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

Vascular function and structure is maintained or improved by long term administration of physiologically acceptable compounds, namely L-arginine, L-lysine, physiologically acceptable salts thereof, and polypeptide precursors thereof, which enhance the level of endogenous nitric oxide or other intermediates in the NO induced relaxation pathway in the host. In or in combination, other compounds, such as B.sub.6, folate, B.sub.12, or an antioxidant, which provide for short term enhancement of nitric oxide, either directly or by physiological processes may be employed.

INTRODUCTION 
This invention was supported in part by the United States Government under 
Grant 1KO7HCO2660 (NHLBI). The U.S. Government may have an interest in 
this application. 
1. Technical Field 
The field of this invention is the modulation of NO activity, which finds 
application in maintaining and improving vascular function and thereby 
preventing or improving vascular degenerative diseases. 
2. Background 
Atherosclerosis and vascular thrombosis are a major cause of morbidity and 
mortality, leading to coronary artery disease, myocardial infarction, and 
stroke. Atherosclerosis begins with an alteration in the endothelium, 
which lines the blood vessels. The endothelial alteration results in 
adherence of monocytes, which penetrate the endothelial lining and take up 
residence in the subintimal space between the endothelium and the vascular 
smooth muscle of the blood vessels. The monocytes absorb increasing 
amounts of cholesterol (largely in the form of oxidized or modified 
low-density lipoprotein) to form foam cells. Oxidized low-density 
lipoprotein (LDL) cholesterol alters the endothelium, and the underlying 
foam cells distort and eventually may even rupture through the 
endothelium. 
Platelets adhere to the area of endothelial disruption and release a number 
of growth factors, including platelet derived growth factor (PDGF). PDGF, 
which is also released by foam cells and altered endothelial cells, 
stimulates migration and proliferation of vascular smooth muscle cells 
into the lesion. These smooth muscle cells release extracellular matrix 
(collagen and elastin) and the lesion continues to expand. Macrophages in 
the lesion elaborate proteases, and the resulting cell damage creates a 
necrotic core filled with cellular debris and lipid. The lesion is then 
referred to as a "complex lesion." Rupture of this lesion can lead to 
thrombosis and occlusion of the blood vessel. In the case of a coronary 
artery, rupture of a complex lesion may precipitate a myocardial 
infarction, whereas in the case of a carotid artery, stroke may ensue. 
One of the treatments that cardiologists and other interventionalists 
employ to reopen a blood vessel which is narrowed by plaque is balloon 
angioplasty (approximately 300,000 coronary and 100,000 peripheral 
angioplasties are performed annually). Although balloon angioplasty is 
successful in a high percentage of the cases in opening the vessel, it 
unfortunately denudes the endothelium and injures the vessel in the 
process. This damage causes the migration and proliferation of vascular 
smooth muscle cells of the blood vessel into the area of injury to form a 
lesion, known as myointimal hyperplasia or restenosis. This new lesion 
leads to a recurrence of symptoms within three to six months after the 
angioplasty in a significant proportion of patients (30-40%). 
In atherosclerosis, thrombosis and restenosis there is also a loss of 
normal vascular function, such that vessels tend to constrict, rather than 
dilate. The excessive vasoconstriction of the vessel causes further 
narrowing of the vessel lumen, limiting blood flow. This can cause 
symptoms such as angina (if a heart artery is involved), or transient 
cerebral ischemia (i.e. a "small stroke", if a brain vessel is involved). 
This abnormal vascular function (excessive vasoconstriction or inadequate 
vasodilation) occurs in other disease states as well. Hypertension (high 
blood pressure) is caused by excessive vasoconstriction, as well as 
thickening, of the vessel wall, particularly in the smaller vessels of the 
circulation. This process may affect the lung vessels as well causing 
pulmonary (lung) hypertension. Other disorders known to be associated with 
excessive vasoconstriction, or inadequate vasodilation include transplant 
atherosclerosis, congestive heart failure, toxemia of pregnancy, Raynaud's 
phenomenon, Prinzmetal's angina (coronary vasospasm), cerebral vasospasm, 
hemolytic-uremia and impotence. 
Because of their great prevalence and serious consequences, it is 
critically important to find therapies which can diminish the incidence of 
atherosclerosis, vascular thrombosis, restenosis, and these other 
disorders characterized by abnormality of vascular function and structure. 
Ideally, such therapies would inhibit the pathological vascular processes 
associated with these disorders, thereby providing prophylaxis, retarding 
the progression of the degenerative process, and restoring normal 
vasodilation. 
As briefly summarized above, these pathological processes are extremely 
complex, involving a variety of different cells which undergo changes in 
their character, composition, and activity, as well as in the nature of 
the factors which they secrete and the receptors that are up- or 
down-regulated. A substance released by the endothelium, "endothelium 
derived relaxing factor" (EDRF), may play an important role in inhibiting 
these pathologic processes. EDRF is now known to be nitric oxide (NO) or a 
labile nitroso compound which liberates NO. (For purposes of the subject 
invention, unless otherwise indicated, nitric oxide (NO) shall intend 
nitric oxide or the labile nitroso compound which liberates NO.) This 
substance relaxes vascular smooth muscle, inhibits platelet aggregation, 
inhibits mitogenesis and proliferation of cultured vascular smooth muscle, 
and leukocyte adherence. Because NO is the most potent endogenous 
vasodilator, and because it is largely responsible for exercise-induced 
vasodilation in the conduit arteries, enhancement of NO synthesis could 
also improve exercise capacity in normal individuals and those with 
vascular disease. NO may have other effects, either direct or indirect, on 
the various cells associated with vascular walls and degenerative diseases 
of the vessel. 
Relevant Literature 
Girerd et al. (1990) Circulation Research 67:1301-1308 report that 
intravenous administration of L-arginine potentiates endothelium-dependent 
relaxation in the hind limb of cholesterol-fed rabbits. The authors 
conclude that synthesis of EDRF can be increased by L-arginine in 
hypercholesterolemia. Rossitch et al. (1991) J. Clin. Invst. 87:1295-1299 
report that in vitro administration of L-arginine to basilar arteries of 
hypercholesterolemic rabbits reverses the impairment of 
endothelium-dependent vasodilation and reduces vasoconstriction. They 
conclude that the abnormal vascular responses in hypercholesterolemic 
animals is due to a reversible reduction in intracellular arginine 
availability for metabolism to nitric oxide. 
Creager et al. (1992) J. Clin. Invest. 90:1248-1253, report that 
intravenous administration of L-arginine improves endothelium-derived 
NO-dependent vasodilation in hypercholesterolemic patients. 
Cooke et al., "Endothelial Dysfunction in Hypercholesterolemia is Corrected 
by L-arginine," Endothelial Mechanisms of Vasomotor Control, eds. Drexler, 
Zeiher, Bassenge, and Just; Steinkopff Verlag Darmstadt, 1991, pp. 
173-181, review the results of the earlier references and suggest, "If the 
result of these investigations may be extrapolated, exogenous 
administration of L-arginine (i.e., in the form of dietary supplements) 
might represent a therapeutic adjunct in the treatment and/or prevention 
of atherosclerosis". 
Cooke (1990) Current Opinion in Cardiology 5:637-644 discusses the role of 
the endothelium in the atherosclerosis and restenosis, and the effect that 
these disorders have on endothelial function. 
Cooke (1992) J. Clin. Invest. 90:1168-1172, describe the effect of chronic 
administration of oral L-arginine in hypercholesterolemic animals on 
atherosclerosis. This is the first demonstration that oral L-arginine 
supplements can improve the release of NO from the vessel wall. The 
increase in NO release from the vessel wall was associated with a striking 
reduction in atherosclerosis in hypercholesterolemic animals. This is the 
first evidence to support the hypothesis that increasing NO production by 
the vessel wall inhibits the development of atherosclerosis. 
Cooke and Tsao (1992) Current Opinion in Cardiology 7:799-804 describe the 
mechanism of the progression of atherosclerosis and suggest that 
inhibition of nitric oxide may disturb vascular homeostasis and contribute 
to atherogenesis. 
Cooke and Santosa (1993) In: Steroid Hormones and Dysfunctional Bleeding, 
AAAS Press, review the activities of EDRF in a variety of roles and 
suggest that reversibility of endothelial dysfunction may be affected by 
the stage of atherosclerosis. They conclude that EDRF is a potent 
vasodilator, plays a key role in modulating conduit and resistance vessel 
tone, has important effects on cell growth and interactions of circulatory 
blood cells with a vessel wall, and that disturbances of EDRF activity may 
initiate or contribute to septic shock, hypertension, vasospasm, toxemia 
and atherosclerosis. 
Fitzpatrick et al., American Journal of Physiology 265 (Heart Circ. 
Physiol. 34):H774-H778, 1993 report that wine and other grape products may 
have endothelium-dependent vasorelaxing activity in vitro. 
Wang et al. (1994) J. Am. Cell. Cardiol. 23:452-458, report that oral 
administration of arginine prevents atherosclerosis in the coronary 
arteries of hypercholesterolemic rabbits. 
Drexler et al. (1994) Circulation 89:1615-1623 describe the effect of 
intravenous arginine upon coronary vascular tone. This was the first 
evidence that intravenous arginine could restore normal NO-dependent 
vasodilation in the coronary arteries of patients with cardiac 
transplants, Tsao et al. (1994) Circulation 89:2176-2182 demonstrates that 
oral administration of arginine to hypercholesterolemic rabbits enhances 
the release of nitric oxide by the vessel wall, and inhibits monocytes 
from sticking to the vessel. 
Tsao et al. (1994) J. Arterioscl. Thromb. 14:1529-1533 reveals that oral 
arginine administration to hypercholesterolemic rabbits inhibits platelet 
aggregation (blood clotting). Platelet aggregation plays an important role 
in causing vascular thrombosis in vascular degenerative disorders. 
