Nitric oxide: actions and roles in arthritis and diabetes

The Foot (2001) 11, 45±51 ß 2001 Harcourt Publishers Ltd doi: 10.1054/foot.2000.0641, available online at http://www.idealibrary.com on

REVIEW ARTICLE

Nitric oxide: actions and roles in arthritis and diabetes M. M. Chan,* J. A. Mattiacci{ *Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA; {Office of the Dean, Temple University School of Podiatric Medicine, Philadelphia, Pennsylvania,USA SUMMARY. Nitric oxide (NO) plays physiological and pathophysiological roles as vasodilator, neurotransmitter, anti-microbial effector molecule, and immunomodulator. Of interest to podiatric medicine is the growing evidence implicating NO in inflammation, arthritis, diabetes, vascular complications and wound healing. Release of NO and formation of peroxynitrite in chronic inflammation have been linked to destruction of chondrocytes, endothelium and pancreatic islet cells. NO may be a potential target for pharmacologic intervention and gene therapy. ß 2001 Harcourt Publishers Ltd

undesired in¯ammation has been linked to tissue damage in the pathogenesis of autoimmune diseases. Here, we discuss the role of NO and the enzymes nitric oxide synthases (NOSs) that produce the molecule in two of such diseases, arthritis and diabetes, which are major concerns of podiatric medicine.2,3

INTRODUCTION In 1998, the Nobel Prize in physiology or medicine was awarded to the seminal discoveries on nitric oxide (NO), subsequent to the naming of NO as `molecule of the year' in 1992 by the journal Science. Nitric oxide is a simple gaseous molecule that consists of an atom of nitrogen and an atom of oxygen, similar to carbon monoxide (Fig. 1). Nonetheless, it is a crucial participant in many vital physiological functions, although it was once considered a noxious pollutant from car emissions. It regulates blood pressure, as endothelial cells produce NO to relax vascular smooth muscle cells, and it also transmits impulse, functioning as a messenger in the nervous system. In the past, it had been known as the endotheliumderived relaxing factor (EDRF). NO molecules are radicals, for they have one unpaired electron (Fig. 1). Although the molecules themselves are stable in the anaerobic liquid phase, in the presence of oxygen free radicals they form reactive nitrogen intermediates (RNIs), such as peroxynitrite and hydroxyl radical. These oxidants are highly reactive and cytotoxic.1 In fact, the immune system produces these molecules as a weapon for destroying infectious microbes: bacteria, yeasts, fungi and parasites in various tissues. Unfortunately, their action is double-edged. Release of NO at a high level during

NITRIC OXIDE SYNTHASES NO is produced by the enzyme nitric oxide synthase (NOS), which is synthesized as three isoforms in different cell types. They have been named according to the order of discovery by gene cloning as NOS1, NOS2 and NOS3 (Table 1). NOS1 was identi®ed from neuronal samples and is also known as nNOS (`n' for neuronal). Many tissues, including macrophages, hepatocytes, chondrocytes and pancreatic islet cells, synthesize the second form, NOS2.4±6 Its production is dependent upon stimulation by external agents; thus, the isoform is also called iNOS (`i' for inducible). NOS3 was initially cloned from endothelial samples and hence also named eNOS (`e' for endothelial). In contrast to iNOS, the genes for NOS1 and NOS3 are always turned on for protein synthesis independent of external stimuli, so they are also referred to as ncNOS and ecNOS (`c' for constitutive). The classi®cation of isoforms soon became increasingly dif®cult as more variations were discovered. For example, studies reveal that endothelial cells can be activated to produce iNOS.7 Isoforms resembling nNOS are found in chondrocytes, pancreatic islet cells, and skeletal muscle cells. Moreover, in addition

Correspondence to: Dr Marion M. Chan, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA. Tel: ‡1 215 625 5270; Fax: ‡1 215 707 7788; E-mail: [email protected] 45

