be achieved by providing R and/or K, other NOprecursor, or NO enhancing agent as a pill, powder, capsule,liquid solution or dispersion, particularly aqueous, or thelike. Various carriers and excipients may find use in formu-lating the NO precursor, such as lactose, terra alba, sucrose,gelatin, aqueous media, physiologically acceptable oils, e.g.peanut oil, and the like. Usually, if daily, the administrationof L-arginine and/or L-lysine for a human host will be about1 to 12 g per day.Furthermore, other agents can be added to the oral for-mulation of the amino acids or polypeptides to enhance theirabsorption, and/or to enhance the activity of NO synthase,e.g. B6 (5050 mg/d), folate (0.410 mg per daily dose),Byp (0.5 mg/d) or calcium (250000mg per daily dose).Furthermore, agents known to protect NO from degradation,such as antioxidants (e.g. cysteine or N-acetyl cysteine2001000 mg/d Vitamin C (250000 mg daily dose),(coenzyme Q 250 mg daily dose, glutathione 5050 mgdaily dose), Vitamin E (2001000 I.U. daily dose), orJI-carotene (1025,000 I.U. daily dose) or other naturallyoccurring plant antioxidants such as tocopherols, phenoliccompounds, thiols, and ubiquinones can be added to the oralor intravenous formulations of R and/or K, or R and/orK-containing peptides. Alternatively, one may include theactive agent in a nutritional supplement, where other addi-tives may include vitamins, amino acids, or the like, wherethe subject active agent will be at least 10weight %, moreusually at least about 25 weight % of the active ingredients.The administration of R and/or K or its physiologic

equivalent in supporting NO can be administered prophy-lactically to improve vascular function, serving to enhancevasodilation and to inhibit atherogenesis or restenosis, ortherapeutically after atherogenesis has been initiated. Thus,for example, a patient who is to undergo balloon angioplastycan have a regimen of R and/or K administered substantiallyprior to the balloon angioplasty, preferably at least about aweek or substantially longer. Alternatively, in a patient, theadministration of R and/or K can begin at any time.


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5,891,459 10particular interest is the incorporation of R and/or K as asupplement in a food, such as a health bar, e.g. granola, othergrains, fruit bars, such as a date bar, fig bar, apricot bar, orthe like. The amount of R and/or K or the equivalent wouldbe about 25 g per dosage or bar, preferably about 315 g.Instead of oral administration, intravascular administra-tion can also be employed, particularly where more rapidenhancement of the nitric oxide level in the vascular system

is desired (i.e. as with acute thrombosis of a critical vessel),so that combinations of oral and parenteral administrationscan be employed in accordance with the needs of the patient.Furthermore, parenteral administration can allow for theadministration of compounds which would not readily betransported across the mucosa from the gastrointestinal tractinto the vascular system.Another approach is to administer the active ingredient of

grape skin extract, which is known to enhance NO activity.See Fitzpatrick et al. (1993), supra. The extract can beenriched for the active component by employing separationtechniques and assaying the activity of each of the fractionsobtained. The grape skin extract can be divided into frac-tions using a first gel permeation separation to divide theextract by the size of the components. The active fraction(s)can be determined by an appropriate assay, see the experi-mental section.The active fraction(s) can be further separated usingHPLC and an appropriate eluent, conveniently either anisocratic eluent of aqueous acetonitrile or propanol or a

linearly varying eluent, using the same solvents. Fractionswhich are shown to be active and substantially pure, e.g. atleast SO weight %, by thin layer chromatography, massspectrometry, gas phase chromatography, or the like canthen be characterized using infra-red, nuclear magneticresonance, mass or other spectroscopy.For oral or intravascular administration, one can provideR and/or K, by itself or in a polypeptide, or its physiologicalequivalent in supporting NO, together with antioxidants orscavengers of oxygen-derived free radicals (such as sulfhy-

dryl containing compounds) or compounds that prevent theproduction of oxygen-derived free radicals (such as super-oxide dismutase), as it is known that oxygen-derived freeradicals (such as superoxide anion) can inactivate nitricoxide. Alternatively, or in addition, one can administercofactors required for NO synthase activity, such as calciumor folate. The amounts of each of these co-agents can bedetermined empirically, using the assays in the experimentalsection to determine NO activity.The various cofactors that may be used with the NOprecursors will vary in amount in relation to the amount ofNO precursor and the elfectiveness of the cofactor, particu-larly for oral administration. Generally, the cofactors may bepresent in amounts that would provide daily doses of folate(0.4-10 mg), B6 (5050 mg), B (0.5 mg) and/orcalcium (2501000 mg). Usually, where the amount of theNO precursor is greater than about 2 g, it may be adminis-tered periodically during the day, being administered 2 to 4times daily. For the most part, the cofactors will be GRASsubstances and will be able to be taken at high dosageswithout adverse elfects on the recipient host.The subject compositions will be for the most part admin-

istered orally and the dosage may take a variety of forms.The dosage may be tablets, pill, capsules, powders,

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upon the mode of administration, the amount of active agentmay be up to about 25 g. For formulations as dietarysupplements, individual dosages will generally range fromabout 0.5 to 5 g, more usually from about 1 to 3 g of the NOprecursor.Alternatively, one can enhance, either in conjunction withthe enhancement of precursors to nitric oxides or

independently, components of the nitric oxide metabolicpathway. For example, one can enhance the amount of nitricoxide synthase present in the vessel wall, particularly at thesite of lesions. This can be done by local administration tothe lesion site or systemically into the vascular system. Thesynthase can be administered using liposomes, slow releaseparticles, or in the form of a depot, e.g. in collagen,hyaluronic acid, biocompatible gels, vascular stents, or othermeans, which will provide the desired concentration of theNO synthase at the lesion site.Instead of providing for the enhancement of NO at the

physiological site of interest, one can choose to extend thelifetime of the signal transduced as a result of the presenceof nitric oxide. Since cGMP is produced intracellularly as aresult of a nitric oxide induced signal, employing agentswhich result in the production of or extending the lifetime ofcGMP can be employed. Illustrative agents include cGMPphosphodiesterase inhibitors or agents which increase theamount of guanylate cyclase.Alternatively, cells can be genetically engineered to pro-

