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*
Merck Research Laboratories, Rahway, NJ 07065;
Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
The Wistar Institute, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Participation of monocytes in the inflammatory aspect of atherosclerosis appears key, because mutations that alter monocyte function have effects on the development of lesions in animal models. Cross-breeding atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice (2) with mice lacking M-CSF (osteopetrotic) results in a reduction in lesion area (3, 4). Similar results are also obtained when LDL receptor-deficient mice are made genetically deficient in monocyte chemoattractant protein-1 (5). In addition, experiments crossing apoE-/- mice with mice deficient in the monocyte chemoattractant protein-1 receptor CCR2 show a 50% decrease in lesion area (6). Therefore, macrophage development and migratory signals may be important to lesion progression, and macrophages themselves serve as a source of inflammatory cytokines.
While cytokines, chemokines, and growth factors have been demonstrated in atherosclerotic lesions, macrophages may also serve as the source of other inflammatory mediators that could contribute to tissue injury in atherosclerosis. NO produced in abundance by the inducible form of NO synthase (iNOS, NOS2) is an example (7). NO produced by the endothelial cell enzyme ecNOS has been associated with prevention of endothelial cell dysfunction (8), an effect that may be beneficial in atherosclerosis. However, iNOS is a high output enzyme that can produce copious amounts of NO for an extended interval of time (7). Large quantities of NO could combine with superoxide to form peroxynitrite, an adduct with enhanced oxidizing capability (9). iNOS has been demonstrated to be present in human atherosclerotic lesions (10, 11, 12) and to contribute to peroxynitrite formation at those sites (10, 13). In addition, oxidation of LDL by reactive nitrogen intermediates may contribute to the development of atherosclerosis (14). Thus NO also has the potential to exacerbate atherosclerosis by promoting inflammation and necrosis at the site of lesions.
To directly test whether iNOS contributes to the development of atherosclerosis, we cross-bred iNOS-/- mice (15) with apoE-/- mice to create mice deficient in both iNOS and apoE. Both apoE-/-/iNOS-/- and apoE-/-/iNOS+/+ mice were fed a high fat diet, and atherosclerosis was evaluated by two different methods, morphometry of lesions in the aortic root and biochemical determination of aortic cholesterol. Our results indicate that the absence of iNOS has a significant beneficial effect on lesion progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were barrier housed, specific pathogen free, and maintained in static microisolator cages. Autoclaved food and water were provided ad libitum. The Institutional Animal Care and Use Committee of Merck Research Laboratories (Rahway, NJ) approved the animal use for experimentation, and all animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (revised 1996, National Academy Press, Washington D.C.).
Mice homozygous for the targeted iNOS-/- gene (129SvEv x C57BL/6) (15) were crossed with apoE-/- mice on a 50% C57BL/6, 50% 129 background (The Jackson Laboratory, Bar Harbor, ME). F1 offspring, by definition apoE+/-/iNOS+/-, were interbred. F2 weanlings were genotyped for the iNOS allele by Southern analysis as described (15) and for the apoE allele as described (16). The serum cholesterol levels of apoE-/- mice were measured (see below) to confirm the apoE null genotype. Homozygous apoE-/- null mice from the F2 progeny containing either the wild-type (iNOS+/+) or knockout (iNOS-/-) alleles were used for establishing matings for the generation of experimental apoE-/-/iNOS+/+ and apoE-/-/iNOS-/- animals. The genotype of iNOS-/- breeders was confirmed using a published protocol (15) to detect the mutated iNOS allele by PCR. In addition, breeders were screened by PCR to confirm the absence of the wild-type iNOS gene using the 5' primer 5'-ATC AgC CTT TCT CTg TCT CC-3' and the 3' primer 5'-ggC TTT CTg TCT gTT CTC TC-3'.
