(Circulation. 1997;95:430-437.)
© 1997 American Heart Association, Inc.
Articles |
L-Arginine Prevents Xanthoma Development and Inhibits Atherosclerosis in LDL Receptor Knockout Mice
the Departments of Medicine (W.A., S.R., X.J., R.R.S., P.J.C.), Surgery (R.E.M.), and Pathology (M.S.), Columbia University College of Physicians and Surgeons, New York, NY.
Correspondence to Paul J. Cannon, MD, Department of Medicine, Division of Cardiology, Columbia University, 630 W 168th St, New York, NY 10032.
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Abstract |
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Background The potential antiatherosclerotic actions of NO were investigated in four groups of mice (n=10 per group) lacking functional LDL receptor genes, an animal model of familial hypercholesterolemia. Group 1 was fed a regular chow diet. Groups 2 through 4 were fed a 1.25% high-cholesterol diet. In addition, group 3 received supplemental L-arginine and group 4 received L-arginine and N
-nitro-L-arginine (L-NA), an inhibitor of NO synthase (NOS). Methods and Results Animals were killed at 6 months; aortas were stained with oil red O for planimetry and with antibodies against constitutive and inducible NOSs. Plasma cholesterol was markedly increased in the animals receiving the high-cholesterol diet. Xanthomas appeared in all mice fed the high-cholesterol diet alone but not in those receiving L-arginine. Aortic atherosclerosis was present in all mice on the high-cholesterol diet. The mean atherosclerotic lesion area was reduced significantly (P<.01) in the cholesterol-fed mice given L-arginine compared with those receiving the high-cholesterol diet alone. The mean atherosclerotic lesion area was significantly larger (P<.01) in cholesterol-fed mice receiving L-arginine + L-NA than in those on the high-cholesterol diet alone. Within the atherosclerotic plaques, endothelial cells immunoreacted for endothelial cell NOS; macrophages, foam cells, and smooth muscle cells immunostained strongly for inducible NOS and nitrotyrosine residues.
Conclusions The data indicate that L-arginine prevents xanthoma formation and reduces atherosclerosis in LDL receptor knockout mice fed a high-cholesterol diet. The abrogation of the beneficial effects of L-arginine by L-NA suggests that the antiatherosclerotic actions of L-arginine are mediated by NOS. The data suggest that L-arginine may be beneficial in familial hypercholesterolemia.
Key Words: atherosclerosis • genes • L-arginine • nitric oxide synthase • hypercholesterolemia
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Introduction |
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Atherogenesis is a complex process involving interaction between lipids and cells of the vascular wall.1 According to the formulation of Witztum and Steinberg,2 3 LDL penetrates and is retained in the intima of the vascular wall, where it undergoes minimal oxidative modification. Monocytes are attracted to and adhere to the endothelium, penetrate into the subendothelium, and differentiate into macrophages that express the scavenger receptor. LDL undergoes more extensive oxidative modification by macrophages and other cells in the intima and is taken up via the scavenger receptor to form lipid-rich "foam cells."
Patients homozygous for FH have deficient or defective LDL receptors leading to marked accumulation of cholesterol-rich lipoproteins in plasma and the development of cutaneous xanthomas and extensive premature atherosclerosis of the aortic root and proximal coronary arteries.4 Similarly, genetic defects in LDL receptors produce hypercholesterolemia and atherosclerosis in Watanabe rabbits and rhesus monkeys.5 6 More recently, Ishibashi and coworkers7 8 produced LDLR-ko mice and showed that in response to a high-cholesterol diet, the LDLR-ko mice developed marked elevations of cholesterol-rich VLDL, IDL, and LDL in plasma along with massive xanthomatosis and atherosclerosis.