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The Foot in the hydrophilic cytosol. Correspondingly, gene sequences for the various NOS isoforms are only identical by 50±60%. The principal difference among the NOS isoforms, however, lies in how they are regulated. The nNOS and eNOS isoforms are controlled at the level of enzyme activity and the activating stimulant is in¯ux of calcium into the cells (Fig. 3). The enzymes are presynthesized and always present in neuronal and endothelial cells. However, they are inactive and do not produce NO until calcium becomes available and binds to their calmodulin co-factor. Thus, they have also been referred to as the calcium-dependent isoforms. In contrast, iNOS is regulated at the level of gene expression. External stimuli, microbial components and proteins that activate in¯ammation, turn on the gene that synthesizes the iNOS enzyme. Once synthesized, the enzyme binds calcium tightly (also via calmodulin) and produces NO without the need for further in¯ux of the ion. It is known as the calcium-independent NOS isoform.12 The isoforms also differ in the quantity of NO they produce. The calcium-dependent nNOS and eNOS produce NO in low amounts (picomolar) that are suf®cient for mediating homeostatic physiological functions, i.e. dilation of blood vessels and transmission of nerve impulses. The calcium-independent iNOS isoform produces NO in high amounts (micromolar), so that there are suf®cient free radicals to destroy invading pathogens. This characteristic corresponds to the fact that its gene expression is activated by lipopolysaccharides from cell wall of Gram-negative bacteria and by in¯ammation potentiating proteins, such as the cytokines, tumor necrosis factor- and interleukin-1. It is well established that NO is crucial for protection against many bacteria, fungi and parasites.13

N=O Nitric oxide Fig. 1

Nitric oxide.

Table 1 Isoforms of NOS Endothelial NOS (eNOS, ecNOS, NOS3) Neuronal NOS (nNOS, ncNOS, NOS1) Inducible NOS (iNOS, NOS2)

Regulated by Ca‡‡

Low output

‡‡

Regulated by Ca

Low output

Regulated by gene expression

High output

to nNOS, eNOS is also present in a cell population in certain areas of the brain.5,6 All NOS enzymes are relatively large proteins that resemble cytochrome P450 structurally. They are homodimers composed of two identical peptides, each peptide containing an oxygenase domain and a reductase domain. At the oxygenase domain, there are binding sites for heme, tetrahydrobiopterin, and arginine. At the reductase domain, there are binding sites for the co-factors, ¯avin adenine dinucleotide (FAD), ¯avin mononucleotide (FMN), and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH).8 The peptides are held together at the oxygenase domain to form an active enzyme (Fig. 2). Thus the activity of the enzymes can be regulated by the bioavailability of tetrahydrobiopterin and arginine.9,10 Functionally, NOS enzymes catalyze the conversion of the substrate, arginine, to the products, NO and citrulline. In the presence of oxygen and NADPH, the terminal guanidinium nitrogen of arginine is converted into a gaseous NO radical, via ®ve-electron oxidative reaction carried through the co-factors. In spite of these similarities, the isoforms have differences. They range in size from a typical nNOS with 1433 amino acid residues, to eNOS with 1203, and to iNOS with 1153 residues. The neuronal isoform has a recognition site for protein±protein interaction that is not shared by the other two isoforms.11 While eNOS contains amino acids that direct it to bind to the plasma membrane via covalent attachment to lipid chains, nNOS and iNOS do not and they are located

NITRIC OXIDE ACTION As a gaseous molecule, NO diffuses freely into the plasma membrane and the cytosol. It exists in three forms: NO.; NO‡ when it loses the unpaired electron to become a nitrosonium cation; and NOÿ when Calmodulin

H4B

FMN FAD Heme

Fe

Arginine H4B Fig. 2

Nitric oxide synthase homodimer.

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Roles of NO in arthritis and diabetes 47 (A) eNOS Calcium

NO•

NO•

NO•

(B) iNOS

NO• NO• Microbes NO– NO•

Inflammation

NO• Fig. 3 Calcium-dependent and calcium-independent regulated NOS activity. The dark solid bars represent presence of the enzyme in the cell.