Study design
Offspring were weaned at 4 wk of age onto a high fat, Western-type diet containing 21.22% (g/100 g) fat, 17.01% protein, 48.48% carbohydrate, and 0.15% cholesterol (TD88137, Harlan Teklad, Madison, WI) and maintained on the diet until 16 or 22 wk. At the time the mice were euthanized, they were weighed. Blood was collected from the vena cava into syringes containing EDTA as an anticoagulent. Plasma was prepared by centrifugation at 850 x g for 15 min at 4°C and stored at -20°C for later evaluation of plasma cholesterol and triglyceride levels and plasma nitrate concentrations. Plasma cholesterol and triglyceride measurements were made using standard enzymatic kits (Sigma, St. Louis, MO).
Histology and morphometry
Mice that had been fed high fat diet until 16 wk of age were gently perfused through the left ventricle with cold PBS. Perfused hearts were removed with about 1 mm of proximal aorta attached, and the portion distal to the tips of the auricles was excised and discarded. The remaining heart with aortic root was stored briefly on ice in PBS, 0.02% NaN3 then frozen in OCT (Optimal Cutting Temperature) embedding medium (Fisher Scientific, Springfield, NJ) over liquid nitrogen-isopentane.
The fresh-frozen OCT-embedded hearts were used for immunohistochemistry and quantitation of lesion area in the aortic root. Serial sections (8 µM) of the aortic root were mounted on 10-well masked slides (Erie Scientific, Portsmouth, NH). Sections were fixed in acetone, air dried, rehydrated in PBS containing 0.02% NaN3, and blocked with 1% BSA in PBS/NaN3. For detection of laminin, CD18, CD11c, and iNOS sections were reacted with rabbit anti-laminin Ab (Sigma), monoclonal hamster anti-CD18 Ab (clone 2E6) (17), monoclonal hamster anti-CD11c Ab (clone N418) (17), or rabbit anti-iNOS COOH-terminal peptide (NO16) provided by Rick Mumford (Merck Research Laboratories) (18). As a control, sections were incubated with anti-iNOS in the presence of an excess of the octomeric iNOS peptide against which the Ab was raised. Primary Ab was followed by incubation with biotinylated goat anti-rabbit IgG Ab or goat anti-hamster IgG Ab, as appropriate, in the presence of 200 µg/ml normal mouse IgG. Ab reactivity was detected using HRP-conjugated biotin-streptavidin complexes and developed with diaminobenzidine tetrahydrochloride as substrate.
Lesions in the aortic root were quantitated on five sections, each separated by 32 µm (thus spanning a total of 200 µm of the root), using methods similar to those previously described (19). The entire intimal lesion area in each section was manually traced and quantitated using a Phase3 Image analysis system (Phase 3 Imaging Systems, Glen Mills, PA) in a blinded fashion. The mean lesion area per section was determined for each mouse. Fibrotic lesion area was quantitated on multiple sections for each aortic root stained with anti-laminin Ab to mark areas rich in extracellular matrix. In all cases, staining for laminin coincided with staining for collagen in serial sections, although the staining with anti-collagen was less intense than with anti-laminin. These areas were also largely negative for staining with leukocyte markers in serial sections. Foam cell-rich and necrotic lesion areas were quantitated on sections stained with anti-CD18 and anti-CD11c Ab. Foam cell-rich areas were further validated by staining for CD11b, and total leukocyte areas were demonstrated by staining for CD45 (not shown). Areas that contained a complex mixture of foam cells and extracellular matrix were not scored, but these represented <5–10% of the total lesion area. Results are reported as the mean percent of the total lesion area for each group, with SEM.
Aortic cholesterol measurements
Mice that had been fed high fat diet until 22 wk of age were perfused as above with cold PBS, 5 mM EDTA. All branches and any adipose tissue connected to the aorta were removed, and each aorta was carefully excised from the aortic root to the right renal artery. The dissected aorta included the aortic root, and therefore hearts from these mice were not used for histological examination and morphometric measurements. The aortas were stored on ice in PBS for not longer than 8 h, but typically only 2 h, blotted dry, weighed, minced, and extracted with chloroform:methanol (2:1) by the method of Folch et al. (20). The lipid extracts were dried down, resuspended quantitatively in chloroform:methanol (2:1), and stored at -20°C until the time of assay.