NO and citrulline are the products of the five-electron oxidation of L-arginine by the NOS isoforms.9 10 11 Both constitutive ecNOS and iNOS are expressed in human atherosclerotic lesions.12 ecNOS, found in endothelial cells, produces small amounts of NO in response to shear stress or agonists such as bradykinin.9 10 11 iNOS, induced in macrophages, smooth muscle cells, and other cells by cytokines, produces large amounts of NO for long periods of time.9 10 11 NO can activate soluble guanylyl cyclase in target cells, increasing the formation of cGMP, or it can be degraded in physiological solutions to form nitrite and nitrate.9 10 11 NO, particularly when produced in larger amounts by iNOS, also has a variety of other effects, some of which can be cytotoxic. It can cause nitration of proteins, inhibit enzymes involved in mitochondrial respiration, bind to FeS clusters in proteins, cause ADP ribosylation, and produce fragmentation of DNA.9 10 11 When NO combines with equimolar amounts of superoxide, it forms peroxynitrite, a strong oxidant that can lead to nitration of proteins and can decompose to form toxic hydroxyl radicals.13 14
In experimental animals and patients with hypercholesterolemia and atherosclerosis, endothelium-dependent vasodilator responses of coronary and peripheral blood vessels are diminished.15 16 17 18 These responses, believed to be due to NO or a closely related nitrosothiol, can be restored toward normal by infusions of L-arginine and by antioxidant drugs such as superoxide dismutase, probucol, and vitamin C.19 20 21 22 23 24 In 1992, Cooke and coworkers25 reported that L-arginine supplementation to hypercholesterolemic rabbits not only partially restored endothelium-dependent vasorelaxation but also was associated with a significant reduction in the extent of atherosclerosis. Since L-arginine is known to have antioxidant properties, it was not clear from that study whether the modulation of atherosclerosis was a direct effect of the L-arginine or mediated by NO. Accordingly, the objective of the present study was to investigate the antiatherosclerotic effects of chronic supplementation of L-arginine in another model, mice with targeted disruption of the LDL receptor fed a high-cholesterol diet. In addition, the ability to reverse the L-arginine effect was studied with L-NA, an inhibitor of NOS.
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Methods |
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Animals
LDLR-ko mice were obtained from the Jackson Laboratory. The LDLR-ko mice had been created by homologous recombination as described by Ishibashi et al.7 Four groups of 10 LDLR-ko mice were studied. They were descendants of the F2 generation and were hybrids between the C57BL/6J and 129Sv strains. The mice were about 22 weeks old when entered into the study. They were maintained on 12-hour-dark/12-hour-light cycles and were allowed access to food and water ad libitum.
Diets
The LDLR-ko mice were fed for 6 months and then were killed. Four diets were used. Group 1 received a normal mouse chow diet (ICN) that contained 4.5% fat and 0.022% cholesterol. Groups 2 through 4 received a high-cholesterol diet (Harlan Teklad Premier Laboratory Diets) that contained 1.25% cholesterol, 7.5% (wt/wt) cocoa butter, 7.5% casein, and 0.5% (wt/wt) sodium cholate and 0.75% L-arginine. Group 3 received the high-cholesterol diet supplemented with L-arginine (2.25%) in the drinking water. Group 4 received the high-cholesterol diet supplemented with L-arginine (2.25%) and the NOS inhibitor L-NA (10 mg/100 mL, Sigma) in the drinking water.
Preparation of Aortic Sections
The mice were anesthetized with ketamine, and the hearts were exposed by thoracotomy. After the inferior vena cava was nicked to obtain blood samples, the animals were perfused via ventricular puncture, first with PBS to flush out the blood and then with 10% neutral buffered formalin for 5 minutes to fix the aorta. The hearts and the thoracic aortas were dissected away from the thorax en bloc and stored in 10% formalin at 4°C before sectioning. The hearts were then processed according to a modification of the method described by Paigen et al.26 The formalin-fixed hearts were incubated at 37°C in 5% gelatin for 2 hours, in 10% gelatin for 2 hours, and in 25% gelatin overnight. The gelatin was then hardened by refrigeration at 4°C for 4 hours. The hearts were trimmed into blocks and stored in 10% buffered formalin until sectioning. The distal half of the heart was sectioned at a plane parallel to that of a line drawn between the tips of the atria and then discarded. The remaining proximal half was then mounted in OCT compound, snap-frozen, and cut in a cryostat. Sequential cross sections 10 µm thick were cut until the appearance of the aortic sinus, recognized on unstained sections by the presence of the three leaflets of the aortic valve and by the bulging shape of the aorta. From this point on, every other section was collected on gelatin-coated slides until the valve cusps were no longer visible and the aorta was uniformly round. Sections were then stained with oil red O and counterstained with hematoxylin and light green. For optimal morphological evaluation, one heart per group was embedded in paraffin after fixation; sections 4 µm thick were cut and stained with hematoxylin-eosin, Masson's trichrome stain for collagen, and Verhoeff–van Gieson's stain for elastic tissue. Every other section was saved for immunohistochemistry.