Reactive nitrogen species

Lipids

Proteins (aromatic, amino and -SH groups, Fe-clusters)

DNA

Fig. 4 Damaging actions of nitric oxide and reactive nitrogen intermediates.

it gains an electron to form nitrosyl anion. NO. molecules are very reactive and readily interact with reactive oxygen molecules. For example, it can react with superoxide anions (O2. ÿ) and hydroxyl radicals (OH.) at diffusion rate to form peroxynitrite and various reactive nitrogen intermediates (RNIs), which can rearrange into nitrate, toxic hydroxyl radicals, nitrous oxide and peroxynitrous acid.14 This arsenal of products may cause cellular damage through disruption of cellular membrane, mutation of DNA and inactivation of proteins (Fig. 4). Among them, one of the strongest oxidizing and nitrating agents is peroxynitrite (ONOOÿ), whose formation is favoured when NO and superoxide are synthesized concomitantly, e.g. during in¯ammation. This molecule can destroy protein structure by converting tyrosine to 3-nitrotyrosine and reacting with the sulfur hydryl (-SH) groups on cysteine to destroy the disul®de bond. It can also ß 2001 Harcourt Publishers Ltd

inactivate enzymes that are essential for basal cellular metabolism by reacting with transition metal ions, e.g. those on glyceraldehyde-3-phosphate dehydrogenase and iron-sulfur proteins in the mitochondria.1,13±17 Peroxynitrite-mediated damages have been linked to death of neuronal cells in Alzheimer's, chondrocytes in rheumatoid arthritis, endothelial cells in athersclerosis, and epithelial cells in lung injury.18±23 Paradoxically, there are indications that NO may also be protective. The action of NO is biphasic, triggering responses differently depending on the NO./O2. ÿratio. When in excess, NO can attenuate peroxynitrite and superoxide-mediated damages. Moreover, it can scavenge peroxyl and alkoxyl radicals and inhibit lipid peroxidation in some systems by removing chain propagation.12 Furthermore, when high amount of NO accumulates during the in¯ammation processes, it may play a role in turning off the The Foot (2001) 11, 45 ± 51

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response by feedback inhibition, inactivating the proteins that trigger its production.14,24 NITRIC OXIDE IN ARTHRITIS Increasing evidence indicates that iNOS may contribute to the pathophysiology of joint disorders,22±24 including rheumatoid arthritis (RA) and osteoarthritis (OA). RA is an autoimmune disorder manifested as in¯ammation and erosion of peripheral joints. NO is over-produced in affected joints from patients with RA. Compared with normal individuals, the level of NO is elevated in synovial ¯uid and serum samples from these patients.25 Immunohistochemistry shows that iNOS is present at a high level in their cartilage and synovia of the joints. The cells that produce NO have been identi®ed as the in®ltrating leukocytes, endothelial cells of the blood vessel, ®broblasts of the synovial membrane, and chondrocytes embedded in the cartilage.26 OA is a manifestation of age-related joint impairment due to progressive loss of articular cartilage. Similar to RA, ¯uid from affected joints of osteoarthritic patients also has abnormally high levels of NO. In¯ammation leads to the pro-in¯ammatory cytokines, interleukin-1, interferon- and tumour necrosis factor-, which, in combination, activates the chondrocytes, cells that are responsible for the synthesis and maintenance of cartilage matrix, in the joints to turn on the gene for iNOS.27 It is well established that interleukin-1 induces catabolism and inhibits anabolism in chondrocytes. Recent studies showed that NO is one of the key molecules that affect the action of interleukin-1. NO mediates its destructive actions on cartilage matrix and joints by several modes of action. NO molecules bind to the enzyme cyclooxygenase and enhance its production of arachidonic acid metabolites.28,29 One group of these metabolites is made up of the prostaglandins that induce pain and enhance the production of metalloproteinases, another group of enzymes that destroy cartilage by degrading collagen and proteoglycan. Cyclooxygenase is a target of many nonsteroid antiin¯ammatory drugs (NSAIDs). However, the most detrimental effect of NO is the induction of apoptotic death of chondrocytes.30 Evidently, apoptotic bodies, breakdown products from dead cells, are found in acellular calci®ed cartilage. On the other hand, with respect to the anabolic actions, studies on chondrocytes have shown that interleukin-1 inhibits the synthesis of proteoglycan, a component of cartilage that is constantly being turned over by chondrocytes in the joints. Loss of proteoglycan will lead to cartilage dysfunction. Reducing NO production by adding NOS inhibitors (N-monomethyl-L-arginine and Niminoethyl-L-ornithine) can partially recovered proteoglycan synthesis, as shown in a rat model.31 Finally, the most de®nitive evidence indicating that iNOS plays an essential role in the destruction of The Foot (2001) 11, 45 ± 51