vide for constitutive or inducible expression of one or moregenes, which will provide for the desired relaxationresponse, by expressing NO synthase, or other enzyme orprotein which is secreted and acts extracellularly. Thus,expression vectors (viral or plasmid) can be prepared whichcontain the appropriate gene(s) and which can be introducedinto host cells which will then produce high concentrationsof nitric oxide or other intermediate in the relaxation path-way. These cells can be introduced at the lesion site or atanother site in the host, where the expression will induce theappropriate response as to relaxation, proliferation, etc. TheNO synthase or cells expressing the NO synthase can bepresent as depots by encapsulation and positioning at the siteof interest. For example, porous stents can be producedwhich encapsulate the enzyme or cells to protect the enzymefrom degradation or being washed away.Cultured cells can be transfected with expression vectors

containing the NO synthase or other gene ex-vivo and thenintroduced into the vessel wall, using various intra-arterialor intra-venous catheter delivery systems. Alternatively,techniques of in vivo gene transfer can be employed totransfect vascular cells within the intact vessel in vivo. Thegene(s) can be expressed at high constitutive levels or can belinked to an inducible promoter (which can have tissuespecificity) to allow for regulation of expression.DNA constructs are prepared, where the appropriate gene,e.g. a NO synthase gene, is joined to an appropriatepromoter, either with its native termination region or adilferent termination region, which can provide forenhanced stability of the messenger RNA. Constitutive

promoters of particular interest will come from viruses, suchas Simian virus, papilloma virus, adenovirus, HIV, Roussarcoma virus, cytomegalovirus or the like, where the pro-moters include promoters for early or late genes, or longterminal repeats. Endogenous promoters can include theJI-actin or promoters.


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capable of replication in a bacterial and/or eucaryotic host.Normally, the vector will include a marker, which allows forselection of cells carrying the vector, e.g. antibiotic resis-tance. The vector will normally also include an origin whichis functional in the host for replication. Other functionalelements can also be present in the vector.Once the vector has been prepared and replicated, it canthen be used for introduction into host cells. The plasmidvector construct can be further modified by being joined toviral elements which allow for ease of transfection, canprovide a marker for selection, e.g. antibiotic resistance, orother functional elements. Introduction of the plasmid vectorconstruct into the host cells can be achieved by calciumphosphate precipitated DNA, transfection, electroporation,fusion, lipofection, viral capsid-mediated transfer, or thelike. Alternatively, the expression construct within viralvectors can be introduced by standard infection techniques.For somatic cell gene therapy, autologous cells will gener-ally be employed, although in some instances allogeneiccells or recombinantly modified cells can be employed.Usually the cells employed for genetic modification will bemature endothelial or vascular smooth muscle cells.Occasionally, the cells employed for genetic modificationwill be progenitor cells, particularly early progenitor cells.For example, myoblasts can be employed for muscle cells orhematopoietic stem cells or high proliferative potential cellscan be employed for lymphoid and/or myelomonocyticcells.Depending upon the nature of the cells, they can be

injected into tissue of the same or dilferent cellular nature,they can be injected into the vascular system, where theymay remain as mobile cells or home to a particular site (i.e.the lesion). For the NO synthase gene, the number of cellswhich are administered will depend upon the nature of thecells, the level of production of the NO synthase, the desiredlevel of NO synthase in the host vascular system, at thelesion site, or the like, whether the enhanced level of NOsynthase is the only treatment or is used in conjunction withother components of the nitric oxide synthetic pathway, andthe like. Therefore, the particular number of cells to beemployed will be determined empirically in accordance withthe requirements of the particular patient.These cells can also be introduced into the circulation byfirst growing them on the surface of standard vascular graft

material (i.e. DACRON or polytetrafluoroethylene grafts)or other synthetic vascular conduits or vascular bioprosthe-ses.Alternatively, one can use viral vectors, which are capableof infecting cells in vivo, such as adenovirus or retroviruses.

In this case, the viral vector containing the NO synthasegene or other gene involved with the relaxation pathway willbe administered directly to the site of interest, where it willenter into a number of cells and become integrated into thecell genome. Thus, one can titer the desired level of nitricoxide synthase which is secreted or other protein which isexpressed, by providing for one or more administrations ofthe virus, thus incrementally increasing the amount of syn-thase which is secreted or other protein which is produced.Alternatively, one can use modified liposomes as a vehicle

for endovascular administration of the vector containing theNO synthase or other gene. One such modified liposometechnique involves mixing the liposomes with the vectorcontaining NO synthase. Once the gene expression

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12In some situations, the NO synthase or other gene in therelaxation pathway can be co-transfected with an artificial

gene encoding an arginine and/or lysine rich polypeptidesusceptible to proteolytic cleavage as an intracellular sourceof L-arginine and/or L-lysine. In other situations, the NOsynthase or other gene can be co-transfected with thesuperoxide dismutase gene, so as to inhibit the degradationof the nitric oxide.In some situations, acute treatment may be involved,

requiring one or a few administrations. This will normally beassociated with compounds which can act as nitric oxideprecursors and are other than naturally occurring compoundsor are compounds which can be added with naturally occur-ring compounds to enhance the rate of formation of nitricoxide. Thus, one can provide for acute administration ofL-arginine and/or L-lysine and superoxide dismutase toincrease the nitric oxide concentration over a restrictedperiod of time. These administrations can be independent ofor in conjunction with long term regimens.The following examples are offered by way of illustration