Von de Leyen et al. (1995) PNAS USA, show that the gene encoding nitric 
oxide synthase (the enzyme that produces NO) can be inserted into the 
carotid artery of the rat. This causes the rat carotid artery to make more 
NO, and thereby enhances vasodilation and inhibits thickening of the 
vessel wall after balloon angioplasty. 
Noruse et al. (1994) Arterioscler. Thromb. 14:746-752, report that oral 
administration of an antagonist of NO production accelerates atherogenesis 
in hypercholesterolemic rabbits. 
Cayette et al. (1994) Arterioscler. Thromb. 14:753-759, also report that 
oral administration of an antagonist of NO production accelerates plaque 
development in hypercholesterolemic rabbits. 
Other references which refer to activities attributed to NO or its 
precursor include: Pohl and Busse (1989) Circ. Res. 65:1798-1803; Radomski 
et al. (1987) Br. J. Pharmacol. 92:181-187; Stamler et al. (1989) Circ. 
Res. 65:789-795; anti-platelet activity); Garg and Hassid (1989) J. Clin. 
Invest. 83:1774-1777; Weidinger et al. (1990) Circulation 81:1667-1679; NO 
activity in relation to vascular smooth muscle growth); Ross (1986) N. 
Engl. J. Med. 314:488-500; Bath et al. (1991) Arterioscler. Thromb. 
11:254-260; Kubes et al. (1991) Proc. Natl. Acad. Sci. USA 89:6348-6352; 
Lefer et al. (1990) In: Endothelium-Derived Contracting Factors. Basel, S. 
Karger, pp.190-197; NO activity in relation to leukocyte adhesion and 
migration); Heistad et al. (1984) Circ. Res. 43:711-718; Rossitch et al. 
(1991) J. Clin. Invest. 87:1295-1299; Yamamoto et al. (1988) ibid 
81:1752-1758; Andrews et al. (1987) Nature 327:237-239; Tomita et al. 
(1990) Circ. Res. 66:18-27; Kugiyama et al. (1990) Nature 344:160-162; 
Mitchell et al. (1992) J. Vasc. Res. 29:169 (abst.); Minor et al. (1990) 
J. Clin. Invest. 86:2109-2116; NO activity in relation to 
hypercholesterolemia); Tanner et al. (1991) Circulation 83:2012-2020; Kuo 
et al. (1992) Circ. Res. 70:f465-476; Drexler et al. (1991) Lancet 
338:1546-1550; Schuschke et al. (1994) Int. J. of Microcircu: Clin. and 
Exper. 14(4):204-211; Yao et al. (1992) Circulation 86:1302-1309; Higashi 
et al. (1995) Hypertension 25(4 Pt 2):898-902; Kharitonov et al. (1995) 
Clin. Sci. 88(2):135-139; Smulders et al. (1994) Clin. Sci. 87(1):37-43; 
Bode-boger et al. (1994) Clin. Sci. 87(3):303-310; Bode-Boger et al. 
(1994) Clin. Sci.; Randall et al. (1994) Clin. Sci. 87(1):53-59; 
Dubois-Rande et al. (1992) J. Card. Pharm. 20 Suppl. 12:S211-3; Otsuji et 
al. (1995) Am. Heart J. 129(6): 1094-1100; Nakanishi et al. (1992) Am. J. 
of Physio. 263(6 Pt 2):H1650-8; Kuo et al. (1992) Circ. Research 70(3): 
465-476; Tanner et al. (1991) Circulation 83(6):2012-2020; Meng et al. 
(1995) J. Am. Col. Card. 25(1):269-275; Lefer and Ma (1993) Arterioscl. 
and Thromb. 13(6):771-776; McNamara et al. (1993) Biochem. and Biophys. 
Res. Comm. 193(1):291-296; Tarry and Makhoul (1994) Arter. and Thromb. 
14(6):983-943; Davies et al. (1994) Surgery 116(3):557-568; and Raij 
(1994) Kidney Institute 45:775-781. 
SUMMARY OF THE INVENTION 
Methods are provided for improving vascular function and structure, 
particularly modulating vascular relaxation, cellular adhesion, 
infiltration and proliferation by modulating the level of nitric oxide or 
active precursor at a physiological site. The methods find use in 
preventing the degradation of vascular function, particularly as involved 
with the occurrence of atherosclerosis, restenosis, thrombosis, 
hypertension, impotence, and other disorders characterized by reduced or 
inadequate vasodilation. The enhancement of endogenous nitric oxide or 
secondary messenger availability at a physiological site improves vascular 
relaxation and thereby relieves symptoms due to inadequate blood flow 
(such as angina) and can counteract inappropriate elevation of blood 
pressure. The enhancement of endogenous nitric oxide also inhibits 
initiation and the progression of atherosclerosis, restenosis, vascular 
hypertrophy or hyperplasia and thrombosis. This is due to the fact that 
nitric oxide is not only a potent modulator, but can also inhibit 
platelets and white blood cells from adhering to the vessel wall. As a 
prophylaxis or treatment for vascular function deterioration, particularly 
in atherosclerotic susceptible hosts, the agent is chronically 
administered at an effective dosage. For restenosis, the agent may be 
administered for a limited period since this pathological process 
generally abates 3-6 months after the vascular injury (i.e. angioplasty or 
atherectomy). Oral administration of L-arginine, precursors to L-arginine, 
e.g. oligopeptides or polypeptides comprising L-arginine, or proteins 
comprising high levels of L-arginine, by itself or in combination with 
L-lysine, particularly further supplemented with GRAS substances which 
enhance the effectiveness of the active agents, as a dietary supplement 
will increase NO elaboration and thereby diminish the effects of 
atherogenesis. Other techniques to enhance NO or secondary messenger 
availability may be utilized such as increasing the availability of NO 
synthase, for example, as a result of enhanced expression of NO synthase 
in the vessel wall, particularly at the lesion site, release of NO from 
the vessel wall or reduction of degradation of NO or the secondary 
messenger, cyclic guanosine monophosphate ("cGMP").

DESCRIPTION OF SPECIFIC EMBODIMENTS 
In accordance with the subject invention, vascular function is maintained 
or its deterioration inhibited or retarded by enhancing the level or 
activity of endogenous nitric oxide. By enhancing the level or activity of 
endogenous nitric oxide, common vascular degenerative diseases such as 
atherosclerosis, restenosis, hypertension, vasospasm, impotence, angina, 
and vascular thrombosis, can be treated prophylactically and/or 
therapeutically. The enhanced level or activity of nitric oxide (which is 
intended to include any precursor of nitric oxide which results in such 
enhanced level) can be achieved by modulating the activity, synthesis or 
concentration of any of the components associated with the formation of 
nitric oxide in the nitric oxide synthetic pathway, or inhibiting the rate 
of degradation of nitric oxide, its precursors, or the secondary 
messengers associated with the relaxation signal. In referring to the 
enhanced level or activity, the term "effect" will be used to encompass 
the two situations. The enhanced effect of nitric oxide may be a result of 
oral or intravenous administration to the patient of a precursor in the 
metabolic pathway to the production of nitric oxide (such as L-arginine, 
L-lysine, polypeptides comprising these amino acids, and the like), 
providing an enzyme in the metabolic pathway to NO, particularly NO 
synthase, by introduction of the gene for NO synthase under conditions for 
integration of the gene into the endothelial or other cells and expression 
of the gene, or by directly adding an enzyme associated with the 
production of nitric oxide. The enhanced level of nitric oxide may also 
result from administration of an agent to protect the NO from degradation, 
such as an oxidant, reductant or superoxide dismutase. Alternatively, the 
agent may serve to enhance the bioavailability or effectiveness of the 
primary active agent, such as L-arginine or L-lysine. The agent, 
individually or in combination, will be administered in a form of other 
than a natural food source, such as meat or plants as natural protein 
sources, 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 to L-arginine, e. g. a monomer or 
a polypeptide, as a dietary supplement. The amino acid(s) are administered 
as any physiologically acceptable salt, such as the hydrochloride salt, 
glutamate salt, etc. They can also be administered as a peptide (e.g., 
poly-L-arginine, poly-L-lysine, or combinations thereof) so as to increase 
plasma levels of the NO precursor. Oligopeptides of particular interest 
include oligopeptides of from 2 to 30, usually 2 to 20, preferably 2 to 10 
amino acids, having at least 50 mol % of L-arginine and/or L-lysine, 
preferably at least about 75 mol % of L-arginine and/or L-lysine, more 
preferably having at least about 75 mol % of L-arginine and/or L-lysine. 
The oligopeptides can be modified by being ligated to other compounds, 
which can enhance absorption from the gut, provide for enhancement of NO 
synthesis or stability, e.g. reducing agents 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 about 50%. 
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 
extended period of time, generally at least one month, more usually at 
least three months, and as a chronic treatment, could last for one year or 
more, including the life of the host. The dosage administered will depend 
upon the frequency of the administration, the blood level desired, other 
concurrent therapeutic treatments, the severity of the condition, whether 
the treatment is for prophylaxis or therapy, the age of the patient, the 
natural level of NO in the patient, and the like. Desirably, the amount of 
L-arginine and/or L-lysine (R and/or K) or biologically equivalent 
compound which is used would generally provide a plasma level in the range 
of about 0.15 to 30 mM. The oral administration of R and/or K can be 
achieved by providing R and/or K, other NO precursor, or NO enhancing 
agent as a pill, powder, capsule, liquid solution or dispersion, 
particularly aqueous, or the like. Various carriers and excipients may 
find use in formulating 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 administration of 
L-arginine and/or L-lysine for a human host will be about 1 to 12 g per 
day. 