Total and free cholesterol in the aortic extracts were determined using an enzymatic fluorometric assay based on a modification of previously described methods (21, 22). Briefly, the solvent was evaporated from aliquots containing 1–16 nmol of cholesterol, and the lipid residue was resolubilized in 100 µl of reagent-grade ethanol. Aliquots of cholesterol (Aldrich, Milwaukee, WI) and cholesteryl oleate (Aldrich) standard solutions prepared in chloroform:methanol (1:1) were treated similarly. To determine free cholesterol, samples and standards were incubated 1 h at 37°C in a total volume of 1.01 ml with 0.03% Triton X-100, 0.9 mM sodium cholate, and 0.1 M potassium phosphate buffer, pH 7.4. Cholesterol oxidase (0.18 U) (Boehringer Mannheim, Indianapolis, IN), peroxidase (2 U) (Boehringer Mannheim), and p-hydroxyphenylacetic acid (0.5 mg/ml) (Aldrich) were added for an additional hour incubation at 37°C. The fluorescent product was measured in a Spex FluoroMax (Spex Industries, Edison, NJ) (excitation 325 nm, emission 415 nm) using acrylic UV transparency semimicro cuvettes (Evergreen Scientific, Los Angeles, CA). For total cholesterol determinations, cholesterol esterase (10 U) (Calbiochem, La Jolla, CA) was included in the first incubation step, and cholesteryl oleate was used as a standard. The cholesteryl ester in each sample was calculated by subtracting the value of free cholesterol from that for total cholesterol. Samples for each aorta were run in duplicate at two different concentrations. All values are expressed as nmol/mg aorta (wet weight) for each mouse, with means and SEM determined for each group.
Plasma nitrate levels
Plasma nitrates were reduced to nitrite with nitrate reductase, and nitrates were quantitated exactly as previously described (23).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To directly test whether iNOS contributes to the development of
atherosclerotic lesions, apoE-/- mice
were cross-bred with iNOS-/- mice to produce
animals deficient in both apoE and iNOS. To confirm that iNOS
deficiency had no direct effect on plasma cholesterol levels,
apoE-/-/iNOS-/- males
(n = 18) and females (n = 26), and
apoE-/-/iNOS+/+ males
(n = 21) and females (n = 25) were fed
a high fat diet from 4 to 22 wk, and plasma cholesterol was quantitated
as described in Materials and Methods. There were no
significant differences in plasma cholesterol between
apoE-/-/iNOS-/- and
controls for male or female mice (Fig. 1
A). Plasma triglyceride and
nitrate levels were measured in a subset of the animals used for
cholesterol measurements. Neither male (n = 11) nor
female (n = 14)
apoE-/-/iNOS-/- mice
showed a significant difference in triglyceride levels from controls
(males, n = 11; females, n = 12) (Fig. 1B). Plasma nitrate values in male (n = 10)
and female (n = 10)
apoE-/-/iNOS-/- mice
also did not differ from controls (males, n = 10;
females, n = 8) (Fig. 1D). In addition, the
high fat diet had no effect on plasma nitrate levels in
apoE-/-/iNOS-/- mice
compared with
apoE-/-/iNOS-/- mice
fed a chow diet (data not shown). While there was no difference in body
weight for male mice, the female
apoE-/-/iNOS-/- mice
were slightly heavier than the
apoE-/-/iNOS+/+ mice
(Fig. 1C). Thus iNOS deficiency did not affect plasma
cholesterol, triglycerides, or nitrates and had only a small effect on
the weight of female mice.