and not by way of limitation.EXPERIMENTALEXAMPLE 1

Anti-atherogenic elfects of oral arginineStudy design(See, Cooke et al. , 1992, supra) Male New Zealand whiterabbits (n=49) were assigned to one of three treatmentgroups: 10were fed with normal rabbit chow for ten weeks(Control); 19 received chow enriched with I/o cholesterol(Chol); and 20 received a 1/o cholesterol diet supplementedwith 2.25/o L-arginine hydrochloride in the drinking water(Arg. ). Following ten weeks of the dietary intervention,animals were lightly sedated and the central ear arterycannulated for measurement of intra-arterial blood pressure,followed by collection of blood samples for serum chemis-tries and plasma arginine. Subsequently the animals weresacrificed and the left main coronary artery and the thoracicaorta were harvested for studies of vascular reactivity andhistomorphometry. In some animals, blood was collected forstudies of platelet and monocyte reactivity.ResultsBiochemical and physiological measurements. Hypercho-lesterolemic animals maintained on oral L-arginine supple-mentation (Arg) experienced a twofold elevation in plasmaarginine levels in comparison to animals on a normal(Control) or I /o cholesterol (Chol) diet alone; the elevationin plasma arginine was maintained throughout the course ofthe study. Serum cholesterol measurements were elevatedequally in both groups receiving the I/o cholesterol diet[50+6 vs. 1629+422 vs. 1852+356 mg/dl respectively forControl (=10), Chol (=13), and Arg (=14)].There were nosignificant dilferences in hemodynamic measurementsbetween groups.Organ chamber studies of isolated vesselsFor NO-independent responses, there were no differencesbetween the treatment groups in maximal response or sen-sitivity to norepinephrine (a vasoconstrictor), or to nitro-glycerin (a nitrovasodilator). By contrast, NO-dependentrelaxations were attenuated in vessels harvested from hyper-cholesterolemic animals with a reduction in the maximalresponse to acetylcholine. In comparison, vessels harvested


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13 5,891,459 14ation was confirmed in the hypercholesterolemic rabbitabdominal aorta.Histomorphometric studies (planimetry of EVG-stainedsections)A blinded histomorphometric analysis revealed that

medial cross-sectional areas of thoracic aortae were notdilferent between the groups. By contrast, the intimal cross-sectional area (i.e. amount of atherosclerotic plaque) ofvessels from hypercholesterolemic animals receivingL-arginine supplementation was reduced in comparison tothose from animals receiving cholesterol diet alone. In theArg animals the reduction in the intimal lesion was mostpronounced in the ascending thoracic aorta and left maincoronary artery. In the left main coronary artery of hyperc-holesterolemic animals receiving arginine, essentially noatherosclerotic plaque developed.Changes in lesion surface areaIn a second series of studies, the extent of the thoracic

aorta involved by lesions was examined. In hypercholester-olemic rabbits receiving vehicle (n=6) or L-arginine supple-ment (n=6), thoracic aortae (from left subclavian artery todiaphragm) were harvested after ten weeks of treatment,bisected longitudinally, and stained with oil-red O. Vesselswere photographed and vessel and lesion surface area deter-mined by planimetry. Approximately 40% of the total sur-face area was covered with plaque in thoracic aortae fromhypercholesterolemic animals receiving vehicle, whereas inthoracic aortae from arginine-treated hypercholesterolemicanimals, less than 10%of the surface area was covered withplaque (FIG. 1).To summarize, dietary arginine supplementation increases

plasma arginine levels, but does not alter serum cholesterol.This is associated with significant improvement inNO-dependent vasodilation as judged by bioassay. Finally,the improvement in NO-dependent vasodilation is associ-ated with reduction in thickness and area of the lesions invessels from hypercholesterolemic animals.

EXAMPLE 2Inhibition of platelet aggregation by oral L-arginineThe elfect of L-arginine supplementation on platelet reac-

tivity in rabbits that had normal chow (Control; n=6), a 1%cholesterol diet (Chol; n=5), or a 1%cholesterol diet supple-mented with oral arginine (Arg; n=6), as detailed above, wasexamined. Arterial blood obtained after central ear arterycannulation was anticoagulated with 13mM sodium citrate.Platelet-rich suspension was prepared by washing plateletsin calcium-free Krebs-Henseleit solution and resuspendingthem in Tyrode's solution with albumin. Aggregation wasinitiated by addition of adenosine diphosphate and moni-tored by standard nephelometric techniques. In plateletsderived from Chol animals, aggregation was not dilferent inrate or maximum extent in comparison to platelets fromControl animals (A, in FIG. 2). By contrast, aggregation ofplatelets from Arg animals was reduced by 50% (B, in FIG.2).This reduction in platelet aggregation was associated with

a two-fold greater cGMP content in aggregated plateletsfrom arginine-treated animals. The reduction of plateletreactivity could be reversed by administration ofN-methylarginine (10 M) in vitro (C, in FIG. 2).Therefore,

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EXAMPLE 3Inhibition of monocyte adherenceA. Functional Binding AssayTo determine if oral arginine supplementation alfectsmonocyte adherence, blood was collected from rabbits fednormal chow (=6) a 1% cholesterol diet (=6), or a 1%cholesterol diet supplemented with L-arginine (=6), asdescribed above. Mononuclear cells were purified fromblood by Ficoll-paque density gradient centrifugation. Inthese preliminary studies, adhesion was examined of bloodleukocytes to a transformed endothelial cell line, bEnd3(mouse brain-derived polyoma middle T antigen trans-formed endothelial cells). The Bend3 cells display themorphology of endothelial cells, and like human endothelialcells are capable of uptake of acetylated low-density lipo-protein and express adhesion molecules in a cytokine-regulatable fashion. Cultured cells were grown to confluencein 0.5 cm Lab-Tek chamber slides (MilesScientific) andtreated with control medium or with LPS (1 mg/ml) orTNFu (25 U/ml) for 18 hours. Cultures were washed withfresh assay bulfer, and low, medium, or high concentrationsof leukocytes (0.75, 1.5, or 3x10 cells/ml, respectively)were added per well. Following a 30-minute incubation ona rocking platform at room temperature to allow binding, theslides were inverted and immersed in bulfer containing 2%(v/v) glutaraldehyde, such that non-adherent cells were lostand adherent cells were fixed to the monolayer. The adherentmononuclear cells were enumerated using video-lightmicroscopy.Monocytes from hypercholesterolemic animals (Chol)exhibited greater adherence, consistent with observation byothers, that monocytes from hypercholesterolemic cats or

humans exhibit greater adherence to cultured endothelialcells. (deGruijter et al. (1991) Metabol. Clin. Exp.40:11191121; Fan et al. (1991) Irtrchows Arch. B CellPathol. 61:1927).In comparison to monocytes derived from vehicle-treatedhypercholesterolemic animals (Chol), those from arginine-treated hypercholesterolemic animals (Arg) were much lessadherent. This data shows that the arginine treatment inhibitsadhesion ofmonocytes to the endothelium, which is the firstobservable event in atherogenesis.