cartilage comes from experiments employing mice whose gene for encoding the enzyme has been deleted (iNOS knockout mice). Without iNOS, injection of interleukin-1 or zymosan fails to cause arthritis in these mice; they do not lose proteoglycan or cartilage after induction by these stimuli.32 Nonetheless, NO is not unequivocally detrimental in all forms of arthritis. Bacterial septic arthritis induced by Staphylococcus aureus is exacerbated by inhibition of NO, since NO is needed for anti-microbial defense.33,34 In addition, arthritis associated with the disease systemic lupus erythematosus is not reduced by removal of the iNOS gene from disease prone mice (MRL-lpr/lpr) although iNOS inhibitors attenuate the condition.35,36 The reason for this discrepancy remains to be determined and is being actively investigated. NITRIC OXIDE IN DIABETES Diabetes is de®ned by the presence of high levels of sugars in the blood stream and urine due to defects in insulin metabolism. In individuals who suffer from insulin-dependent diabetes mellitus (IDDM, type I diabetes), iNOS has also been implicated in the destruction of pancreatic islet cells. In this disease, the immune system mistakenly reacts to self-molecules and destroys the insulin-producing beta cells in islets of Langerhans. The events in the diabetic pancreas are similar to those in the arthritic joints. Autoimmune responses lead to chronic in¯ammation and macrophages in®ltrate the in¯amed islets and secrete interlelukin-1 and NO. As with chondrocytes, interleukin-1 stimulates the islet cells to produce NO and the NO and RNI from macrophages, as well as from islet cells themselves, induces DNA damage and apoptotic death of the beta cells, depriving the patients of the ability to synthesize insulin.37,38 Studies with explanted pancreas and animal models support the hypothesis. When human islets are isolated and cultured with a mixture of cytokines (interleukin-1, tumour necrosis factor- and interferon- ), they synthesize iNOS, produce NO, and correspondingly have damaged DNA.39 In animals, evidently, a strain of diabetes-prone rats (Biobreeding rats) overproduces iNOS,40 and mice engineered to produce an exceedingly high level of iNOS in the pancreas (iNOS transgenic mice) develop IDDM.41 Conversely, mice genetically unable to synthesize iNOS (iNOS knockout mice) do not develop diabetes after receiving interleukin-1 or streptozotocin, a compound commonly used to induce this disease in rats and mice. Correspondingly, they have a lower level of hyperglycemia than the wild type mice.42,43 Diabetic patients are under increased oxidative stress for excess glucose molecules form toxic aldehydes that slowly glycosylate biomolecules, proteins, lipids and DNA.14,44 In hyperglycaemia, glycosylated ß 2001 Harcourt Publishers Ltd

