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Am J Physiol Heart Circ Physiol 293: H2878-H2887, 2007. First published August 31, 2007; doi:10.1152/ajpheart.01144.2006
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Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase

Rita K. Upmacis,1,2,* Mark J. Crabtree,3,* Ruba S. Deeb,1,2 Hao Shen,1,2 Paul B. Lane,3 Lea Esther S. Benguigui,1,2 Nobuyo Maeda,4 David P. Hajjar,1,2 and Steven S. Gross1,3

1Center of Vascular Biology, 2Department of Pathology and Laboratory Medicine, and 3Department of Pharmacology, Weill Medical College of Cornell University, New York, New York; and 4Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina

Submitted 17 October 2006 ; accepted in final form 25 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH4). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH4 becomes oxidized in organs of apolipoprotein E-deficient (ApoE–/–) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH4 were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE–/– mice or ApoE–/– mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE–/– mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH4 oxidation in heart, lung, and kidney of atherogenic ApoE–/– mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE–/– mice and that these species differentially contribute to protein 3-NT accumulation and BH4 oxidation in a tissue-selective manner. Since BH4 oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.

inducible nitric oxide synthase; high-fat diet

NITRIC OXIDE (NO) has important antiatherogenic activities, including inhibition of vascular smooth muscle cell proliferation (14), platelet-endothelial adhesion (8), leukocyte extravasation (25), and suppression of NF-{kappa}B-mediated proinflammatory gene expression (17, 22). NO is produced by a family of three NO synthase (NOS) enzymes, encoded by distinct genes and termed neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Both eNOS and nNOS are constitutively expressed in cognate cell types, whereas iNOS expression is broadly induced in cells by proinflammatory cytokines, in pathophysiological settings that include atherosclerotic lesions (7). Each NOS isoform is expressed in atherosclerotic vascular lesions (54), and isoform-specific NOS gene deletion in the apolipoprotein E (ApoE)-null genetic model of atherosclerosis has demonstrated opposing consequences for disease progression; whereas expression of eNOS and nNOS is disease limiting (19, 23, 32), iNOS expression is disease promoting (13, 26). The basis for the opposing roles of NOS isoforms in atherogenesis is incompletely understood, but differences in NO generation rates, cell source, and secondary reaction products are likely to be the major determinants. A further level of complexity arises from the likelihood that each of these putative determinants will vary in a tissue-specific manner during the various stages of atherogenesis.

All NOS isoforms have identical cofactor requirements [heme, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin (BH4), and calmodulin] (47). The reaction mechanism requires electron flow from NADPH for activation of heme-bound O2 in two successive monooxygenation reactions (46) that each requires BH4 (43, 45). Importantly, O2 consumption must be tightly coupled to electron transfer steps during the NOS catalytic cycle to limit uncoupled O2 reduction and release as superoxide anion radical (O2bullet–). eNOS (49, 53, 55), nNOS (18, 40), and iNOS (56) all have the capacity to release O2bullet–.

With deficiency of BH4, NOS uncoupling can predominate, manifested by O2bullet– production in lieu of NO (46). Given the near-diffusion limited reaction of NO and O2bullet– to produce peroxynitrite (ONOO–), formation of ONOO– can outcompete the ability of superoxide dismutase to consume O2bullet– (2, 20). Importantly, ONOO– has been shown to efficiently oxidize BH4 to 7,8-dihydrobipterin (BH2) in vitro and in vivo (24, 27). Oxidation of BH4 has been demonstrated to occur in blood vessels, presumably mediated by ONOO–, under conditions associated with chronic inflammation (1), and the resulting BH4 deficiency is implicated as a molecular basis for eNOS uncoupling (28, 49).

A hallmark reaction of ONOO– production is nitration of tyrosine residues, leading to 3-nitrotyrosine (3-NT) accumulation in proteins (3). An alternative pathway that leads to Tyr nitration involves nitrite metabolism by a peroxidase enzyme in the presence of hydrogen peroxide (36, 39). In human atherosclerotic blood vessels, increased production of vascular O2bullet– (35, 37) and expression of iNOS (7, 54) have been observed in association with elevated levels of protein 3-NT (30, 44, 48). Formation of ONOO– would necessarily abbreviate the bioactive lifetime of NO, dampening the vasoprotective actions afforded by NO. Whereas multiple biochemical sources of O2bullet– may contribute to NO scavenging in the early stages of chronic vasoinflammatory conditions [perhaps most importantly, activation of NADPH oxidase (15)], subsequent oxidation of BH4 can cause uncoupled eNOS to become a sustained source of O2bullet– (28, 49). Consequent production of BH4-oxidizing species like ONOO– can significantly promote further ONOO– formation, via uncoupling of NOSs (56), and can result in progressive vascular dysfunctions during atherogenesis.

To explore molecular mechanisms in atherogenesis, the ApoE-deficient (ApoE–/–) mouse has provided a useful genetic model that faithfully represents several aspects of the human condition (57). Notably, the ApoE amphipathic protein plays a pivotal role in lipoprotein trafficking by stabilizing and solubilizing lipoprotein particles. ApoE, a constituent of chylomicrons, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein, acts as a ligand for the receptor-mediated clearance of these particles (33). Normotensive ApoE–/– mice exhibit plasma cholesterol levels that are four to five times greater than ApoE+/+ mice and spontaneously develop atherosclerotic lesions even when fed a low-cholesterol diet (57). When fed a high-fat Western diet, the development of atherosclerotic lesions in ApoE–/– mice is exacerbated (5) in association with accelerated endothelial dysfunction (21) and increased formation of proatherogenic reactive nitrogen intermediates (e.g., OONO–) (30).