EXAMPLE 4Dietary L-Arginine Inhibits the Enhanced Endothelial-Monocyte Interaction In HypercholesterolemiaThe earliest observable abnormality of the vessel wall in

hypercholesterolemic animals is enhanced monocyte adher-ence to the endothelium, which occurs within one week ofa high cholesterol diet. This event is thought to be mediatedby the surface expression of endothelial adhesion moleculesand chemotactic proteins induced by hypercholesterolemia.Another endothelial alteration that occurs in parallel is areduced activity of nitric oxide (i.e., NO), derived frommetabolism of L-arginine. As shown above chronic dietarysupplementation with L-arginine restores NO-dependentvasodilatation in hypercholesterolemic rabbits, and that thisimprovement in NO activity is associated with a strikinganti-atherogenic elfect. In the following study was tested thehypothesis that the anti-atherogenic elfect of dietary argininewas mediated by endothelial derived NO which inhibitsmonocyte-endothelial cell interaction.MethodsAnimals.


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15mented with 2.25% L-arginine HCI in the drinking water(Arg, n=7) ad libitum throughout the course of the study. Ina second series of studies designed to further explore the roleof endogenous NO on monocyte-endothelial cell interaction,another group of animals were pair fed, receiving a normalrabbit diet supplemented with either vehicle control (N=5)or the NO synthase antagonist, nitro-L-arginine (L-NA, 10mg/100 ml; n=5), administered in the drinking water adlibitum throughout the course of the study (for an averagedaily dose of 13.5 mg/kg/day). In a third series of experi-ments animals received a normal diet and either vehicle(n=4), L-NA (13.5 mg/kg/day; n=4), or L-NA and hydrala-zine (n=4) added to the drinking water for two weeks. At thisdose, hydralazine (5 mg/kg/day) reversed the increase inblood pressure induced by L-NA. One day before sacrifice(after 2 weeks of dietary intervention), animals were lightlysedated and the central ear artery was cannulated for col-lection of blood samples.Mononuclear cell culture and isolation.Murine monocytoid cells, WEHI 78/24 cells were grownin Dulbecco's Modified Eagle's Medium supplemented 10%

fetal calf serum (vol/vol) and were kept in an atmosphere of5% CO~/95% air. Prior to binding studies, mononuclear cellswere fluorescently labeled with TRITC (3 pg/ml). To con-firm the results using WEHI cells, in some studies bindingstudies were performed in parallel using rabbit mononuclearcells. Mononuclear cells were isolated from fresh wholeblood of Control rabbits before sacrifice.Preparation of aortic endothelium and binding assay.After 2 weeks of the dietary intervention, the thoracic

aortae were removed and placed in cold, oxygenated saline.A 15mm segment of thoracic aorta was excised from a pointimmediately distal to the left subclavian artery to the mid-thoracic aorta. The segments were then carefully openedlongitudinally and placed into culture dishes containingHBSS medium. Aortic strips were fixed to the culture dishusing 25 gauge needles so as to expose the endothelialsurface to the medium. Culture dishes were then placed ona rocking platform at room temperature.After 10 minutes the HBSS medium was replaced bybinding medium containing WEHI cells. The aortic stripswere incubated with the mononuclear cells for 30 minutes.The medium was then replaced by fresh binding mediumwithout cells to remove non-adherent cells. The aorticsegments were then removed and placed on a glass slide, andadherent cells counted under epifluorescent microscopyfrom at least 30 sites on each segment.ResultsMonocyte adhesion to rabbit aortic endothelium.Exposure of WEHI 78/24 cells to normal rabbit aortic

endothelium results in a minimal cell binding in this ex vivoadhesion assay. However, when WEHI cells were incubatedwith aortic endothelium from hypercholesterolemic animals(Chol; n=7), cell binding was enhanced 3-fold in comparisonto Cont (n=7). The increased cell binding manifested byaortic endothelium of hypercholesterolemic animals wassignificantly attenuated by L-arginine supplementation(n=7). (FIG. 3) Similar results were achieved when adhesionassays were performed in parallel with mononuclear cellsthat were freshly isolated from Cont animals (n=2) in eachof the three groups.EIfect of chronic NO synthase inhibition on endothelialadhesiveness.

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16mented with vehicle (n=5) or the NO synthase inhibitor,L-NA (n=5). The adhesion of WEHI cells was markedlyincreased when incubated with aortic endothelium fromL-NA animals compared to control endothelium. This elfectcould not be attributed to hypertension caused by L-NAsince concomitant administration of hydralazine normalizedblood pressure but did not reverse the augmentation of cellbinding induced by L-NA.In a separate series of studies it was confirmed thatchronic administration of L-NA (the inhibitor of NOsynthase) significantly inhibited generation and release ofNO from the vessel wall (as measured bychemiluminescence), compared to vessels from animalstreated with vehicle or arginine.The salient findings of this investigation are: I) monocyte

binding to the endothelium ex vivo is increased in vesselsfrom hypercholesterolemic animals; 2) this increase inmonocyte binding is attenuated in hypercholesterolemicanimals treated chronically with the NO precursorL-arginine; 3) monocyte binding to the endothelium isincreased in vessels from normocholesterolemic animalstreated with the NO synthase antagonist L-nitro-arginine;and 4) this elfect of NO synthase antagonism was notreversed by administration of hydralazine in doses sulficientto normalize blood pressure. These findings are consistentwith the hypothesis that NO inhibits monocyte-endothelialcell interaction.To conclude, an ex vivo model of monocyte binding hasbeen used to study the increase in endothelial adhesiveness

induced by hypercholesterolemia. Endothelial adhesivenessis attenuated by oral administration of the NO precursorL-arginine is shown. Conversely, inhibition of NO synthaseactivity by oral administration of nitro-arginine strikinglyincreases endothelial alfinity for monocytes ex vivo. Thedata are consistent with NO being an endogenous anti-atherogenic molecule.