Roles of NO in arthritis and diabetes 49 end-products induce production of oxygen free radicals.45,46 These radicals may lead to loss of control over the NO production pathway and hinder endothelium-dependent relaxation, thus inducing microvascular impairment and neuropathy which cause damages in many organs, including the eye, kidney and cardiovascular system in both type I and type II (non-insulin-dependent) diabetes.47,48 In the cardiovascular system, for normal homeostasis, it is vital that the endothelium can generate NO in small amounts through the eNOS isoform to maintain desired vessel dilation and to inhibit aggregation of leukocytes and platelets onto the vascular wall. Superoxides and hydroxyl radicals can react with NO at diffusion rate, preventing NO from reaching the smooth muscle cells to promote vessel dilation. Consequently, the level of NO that supplies the microcirculation of the organs of diabetic individuals is de®cient, leading to impaired vasodilation (hypertension), and enhanced the attachment of platelets and leukocytes to the vessel wall (initiation of atherosclerosis).14,44,48±50 Atherosclerosis is another major complication in diabetics. At the in¯amed sites, interestingly, gene expression of the high output isoform, iNOS, is increased in blood vessels even though eNOS activity is decreased. Activated macrophages in®ltrate the intima of the blood vessels and induce endothelial damage by producing pro-in¯ammatory cytokines that further promote attachment and in®ltration of more leukocytes in a cyclic manner, as well as producing superoxide and NO to form peroxynitrite (Fig. 4), and thus impairing NO-mediated endothelium-dependent relaxation, decreasing blood ¯ow, and exacerbating hypertension and atherosclerosis.7 Supplementing the diet with arginine has been shown to improve endothelial dysfunction in animals and man.49,51±55 Increasing the intake of arginine, the substrate, increases the synthesis of NO by eNOS and replenishes the molecules that are removed by reaction with other free radicals, such as superoxides. Similarly, administration of sodium nitroprusside, a compound that liberates NO,56 can reverse hypertension, whereas, on the other hand, depletion of arginine by arginase, an arginine-metabolizing enzyme that competes with eNOS for the amino acid, has the opposite effect. It has been shown that arginase activity is increased in macrophages and endothelial cells after experimental activation with Gram-negative bacterial lipopolysaccharide, in diabetic foot ulcers of humans, and in endothelium of diabetic rats.57±59 Diabetic animals have been found to be de®cient in the NOS enzyme cofactor tetrahydrobiopterin. Another approach is to increase its bioavailibity by dietary supplementation with its precursors. Increasing the level of tetrahydrobiopterin has been shown to increase NO production, alter NO./O2. ± ratio, reduce superoxide-mediated hypertension, and decrease phagocyte adherence to blood vessel. Tetrahydrobiopterin has a strong ß 2001 Harcourt Publishers Ltd

scavenging activity; it may also remove superoxide anion produced from the dysfunctional endothelium, thus reducing the formation of peroxynitrite.60,61 DIABETIC FOOT ULCERS As in the microcirculation of other organs, decreased eNOS and increased iNOS activity occur in diabetic foot ulcers. Chronic foot ulcer is a major problem in 15% of diabetic patients. The condition may be so severe as to require amputation of the foot in 12±24% of the affected. Both peripheral neuropathy and poor blood ¯ow due to impaired vasodilation have been implicated as the underlying causes of these wounds. With skin biopsies, it has been shown that eNOS is decreased in the microvasculature that supplies the diabetic foot ulcers of patients with neuropathy, except at the edge of ulcers where angiogenesis occurs. At the centre of the ulcer, similar to the atherosclerotic lesions, macrophages have in®ltrated and a high level of iNOS is detected instead.62 Since macrophages produce superoxide and NO in high amounts, peroxynitrite will be generated to cause destruction of the microcirculation immediate to the ulcers and hence loss of angiogenesis. Consequently, an imbalance in tissue regeneration and destruction leads to impairment of healing and chronic wounds. However, with respect to wound healing, whether production of iNOS is detrimental or bene®cial is not straightforward. INOS de®cient (non-diabetic) knockout mice have delayed wound healing and the process can be reversed by replacement of the iNOS gene.63 A possible explanation for this observation is that iNOS is important for generating large amounts of NO for anti-microbial defense in super®cial wounds. Thus, although NO is destructive to tissues, it may be necessary for microbial defense in diabetic foot ulcers. Furthermore, as described previously (NO action), the feedback inhibition mechanism of NO may be necessary to curb tissue destruction by excessive in¯ammation response and make way for tissue repair. THERAPEUTIC PROSPECTS NO is a popular therapeutic target for many in¯ammatory and autoimmune diseases. Thus, thorough understanding of the roles of nitric oxide and the identi®cation of the participating isoforms are important for designing therapeutic measures. Depending on the concentration and location, NO may be bene®cial or deleterious. In arthritis, iNOS inhibitors, such as arginine analogues, are effective in attenuating in¯ammation and joint destruction, while certain natural products, e.g. turmeric, known as anti-arthritic, also have this property. In fact, many of the commonly prescribed NSAID, e.g. aspirin, have inhibition of iNOS synthesis as one of their mechanisms of action.64,65 The Foot (2001) 11, 45 ± 51