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Preliminary histological studies using a few mice fed a high fat
diet over a time course of 14–18 wk indicated that at 16 wk lesions in
the aortic root had progressed beyond the fatty streak stage and ranged
from intermediate to more complex lesions with necrotic cores. At times
longer than 16 wk, many lesions had reached a maximal occluding volume.
Therefore, morphometry was used to evaluate the lesion area in the
aortic root of
apoE-/-/iNOS-/- males
(n = 9) and females (n = 10) and
apoE-/-/iNOS+/+ males
(n = 9) and females (n = 5) that were
fed a high fat diet until 16 wk of age (Fig. 2). In the male
apoE-/-/iNOS-/- mice,
there was a 50% reduction in lesion area compared with
apoE-/-/iNOS+/+ mice
(p < 0.02). In the female mice, there was a
30% reduction in lesion area that did not reach statistical
significance (p > 0.2).
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). However, a quantitative comparison of
the lesions by types of area revealed that the distribution of
fibrotic, foam cell-rich, and necrotic areas in lesions from
apoE-/-/iNOS-/- mice
was not significantly different from that of
apoE-/-/iNOS+/+ mice
(Table I). Some small areas of the
lesions were classified as complex, containing a mixture of fibrotic
and foamy areas that were too difficult to score as either.
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), and staining with anti-iNOS
was eliminated by the presence of excess iNOS peptide, demonstrating
the specificity of the labeling. Sections of aortic root from
apoE-/-/iNOS-/- mice
also did not stain with anti-iNOS (data not shown). This result
indicates that, like human lesions, murine atherosclerotic lesions also
contain iNOS.
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Time course studies of the accumulation of cholesterol in the
aorta of apoE-/- mice indicated that
cholesterol and cholesteryl ester could be easily detected
biochemically by 22 wk of age in mice fed a high fat diet
(24). Therefore, this time was chosen for evaluation to
provide a different time point from that evaluated morphometrically.
Aortic atherosclerosis was quantitated in
apoE-/-/iNOS-/- males
(n = 13) and females (n = 19) and
apoE-/-/iNOS+/+ males
(n = 21) and females (n = 26) fed a
high fat diet until 22 wk of age by biochemical measurements of the
aortic total cholesterol, free cholesterol, and cholesteryl ester
content, as described in Materials and Methods. The
enzymatic method used for cholesterol determinations permits samples to
be analyzed rapidly, with great efficiency and excellent
reproducibility. All three parameters showed a significant reduction in
both male and female
apoE-/-/iNOS-/- mice
compared with
apoE-/-/iNOS+/+ mice
(Fig. 5). Total cholesterol and free
cholesterol were both reduced about 40% for males and about 50% for
females (p < 0.0005). Cholesteryl ester showed
the largest decline, 57% for males and 72% for females
(p < 0.0005). Thus a deficiency in iNOS was
able to ameliorate lesion development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While the argument for the beneficial effects of ecNOS-derived NO on atherosclerosis is quite strong, NO has additional characteristics that could contribute to tissue damage at sites of inflammation, such as atherosclerotic lesions. The form of NOS expressed in macrophages, iNOS, is a high output enzyme whose function is regulated transcriptionally, rather than by intracellular calcium concentration (7). iNOS produces NO at a higher rate and for longer periods than does ecNOS (7), and NO produced in large quantities has the potential to injure tissue. In combination with superoxide, NO forms peroxynitrite (9), and it has been suggested that most of the NO produced by activated macrophages is converted to peroxynitrite (43). This adduct has enhanced oxidative capacity and is capable of modifying cellular proteins and lipids. Nitrosylation or other modification (44) of tyrosines by peroxynitrite or its more reactive radical products ·OH and ·NO2 (9) may interfere with intracellular signaling by blocking the normal cycle of phosphorylation and dephosphorylation. iNOS may also compete with ecNOS for substrate, depleting available arginine from supplying the steady production of NO that appears so beneficial. This has been proposed to account for the benefits of an arginine-supplemented diet on atherosclerosis in animal models of the disease, even though arginine is not thought to be limiting in the plasma. In addition, a known risk factor for the development of atherosclerosis, homocysteine, induces iNOS transcription in vascular smooth muscle cells in vitro (45, 46). Thus the potential for tissue destruction exists at sites where iNOS is expressed.