EXAMPLE 5Oral Arginine causes regression of atherosclerosis in hyper-cholesterolemic rabbitsOur previous work demonstrated that oral arginine could

prevent the development of plaque in hypercholesterolemicanimals but it was not known if pre-existing plaque could bealfected by arginine treatment. This is clinically important ifarginine is to be useful in the treatment of pre-existingatherosclerosis in humans. Accordingly, New Zealand whiterabbits (n=85) received normal chow or 0.5% cholesterolchow for 10 weeks. Subsequently, half of the hypercholes-terolemic rabbits were given 2.25% (W/V) L-arginine intheir drinking water. Thoracic aortae were harvested atweeks 10, 14, 18, or 23. Rings of aorta were used to assessNO-dependent vasodilation to acetylcholine (ACh). Maxi-mal relaxation to ACh in the hypercholesterolemic rabbitsreceiving vehicle (CHOL) became progressively attenuatedfrom 53.4% (at week 10) to 17.4% (by week 23). Planimetryof the luminal surface of the aortae from CHOL animalsrevealed a progressive increase in plaque area from 30.3%(at week 10) to 56.5% (by week 23) of the total surface ofthe thoracic aorta. By contrast, hypercholesterolemic ani-mals receiving arginine (ARG) manifested improvedendothelium-dependent relaxation associated with a reduc-tion of plaque area at 14 and 18 weeks. Lesion surface areain all arginine treated hypercholesterolemic animals (weeks143) was significantly reduced in comparison to vehicle-


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17oxide anion. By 23 weeks, 3 of 7 ARG animals hadpersistent improvement in NO-dependent vasodilation andexhibited a further reduction of plaque area to 5.4%Conclusionshypercholesterolemia induces a progressive loss of

NO-dependent vasodilation associated with progressive inti-mal lesion formation. Administration of L-arginine to ani-mals with pre-existing intimal lesions augments vascularNO elaboration, reduces superoxide anion generation, and isassociated with a reduction in plaque area. This is the firstdemonstration that restoration of NO activity can induceregression of pre-existing intimal lesions, and providesevidence that L-arginine therapy may be of potential clinicalbenefit.

EXAMPLE 6Oral arginine administration restores vascular NO activityand inhibits myointimal hyperplasia after balloon injury inhypercholesterolemic rabbits.Purpose.The purpose of this study was to determine if the alter-ations in vascular function and structure following balloon

angioplasty in hypercholesterolemic rabbits could be inhib-ited by restoration of endogenous nitric oxide (NO) activity.MethodsTwenty-eight New Zealand white rabbits were random-

ized into one of three dietary groups and received eithernormal rabbit chow, 0.5% cholesterol diet, or 0.5% choles-terol diet plus L-arginine hydrochloride (2.25%W/V) in thedrinking water. After six weeks of dietary intervention, theleft iliac artery of each animal was subjected to a balloonangioplasty. Four weeks later, the iliac arteries were har-vested for vascular reactivity studies and immunohis-tochemistry.ResultsThe bioassay studies indicated that endothelium-derived

NO activity was inhibited in hypercholesterolemic animalsin comparison to normocholesterolemic animals. Theadministration of arginine partially restored endothelium-derived NO activity. Balloon angioplasty induced intimalthickening which was largely composed of vascular smoothmuscle cells and extracellular matrix. In the setting ofhypercholesterolemia, vascular injury induced an exuberantmyointimal lesion that was augmented by the accumulationof lipid-laden macrophages. Administration of L-arginineinduced a quantitative as well as qualitative change in thelesion. Dietary arginine reduced intimal thickening in theinjured vessels of hypercholesterolemic animals, and sub-stantially inhibited the accumulation of macrophages in thelesion (FIG. 5).ConclusionsWe report that the lesions induced by balloon angioplasty

in hypercholesterolemic animals are markedly reduced byoral administration of arginine. Moreover, we find that thenature of the lesion is altered, with a striking reduction in thepercentage of macrophages comprising the lesion. Hyperc-holesterolemia induces an endothelial vasodilator dysfunc-tion in the rabbit iliac artery that is reversible by chronic oraladministration of arginine.

EXAMPLE 7Nitric oxide regulates monocyte chemotactic protein-1Our previous studies had established that oral arginineadministration could enhance vascular NO synthesis. This

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18"How does vascular nitric oxide inhibit monocyte adherenceand accumulation in the vessel wall?"Monocyte chemotactic protein-1 (MCP-I) is a 76-aminoacid chemokine thought to be the major chemotactic factorfor monocytes (chemotactic factors are proteins that attract

white blood cells). We hypothesized that the anti-atherogenic elfect of NO may be due in part to its inhibitionof MCP-1 expression.Methods and ResultsSmooth muscle cells (SMC) were isolated from normalrabbit aortae by explant method. Cells were then exposed tooxidized LDL (30pml) (which is known to induce vascularcells to synthesize MCP-I). The expression of MCP-1 inSMC was associated with an increased generation of super-oxide anion by the SMC, and increased activity of thetranscriptional protein NFKB.All of these elfects of oxidizedLDL cholesterol were reduced by previous exposure of theSMC to the NO-donor DETA-NONOate (100pM) (p&0.05).To determine if NO exerted its elfect at a transcriptionallevel, SMC and COS cells were transfected with a 400 bpfragment of the MCP-1 promoter. Enhanced promoter activ-ity by oxLDL was inhibited by DETA-NO.To investigate the role of endogenous NO in the regula-tion ofMCP-1 in vivo, NZW rabbits were fed normal chow,normal chow plus nitro-L-arginine (L-NA) (to inhibit vas-cular NO synthesis), high cholesterol diet (Chol), or highcholesterol diet supplemented with L-arginine (Arg) (toenhance NO synthesis). After two weeks, thoracic aortaewere harvested and total RNA was isolated. Northern analy-sis demonstrated increased expression of MCP-1 in Choland L-NA aortae; this expression was decreased in aortaefrom Arg animals. These studies indicate that the anti-atherogenic elfect of NO may be mediated in part by itsinhibition of MCP-1 expression. NO inhibits the generationof superoxide anion by the vascular cells and thereby turnsolf an oxidant-responsive transcriptional pathway (i.e.NFKB-mediated transcription) activating MCP-1 expres-sion.