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The role of NO in diabetes, however, is more complex. A possible therapeutic approach may be to simultaneously promote eNOS synthesis of NO so as to improve microvascular circulation in non-in¯ammatory hypertensive conditions, and decrease formation of peroxynitrite and endothelial cell damages, such as by removal of oxygen free radicals, in in¯ammation-associated atherosclerosis. However, for diabetic foot ulcers, unlike atherosclerotic lesions, microbial infection is an important concern, thus measures to eliminate microbes, such as application of antibiotics, will also be required in association with the reduction of oxidative radicals.59 REFERENCES 1. Butler A R, Flitney F W, Williams D L. NO, nitrosonium ions, nitroxide ions, nitrosothiols and iron-nitrosyls in biology: a chemist's perspective. Trends Pharmacol Sci 1995; 16: 18. 2. Bouysset M. Bone and Joint Disorders of the Foot and Ankle: A Rheumatological Approach. Paris: Springer-Verlag, 1998. 3. Bowker J H, Pfeifer M A. Levin and O'Neal's The Diabetic Foot. St Louis, MO: Mosby-Year Book, 1999. 4. Hobbs A J, Higgs A, Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol 1999; 39: 191. 5. Laskin J D, Laskin D L. Cellular and Molecular Biology of Nitric Oxide. New York: Marcel Dekker, 1999. 6. Murad F. Nitric oxide signaling: would you believe that a simple free radical could be a second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent Prog Horm Res 1998; 53: 43. 7. Wilcox J N, Subramanian R R, Sundell C L et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 1997; 17: 2479. 8. Stuehr D J. Structure-function aspects in the nitric oxide synthases. Annu Rev Pharmacol Toxicol 1997; 37: 339. 9. Hevel J M, Marletta M A. Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity. Biochemistry 1992; 31: 7160. 10. Crane B R, Arvai A S, Ghosh K et al. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 1998; 279: 2121. 11. Geller D A, Billiar T R. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev 1998; 17: 7. 12. MacMicking J, Xie Q W, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15: 323. 13. Fang F C. Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial. J Clin Invest 1997; 99: 2818. 14. Halliwell B, Gutteridge J M C. Free Radicals in Biology and Medicine, 3rd edn. Oxford: Oxford Science Publications, 1999. 15. Packer L. Methods in Enzymology. Vol. 301. Nitric Oxide. San Diego, CA: Academic Press, 1999. 16. Ducrocq C, Blanchard B, Pignatelli B et al. Peroxynitrite: an endogenous oxidizing and nitrating agent. Cell Mol Life Sci 1999; 55: 1068. 17. Squadrito G L, Pryor W A. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic Biol Med 1998; 25: 392. 18. Smith M A, Richey Harris P L, Sayre L M et al. Widespread peroxynitrite-mediated damage in Alzheimer's disease. J Neurosci 1997; 17: 2653. 19. Beckmann J S, Ye Y Z, Anderson P G et al. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler 1994; 375: 81. 20. Lamb N J, Gutteridge J M, Baker C et al. Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophilmediated hydroxylation, nitration, and chlorination. Crit Care Med 1999; 27: 1738.