Quantification of Atherosclerosis in the Proximal Ascending Aorta
A computer-assisted image analysis system was used to quantify the area of atherosclerotic lesions within the sections (Bioscan, Optimetric Software). The borders of the lesions apparent from the oil red O staining were traced, and the total lesion area for each section was calculated by planimetry. At least five sequential alternate sections from each animal were analyzed, and the mean lesion area was calculated for each animal and subsequently for each group. The coefficient of variation of the lesion size within individual animals averaged 6.4%.
Plasma Lipid and Lipoprotein Analysis
Total cholesterol in plasma samples and column fractions was measured by an enzymatic method (Wako Chemicals). Lipoprotein profiles were obtained by means of fast-phase liquid chromatography with a Superose 6B column.27 A 200-µL aliquot of pooled plasma was loaded onto the column and eluted with TSE buffer (50 mmol/L Tris, 0.15 mol/L NaCl, and 2 mmol/L EDTA, pH 7.4) at a constant flow rate of 0.35 mL/min. An aliquot of 80 µL from each fraction was used for the total cholesterol.
Immunohistochemistry
Paraffin sections were cleared with xylene, rehydrated in sequential alcohol baths, and then washed in PBS. Endogenous peroxidase was inactivated with 3% hydrogen peroxide in ethanol for 30 minutes; nonspecific antibody binding was suppressed with 20% goat serum in PBS for 30 minutes. Sections were then incubated for 1 hour at room temperature with rabbit antibodies to macrophages (Accurate Chemical), iNOS (a generous gift from Dr Susan A. Gregory of GD Searle Company), ecNOS (generously provided by Dr David Harrison, Emory University), and nitrotyrosine (Upstate Biotechnology). Pc12 cells exposed to peroxynitrate served as positive controls for nitrotyrosine immunolabeling. With intervening washes in PBS, sections were then incubated for 30 minutes at room temperature with a biotin-conjugated goat anti-rabbit IgG (Vector Laboratories) and the avidin-biotin immunoperoxidase complex (ABC Elite, Vector). Peroxidase activity was visualized with 3-amino-9-ethyl carbazole (Vector) or with diaminobenzidine (Sigma). Sections were washed in tap water, counterstained with Meyer's hematoxylin, dehydrated in sequential alcohols and xylene, and mounted with coverslips. As negative controls, the immunostaining was performed after the primary antibodies were substituted with nonimmune rabbit serum.
Statistical Analysis
Continuous variables are reported as mean±SD. Differences in cholesterol levels among groups were tested by one-way ANOVA. Post hoc tests of individual means were performed with Tukey's procedure. Differences in atherosclerotic lesion area among groups were tested with the nonparametric Kruskal-Wallis procedure.
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Results |
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Four groups of 10 LDLR-ko mice were entered into the study. Two group 3 mice died of infection early in the study; 1 group 4 mouse died of xanthomas that impeded feeding. The tissues from 1 female and 1 male in group 2 and 1 male in group 3 were damaged in processing and were excluded from analysis. Pathological examination was therefore performed on 34 mice that completed the 6-month protocol (17 females and 17 males). Of these 34 specimens, tissues from 1 animal in each group were used for hematoxylin-eosin and immunostaining, and measurements of mean lesion area were performed on sections from the remaining 30 animals. In the control LDLR-ko mice fed a normal chow diet, group 1, total plasma cholesterol was 140±71 mg/dL. Total plasma cholesterol was significantly increased in the animals fed a high-cholesterol diet: 803±206, 913±41, and 859±61 mg/dL, respectively, for groups 2, 3, and 4. Neither total plasma cholesterol nor the distribution of cholesterol in lipoprotein fractions differed significantly among the mice of groups 2 through 4 that exhibited large increases in the cholesterol contained in the VLDL, IDL, and LDL fractions (Fig 1
).
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Among the control LDLR-ko mice fed a normal chow diet for 6 months, group 1, no gross xanthomatous lesions were observed. In the cholesterol-fed LDLR-ko mice, group 2, xanthomatous lesions of the face, ears, ventral surfaces, and extremities began to appear at 2 months and were present in all animals by 6 months. In the cholesterol-fed LDLR-ko mice that received supplemental L-arginine, group 3, no xanthomatous lesions were apparent in any animal by 6 months. In the cholesterol-fed LDLR-ko mice of group 4 that received supplemental L-arginine but also the NOS inhibitor L-NA, xanthomatous lesions were apparent in all animals by 2 months and were larger and more extensive at 6 months than in the animals of group 2. A representative example from each animal group is shown in Fig 2.