Furthermore, other agents can be added to the oral formulation of the amino 
acids or polypeptides to enhance their absorption, and/or to enhance the 
activity of NO synthase, e.g. B.sub.6 (50-250 mg/d), folate (0.4-10 mg per 
daily dose), B.sub.12 (0.5-1 mg/d) or calcium (250-1000 mg per daily 
dose). Furthermore, agents known to protect NO from degradation, such as 
antioxidants (e.g. cysteine or N-acetyl cysteine 200-1000 mg/d Vitamin C 
(250-2000 mg daily dose), (coenzyme Q 25-90 mg daily dose, glutathione 
50-250 mg daily dose), Vitamin E (200-1000 I.U. daily dose), or 
.beta.-carotene (10-25,000 I.U. daily dose) or other naturally occurring 
plant antioxidants such as tocopherols, phenolic compounds, thiols, and 
ubiquinones can be added to the oral or intravenous formulations of R 
and/or K, or R and/or K-containing peptides. Alternatively, one may 
include the active agent in a nutritional supplement, where other 
additives may include vitamins, amino acids, or the like, where the 
subject active agent will be at least 10 weight %, more usually 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 prophylactically to improve vascular 
function, serving to enhance vasodilation and to inhibit atherogenesis or 
restenosis, or therapeutically after atherogenesis has been initiated. 
Thus, for example, a patient who is to undergo balloon angioplasty can 
have a regimen of R and/or K administered substantially prior to the 
balloon angioplasty, preferably at least about a week or substantially 
longer. Alternatively, in a patient, the administration of R and/or K can 
begin at any time. Conveniently, the amino acid composition can be 
administered by incorporating the appropriate dose in a prepared food. 
Types of foods include gelatins, ice creams, cereals, candies, sugar 
substitutes, soft drinks, and the like. Of particular interest is the 
incorporation of R and/or K as a supplement in a food, such as a health 
bar, e.g. granola, other grains, fruit bars, such as a date bar, fig bar, 
apricot bar, or the like. The amount of R and/or K or the equivalent would 
be about 2-25 g per dosage or bar, preferably about 3-15 g. 
Instead of oral administration, intravascular administration can also be 
employed, particularly where more rapid enhancement 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 
administrations can be employed in accordance with the needs of the 
patient. Furthermore, parenteral administration can allow for the 
administration of compounds which would not readily be transported across 
the mucosa from the gastrointestinal tract into 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 be enriched for the active component by 
employing separation techniques and assaying the activity of each of the 
fractions obtained. The grape skin extract can be divided into fractions 
using a first gel permeation separation to divide the extract by the size 
of the components. The active fraction(s) can be determined by an 
appropriate assay, see the experimental section. 
The active fraction(s) can be further separated using HPLC and an 
appropriate eluent, conveniently either an isocratic eluent of aqueous 
acetonitrile or propanol or a linearly varying eluent, using the same 
solvents. Fractions which are shown to be active and substantially pure, 
e.g. at least 80 weight %, by thin layer chromatography, mass 
spectrometry, gas phase chromatography, or the like can then be 
characterized using infra-red, nuclear magnetic resonance, mass or other 
spectroscopy. 
For oral or intravascular administration, one can provide R and/or K, by 
itself or in a polypeptide, or its physiological equivalent in supporting 
NO, together with antioxidants or scavengers of oxygen-derived free 
radicals (such as sulfhydryl containing compounds) or compounds that 
prevent the production of oxygen-derived free radicals (such as superoxide 
dismutase), as it is known that oxygen-derived free radicals (such as 
superoxide anion) can inactivate nitric oxide. Alternatively, or in 
addition, one can administer cofactors required for NO synthase activity, 
such as calcium or folate. The amounts of each of these co-agents can be 
determined empirically, using the assays in the experimental section to 
determine NO activity. 
The various cofactors that may be used with the NO precursors will vary in 
amount in relation to the amount of NO precursor and the effectiveness of 
the cofactor, particularly for oral administration. Generally, the 
cofactors may be present in amounts that would provide daily doses of 
folate (0.4-10 mg), B.sub.6 (50-250 mg), B.sub.12 (0.5-1 mg) and/or 
calcium (250-1000 mg). Usually, where the amount of the NO precursor is 
greater than about 2 g, it may be administered periodically during the 
day, being administered 2 to 4 times daily. For the most part, the 
cofactors will be GRAS substances and will be able to be taken at high 
dosages without adverse effects on the recipient host. 
The subject compositions will be for the most part administered orally and 
the dosage may take a variety of forms. The dosage may be tablets, pill, 
capsules, powders, solutions, dispersions, bars, ice creams, gelatins, and 
the like, formulated with physiologically acceptable carriers, and 
optionally stabilizers, colorants, flavoring agents, excipients, 
tabletting additives, and the like. Depending upon the mode of 
administration, the amount of active agent may be up to about 25 g. For 
formulations as dietary supplements, individual dosages will generally 
range from about 0.5 to 5 g, more usually from about 1 to 3 g of the NO 
precursor. 
Alternatively, one can enhance, either in conjunction with the enhancement 
of precursors to nitric oxides or independently, components of the nitric 
oxide metabolic pathway. For example, one can enhance the amount of nitric 
oxide synthase present in the vessel wall, particularly at the site of 
lesions. This can be done by local administration to the lesion site or 
systemically into the vascular system. The synthase can be administered 
using liposomes, slow release particles, or in the form of a depot, e.g. 
in collagen, hyaluronic acid, biocompatible gels, vascular stents, or 
other means, which will provide the desired concentration of the NO 
synthase at the lesion site. 
Instead of providing for the enhancement of NO at the physiological site of 
interest, one can choose to extend the lifetime of the signal transduced 
as a result of the presence of nitric oxide. Since cGMP is produced 
intracellularly as a result of a nitric oxide induced signal, employing 
agents which result in the production of or extending the lifetime of cGMP 
can be employed. Illustrative agents include cGMP phosphodiesterase 
inhibitors or agents which increase the amount of guanylate cyclase. 
Alternatively, cells can be genetically engineered to provide for 
constitutive or inducible expression of one or more genes, which will 
provide for the desired relaxation response, by expressing NO synthase, or 
other enzyme or protein which is secreted and acts extracellularly. Thus, 
expression vectors (viral or plasmid) can be prepared which contain the 
appropriate gene(s) and which can be introduced into host cells which will 
then produce high concentrations of nitric oxide or other intermediate in 
the relaxation pathway. These cells can be introduced at the lesion site 
or at another site in the host, where the expression will induce the 
appropriate response as to relaxation, proliferation, etc. The NO synthase 
or cells expressing the NO synthase can be present as depots by 
encapsulation and positioning at the site of interest. For example, porous 
stents can be produced which encapsulate the enzyme or cells to protect 
the enzyme from degradation or being washed away. 
Cultured cells can be transfected with expression vectors containing the NO 
synthase or other gene ex-vivo and then introduced into the vessel wall, 
using various intra-arterial or intra-venous catheter delivery systems. 
Alternatively, techniques of in vivo gene transfer can be employed to 
transfect vascular cells within the intact vessel in vivo. The gene(s) can 
be expressed at high constitutive levels or can be linked to an inducible 
promoter (which can have tissue specificity) to allow for regulation of 
expression. 
DNA constructs are prepared, where the appropriate gene, e.g. a NO synthase 
gene, is joined to an appropriate promoter, either with its native 
termination region or a different termination region, which can provide 
for enhanced stability of the messenger RNA. Constitutive promoters of 
particular interest will come from viruses, such as Simian virus, 
papilloma virus, adenovirus, HIV, Rous sarcoma virus, cytomegalovirus or 
the like, where the promoters include promoters for early or late genes, 
or long terminal repeats. Endogenous promoters can include the 
.beta.-actin promoter, or cell-type specific promoters. 
A construct is prepared in accordance with conventional techniques, the 
various DNA fragments being introduced into an appropriate plasmid or 
viral vector, normally a vector capable of replication in a bacterial 
and/or eucaryotic host. Normally, the vector will include a marker, which 
allows for selection of cells carrying the vector, e.g. antibiotic 
resistance. The vector will normally also include an origin which is 
functional in the host for replication. Other functional elements can also 
be present in the vector. 
Once the vector has been prepared and replicated, it can then be used for 
introduction into host cells. The plasmid vector construct can be further 
modified by being joined to viral elements which allow for ease of 
transfection, can provide a marker for selection, e.g. antibiotic 
resistance, or other functional elements. Introduction of the plasmid 
vector construct into the host cells can be achieved by calcium phosphate 
precipitated DNA, transfection, electroporation, fusion, lipofection, 
viral capsid-mediated transfer, or the like. Alternatively, the expression 
construct within viral vectors can be introduced by standard infection 
techniques. For somatic cell gene therapy, autologous cells will generally 
be employed, although in some instances allogeneic cells or recombinantly 
modified cells can be employed. Usually the cells employed for genetic 
modification will be mature endothelial or vascular smooth muscle cells. 
Occasionally, the cells employed for genetic modification will be 
progenitor cells, particularly early progenitor cells. For example, 
myoblasts can be employed for muscle cells or hematopoietic stem cells or 
high proliferative potential cells can be employed for lymphoid and/or 
myelomonocytic cells. 
Depending upon the nature of the cells, they can be injected into tissue of 
the same or different cellular nature, they can be injected into the 
vascular system, where they may remain as mobile cells or home to a 
particular site (i.e. the lesion). For the NO synthase gene, the number of 
cells which are administered will depend upon the nature of the cells, the 
level of production of the NO synthase, the desired level of NO synthase 
in the host vascular system, at the lesion site, or the like, whether the 
enhanced level of NO synthase is the only treatment or is used in 
conjunction with other components of the nitric oxide synthetic pathway, 
and the like. Therefore, the particular number of cells to be employed 
will be determined empirically in accordance with the requirements of the 
particular patient. 