EXAMPLE SNitric Oxide inhibits the expression of an endothelial adhe-sion molecule known to be involved in atherosclerosisVascular cell adhesion molecule (VCAM-I) is an endot-

helial adhesion molecule that binds monocytes. This mol-ecule is expressed by the endothelium of hypercholester-olemic animals, and is expressed by endothelial cellsoverlying plaque in animals and humans. This adhesionmolecule is believed to participate in monocyte adherenceand accumulation in the vessel wall during the developmentof plaque. Other workers have shown that the expression ofthis molecule is regulated by an oxidant-responsive tran-scriptional pathway mediated by the transcriptional factorNFKB. Endothelial cells exposed to oxidized LDL choles-terol (or cytokines like TNF-u) begin to generate superoxideanion. Superoxide anion turns on oxidant-responsive tran-scription leading to the expression of VCAM-1 and MCP-1(and probably other genes that participate inatherosclerosis). Our data indicates that NO inhibits thegeneration of superoxide anion, thereby turning off theseoxidant-responsive transcriptional pathways.Methods and ResultsConfluent monolayers of human aortic endothelial cells

(HAEC) were exposed to static or fluid flow conditions for4 hours (fluid flow stimulates the production of endogenous


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19 5,891,459 20THP-1 monocytes were then performed. Superoxide pro-duction by HAECs was monitored by lucigenin chemilumi-nescence and expression of the adhesion moleculesVCAM-1 and ICAM-1 was quantitated by flow cytometry.Whereas native LDL had little elfect, incubation with eitheroxLDL or LPS/TNF significantly increased superoxideproduction, NF-KB activity, VCAM-1 expression and endot-helial adhesiveness for monocytes. Previous exposure tofluid flow inhibited endothelial adhesiveness for monocytes(FIG. 6) and the other sequelae of exposure to cytokines oroxidized lipoprotein. The elfect of fluid flow was due toshear-induced release of nitric oxide since coincubation withL-nitro-arginine completely abolished these elfects of flow.Furthermore, the NO donor PAPA-NONOate mimicked theelfects of flow.ConclusionsPrevious exposure to fluid flow decreased cytokine orlipoprotein-stimulated endothelial cell superoxide

production, VCAM-1 expression and monocyte binding; theelfects of flow are due at least in part to nitric oxide.NO participates in the regulation of the endothelial gen-eration of superoxide anion and thereby inhibits oxidant-responsive transcription of genes (i.e.VCAM-1 and MCP-I)that are involved in atherogenesis.

EXAMPLE 9Transfection of the gene encoding NO synthase increasesNO generation and inhibits monocyte adherence.The following experiment was done to determine iftransfer of the gene encoding NO synthase (the enzyme thatproduces NO) could increase generation of nitric oxide andthereby inhibit monocyte adherence. Cultured endothelialcells (bEnd-3; a murine endothelial cell line) were trans-fected with a plasmid construct encoding the NO synthasegene, using lipofectamine liposomal technique. Forty-eighthours later, generation of nitric oxide was measured usingchemiluminescence. Nitric oxide generation was increased2-fold in cells transfected with the NO synthase construct(but not in cells transfected with a control construct). Inparallel, binding assays were performed using a murinemonocytoid cell line. The binding of monocytoid cells to theendothelial cells was reduced by 30% in those cells trans-fected with the NO synthase construct.Conclusionendothelial cells transfected with a plasmid construct

containing the NO synthase gene were able to elaboratemore nitric oxide. The increased elaboration of nitric oxidewas associated with an inhibition ofmonocyte binding to theendothelial cells.

EXAMPLE 10EIfect of NO synthase expression on proliferation of vas-cular smooth muscle cellsCultured rat aortic vascular smooth muscle cells under

confluent quiescent conditions were studied. An elficientviral coat protein-mediated DNA transfer method wasemployed to transfect the cells with the NO synthase genedriven by the JI-actin promoter and CMV enhancer. Thisresulted in increased NO synthase activity (as measured bythe arginine-to-citrulline conversion assay) in comparison tocontrol vector transfected cells. Transfection of the NOsynthase gene completely abolished serum-stimulated DNAsynthesis compared to control vector transfection. Theseresults indicated that increased expression of NO synthase

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EXAMPLE 11Gene Therapy Using NO Synthase cDNA Prevents Rest-enosisThe study above indicated that NO inhibits proliferationof vascular smooth muscle cells. In atherogenesis andrestenosis, excessive proliferation of vascular smoothmuscle cells contributes to lesion formation. Injury to theendothelium in atherosclerosis and after catheter interven-tions apparently reduces or removes the salutary influence ofNO. The following study shows delivery of the gene for NOsynthase to the vessel wall inhibits lesion formation.A plasmid construct encoding the cDNA of endothelial-type NO synthase (EC-NOS) was synthesized. A full lengthcDNA encoding for EC-NOS was inserted into the EcoRIsite of the pUCcaggs expression vector. Balloon angioplas-ties of the carotid artery in Sprague-Dawley rats wereperformed and HVJ-Iiposomes with plasmids encodingEC-NOS cDNA infused, or plasmids lacking EC-NOScDNA(control vector) infused. After 4 days to 2 weeks, therats were sacrificed and the carotid arteries harvested for: I)histomorphometry; 2) measurement of DNA synthesis; and3) ex vivo determination of NO synthesis and release bybioassay and by chemiluminescence.ResultsMorphometric measurements 2 weeks after injuryrevealed a significant (68%) reduction of intimal lesionthickness in EC-NOS treated (Inj+NOS) in comparison tocontrol vector treated (Inj+CV) or untreated (Inj) injuredvessels. (FIG. 7) Measurements of DNA synthesis wereperformed four days after injury using bromodeoxyuridine.EC-NOS transfection significantly limited bromodeoxyuri-dine incorporation (by 25%) in comparison to control vectortreated or untreated injured vessels. Vessel segments werestudied ex vivo using organ chamber technique to bioassayfor NO release. Calcium ionophore increases intracellularcalcium and activates NO synthase to produce NO. Calciumionophore induced relaxations in injured carotid arteriestransfected with control vector that were only 15% ofuninjured vessels. Injured arteries that had been transfectedwith EC-NOS relaxed to a much greater degree, approxi-mately 50% of that observed in uninjured vessels. Directmeasurement of NO (by chemiluminescence) released intothe medium revealed that NO released by injured tissues(transfected with the control vector) was only 20% of thatreleased by normal uninjured tissues. By contrast, injuredtissues transfected with EC-NOS released more NO (about75% of normal).To conclude, balloon angioplasty of the rat carotid arteryremoves the endothelial source of NO, induces excessivevascular smooth muscle DNA synthesis and proliferation,

resulting in an intimal lesion (restenosis). Transfection of thevessel with EC-NOS at the time of balloon injury partiallyrestores NO production by the vessel, and this is associatedwith reduced DNA synthesis and vascular smooth muscleproliferation, thereby reducing lesion formation. Theseresults are consistent with the conclusion that NO is anendogenous anti-atherogenic molecule.