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Roles of NO in arthritis and diabetes 51 43. Zumsteg U, Frigerio S, Hollander G A. Nitric oxide production and Fas surface expression mediate two independent pathways of cytokine-induced murine beta-cell damage. Diabetes 2000; 49: 39. 44. Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988; 318: 1315. 45. Huie R E, Padmaja S. The reaction of no with superoxide. Free Radic Res Commun 1993; 18: 195. 46. Cosentino F, Hishikawa K, Katusic Z S et al. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 1997; 96: 25. 47. Graves P M, Eisenbarth G S. Pathogenesis, prediction and trials for the prevention of insulin-dependent (type 1) diabetes mellitus. Adv Drug Delivery Rev 1999; 35: 143. 48. Williams S B, Cusco J A, Roddy M A et al. Impaired nitric oxide-mediated vasodilation in patients with noninsulin-dependent diabetes mellitus. J Am Coll Cardiol 1996; 27: 567. 49. Li H, Fostermann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000; 190: 244. 50. Rabini R A, Staffolani R, Martarelli D et al. Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions. J Clin Endocrinol Metab 1999; 84: 3770. 51. Honing M L, Morrison P J, Banga J D et al. Nitric oxide availability in diabetes mellitus. Diabetes Metab Rev 1998; 14: 241. 52. Pieper G M. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 1998; 31: 1047. 53. Pieper G M and Peltier B A. Amelioration by L-arginine of a dysfunctional arginine/nitric oxide pathway in diabetic endothelium. J Cardiovasc Pharmacol 1995; 25: 397. 54. Tsao P S, Theilmeier G, Singer A H et al. L-arginine attenuates platelet reactivity in hypercholesterolemic rabbits. Arterioscler Thromb 1994; 14: 1529. 55. Wolf A, Zalpour C, Theilmeier G et al. Dietary L-arginine supplementation normalizes platelet aggregation in hypercholesterolemic humans. J Am Coll Cardiol 1997; 29: 479.

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56. Mohan I K, Das U N. Effect of L-arginine-nitric oxide system on chemical-induced diabetes mellitus. Free Radic Biol Med 1998; 25: 757. 57. Wu G, Meininger C J. Impaired arginine metabolism and NO synthesis in coronary endothelial cells of the spontaneously diabetic BB rat. Am J Physiol 1995; 269: H1312. 58. Buga G M, Singh R, Pervin S et al. Arginase activity in endothelial cells: inhibition by NG-hydroxy-L-arginine duringhigh-output NO production. Am J Physiol 1996; 71: H1988. 59. Jude E B, Boulton A J, Ferguson M W, et al. The role of nitric oxide synthase isoforms and arginase in the pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming gorwth factor beta 1. Diabetologia 1999; 42: 748. 60. Pieper G M. Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J Cardiovasc Pharmacol 1997; 29: 8. 61. Wever R M F, van Dam T, van Rijn H J et al. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun 1997; 237: 340. 62. Veves A, Akbari C M, Primavera J et al. Endothelial dysfunction and the expression of endothelial nitric oxide synthetase in diabetic neuropathy, vascular disease, and foot ulceration. Diabetes 1998; 47: 457. 63. Yamasaki K, Edington H D, McClosky C et al. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998; 101: 967. 64. Chan M M, Ho C T, Huang H I. Effects of three dietary phytochemicals from tea, rosemary and turmeric on inflammation-induced nitrite production. Cancer Lett 1995; 96: 23. 65. Amin A R, Vyas P, Attur M et al. The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase. Proc Natl Acad Sci USA 1995; 92: 7926.

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