These cells can also be introduced into the circulation by first growing 
them on the surface of standard vascular graft material (i.e. DACRON.RTM. 
or polytetrafluoroethylene grafts) or other synthetic vascular conduits or 
vascular bioprostheses. 
Alternatively, one can use viral vectors, which are capable of infecting 
cells in vivo, such as adenovirus or retroviruses. In this case, the viral 
vector containing the NO synthase gene or other gene involved with the 
relaxation pathway will be administered directly to the site of interest, 
where it will enter into a number of cells and become integrated into the 
cell genome. Thus, one can titer the desired level of nitric oxide 
synthase which is secreted or other protein which is expressed, by 
providing for one or more administrations of the virus, thus incrementally 
increasing the amount of synthase 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 the NO synthase or other gene. One 
such modified liposome technique involves mixing the liposomes with the 
vector containing NO synthase. Once the gene expression 
construct-containing vector is incorporated into the liposome, the 
liposomes are coated with a protein (e.g. the viral coat protein of the 
Hemagglutinating Virus of Japan) that increases the affinity of the 
liposome for the vessel wall. 
In some situations, the NO synthase or other gene in the relaxation pathway 
can be co-transfected with an artificial gene encoding an arginine and/or 
lysine rich polypeptide susceptible to proteolytic cleavage as an 
intracellular source of L-arginine and/or L-lysine. In other situations, 
the NO synthase or other gene can be co-transfected with the superoxide 
dismutase gene, so as to inhibit the degradation of the nitric oxide. 
In some situations, acute treatment may be involved, requiring one or a few 
administrations. This will normally be associated with compounds which can 
act as nitric oxide precursors and are other than naturally occurring 
compounds or are compounds which can be added with naturally occurring 
compounds to enhance the rate of formation of nitric oxide. Thus, one can 
provide for acute administration of L-arginine and/or L-lysine and 
superoxide dismutase to increase the nitric oxide concentration over a 
restricted period of time. These administrations can be independent of or 
in conjunction with long term regimens. 
The following examples are offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL 
EXAMPLE 1 
Anti-atherogenic effects of oral arginine 
Study design 
(See, Cooke et al., 1992, supra) Male New Zealand white rabbits (n=49) were 
assigned to one of three treatment groups: 10 were fed with normal rabbit 
chow for ten weeks (Control); 19 received chow enriched with 1% 
cholesterol (Chol); and 20 received a 1% cholesterol diet supplemented 
with 2.25% L-arginine hydrochloride in the drinking water (Arg.). 
Following ten weeks of the dietary intervention, animals were lightly 
sedated and the central ear artery cannulated for measurement of 
intra-arterial blood pressure, followed by collection of blood samples for 
serum chemistries and plasma arginine. Subsequently the animals were 
sacrificed and the left main coronary artery and the thoracic aorta were 
harvested for studies of vascular reactivity and histomorphometry. In some 
animals, blood was collected for studies of platelet and monocyte 
reactivity. 
Results 
Biochemical and physiological measurements. Hypercholesterolemic animals 
maintained on oral L-arginine supplementation (Arg) experienced a twofold 
elevation in plasma arginine levels in comparison to animals on a normal 
(Control) or 1% cholesterol (Chol) diet alone; the elevation in plasma 
arginine was maintained throughout the course of the study. Serum 
cholesterol measurements were elevated equally in both groups receiving 
the 1% cholesterol diet 50.+-.6 vs. 1629.+-.422 vs. 1852.+-.356 mg/dl 
respectively for Control (=10), Chol (=13), and Arg (=14)!. There were no 
significant differences in hemodynamic measurements between groups. 
Organ chamber studies of isolated vessels 
For NO-independent responses, there were no differences between the 
treatment groups in maximal response or sensitivity to norepinephrine (a 
vasoconstrictor), or to nitroglycerin (a nitrovasodilator). By contrast, 
NO-dependent relaxations were attenuated in vessels harvested from 
hypercholesterolemic animals with a reduction in the maximal response to 
acetylcholine. In comparison, vessels harvested from hypercholesterolemic 
animals receiving L-arginine supplementation had improved NO-dependent 
relaxation to acetylcholine. In a separate study, the effect of chronic 
arginine supplementation to improve NO-dependent relaxation was confirmed 
in the hypercholesterolemic rabbit abdominal aorta. 
Histomorphometric studies (planimetry of EVG-stained sections) 
A blinded histomorphometric analysis revealed that medial cross-sectional 
areas of thoracic aortae were not different between the groups. By 
contrast, the intimal cross-sectional area (i.e. amount of atherosclerotic 
plaque) of vessels from hypercholesterolemic animals receiving L-arginine 
supplementation was reduced in comparison to those from animals receiving 
cholesterol diet alone. In the Arg animals the reduction in the intimal 
lesion was most pronounced in the ascending thoracic aorta and left main 
coronary artery. In the left main coronary artery of hypercholesterolemic 
animals receiving arginine, essentially no atherosclerotic plaque 
developed. 
Changes in lesion surface area 
In a second series of studies, the extent of the thoracic aorta involved by 
lesions was examined. In hypercholesterolemic rabbits receiving vehicle 
(n=6) or L-arginine supplement (n=6), thoracic aortae (from left 
subclavian artery to diaphragm) were harvested after ten weeks of 
treatment, bisected longitudinally, and stained with oil-red O. Vessels 
were photographed and vessel and lesion surface area determined by 
planimetry. Approximately 40% of the total surface area was covered with 
plaque in thoracic aortae from hypercholesterolemic animals receiving 
vehicle, whereas in thoracic aortae from arginine-treated 
hypercholesterolemic animals, less than 10% of the surface area was 
covered with plaque (FIG. 1). 
To summarize, dietary arginine supplementation increases plasma arginine 
levels, but does not alter serum cholesterol. This is associated with 
significant improvement in NO-dependent vasodilation as judged by 
bioassay. Finally, the improvement in NO-dependent vasodilation is 
associated with reduction in thickness and area of the lesions in vessels 
from hypercholesterolemic animals. 
EXAMPLE 2 
Inhibition of platelet aggregation by oral L-arginine 
The effect of L-arginine supplementation on platelet reactivity in rabbits 
that had normal chow (Control; n=6), a 1% cholesterol diet (Chol; n=5), or 
a 1% cholesterol diet supplemented with oral arginine (Arg; n=6), as 
detailed above, was examined. Arterial blood obtained after central ear 
artery cannulation was anticoagulated with 13 mM sodium citrate. 
Platelet-rich suspension was prepared by washing platelets in calcium-free 
Krebs-Henseleit solution and resuspending them in Tyrode's solution with 
albumin. Aggregation was initiated by addition of adenosine diphosphate 
and monitored by standard nephelometric techniques. In platelets derived 
from Chol animals, aggregation was not different in rate or maximum extent 
in comparison to platelets from Control animals (A, in FIG. 2). By 
contrast, aggregation of platelets 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 platelets from arginine-treated 
animals. The reduction of platelet reactivity could be reversed by 
administration of N-methylarginine (10.sup.-4 M) in vitro (C, in FIG. 2). 
Therefore, the anti-platelet effect of chronic oral arginine 
administration can be credited to an increased synthesis of endogenous NO. 
Furthermore, NO synthesis may be induced in both the platelets and the 
endothelium. 
EXAMPLE 3 
Inhibition of monocyte adherence 
A. Functional Binding Assay 
To determine if oral arginine supplementation affects monocyte adherence, 
blood was collected from rabbits fed normal chow (=6) a 1% cholesterol 
diet (=6), or a 1% cholesterol diet supplemented with L-arginine (=6), as 
described above. Mononuclear cells were purified from blood by 
Ficoll-paque density gradient centrifugation. In these preliminary 
studies, adhesion was examined of blood leukocytes to a transformed 
endothelial cell line, bEnd3 (mouse brain-derived polyoma middle T antigen 
transformed endothelial cells). The Bend3 cells display the morphology of 
endothelial cells, and like human endothelial cells are capable of uptake 
of acetylated low-density lipoprotein and express adhesion molecules in a 
cytokine-regulatable fashion. Cultured cells were grown to confluence in 
0.5 cm.sup.2 Lab-Tek chamber slides (MilesScientific) and treated with 
control medium or with LPS (1 mg/ml) or TNF.alpha. (25 U/ml) for 18 hours. 
Cultures were washed with fresh assay buffer, and low, medium, or high 
concentrations of leukocytes (0.75, 1.5, or 3.times.10.sup.5 cells/ml, 
respectively) were added per well. Following a 30-minute incubation on a 
rocking platform at room temperature to allow binding, the slides were 
inverted and immersed in buffer containing 2% (v/v) glutaraldehyde, such 
that non-adherent cells were lost and adherent cells were fixed to the 
monolayer. The adherent mononuclear cells were enumerated using 
video-light microscopy. 
Monocytes from hypercholesterolemic animals (Chol) exhibited greater 
adherence, consistent with observation by others, that monocytes from 
hypercholesterolemic cats or humans exhibit greater adherence to cultured 
endothelial cells. (deGruijter et al. (1991) Metabol. Clin. Exp. 
40:1119-1121; Fan et al. (1991) Virchows Arch. B Cell Pathol. 61:19-27). 
In comparison to monocytes derived from vehicle-treated 
hypercholesterolemic animals (Chol), those from arginine-treated 
hypercholesterolemic animals (Arg) were much less adherent. This data 
shows that the arginine treatment inhibits adhesion of monocytes to the 
endothelium, which is the first observable event in atherogenesis. 