Both iNOS and products of peroxynitrite have been localized in human atheromatous tissue and in atherosclerotic lesions from animals. iNOS has been identified in macrophages, smooth muscle cells, and lymphocytes in human lesions by immunohistochemistry and in situ hybridization (10, 11, 12). Here we demonstrate that iNOS is expressed in foam cell-rich regions of atherosclerotic lesions from apoE-/- mice. It has also been found in macrophages and lymphocytes in lesions from rabbits fed a high cholesterol diet (47, 48), and iNOS expression correlated positively with lesion size in this model (49). Nitrosylated tyrosines colocalize with iNOS in areas containing macrophages and surrounding the necrotic core (13). The proximal nature of the interaction of peroxynitrite products with the necrotic core suggests that this chemical modification could contribute to the process of necrosis.
Here we report studies that directly test the role of iNOS in the development of atherosclerosis in a murine model of the disease. ApoE-/- mice develop lesions that resemble human lesions in many respects, with a large involvement of foam cells and a fibrous cap (50). When these mice were cross-bred with iNOS-/- mice, and the resulting apoE-/-iNOS-/- mice were fed a high fat, Western-type diet, they developed significantly less atherosclerosis than did the apoE-/-iNOS+/+ controls. These results were obtained with both males and females. The strongest results occurred with mice fed a high fat diet until 22 wk of age, suggesting that iNOS may contribute to the formation of advanced lesions rather than to the establishment of initial lesions. This interpretation is supported by the recent observation that when apoE-/-iNOS-/- mice are evaluated for atherosclerotic lesions after 16 wk on a chow diet, there is no difference from apoE-/-iNOS+/+ mice (42). Under those conditions, the lesions are less developed than those observed with mice on a high fat diet. Our results indicate that, while NO could have beneficial, anti-inflammatory properties for atherosclerosis, it appears rather that the production of large amounts of NO by iNOS may be proinflammatory in the atherosclerotic setting and may contribute to lesion development.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LDL, low density lipoprotein; iNOS, inducible NO synthase; apoE, apolipoprotein E; ecNOS, endothelial constitutive NO synthase.
Received for publication June 19, 2000. Accepted for publication July 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent transcriptional activation of Nos2. Proc. Assoc. Am. Phys. 110:22.[Medline]
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 M. Zanetti, L. V. d'Uscio, I. Kovesdi, Z. S. Katusic, and T. O'Brien In Vivo Gene Transfer of Inducible Nitric Oxide Synthase to Carotid Arteries From Hypercholesterolemic Rabbits Stroke, May 1, 2003; 34(5): 1293 - 1298. [Abstract] [Full Text] [PDF]
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M. M. Kockx, K. M. Cromheeke, M. W.M. Knaapen, J. M. Bosmans, G. R.Y. De Meyer, A. G. Herman, and H. Bult Phagocytosis and Macrophage Activation Associated With Hemorrhagic Microvessels in Human Atherosclerosis Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 440 - 446. [Abstract] [Full Text] [PDF]
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J. Loscalzo L-Arginine in Atherosclerosis: Consequences of Methylation Stress in a Complex Catabolism? Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 3 - 5. [Full Text] [PDF]
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J. Chen, P. Kuhlencordt, F. Urano, H. Ichinose, J. Astern, and P. L. Huang L-Arginine on Atherosclerosis in ApoE Knockout and ApoE/Inducible NO Synthase Double-Knockout Mice Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 97 - 103. [Abstract] [Full Text] [PDF]