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At the time the animals were killed, none of the group 1 LDLR-ko mice fed a normal chow diet had atherosclerotic lesions of the aorta, whereas all of the LDLR-ko mice fed a high-cholesterol diet (groups 2 through 4) had atherosclerotic lesions of the aortas and coronary ostia. Histological examination revealed the aortic lesions to be composed of multilayered deposits of foam cells beneath an intact endothelium frequently overlying an acellular core composed of cellular debris, cholesterol crystals, and occasional calcifications (Fig 3A). Immunostaining with a macrophage marker revealed that most of the lipid-filled foam cells were of macrophage lineage (Fig 3B).
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Multiple transverse sections of the aorta from each animal stained with oil red O were planimetered with an image analysis system according to a well-established method originally described by Paigen and collaborators.26 The mean lesion area was calculated for each animal, and the average lesion area in each group of LDL-ko mice was calculated. There were no atherosclerotic lesions stained with oil red O in group 1. In each of the three groups of cholesterol-fed LDLR-ko mice, groups 2 through 4, the mean lesion area was larger in female than in male mice (Fig 4). In group 3 mice that received supplemental L-arginine, the mean atherosclerotic lesion area in males and in females was reduced significantly (P<.01) below the mean lesion area observed in the group 2 animals that received a high-cholesterol diet without supplemental L-arginine (Fig 4; females, 41 965±5699 versus 79 705±9953 µm2; males, 17 960±3778 versus 57 205±3192 µm2). In group 4 mice that received L-arginine supplements and L-NA, the mean lesion area (females, 116 183±9814 µm2; males, 89 898±5618 µm2) was significantly (P<.01) larger in each sex than that observed in the group 2 or group 3 animals.
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Immunostaining of the atherosclerotic lesions in the cholesterol-fed LDLR-ko mice with a rabbit polyclonal antibody to ecNOS revealed positive staining for ecNOS in the intact endothelial cells overlying the lesions (Fig 5). When the atheromatous lesions were immunostained with a highly specific antibody raised against iNOS, there was significant and abundant iNOS immunostaining of intimal macrophages, smooth muscle cells, and foam cells (Fig 6). Immunostaining for nitrotyrosine has been used as a marker for the effects of peroxynitrite.14 Immunostaining for nitrotyrosine was present in cellular debris and in macrophages, smooth muscle cells, and foam cells within the atherosclerotic plaques near regions where iNOS was apparent (Fig 7).
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Discussion |
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Genetically altered mice have become useful experimental models for the study of atherogenesis.28 Ishibashi and coworkers7 8 first developed LDLR-ko mice and showed that these animals had defective clearance from plasma of cholesterol-rich IDL and LDL and developed marked hypercholesterolemia and xanthomas along with extensive atherosclerosis when fed a cholesterol-enriched diet for 5 to 8 months. In the present study, both male and female LDLR-ko mice were fed an identical cholesterol-enriched diet for 6 months. Control animals received a normal chow diet. Blood samples taken from a group of female mice at 6 months indicated that there were similar significantly elevated plasma cholesterol levels in the mice fed the high-cholesterol diet (groups 2 through 4). The distributions of increased IDL and LDL cholesterol did not differ in animals of groups 2 through 4. Although plasma arginine levels were not measured in the present study, the amount of L-arginine added to the drinking water (2.25%) was similar to that associated with twofold to threefold increases in plasma arginine concentrations when given to cholesterol-fed rabbits.25 29
Cutaneous xanthomas and xanthelasma are prominent clinical features of homozygous familial hypercholesterolemia.4 In the group 2 LDLR-ko mice fed the high-cholesterol diet alone, cutaneous xanthomas were observed at 2 months in every animal. This confirms the observations of Ishibashi et al,8 who also demonstrated that the xanthomatous lesions were composed of lipid-engorged macrophages along with extracellular deposits of cholesterol crystals. Interestingly, despite significant hypercholesterolemia when these mice are fed a Western diet and despite the development of atherosclerosis, mice deficient in apo E do not develop xanthomas; this raises the question whether apo E is required for xanthoma formation.30 No xanthomatous lesions were visible by 6 months in any of the group 3 animals that were fed cholesterol but also received L-arginine supplements. To the best of our knowledge, this effect of L-arginine to inhibit xanthoma formation has not been reported previously. The mechanism of this antixanthoma action of L-arginine is unclear. L-Arginine has antioxidant properties and has been shown to reduce Cu2+ oxidation of LDL in vitro.31 However, in the cholesterol-fed group 4 mice that received L-arginine plus an inhibitor of NOS, xanthomas developed earlier than 2 months in all animals and by 6 months were more extensive than in group 2. This finding suggests that the antixanthoma actions of L-arginine are not due to its antioxidant properties but may be mediated by NO.