EXAMPLE 4 
Dietary L-Arginine Inhibits the Enhanced Endothelial-Monocyte Interaction 
In Hypercholesterolemia 
The earliest observable abnormality of the vessel wall in 
hypercholesterolemic animals is enhanced monocyte adherence to the 
endothelium, which occurs within one week of a high cholesterol diet. This 
event is thought to be mediated by the surface expression of endothelial 
adhesion molecules and chemotactic proteins induced by 
hypercholesterolemia. 
Another endothelial alteration that occurs in parallel is a reduced 
activity of nitric oxide (i.e., NO), derived from metabolism of 
L-arginine. As shown above chronic dietary supplementation with L-arginine 
restores NO-dependent vasodilatation in hypercholesterolemic rabbits, and 
that this improvement in NO activity is associated with a striking 
anti-atherogenic effect. In the following study was tested the hypothesis 
that the anti-atherogenic effect of dietary arginine was mediated by 
endothelial derived NO which inhibits monocyte-endothelial cell 
interaction. 
Methods 
Animals. 
Male New Zealand White rabbits were pair fed, receiving one of the 
following dietary interventions for two weeks: normal rabbit chow (Cont, 
n=7); rabbit chow enriched with 1% cholesterol (Chol, n=7); or 1% 
cholesterol chow supplemented with 2.25% L-arginine HCl in the drinking 
water (Arg, n=7) ad libitum throughout the course of the study. In a 
second series of studies designed to further explore the role of 
endogenous NO on monocyte-endothelial cell interaction, another group of 
animals were pair fed, receiving a normal rabbit diet supplemented with 
either vehicle control (N=5) or the NO synthase antagonist, 
nitro-L-arginine (L-NA, 10 mg/100 ml; n=5), administered in the drinking 
water ad libitum throughout the course of the study (for an average daily 
dose of 13.5 mg/kg/day). In a third series of experiments animals received 
a normal diet and either vehicle (n=4), L-NA (13.5 mg/kg/day; n=4), or 
L-NA and hydralazine (n=4) added to the drinking water for two weeks. At 
this dose, hydralazine (5 mg/kg/day) reversed the increase in blood 
pressure induced by L-NA. One day before sacrifice (after 2 weeks of 
dietary intervention), animals were lightly sedated and the central ear 
artery was cannulated for collection of blood samples. 
Mononuclear cell culture and isolation. 
Murine monocytoid cells, WEHI 78/24 cells were grown in Dulbecco's Modified 
Eagle's Medium supplemented 10% fetal calf serum (vol/vol) and were kept 
in an atmosphere of 5% CO.sub.2 /95% air. Prior to binding studies, 
mononuclear cells were fluorescently labeled with TRITC (3 .mu.g/ml). To 
confirm the results using WEHI cells, in some studies binding studies were 
performed in parallel using rabbit mononuclear cells. Mononuclear cells 
were isolated from fresh whole blood 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 15 mm segment of thoracic aorta 
was excised from a point immediately distal to the left subclavian artery 
to the mid-thoracic aorta. The segments were then carefully opened 
longitudinally and placed into culture dishes containing HBSS medium. 
Aortic strips were fixed to the culture dish using 25 gauge needles so as 
to expose the endothelial surface to the medium. Culture dishes were then 
placed on a rocking platform at room temperature. 
After 10 minutes the HBSS medium was replaced by binding medium containing 
WEHI cells. The aortic strips were incubated with the mononuclear cells 
for 30 minutes. The medium was then replaced by fresh binding medium 
without cells to remove non-adherent cells. The aortic segments were then 
removed and placed on a glass slide, and adherent cells counted under 
epifluorescent microscopy from at least 30 sites on each segment. 
Results 
Monocyte 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 vivo adhesion assay. However, when WEHI 
cells were incubated with aortic endothelium from hypercholesterolemic 
animals (Chol; n=7), cell binding was enhanced 3-fold in comparison to 
Cont (n=7). The increased cell binding manifested by aortic endothelium of 
hypercholesterolemic animals was significantly attenuated by L-arginine 
supplementation (n=7). (FIG. 3) Similar results were achieved when 
adhesion assays were performed in parallel with mononuclear cells that 
were freshly isolated from Cont animals (n=2) in each of the three groups. 
Effect of chronic NO synthase inhibition on endothelial adhesiveness. 
To further investigate the role of endothelium-derived NO in modulating 
endothelial-monocyte interaction, an additional series of binding studies 
were performed using thoracic aorta from animals that received regular 
chow supplemented with vehicle (n=5) or the NO synthase inhibitor, L-NA 
(n=5). The adhesion of WEHI cells was markedly increased when incubated 
with aortic endothelium from L-NA animals compared to control endothelium. 
This effect could not be attributed to hypertension caused by L-NA since 
concomitant administration of hydralazine normalized blood pressure but 
did not reverse the augmentation of cell binding induced by L-NA. 
In a separate series of studies it was confirmed that chronic 
administration of L-NA (the inhibitor of NO synthase) significantly 
inhibited generation and release of NO from the vessel wall (as measured 
by chemiluminescence), compared to vessels from animals treated with 
vehicle or arginine. 
The salient findings of this investigation are: 1) monocyte binding to the 
endothelium ex vivo is increased in vessels from hypercholesterolemic 
animals; 2) this increase in monocyte binding is attenuated in 
hypercholesterolemic animals treated chronically with the NO precursor 
L-arginine; 3) monocyte binding to the endothelium is increased in vessels 
from normocholesterolemic animals treated with the NO synthase antagonist 
L-nitro-arginine; and 4) this effect of NO synthase antagonism was not 
reversed by administration of hydralazine in doses sufficient to normalize 
blood pressure. These findings are consistent with the hypothesis that NO 
inhibits monocyte-endothelial cell interaction. 
To conclude, an ex vivo model of monocyte binding has been used to study 
the increase in endothelial adhesiveness induced by hypercholesterolemia. 
Endothelial adhesiveness is attenuated by oral administration of the NO 
precursor L-arginine is shown. Conversely, inhibition of NO synthase 
activity by oral administration of nitro-arginine strikingly increases 
endothelial affinity for monocytes ex vivo. The data are consistent with 
NO being an endogenous anti-atherogenic molecule. 
EXAMPLE 5 
Oral Arginine causes regression of atherosclerosis in hypercholesterolemic 
rabbits 
Our previous work demonstrated that oral arginine could prevent the 
development of plaque in hypercholesterolemic animals but it was not known 
if pre-existing plaque could be affected by arginine treatment. This is 
clinically important if arginine is to be useful in the treatment of 
pre-existing atherosclerosis in humans. Accordingly, New Zealand white 
rabbits (n=85) received normal chow or 0.5% cholesterol chow for 10 weeks. 
Subsequently, half of the hypercholesterolemic rabbits were given 2.25% 
(W/V) L-arginine in their drinking water. Thoracic aortae were harvested 
at weeks 10, 14, 18, or 23. Rings of aorta were used to assess 
NO-dependent vasodilation to acetylcholine (ACh). Maximal relaxation to 
ACh in the hypercholesterolemic rabbits receiving vehicle (CHOL) became 
progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). 
Planimetry of the luminal surface of the aortae from CHOL animals revealed 
a progressive increase in plaque area from 30.3% (at week 10) to 56.5% (by 
week 23) of the total surface of the thoracic aorta. By contrast, 
hypercholesterolemic animals receiving arginine (ARG) manifested improved 
endothelium-dependent relaxation associated with a reduction of plaque 
area at 14 and 18 weeks. Lesion surface area in all arginine treated 
hypercholesterolemic animals (weeks 14-23) was significantly reduced in 
comparison to vehicle-treated hypercholesterolemic animals (FIG. 4). The 
arginine-induced improvement in endothelium-dependent relaxation was 
associated with an increased generation of vascular NO, and a reduced 
generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals 
had persistent improvement in NO-dependent vasodilation and exhibited a 
further reduction of plaque area to 5.4% 
Conclusions 
hypercholesterolemia induces a progressive loss of NO-dependent 
vasodilation associated with progressive intimal lesion formation. 
Administration of L-arginine to animals with pre-existing intimal lesions 
augments vascular NO elaboration, reduces superoxide anion generation, and 
is associated with a reduction in plaque area. This is the first 
demonstration that restoration of NO activity can induce regression of 
pre-existing intimal lesions, and provides evidence that L-arginine 
therapy may be of potential clinical benefit. 
EXAMPLE 6 
Oral arginine administration restores vascular NO activity and inhibits 
myointimal hyperplasia after balloon injury in hypercholesterolemic 
rabbits. 
Purpose. 
The purpose of this study was to determine if the alterations in vascular 
function and structure following balloon angioplasty in 
hypercholesterolemic rabbits could be inhibited by restoration of 
endogenous nitric oxide (NO) activity. 
Methods 
Twenty-eight New Zealand white rabbits were randomized into one of three 
dietary groups and received either normal rabbit chow, 0.5% cholesterol 
diet, or 0.5% cholesterol diet plus L-arginine hydrochloride (2.25% W/V) 
in the drinking water. After six weeks of dietary intervention, the left 
iliac artery of each animal was subjected to a balloon angioplasty. Four 
weeks later, the iliac arteries were harvested for vascular reactivity 
studies and immunohistochemistry. 
Results 
The bioassay studies indicated that endothelium-derived NO activity was 
inhibited in hypercholesterolemic animals in comparison to 
normocholesterolemic animals. The administration of arginine partially 
restored endothelium-derived NO activity. Balloon angioplasty induced 
intimal thickening which was largely composed of vascular smooth muscle 
cells and extracellular matrix. In the setting of hypercholesterolemia, 
vascular injury induced an exuberant myointimal lesion that was augmented 
by the accumulation of lipid-laden macrophages. Administration of 
L-arginine induced a quantitative as well as qualitative change in the 
lesion. Dietary arginine reduced intimal thickening in the injured vessels 
of hypercholesterolemic animals, and substantially inhibited the 
accumulation of macrophages in the lesion (FIG. 5). 