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D. Eguchi, L. V. d'Uscio, C. Wambi, D. Weiler, I. Kovesdi, T. O'Brien, and Z. S. Katusic Inhibitory effect of recombinant iNOS gene expression on vasomotor function of canine basilar artery Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2560 - H2566. [Abstract] [Full Text] [PDF]
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C.A. Gunnett, D.D. Lund, M.A. Howard III, Y. Chu, F.M. Faraci, and D.D. Heistad Gene Transfer of Inducible Nitric Oxide Synthase Impairs Relaxation in Human and Rabbit Cerebral Arteries Stroke, September 1, 2002; 33(9): 2292 - 2296. [Abstract] [Full Text] [PDF]
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J. Huang, F. J. DeGraves, S. D. Lenz, D. Gao, P. Feng, D. Li, T. Schlapp, and B. Kaltenboeck The quantity of nitric oxide released by macrophages regulates Chlamydia-induced disease PNAS, March 19, 2002; 99(6): 3914 - 3919. [Abstract] [Full Text] [PDF]
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J. K. Lee, M. Borhani, T. L. Ennis, G. R. Upchurch Jr, and R. W. Thompson Experimental Abdominal Aortic Aneurysms in Mice Lacking Expression of Inducible Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1393 - 1401. [Abstract] [Full Text] [PDF]
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 J. M. STULAK, A. LERMAN, M. R. PORCEL, J. A. CACCITOLO, J. C. ROMERO, H. V. SCHAFF, C. NAPOLI, and L. O. LERMAN Renal Vascular Function in Hypercholesterolemia Is Preserved by Chronic Antioxidant Supplementation J. Am. Soc. Nephrol., September 1, 2001; 12(9): 1882 - 1891. [Abstract] [Full Text] [PDF]
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B. C Kone Molecular biology of natriuretic peptides and nitric oxide synthases Cardiovasc Res, August 15, 2001; 51(3): 429 - 441. [Abstract] [Full Text] [PDF]
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J. Husemann, A. Obstfeld, M. Febbraio, T. Kodama, and S. C. Silverstein CD11b/CD18 Mediates Production of Reactive Oxygen Species by Mouse and Human Macrophages Adherent to Matrixes Containing Oxidized LDL Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1301 - 1305. [Abstract] [Full Text] [PDF]
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P. J. Kuhlencordt, J. Chen, F. Han, J. Astern, and P. L. Huang Genetic Deficiency of Inducible Nitric Oxide Synthase Reduces Atherosclerosis and Lowers Plasma Lipid Peroxides in Apolipoprotein E-Knockout Mice Circulation, June 26, 2001; 103(25): 3099 - 3104. [Abstract] [Full Text] [PDF]
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B. Nicholson, C. K. Manner, J. Kleeman, and C. L. MacLeod Sustained Nitric Oxide Production in Macrophages Requires the Arginine Transporter CAT2 J. Biol. Chem., May 4, 2001; 276(19): 15881 - 15885. [Abstract] [Full Text] [PDF]
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D. H. McDermott, J. P.J. Halcox, W. H. Schenke, M. A. Waclawiw, M. N. Merrell, N. Epstein, A. A. Quyyumi, and P. M. Murphy Association Between Polymorphism in the Chemokine Receptor CX3CR1 and Coronary Vascular Endothelial Dysfunction and Atherosclerosis Circ. Res., August 31, 2001; 89(5): 401 - 407. [Abstract] [Full Text] [PDF]
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) and
apoE-/-/iNOS-/- (
) mice were fed a high
fat diet until 22 wk of age then euthanized. The mice were weighed
(C), and plasma cholesterol (A),
triglyceride (B), and nitrate (D) levels
were measured as described in Materials and Methods. The
data represent the mean for each group with SEM. Values of
p for comparison of experimental and control groups were
all >0.3, except * where p = 0.002 (Student’s
t test).