All of the cholesterol-fed LDLR-ko mice in groups 2 through 4 developed extensive atherosclerosis by 6 months, with a distribution similar to that observed in patients with FH, ie, proximal aorta and coronary ostia.4 Histological examination revealed that the lesions were composed primarily of lipid-filled macrophage foam cells, also similar to findings in patients with FH.4 These observations in LDLR-ko mice are in contrast to observations in apo E–knockout mice fed a Western diet, which develop atherosclerotic lesions with a more complex composition not only in the aorta but also in distal coronary arteries.32 In the cholesterol-fed LDLR-ko mice in the present study, the extent of atherosclerotic lesion formation was greater in female than in male animals. The reason for this observation is unclear. Systematic comparisons of the response of plasma lipids to cholesterol feeding in male versus female mice were not done. However, a tendency for higher plasma cholesterol values in female than in male LDLR-ko mice on the same diet has been noted previously.8
A computer-assisted quantification of atherosclerotic lesions was performed on multiple transverse oil red O–stained sections of the aorta of each LDLR-ko mouse. There was no significant atherosclerosis in the control mice fed a normal chow diet, group 1. In the cholesterol-fed mice that received L-arginine supplements, group 3, the mean atherosclerotic lesion area was reduced by >40% below the mean lesion area in the animals that received only the high-cholesterol diet, group 2. In the animals that received L-arginine plus L-NA, group 4, the mean atherosclerotic lesion area was significantly increased above that observed in the animals of groups 2 and 3. Since L-NA, an inhibitor of NOS, abrogated the beneficial action of L-arginine, the data suggest that the beneficial effects of L-arginine are mediated by NO rather than by the amino acid itself. Although atherosclerosis was extensive in the cholesterol-fed LDLR-ko mice, the error of the measurement of mean atherosclerotic lesion area in each mouse was small, and the differences in mean lesion area in groups 2 through 4 were statistically significant even though the number of mice per group was relatively small.
The suggestion that the beneficial effect of L-arginine is mediated via the product of NOS and is abrogated by inhibition of the enzyme is consistent with several published reports demonstrating that (1) L-arginine but not D-arginine improves endothelium-dependent vasorelaxation and diminishes monocyte adhesion in cholesterol-fed rabbits,20 29 (2) L-arginine but not D-arginine reduces the neointimal response to carotid artery balloon injury in a rat carotid model,33 and (3) transfection of ecNOS into endothelial cells reduces the neointimal hyperplasia observed in response to balloon injury in a rat model.34 In the present study, blood pressure was not measured in the LDLR-ko mice, and the possibility exists that lesions in group 4 were worsened by an elevated blood pressure rather than inhibition of NOS. This possibility is rendered less likely, however, by the report of Cayette et al,35 who observed increased neointimal hyperplasia in cholesterol-fed rabbits that received an inhibitor of NOS and maintained blood pressure at control levels. Since inhibition of NOS reduces renal blood flow, it is also unlikely that plasma L-arginine levels were lower in the group 4 animals.