Conclusions 
We report that the lesions induced by balloon angioplasty in 
hypercholesterolemic animals are markedly reduced by oral administration 
of arginine. Moreover, we find that the nature of the lesion is altered, 
with a striking reduction in the percentage of macrophages comprising the 
lesion. Hypercholesterolemia induces an endothelial vasodilator 
dysfunction in the rabbit iliac artery that is reversible by chronic oral 
administration of arginine. 
EXAMPLE 7 
Nitric oxide regulates monocyte chemotactic protein-1 
Our previous studies had established that oral arginine administration 
could enhance vascular NO synthesis. This increase in vascular NO 
synthesis was associated with inhibition of monocyte adherence and 
accumulation in the vessel wall (thereby reducing the progression, and 
even inducing regression, of plaque). The question remained: "How does 
vascular nitric oxide inhibit monocyte adherence and accumulation in the 
vessel wall?" 
Monocyte chemotactic protein-1 (MCP-1) is a 76-amino acid chemokine thought 
to be the major chemotactic factor for monocytes (chemotactic factors are 
proteins that attract white blood cells). We hypothesized that the 
anti-atherogenic effect of NO may be due in part to its inhibition of 
MCP-1 expression. 
Methods and Results 
Smooth muscle cells (SMC) were isolated from normal rabbit aortae by 
explant method. Cells were then exposed to oxidized LDL (30 .mu.ml) (which 
is known to induce vascular cells to synthesize MCP-1). The expression of 
MCP-1 in SMC was associated with an increased generation of superoxide 
anion by the SMC, and increased activity of the transcriptional protein 
NF.kappa.B. All of these effects of oxidized LDL cholesterol were reduced 
by previous exposure of the SMC to the NO-donor DETA-NONOate (100 .mu.M) 
(p&lt;0.05). To determine if NO exerted its effect at a transcriptional 
level, SMC and COS cells were transfected with a 400 bp fragment of the 
MCP-1 promoter. Enhanced promoter activity by oxLDL was inhibited by 
DETA-NO. 
To investigate the role of endogenous NO in the regulation of MCP-1 in 
vivo, NZW rabbits were fed normal chow, normal chow plus nitro-L-arginine 
(L-NA) (to inhibit vascular NO synthesis), high cholesterol diet (Chol), 
or high cholesterol diet supplemented with L-arginine (Arg) (to enhance NO 
synthesis). After two weeks, thoracic aortae were harvested and total RNA 
was isolated. Northern analysis demonstrated increased expression of MCP-1 
in Chol and L-NA aortae; this expression was decreased in aortae from Arg 
animals. These studies indicate that the anti-atherogenic effect of NO may 
be mediated in part by its inhibition of MCP-1 expression. NO inhibits the 
generation of superoxide anion by the vascular cells and thereby turns off 
an oxidant-responsive transcriptional pathway (i.e. NF.kappa.B-mediated 
transcription) activating MCP-1 expression. 
EXAMPLE 8 
Nitric Oxide inhibits the expression of an endothelial adhesion molecule 
known to be involved in atherosclerosis 
Vascular cell adhesion molecule (VCAM-1) is an endothelial adhesion 
molecule that binds monocytes. This molecule is expressed by the 
endothelium of hypercholesterolemic animals, and is expressed by 
endothelial cells overlying plaque in animals and humans. This adhesion 
molecule is believed to participate in monocyte adherence and accumulation 
in the vessel wall during the development of plaque. Other workers have 
shown that the expression of this molecule is regulated by an 
oxidant-responsive transcriptional pathway mediated by the transcriptional 
factor NF.kappa.B. Endothelial cells exposed to oxidized LDL cholesterol 
(or cytokines like TNF-.alpha.) begin to generate superoxide anion. 
Superoxide anion turns on oxidant-responsive transcription leading to the 
expression of VCAM-1 and MCP-1 (and probably other genes that participate 
in atherosclerosis). Our data indicates that NO inhibits the generation of 
superoxide anion, thereby turning off these oxidant-responsive 
transcriptional pathways. 
Methods and Results 
Confluent monolayers of human aortic endothelial cells (HAEC) were exposed 
to static or fluid flow conditions for 4 hours (fluid flow stimulates the 
production of endogenous nitric oxide). Medium was then replaced and cells 
were then incubated with native LDL (50 .mu.ml), oxidized LDL (30 
.mu.g/ml), or LPS (10 ng/ml) +TNF-.alpha. (10 .mu.U/ml) for an additional 
4 hours. Functional binding assays utilizing THP-1 monocytes were then 
performed. Superoxide production by HAECs was monitored by lucigenin 
chemiluminescence and expression of the adhesion molecules VCAM-1 and 
ICAM-1 was quantitated by flow cytometry. Whereas native LDL had little 
effect, incubation with either oxLDL or LPS/TNF significantly increased 
superoxide production, NF-.kappa.B activity, VCAM-1 expression and 
endothelial adhesiveness for monocytes. Previous exposure to fluid flow 
inhibited endothelial adhesiveness for monocytes (FIG. 6) and the other 
sequelae of exposure to cytokines or oxidized lipoprotein. The effect of 
fluid flow was due to shear-induced release of nitric oxide since 
coincubation with L-nitro-arginine completely abolished these effects of 
flow. Furthermore, the NO donor PAPA-NONOate mimicked the effects of flow. 
Conclusions 
Previous exposure to fluid flow decreased cytokine or 
lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 
expression and monocyte binding; the effects of flow are due at least in 
part to nitric oxide. 
NO participates in the regulation of the endothelial generation of 
superoxide anion and thereby inhibits oxidant-responsive transcription of 
genes (i.e. VCAM-1 and MCP-1) that are involved in atherogenesis. 
EXAMPLE 9 
Transfection of the gene encoding NO synthase increases NO generation and 
inhibits monocyte adherence. 
The following experiment was done to determine if transfer of the gene 
encoding NO synthase (the enzyme that produces NO) could increase 
generation of nitric oxide and thereby inhibit monocyte adherence. 
Cultured endothelial cells (bEnd-3; a murine endothelial cell line) were 
transfected with a plasmid construct encoding the NO synthase gene, using 
lipofectamine liposomal technique. Forty-eight hours later, generation of 
nitric oxide was measured using chemiluminescence. Nitric oxide generation 
was increased 2-fold in cells transfected with the NO synthase construct 
(but not in cells transfected with a control construct). In parallel, 
binding assays were performed using a murine monocytoid cell line. The 
binding of monocytoid cells to the endothelial cells was reduced by 30% in 
those cells transfected with the NO synthase construct. 
Conclusion 
endothelial cells transfected with a plasmid construct containing the NO 
synthase gene were able to elaborate more nitric oxide. The increased 
elaboration of nitric oxide was associated with an inhibition of monocyte 
binding to the endothelial cells. 
EXAMPLE 10 
Effect of NO synthase expression on proliferation of vascular smooth muscle 
cells 
Cultured rat aortic vascular smooth muscle cells under confluent quiescent 
conditions were studied. An efficient viral coat protein-mediated DNA 
transfer method was employed to transfect the cells with the NO synthase 
gene driven by the .beta.-actin promoter and CMV enhancer. This resulted 
in increased NO synthase activity (as measured by the 
arginine-to-citrulline conversion assay) in comparison to control vector 
transfected cells. Transfection of the NO synthase gene completely 
abolished serum-stimulated DNA synthesis compared to control vector 
transfection. These results indicated that increased expression of NO 
synthase (associated with increased production of NO) inhibits excessive 
proliferation of vascular smooth muscle cells. This inhibition can be 
correlated with treatment of atherosclerosis and restenosis. 
EXAMPLE 11 
Gene Therapy Using NO Synthase cDNA Prevents Restenosis 
The study above indicated that NO inhibits proliferation of vascular smooth 
muscle cells. In atherogenesis and restenosis, excessive proliferation of 
vascular smooth muscle cells contributes to lesion formation. Injury to 
the endothelium in atherosclerosis and after catheter interventions 
apparently reduces or removes the salutary influence of NO. The following 
study shows delivery of the gene for NO synthase to the vessel wall 
inhibits lesion formation. 
A plasmid construct encoding the cDNA of endothelial-type NO synthase 
(EC-NOS) was synthesized. A full length cDNA encoding for EC-NOS was 
inserted into the EcoRI site of the pUCcaggs expression vector. Balloon 
angioplasties of the carotid artery in Sprague-Dawley rats were performed 
and HVJ-liposomes with plasmids encoding EC-NOS cDNA infused, or plasmids 
lacking EC-NOS cDNA (control vector) infused. After 4 days to 2 weeks, the 
rats were sacrificed and the carotid arteries harvested for: 1) 
histomorphometry; 2) measurement of DNA synthesis; and 3) ex vivo 
determination of NO synthesis and release by bioassay and by 
chemiluminescence. 
Results 
Morphometric measurements 2 weeks after injury revealed a significant (68%) 
reduction of intimal lesion thickness in EC-NOS treated (Inj+NOS) in 
comparison to control vector treated (Inj+CV) or untreated (Inj) injured 
vessels. (FIG. 7) Measurements of DNA synthesis were performed four days 
after injury using bromodeoxyuridine. EC-NOS transfection significantly 
limited bromodeoxyuridine incorporation (by 25%) in comparison to control 
vector treated or untreated injured vessels. Vessel segments were studied 
ex vivo using organ chamber technique to bioassay for NO release. Calcium 
ionophore increases intracellular calcium and activates NO synthase to 
produce NO. Calcium ionophore induced relaxations in injured carotid 
arteries transfected with control vector that were only 15% of uninjured 
vessels. Injured arteries that had been transfected with EC-NOS relaxed to 
a much greater degree, approximately 50% of that observed in uninjured 
vessels. Direct measurement of NO (by chemiluminescence) released into the 
medium revealed that NO released by injured tissues (transfected with the 
control vector) was only 20% of that released by normal uninjured tissues. 