EXAMPLE 12Local application of L-arginine to the vessel wall inhibitsmyointimal hyperplasiaThe previous studies revealed that oral administration ofarginine could enhance vascular NO activity and inhibitlesion formation induced by a high cholesterol diet and/or


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21 5,891,459 22a 1% cholesterol diet. After one week, angioplasty of theiliac arteries was performed. After angioplasty of one iliacartery, a local infusion catheter was used to expose theinjured area to a high concentration of arginine (6 mM). Theother iliac artery was subjected to balloon angioplasty, butnot treated with a local infusion. After four weeks, thevessels were harvested, and segments of the arteries pro-cessed for histomorphometry. Initial thickening in thearginine-treated vessels was significantly reduced (FIG. 8).This study indicates that the local intraluminal application ofhigh doses of arginine can reduce myointimal hyperplasiaafter vascular injury.

EXAMPLE 13Exclusion of the EIfect of Enhanced Nitrogen or CaloricBalance as Causing the Observed ResultsTo exclude an elfect of L-arginine on nitrogen or caloricbalance as the cause of these results, six animals received1% cholesterol diet supplemented by additional methionine

to increase the dietary methionine six-fold. At ten weeksanimals were sacrificed for studies of platelet and vascularreactivity, and histomorphometry. Endothelium-dependentrelaxation, platelet aggregation and intimal thickness werenot dilferent from those of animals fed 1% cholesterol dietalone. These results reveal that another amino acid, methion-ine (which is not a precursor of NO) does not mimic theelfect of the amino acid L-arginine. Therefore it seems likelythat the elfect of L-arginine is due to its metabolism to nitricoxide, rather than some other elfect of amino acid admin-istration (i.e. change in nitrogen or caloric balance).

EXAMPLE 14L-lysine enhances vascular NO activity and inhibits

athero genesisL-lysine is a basic amino acid like L-arginine, but is not

known to be metabolized by NO synthase to NO. Therefore,the following results were unexpected. New Zealand whiterabbits were fed a normal or high cholesterol chow (n=18).Half of the animals on the cholesterol diet also received oralL-lysine. After ten weeks, the thoracic aortae were harvestedand bioassayed for vascular NO synthesis, and histomor-phometry to assess lesion formation was performed asdescribed above. The administration of L-lysine was just aselfective as L-arginine to increase vascular NO activity inthe hypercholesterolemic animals as assessed byendothelium-dependent vasorelaxation. (FIG. 9) Theimprovement in vascular NO activity was associated with amarked reduction in vascular lesion formation.This study revealed the unexpected result that L-lysine

can enhance vascular NO activity and inhibit atherosclero-S1S.

EXAMPLE 15Oral L-arginine normalizes monocyte adhesiveness inhypercholesterolemic humansAdherence of monocytes to the endothelium is the first

observable event in the development of atherosclerosis. Wehypothesized that chronic oral administration of L-arginineto hypercholesterolemic humans would enhance the genera-tion of endothelium-derived NO, and thereby inhibit theinteraction of monocytes with the endothelium. In thisinvestigation we have developed a reproducible assay for thebinding of human monocytes to cultured endothelial cells,

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average age of 37~2 yrs. Normalcy was determined by acareful history, physical examination, and laboratory analy-sis to exclude individuals with hematologic, renal, or hepaticdysfunction or clinically evident atherosclerosis. There were20 patients (10males and 10 females) with hypercholester-olemia as defined by a total plasma cholesterol greater than240 mg/dl and a LDL cholesterol level greater than 160mg/dl. These individuals had an average age of 51~2 yrs.None of the subjects were taking diuretics, vasoactivemedications, antiplatelet or hypolipidemic medications. Thisstudy was approved by the Stanford University Administra-tive Panel on Human Subjects in Medical Research and eachsubject gave written informed consent before entry into thestudy. Blood was drawn from each subject in the postab-sorptive state.We isolated human monocytes from citrated venousblood. The blood was centrifuged and the bulfy coat

removed and resuspended with HBSS. The suspension wasthen carefully layered onto a cushion of 1.068-d Histopaque,and centrifuged. After centrifugation, the monocytes wereaspirated.We used the transformed endothelial cell (EC) line, bEnd3to examine monocyte-endothelial binding ex vivo. The

bEnd3 cells express endothelial adhesion molecules andbind monocytes in a cytokine-inducible fashion with kinet-ics similar to those observed with human umbilical veinendothelium. Monocytes were added to the wells containingthe endothelial monolayers to reach a final cell number of3x10 /ml. In some studies, monocytes were exposed in vitrofor 30 minutes to sodium nitroprusside (an NO donor) priorto the binding assay.The six-well plates were transferred to a rocking platform

and rocked for 30 minutes at room temperature. After 30minutes, the cell suspension was aspirated from each welland wells were then rinsed with binding bulfer to removenon-adherent monocytes. Videomicroscopic counting ofadherent cells was performed using a computer aided imageanalysis system.ResultsOral administration of L-arginine (7 g daily for 2 weeks)to hypercholesterolemic humans increased plasma arginine

values by 60% (from 79+10 to 128+12mM; n=7), whereasL-arginine values in the placebo-treated (n=3) and normo-cholesterolemic (n=6) groups remained unchanged. Theadministration of oral L-arginine had no elfect on any of thebiochemical or hematologic parameters and was well toler-ated. Oral L-arginine did not lower total cholesterol or LDLcholesterol. Two patients dropped out of the study; onebecause he did not want to take the pills, and one because ofreactivation of oral herpes during the study.The results of the adhesion assays were highly reproduc-ible. Monocytes derived from hypercholesterolemic indi-

viduals demonstrated a 50~8% increase in bound cells/hpf incomparison to cells from normal individuals (p&0.0001).The degree of adhesiveness was correlated to the plasmalevels ofLDL cholesterol (R=0.7, n=33; p&0.0001; FIG. 10).In an open-label study, 3 hypercholesterolemic individu-als were treated with oral L-arginine supplementation for 2weeks. Arginine treatment resulted in a 38% decrease inmonocyte adhesiveness.To confirm this elfect of L-arginine treatment and tocontrol for any experimental bias, a double-blinded,placebo-controlled, randomized study was performed. Ten