The production of NOx (NO and its metabolites nitrite and nitrate) by vascular rings from the LDLR-ko mice was not measured in the present study. In studies of hypercholesterolemic rabbits by Minor et al,36 Tsao et al,29 and Candipan et al,37 the production of NOx by vascular rings was increased above control in animals fed L-arginine. NO has many actions that can be interpreted to be potentially antiatherosclerotic. It inhibits platelet aggregation and adherence to endothelial cells.9 10 11 NO inhibits monocyte adherence to endothelial cells29 and the expression of endothelial-leukocyte AMs,38 which are upregulated in atherosclerosis39 and by cytokines,38 oxidized LDL,40 or lysophosphatidyl choline,41 and it also inhibits the expression of the monocyte chemoattractant molecule, monocyte chemotactic protein-1.42 NO and drugs that release NO inhibit vascular smooth muscle cell migration and proliferation and inhibit in vivo the intimal proliferative response to balloon injury.33 43 44 NO scavenges superoxide and by so doing reduces oxidant stress in the vascular wall.13 14 45 Reduced oxidant stress, in turn, may lower the rate of LDL oxidation and the expression of redox-sensitive genes (such as vascular cell AM-1, intercellular AM-1, endothelial-leukocyte AM-1, etc) that contribute to atherogenesis.39 46 47
In other settings, however, the product of the reaction of L-arginine with the NOS isoforms may be potentially atherogenic. When deprived of adequate substrate or in the presence of inhibitors, both the cNOS and the iNOS isoforms have been shown to generate superoxide instead of NO, an effect that increases oxidant stress and could promote atherogenesis.48 49 When large amounts of NO are produced (such as by iNOS) in the presence of equimolar superoxide, peroxynitrite is formed.13 14 Peroxynitrite is also a powerful oxidant that can induce lipid peroxidation, oxidize LDL, contribute to oxidant stress, promote nitration of tyrosine residues on proteins, and decompose to form toxic hydroxyl radicals.13 14 50 51 52
In the cholesterol-fed LDLR-ko mice, immunostaining with an anti-ecNOS antibody revealed the presence of abundant ecNOS protein in the intact endothelium overlying the intimal atherosclerotic lesions. This finding is consistent with a preliminary report of ecNOS immunostaining in endothelial cells in human atherosclerotic lesions.12 It is also consistent with observations in atherosclerotic rabbits that NOx synthesis was increased above control values.36 It should be noted, however, that in the study by Minor et al,36 the bioactivity of NO (measured as the vasodilator response to acetylcholine) was diminished, a finding that suggests that the degradation of NO was increased.53 Studies by O'Hara et al54 55 have indicated that in response to hypercholesterolemia and atherosclerosis in rabbits, the endothelial cell synthesis of superoxide anions that can inactivate NO is increased above levels found in control animals with normal cholesterol levels.
Immunostaining of the atherosclerotic lesions in the groups 2 through 4 mice with a highly specific antibody to iNOS indicated the presence of iNOS protein in macrophages, lipid-rich foam cells, and intimal smooth muscle cells. Although iNOS staining of lesions in experimental atherosclerosis in mice has not been reported, a preliminary report has noted iNOS immunostaining of macrophages, smooth muscle cells, and foam cells in human atherosclerotic lesions.12 Immunostaining for nitration of protein tyrosines was also observed in the matrix and cells in the atherosclerotic lesions of the groups 2 through 4 LDLR-ko mice near macrophages and foam cells immunostained for iNOS. This observation suggests that formation of peroxynitrite, with its attendant increase in oxidant stress, may also participate in the pathogenesis of atherosclerosis in hypercholesterolemic LDLR-ko mice.
In the present study, L-arginine supplements inhibited xanthoma formation and reduced the atherosclerotic lesion area in cholesterol-fed LDLR-ko mice. This observation confirms in a mouse model the antiatherosclerotic effects of L-arginine reported by Cooke et al,25 Tsao et al,29 and Candipan et al in rabbits.37 The data extend these effects to the formation of skin lesions and indicate that the beneficial actions of L-arginine are abrogated when NOS is inhibited.