By contrast, injured tissues transfected with EC-NOS released more NO 
(about 75% of normal). 
To conclude, balloon angioplasty of the rat carotid artery removes the 
endothelial source of NO, induces excessive vascular smooth muscle DNA 
synthesis and proliferation, resulting in an intimal lesion (restenosis). 
Transfection of the vessel with EC-NOS at the time of balloon injury 
partially restores NO production by the vessel, and this is associated 
with reduced DNA synthesis and vascular smooth muscle proliferation, 
thereby reducing lesion formation. These results are consistent with the 
conclusion that NO is an endogenous anti-atherogenic molecule. 
EXAMPLE 12 
Local application of L-arginine to the vessel wall inhibits myointimal 
hyperplasia 
The previous studies revealed that oral administration of arginine could 
enhance vascular NO activity and inhibit lesion formation induced by a 
high cholesterol diet and/or vascular injury (with balloon angioplasty). 
To determine if intraluminal application of arginine to the vessel wall at 
the time of balloon angioplasty could inhibit lesion formation, the 
following study was performed. Rabbits (n=7) were fed a 1% cholesterol 
diet. After one week, angioplasty of the iliac arteries was performed. 
After angioplasty of one iliac artery, a local infusion catheter was used 
to expose the injured area to a high concentration of arginine (6 mM). The 
other iliac artery was subjected to balloon angioplasty, but not treated 
with a local infusion. After four weeks, the vessels were harvested, and 
segments of the arteries processed for histomorphometry. Initial 
thickening in the arginine-treated vessels was significantly reduced (FIG. 
8). This study indicates that the local intraluminal application of high 
doses of arginine can reduce myointimal hyperplasia after vascular injury. 
EXAMPLE 13 
Exclusion of the Effect of Enhanced Nitrogen or Caloric Balance as Causing 
the Observed Results 
To exclude an effect of L-arginine on nitrogen or caloric balance as the 
cause of these results, six animals received 1% cholesterol diet 
supplemented by additional methionine to increase the dietary methionine 
six-fold. At ten weeks animals were sacrificed for studies of platelet and 
vascular reactivity, and histomorphometry. Endothelium-dependent 
relaxation, platelet aggregation and intimal thickness were not different 
from those of animals fed 1% cholesterol diet alone. These results reveal 
that another amino acid, methionine (which is not a precursor of NO) does 
not mimic the effect of the amino acid L-arginine. Therefore it seems 
likely that the effect of L-arginine is due to its metabolism to nitric 
oxide, rather than some other effect of amino acid administration (i.e. 
change in nitrogen or caloric balance). 
EXAMPLE 14 
L-lysine enhances vascular NO activity and inhibits atherogenesis 
L-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 white rabbits were fed a normal or high 
cholesterol chow (n=18). Half of the animals on the cholesterol diet also 
received oral L-lysine. After ten weeks, the thoracic aortae were 
harvested and bioassayed for vascular NO synthesis, and histomorphometry 
to assess lesion formation was performed as described above. The 
administration of L-lysine was just as effective as L-arginine to increase 
vascular NO activity in the hypercholesterolemic animals as assessed by 
endothelium-dependent vasorelaxation. (FIG. 9) The improvement in vascular 
NO activity was associated with a marked reduction in vascular lesion 
formation. 
This study revealed the unexpected result that L-lysine can enhance 
vascular NO activity and inhibit atherosclerosis. 
EXAMPLE 15 
Oral L-arginine normalizes monocyte adhesiveness in hypercholesterolemic 
humans 
Adherence of monocytes to the endothelium is the first observable event in 
the development of atherosclerosis. We hypothesized that chronic oral 
administration of L-arginine to hypercholesterolemic humans would enhance 
the generation of endothelium-derived NO, and thereby inhibit the 
interaction of monocytes with the endothelium. In this investigation we 
have developed a reproducible assay for the binding of human monocytes to 
cultured endothelial cells, and we have examined the effect of 
hypercholesterolemia and L-arginine treatment on this interaction. 
The control subject population in this study included 12 normal volunteers, 
(10 males and 2 females), with an average age of 37.+-.2 yrs. Normalcy was 
determined by a careful history, physical examination, and laboratory 
analysis to exclude individuals with hematologic, renal, or hepatic 
dysfunction or clinically evident atherosclerosis. There were 20 patients 
(10 males and 10 females) with hypercholesterolemia as defined by a total 
plasma cholesterol greater than 240 mg/dl and a LDL cholesterol level 
greater than 160 mg/dl. These individuals had an average age of 51.+-.2 
yrs. None of the subjects were taking diuretics, vasoactive medications, 
antiplatelet or hypolipidemic medications. This study was approved by the 
Stanford University Administrative Panel on Human Subjects in Medical 
Research and each subject gave written informed consent before entry into 
the study. Blood was drawn from each subject in the postabsorptive state. 
We isolated human monocytes from citrated venous blood. The blood was 
centrifuged and the buffy coat removed and resuspended with HBSS. The 
suspension was then carefully layered onto a cushion of 1.068-d 
Histopaque, and centrifuged. After centrifugation, the monocytes were 
aspirated. 
We used the transformed endothelial cell (EC) line, bEnd3 to examine 
monocyte-endothelial binding ex vivo. The bEnd3 cells express endothelial 
adhesion molecules and bind monocytes in a cytokine-inducible fashion with 
kinetics similar to those observed with human umbilical vein endothelium. 
Monocytes were added to the wells containing the endothelial monolayers to 
reach a final cell number of 3.times.10.sup.6 /ml. In some studies, 
monocytes were exposed in vitro for 30 minutes to sodium nitroprusside (an 
NO donor) prior to the binding assay. 
The six-well plates were transferred to a rocking platform and rocked for 
30 minutes at room temperature. After 30 minutes, the cell suspension was 
aspirated from each well and wells were then rinsed with binding buffer to 
remove non-adherent monocytes. Videomicroscopic counting of adherent cells 
was performed using a computer aided image analysis system. 
Results 
Oral administration of L-arginine (7 g daily for 2 weeks) to 
hypercholesterolemic humans increased plasma arginine values by 60% (from 
79.+-.10 to 128.+-.12 mM; n=7), whereas L-arginine values in the 
placebo-treated (n=3) and normocholesterolemic (n=6) groups remained 
unchanged. The administration of oral L-arginine had no effect on any of 
the biochemical or hematologic parameters and was well tolerated. Oral 
L-arginine did not lower total cholesterol or LDL cholesterol. Two 
patients dropped out of the study; one because he did not want to take the 
pills, and one because of reactivation of oral herpes during the study. 
The results of the adhesion assays were highly reproducible. Monocytes 
derived from hypercholesterolemic individuals demonstrated a 50.+-.8% 
increase in bound cells/hpf in comparison to cells from normal individuals 
(p&lt;0.0001). The degree of adhesiveness was correlated to the plasma levels 
of LDL cholesterol (R=0.7, n=33; p&lt;0.0001; FIG. 10). 
In an open-label study, 3 hypercholesterolemic individuals were treated 
with oral L-arginine supplementation for 2 weeks. Arginine treatment 
resulted in a 38% decrease in monocyte adhesiveness. 
To confirm this effect of L-arginine treatment and to control for any 
experimental bias, a double-blinded, placebo-controlled, randomized study 
was performed. Ten hypercholesterolemic subjects were randomized (1:2) to 
placebo or L-arginine treatment; 6 normocholesterolemic individuals were 
studied in parallel to control for variation over time in the binding 
assay. At baseline, the adhesion of monocytes from both 
hypercholesterolemic groups was increased in comparison to the 
normocholesterolemic individuals (p&lt;0.001). After 2 weeks of L-arginine 
administration, there was an absolute reduction of 53% in monocyte binding 
(n=7, p&lt;0.005, baseline vs 2 weeks) (FIG. 11). By contrast, there was no 
significant change in the adhesiveness of monocytes isolated from 
hypercholesterolemic individuals treated with placebo. Two weeks after 
discontinuation of the L-arginine treatment, the adhesiveness of the 
monocytes isolated from hypercholesterolemic individuals had significantly 
increased compared to the normocholesterolemic individuals (34.+-.9% 
increase in bound cells/hpf; p&lt;0.05), and was also significantly increased 
in comparison to the binding obtained after 2 weeks of L-arginine therapy 
(an increase of 30.+-.9%, p&lt;0.05). The adhesiveness of monocytes from 
placebo-treated hypercholesterolemic individuals did not change 
significantly 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 individuals with the NO donor sodium nitroprusside 
(10.sup.-5 M) markedly reduced binding (164.+-.9% vs 98.+-.7% vehicle vs 
sodium nitroprusside; n=7, p&lt;0.0005; values expressed as a percent of the 
normocholesterolemic control exposed to vehicle; FIG. 12). 
To conclude, the salient findings of this investigation are that: 1) 
Hypercholesterolemia enhances the adhesiveness of monocytes for 
endothelial cells, 2) oral arginine supplementation reverses the increase 
in adhesiveness of monocytes from hypercholesterolemic individuals, and 3) 
the effect of oral arginine is mimicked in vitro by exposure of the 
monocytes from hypercholesterolemic individuals to sodium nitroprusside, 
an NO donor. 