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24monocytes from both hypercholesterolemic groups wasincreased in comparison to the normocholesterolemic indi-viduals (p&0.001). After 2 weeks of L-arginineadministration, there was an absolute reduction of 53% inmonocyte binding (n=7, p&0.005, baseline vs 2 weeks) (FIG.11). By contrast, there was no significant change in theadhesiveness of monocytes isolated from hypercholester-olemic individuals treated with placebo. Two weeks afterdiscontinuation of the L-arginine treatment, the adhesive-ness of the monocytes isolated from hypercholesterolemicindividuals had significantly increased compared to thenormocholesterolemic individuals (34~9% increase inbound cells/hpf; p&0.05), and was also significantlyincreased in comparison to the binding obtained after 2weeks of L-arginine therapy (an increase of 30~9%,p&0.05). The adhesiveness of monocytes from placebo-treated hypercholesterolemic individuals did not changesignificantly during the washout period.In some studies monocytes were exposed to sodium

nitroprusside or vehicle control for 30 minutes in vitro.Pre-incubation of the cells from hypercholesterolemic indi-viduals with the NO donor sodium nitroprusside (10 M)markedly reduced binding (164+9% vs 98+7% vehicle vssodium nitroprusside; n=7, p&0.0005; values expressed as apercent of the normocholesterolemic control exposed tovehicle; FIG. 12).To conclude, the salient findings of this investigation arethat: I) Hypercholesterolemia enhances the adhesiveness ofmonocytes for endothelial cells, 2) oral arginine supplemen-tation reverses the increase in adhesiveness of monocytesfrom hypercholesterolemic individuals, and 3) the elfect oforal arginine is mimicked in vitro by exposure of themonocytes from hypercholesterolemic individuals tosodium nitroprusside, an NO donor.

EXAMPLE 16Platelet Hyperaggregability in HypercholesterolemicHumansReversal by Oral L-ArginineIn this study we tested the hypothesis that chronicL-arginine supplementation would inhibit platelet reactivityin hypercholesterolemic humans. Venous blood was col-lected from normal (NC; n=11) and hypercholesterolemic(HC; n=22) volunteers for isolation of platelet-rich plasmaand aggregometry. Half the HC group received L-arginine (7g/d) for 2 weeks; aggregometry was performed using col-lagen (5 mg/ml) before and after two weeks of treatment.ResultsHC platelets were hyperaggregable. After two weeks ofL-arginine, the aggregability of HC platelets was reduced(FIG. 13). These studies are consistent with our previousobservations in animals that oral administration of

L-arginine inhibits platelet reactivity.EXAMPLE 17Intravenous Administration of L-Arginine Improves

Endothelium-dependent Vasodilation in Hypercholester-olemic HumansHyperlipoproteinemia impairs endothelium-dependent

vasodilation, even before the development of atherosclero-sis. We hypothesized that administration of L-arginine mayincrease synthesis of NO and thereby improve endothelium-dependent vasodilation in hypercholesterolemia. Indeed, ourearlier studies conducted in cholesterol-fed rabbits supportthis notion. The following data demonstrates that L-arginine

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Their ages ranged from 31 to 49 and averaged 39~2 yr.There were 14 patients with hypercholesterolemia. Hyper-cholesterolemia was defined as a serum LDL cholesterollevel greater than the 75th percentile adjusted for age andsex. These individuals included 11 males and 3 femaleswhose ages ranged from 22 to 48 and averaged 38~2 years.Under local anesthesia and sterile conditions, a polyeth-ylene catheter was inserted into a brachial artery of eachsubject for determination of blood pressure and for infusionof drugs. A separate polyethylene catheter was inserted intothe antecubital vein for infusion of L-arginine. Bilateralforearm blood flow was determined by venous occlusionstrain gauge plethysmography, using calibrated mercury-in-silastic strain gauges, and expressed as ml/100 ml tissue permin.To assess NO-dependent vasodilation, methacholine chlo-

ride (which induces the endothelium to release NO) wasadministered via the brachial artery. Forearm blood flow wasmeasured during infusion of methacholine chloride at con-centrations of 0.3, 3, and 10 pg/min each for 3 min.After completion of the methacholine chloride infusions,all normal subjects and 10 individuals with hypercholester-olemia were given L-arginine intravenously over 30minutesand then the methacholine infusions were repeated.D-arginine, the enantiomer of L-arginine, is not a precursorof NO. Thus, to ensure that any observed elfects ofL-arginine were due to its contribution to the synthesis ofNO and not just secondary to its physiochemical properties,five individuals with hypercholesterolemia receivedD-arginine intravenously.ResultsBaseline blood pressure, heart rate, and forearm bloodflow did not dilfer between normal and hypercholester-olemic subjects. Intraarterial infusion of methacholine chlo-ride caused a dose-dependent increase in forearm bloodflow. In the hypercholesterolemic subjects, however, cho-linergic vasodilation was less than that of normal subjects(p&0.05). The maximal forearm blood flow response tomethacholine in normal subjects is 19.0~L9 ml/100 ml oftissue per min, and in hypercholesterolemic subjects, it was13.7~L7 ml/100 ml of tissue per min (p&0.05).In the normal subjects, L-arginine did not potentiate thevasodilation that occurred during the administration ofmethacholine chloride. In the hypercholesterolemicsubjects, however, the L-arginine infusion augmented thevasodilation to methacholine chloride by 25% (p&0.05).There were no complications or side-elfects of theL-arginine infusions.The important findings in this study are: (a) endothelium-dependent vasodilation (due to the release ofNO) is reducedin forearm resistance vessels of hypercholesterolemichumans; and (b) intravenous administration of L-arginineimproves endothelium-dependent vasodilation in these indi-viduals. NO not only causes vasodilation, but it also inhibitsplatelet aggregation and suppresses monocyte adhesion inhypercholesterolemic humans.

EXAMPLE 18Admini

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