The explanation for the present findings is unclear. However, several hypotheses may be advanced as a framework for future studies. It is known that NO scavenges superoxide and that superoxide scavenges NO.53 In the setting of hypercholesterolemia, vascular superoxide formation is increased via the action of endothelial cell xanthine/xanthine oxidase54 and possibly also, as suggested by Pritchard et al,56 by uncoupling of the activity of NOS from the formation of NO, resulting in the generation of superoxide. In hypercholesterolemic rabbits, the vascular synthesis of NOx is increased and is augmented further by supplemental L-arginine.25 29 36 37 The augmented NO formation may scavenge superoxide and/or directly inhibit superoxide formation53 57 ; it could also reduce peroxynitrite formation, as has been shown in vitro.58 Both effects would reduce net oxidant stress, which in turn reduces the expression of leukocyte adhesion molecules and other genes involved in the formation of atherosclerotic lesions.37 45
In summary, the data of the present study indicate that L-arginine supplementation inhibits xanthoma formation and atherosclerosis in LDLR-ko mice fed a high-cholesterol diet. They also indicate that inhibition of NOS with L-NA abrogates the beneficial effects of L-arginine, suggesting that these actions of L-arginine are mediated by NOS. The isoforms ecNOS and iNOS were identified by immunostaining in the atherosclerotic lesions of the LDLR-ko mice along with nitration of protein tyrosines, which provides indirect evidence for the formation of peroxynitrite. Since the LDLR-ko mouse is a model for familial hypercholesterolemia, the data also raise the possibility that dietary L-arginine supplements may be of therapeutic value in patients homozygous for that disorder.
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This work was supported in part by grants HL-21006 and HL-54764 from the NHLBI.
Received June 12, 1996; revision received August 21, 1996; accepted August 31, 1996.
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References |
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1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]
2. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785-1792.
3. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;793-795.
4. Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Benedet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. New York, NY: McGraw-Hill Publishing Co; 1989:1215-1250.
5. Watanabe Y, Ito T, Shiomi M. The effect of selective breeding on the development of coronary atherosclerosis in WHHL rabbits: an animal model for familial hypercholesterolemia. Atherosclerosis. 1985;56:71-97.[Medline] [Order article via Infotrieve]
6. Scanu AM, Khalil A, Neven L, Tidore M, Dawson G, Pfaffinger D, Jackson E, Carey KD, McGill HC, Fleiss CM. Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor. J Lipid Res. 1988;29:1671-1681.[Abstract]
7. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993;92:883-893.
8. Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994;93:1885-1893.
9. Ignarro LJ. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol. 1990;30:535-560.[Medline] [Order article via Infotrieve]
10. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109-142.[Medline] [Order article via Infotrieve]
11. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992;6:3051-3064.[Abstract]
12. Wilcox JN, Subramanian RR, Ross CE, Tracey WR, Pollack JS, Harrison DG, Marsden PA. Expression of multiple nitric oxide synthase isoforms in human aortic fatty streaks and advanced atherosclerotic plaques. Circulation. 1994;90(suppl I):I-298. Abstract.
13.
Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620-1624.
14. Hogg N, Darley-Usman VM, Wilson MT, Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J. 1992;281:419-424.
15.
Verbeuren TJ, Jordaens FH, Zonnekeyn LL, Vanhove CE, Coene MC, Herman AG. Effect of hypercholesterolemia on vascular reactivity in the rabbit, I: endothelium-dependent and independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res. 1986;58:552-564.
16.
Forstermann U, Mugge A, Acherd U, Haverich A, Frolich JC. Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res. 1988;62:185-191.
17.
Cohen RA, Zitnay KM, Haudenschild CC, Cunningham LD. Loss of selective endothelial cell vasoactive functions in pig coronary arteries during hypercholesterolemia. Circ Res. 1988;63:903-910.
18. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046-1051.[Abstract]
19. Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by L-arginine. Lancet. 1991;338:1546-1550.[Medline] [Order article via Infotrieve]
20.
Cooke JP, Andon NA, Girerd XJ, Hirsch AT, Creager MA. Arginine restores cholinergic relaxation of hypercholesterolemic rabbit thoracic aorta. Circulation. 1991;83:1057-1062.
21.
Girerd XJ, Hirsch AT, Cooke JP, Dzau VJ. L-Arginine augments endothelium-dependent vasodilation in cholesterol-fed rabbits. Circ Res. 1990;67:1301-1308.
22.
Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidant in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks slowing the progression of atherosclerosis in the WHHL rabbit. Proc Natl Acad Sci U S A. 1987;84:7725-7729.
23.
Mugge AJ, Elwell JH, Peterson JE, Hofmeyer TG, Heistad DD, Harrison DG. Chronic treatment with polyethylene-glycolated superoxide dismutase partially restores endothelium-dependent vascular relaxations in cholesterol-fed rabbits. Circ Res. 1991;69:1293-1300.
24. Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, Creager MA. Vitamin C improves endothelium dependent vasodilation in patients with non-insulin dependent diabetes mellitus. J Clin Invest. 1996;97:22-28.[Medline] [Order article via Infotrieve]
25. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.