EXAMPLE 16 
Platelet Hyperaggregability in Hypercholesterolemic Humans 
Reversal by Oral L-Arginine 
In this study we tested the hypothesis that chronic L-arginine 
supplementation would inhibit platelet reactivity in hypercholesterolemic 
humans. Venous blood was collected from normal (NC; n=11) and 
hypercholesterolemic (HC; n=22) volunteers for isolation of platelet-rich 
plasma and aggregometry. Half the HC group received L-arginine (7 g/d) for 
2 weeks; aggregometry was performed using collagen (5 mg/ml) before and 
after two weeks of treatment. 
Results 
HC platelets were hyperaggregable. After two weeks of L-arginine, the 
aggregability of HC platelets was reduced (FIG. 13). These studies are 
consistent with our previous observations in animals that oral 
administration of L-arginine inhibits platelet reactivity. 
EXAMPLE 17 
Intravenous Administration of L-Arginine Improves Endothelium-dependent 
Vasodilation in Hypercholesterolemic Humans 
Hyperlipoproteinemia impairs endothelium-dependent vasodilation, even 
before the development of atherosclerosis. We hypothesized that 
administration of L-arginine may increase synthesis of NO and thereby 
improve endothelium-dependent vasodilation in hypercholesterolemia. 
Indeed, our earlier studies conducted in cholesterol-fed rabbits support 
this notion. The following data demonstrates that L-arginine augments 
endothelium-dependent vasodilation in forearm resistance vessels of 
hypercholesterolemic humans. 
The control subject population in this study included 11 normal volunteers 
comprising (10 males and 1 female). Their ages ranged from 31 to 49 and 
averaged 39.+-.2 yr. There were 14 patients with hypercholesterolemia. 
Hypercholesterolemia was defined as a serum LDL cholesterol level greater 
than the 75th percentile adjusted for age and sex. These individuals 
included 11 males and 3 females whose ages ranged from 22 to 48 and 
averaged 38.+-.2 years. 
Under local anesthesia and sterile conditions, a polyethylene catheter was 
inserted into a brachial artery of each subject for determination of blood 
pressure and for infusion of drugs. A separate polyethylene catheter was 
inserted into the antecubital vein for infusion of L-arginine. Bilateral 
forearm blood flow was determined by venous occlusion strain gauge 
plethysmography, using calibrated mercury-in-silastic strain gauges, and 
expressed as ml/100 ml tissue per min. 
To assess NO-dependent vasodilation, methacholine chloride (which induces 
the endothelium to release NO) was administered via the brachial artery. 
Forearm blood flow was measured during infusion of methacholine chloride 
at concentrations of 0.3, 3, and 10 .mu.g/min each for 3 min. 
After completion of the methacholine chloride infusions, all normal 
subjects and 10 individuals with hypercholesterolemia were given 
L-arginine intravenously over 30 minutes and then the methacholine 
infusions were repeated. D-arginine, the enantiomer of L-arginine, is not 
a precursor of NO. Thus, to ensure that any observed effects of L-arginine 
were due to its contribution to the synthesis of NO and not just secondary 
to its physiochemical properties, five individuals with 
hypercholesterolemia received D-arginine intravenously. 
Results 
Baseline blood pressure, heart rate, and forearm blood flow did not differ 
between normal and hypercholesterolemic subjects. Intraarterial infusion 
of methacholine chloride caused a dose-dependent increase in forearm blood 
flow. In the hypercholesterolemic subjects, however, cholinergic 
vasodilation was less than that of normal subjects (p&lt;0.05). The maximal 
forearm blood flow response to methacholine in normal subjects is 
19.0.+-.1.9 ml/100 ml of tissue per min, and in hypercholesterolemic 
subjects, it was 13.7.+-.1.7 ml/100 ml of tissue per min (p&lt;0.05). 
In the normal subjects, L-arginine did not potentiate the vasodilation that 
occurred during the administration of methacholine chloride. In the 
hypercholesterolemic subjects, however, the L-arginine infusion augmented 
the vasodilation to methacholine chloride by 25% (p&lt;0.05). There were no 
complications or side-effects of the L-arginine infusions. 
The important findings in this study are: (a) endothelium-dependent 
vasodilation (due to the release of NO) is reduced in forearm resistance 
vessels of hypercholesterolemic humans; and (b) intravenous administration 
of L-arginine improves endothelium-dependent vasodilation in these 
individuals. NO not only causes vasodilation, but it also inhibits 
platelet aggregation and suppresses monocyte adhesion in 
hypercholesterolemic humans. 
EXAMPLE 18 
Administration of Intravenous L-Arginine Improves Coronary Endothelial 
Function in Cardiac Transplant Recipients 
A reduction in coronary NO-dependent vasodilation occurs in cardiac 
transplant recipients and may represent an early marker for the 
development of graft atherosclerosis. Reduced NO-dependent vasodilation in 
response to acetylcholine is an indicator of endothelial dysfunction and 
has been attributed to reduced synthesis or accelerated degradation of 
endothelium-derived nitric oxide. We hypothesized that endothelial 
dysfunction of epicardial coronary arteries at an early stage of coronary 
allograft atherosclerosis might be reversed by L-arginine. The present 
study tested the hypothesis that administration of L-arginine, the 
precursor of endothelium-derived NO, improves endothelial vasodilator 
function of coronary conduit and resistance vessels. 
Cardiac transplant recipients scheduled for elective annual coronary 
angiography at Stanford University hospital were screened for possible 
participation in the study. The study protocol was approved by the 
Stanford University Committee on Human Subjects in Medical Research. All 
patients gave written informed consent. Eighteen patients who had cardiac 
transplantation 1 to 13 years previously were studied. 
Vasoactive medications were discontinued at least 12 hours before the 
study. After diagnostic angiography revealed no visually apparent coronary 
stenosis, a guiding catheter was used to cannulate the left main coronary 
artery. An infusion catheter was then advanced over a Doppler flow 
velocity guide wire into a nonbranching segment of the coronary artery for 
infusion of acetylcholine (which stimulates the endothelium to release 
NO). After baseline angiography was performed, increasing concentrations 
of acetylcholine were serially infused over 3 minutes. Infusion of 
acetylcholine continued until the maximum dose (10.sup.-4 mol/L) was 
reached or until total coronary occlusion occurred. Then an intravenous 
infusion of L-arginine (30 g over 15 minutes) was performed. Thereafter, 
the intracoronary infusion of acetylcholine was repeated. Coronary 
angiography and Doppler flow velocity recording was performed at the end 
of the L-arginine infusion and after the infusion of each concentration of 
acetylcholine. 
Results 
In epicardial coronary arteries of these transplant recipients, 
acetylcholine caused vasoconstriction. Epicardial coronary 
vasoconstriction caused by acetylcholine was attenuated by infusion of 
L-arginine (10.sup.-4 mol/L, -6.8% versus -2.8%; p&lt;0.01). In coronary 
resistance vessels, acetylcholine induced vasodilation, reflected by 
increases in blood flow. The increase in coronary blood flow was 
significantly enhanced with L-arginine (p&lt;0.002; FIG. 14). There were no 
complications or side-effects of the L-arginine infusion. 
The coronary vasculature of cardiac transplant recipients exhibits a 
generalized reduction of NO-dependent vasodilation. L-arginine improves 
endothelial-derived NO dependent vasodilation of both coronary 
microvasculature and epicardial coronary arteries. 
It is evident from the above results, that by enhancing the nitric oxide 
levels, by means of nitric oxide precursor compounds or other compounds in 
the nitric oxide pathway, substantial benefits will ensue to patients with 
vascular degenerative diseases. This treatment will restore normal 
vascular tone (preventing excessive vasoconstriction and elevation of 
blood pressure; and will improve blood flow to the heart, brain, and other 
critical tissues thereby enhancing exercise tolerance and relieving 
symptoms such as angina or cerebral ischemia); and will diminish the 
formation of atherosclerotic plaque and restenosis (by inhibiting adhesion 
of monocytes and platelets, and by reducing the proliferation of vascular 
smooth muscle cells). Benefits may also ensue to normal individuals, 
because NO is critically involved in exercise-mediated vasodilation, an 
enhancement of NO synthesis could improve blood flow and exercise capacity 
even in normal individuals. 
By virtue of administering to the host, based on a predetermined regimen, 
or providing in the host a supply of a component in the synthetic pathway 
for production of nitric oxide, so as to maintain a mildly elevated level 
of nitric oxide in the host, particularly at the site to be treated, the 
incidence of plaque formation can be substantially diminished. This can be 
achieved in a variety of ways: by oral administration in accordance with a 
predetermined regimen of various compounds associated with nitric oxide 
formation, e.g. L-arginine and/or L-lysine; by administration at the site, 
in a predetermined regimen of compounds which can produce nitric oxide, 
either directly or as a result of physiologic action of endogenous 
compounds, e.g. enzymes; by employing combinations of compounds, which by 
their action result in the production of nitric oxide; or the like. These 
individual administrations, can be done independently or in conjunction 
with a regimen of other compounds associated with the production of nitric 
oxide. 
Alternatively, one may use genetic engineering to introduce a gene 
associated with a component in the synthetic pathway for production of 
nitric oxide, e.g. nitric oxide synthase, where the enhanced production of 
such compounds will have the effect of driving the equilibrium to an 
enhanced production of nitric oxide. Thus, the subject invention provides 
a plurality of pathways to enhance the synthesis or action of nitric 
oxide, or reduce the degradation of nitric oxide, thereby increasing the 
effect of endogenous nitric oxide to prevent the formation of vascular 
lesions and to inhibit restenosis. 
All publications and patent applications cited in this specification are 
herein incorporated by reference as if each individual publication or 
patent application were specifically and individually indicated to be 
incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be readily apparent to those of ordinary skill in the art in light of 
the teachings of this invention that certain changes and modifications may 
be made thereto without departing from the spirit or scope of the appended 
claims.