26. Paigen B, Morrow A, Holmes PA, Michell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:231-244.[Medline] [Order article via Infotrieve]
27. Yokode M, Hammer RE, Ishibashi S, Brown MS, Goldstein JL. Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors. Science. 1990;250(4985):1273-1275.
28.
Breslow JL. Transgenic mouse models of lipoprotein metabolism and atherosclerosis. Proc Natl Acad Sci U S A. 1993;90:8314-8318.
29.
Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation. 1994;89:2176-2182.
30.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E: evaluation of lesional development and progression. Arterioscler Thromb. 1992;14:141-147.
31. Philis-Tsimikas A, Witztum JL. L-Arginine may inhibit atherosclerosis through inhibition of LDL oxidation. Circulation. 1995;92(suppl I):I-422. Abstract.
32.
Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. Apo E-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994;14:133-140.
33. McNamara DB, Bedi B, Aurora H, Tena L, Ignarro LJ, Kadowitz PJ, Akers DL. L-Arginine inhibits balloon catheter-induced intimal hyperplasia. Biochem Biophys Res Commun. 1993;193:291-296.[Medline] [Order article via Infotrieve]
34.
Von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137-1141.
35.
Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753-759.
36. Minor RL Jr, Myers PR, Guerra R Jr, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest. 1990;86:2109-2116.
37.
Candipan RC, Wang B, Buitrago R, Tsao PS, Cooke JP. Regression or progression: dependency on vascular nitric oxide. Arterioscler Thromb Vasc Biol. 1996;16:44-50.
38. De Caterina R, Libby P, Peng J-B, Thannickal VJ, Gimbrone MA Jr, Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest. 1995;96:60-68.
39. O'Brien KD, Allen MD, McDonald TO, Chait A, Harlan JM, Fishbein D, McCarty J, Ferguson M, Hudkins K, Benjamin CD, Lobb R, Alpers CE. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques: implications for the mode of progression of advanced coronary atherosclerosis. J Clin Invest. 1993;92:945-951.
40. Khan BV, Parthasarathy SS, Alexander RW, Medford RM. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995;95:1262-1270.
41. Kume N, Cybulsky MI, Gimbrone MA Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138-1144.
42.
Zeiher AM, Fisslthaler B, Schray-Utz B, Busse R. Nitric oxide modulates the expression of monocyte chemoattractant protein-1 in cultured human endothelial cells. Circ Res. 1995;76:980-986.
43.
Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res. 1996;78:225-230.
44. Grag LD, Hassid S. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultures in rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
45. Rubanyi GM, Vanhoutte PM. Oxygen derived free radicals, endothelium and responsiveness of vascular smooth muscle. Am J Physiol. 1986;250:H815-H821.
46. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866-1874.
47.
Ahmad M, Marui N, Alexander RW, Medford RM. Cell type-specific transactivation of the VCAM-1 promoter through an NF-kB enhancer motif. J Biol Chem. 1995;270:8976-8983.
48. Pout S, Pou WS, Bredt DS, Snyder SH, Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem. 1992;267:24172-24176.
49. Griffith OW, Stuehr DJ. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol. 1995;57:707-736.[Medline] [Order article via Infotrieve]
50. Darley-Usmar VM, Hogg N, O'Leary VJ, Moncada S. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic Res Commun. 1992;17:19-20.
51.
White CR, Brock TA, Chang L-Y, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MH. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:1044-1048.
52. Beckman JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM, White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler. 1994;375:81-88.[Medline] [Order article via Infotrieve]
53.
Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986;250:H822-H827.
54. Ohara J, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546-2551.
55.
Ohara Y, Peterson TE, Sayegh HS, Subramanian RR, Wilcox JN, Harrison DG. Dietary correction of hypercholesterolemia in the rabbit normalizes endothelial superoxide anion production. Circulation. 1995;92:898-903.
56.
Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Mingdan W, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510-518.
57. Clancy RM, Leszczynska-Piziac J, Abramson SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide production via a direct action on the NADPH oxidase. J Clin Invest. 1992;90:1116-1121.
58.
Miles AM, Bohle S, Glassbrenner P, Hansert B, Wink D, Grisham M. Modulation of superoxide-dependent oxidation and hydroxylation reactions by nitric oxide. J Biol Chem. 1996;271:40-47.
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|
|
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|
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|
|
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|
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