To ascertain the selective contribution of iNOS-derived NO to atherogenesis, an ApoE–/– mouse strain was engineered to also be nullizygous for iNOS (13, 23, 26). Such double-null mice were found to be normotensive and develop atherosclerotic lesions that are indistinguishable in size from those of ApoE–/–-iNOS+/+ mice when fed a normal chow diet (23). This finding led to the initial view that iNOS-derived species do not contribute to the progression of atherogenesis in ApoE–/– mice. However, when ApoE–/– mice were instead fed a Western diet that accelerated lesion development, iNOS gene deletion was found to curtail lesion formation (13, 26). Thus iNOS-derived NO perpetuates diet-accelerated atherosclerotic lesion formation in ApoE–/– mice. Since lesion development is associated with increased nitrative stress, we questioned the extent to which iNOS-derived NO specifically contributes to the genesis of reactive proatherogenic species. To test this, we assessed the contribution of iNOS to ONOO– formation by quantifying protein 3-NT accumulation and BH4 oxidation in organs of Western diet-fed ApoE–/– mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The animal protocol used in these studies was reviewed and approved by the Weill Medical College of Cornell University Care and Use Committee. ApoE–/–-iNOS–/– mice were generated as described previously (23). Mice used in these studies were derived from at least six generations of backcross breeding to C57BL/6J mice (23). ApoE–/–-iNOS–/+ heterozygote mice were bred to generate pups that were then genotyped for the iNOS allele by Southern analysis. Primers that are complimentary to portions of exon 11, exon 12, and neomycin (Neo) were used: AGA GTC CTT CAT GAA GCA CAT GCA (exon 11), TCA GCT TCT CAT TCT GCC AGA TGT (exon 12), and CAA TCC ATC TTG TTC AAT GGC CGA (Neo). The primers were mixed one part Neo, two parts exon 11, and one part exon 12. Wild-type mice contain both exons 11 and 12 and give rise to a larger band (~500 base pairs), whereas iNOS–/– mice contain exon 11 and Neo and give rise to a smaller band (~340 base pairs). iNOS+/– mice give rise to both bands.

Study design. ApoE–/–-iNOS–/– males and females were designated as the experimental group, and ApoE–/–-iNOS+/+ served as controls. ApoE–/–-iNOS–/+ pups were used as breeding mice. At 3 wk, all mice were weaned. Some mice were euthanized at 3 wk, whereas others were placed either on a Western diet comprising 21.2% (g/100 g) fat, 0.2% cholesterol, and 0% cholate (Harlan Teklad) or on a regular chow diet comprising 23.6% protein, 11.9% fat, and 64.5% carbohydrates (PicoLab Rodent Diet 20, LabDiet). After 24 wk on these diets, mice were euthanized by cervical dislocation, and tissues were harvested for indicated biochemical analyses. Both male and female mice were included in this study, and no attempt was made to assess possible sex differences in protein 3-NT accumulation and BH4 oxidation levels. The weights of the Western diet-fed ApoE–/–-iNOS+/+ and ApoE–/–-iNOS–/– mice before euthanasia were not significantly different (30.0 ± 0.3 and 29.2 ± 0.7 g, respectively) (11).

Quantification of protein incorporated 3-NT. Tissues (hearts, lungs, livers, kidneys, brains, and aortae) were rinsed of blood using ice-cold phosphate-buffered saline solution (pH 7.2–7.4), minced with scissors, and then homogenized in 1–5 ml/g wet wt of tissue in buffer containing (in mM) 50 Tris, 150 NaCl, 0.1 EDTA, and 20 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (pH 7.4). For aortae, it was necessary to pool tissues from two to three mice for these measurements. The tissue homogenates (100–500 µl) were ultracentrifuged (100,000 g) for 30 min, and the supernatants were subjected to protein assay and complete proteolytic digestion with proteinase K (10 U/mg protein; 8 h at 55°C). Each digested sample was extracted with three volumes of ice-cold acetonitrile, vortexed, incubated on ice (5 min), and centrifuged (12,000 g; 15 min at 4°C). The supernatants were dried by vacuum centrifugation (~4 h at room temperature; SpeedVac concentrator, Savant Model SV100) and reconstituted in vacuum-filtered (0.2-µm nylon membrane) and degassed HPLC mobile-phase buffer [containing 90 mM sodium acetate, 35 mM citric acid, 130 µM EDTA, and 460 µM sodium octane sulfonate (pH 4.35)], prepared in 18-M{Omega} resistance water (~2,000 µg/injection volume). The samples were centrifuge filtered (molecular mass cutoff = 10 kDa; Microcon Ultracel YM-10) to remove high molecular mass species before analysis for protein-incorporated 3-NT.

An isocratic HPLC system with multichannel electrochemical CoulArray (ESA) detection was used to resolve 3-NT from background species using a 100-mm C18 column (Microsorb-MV, Varian) running an isocratic mobile phase (90 mM sodium acetate, 35 mM citric acid, 130 µM EDTA, and 460 µM sodium octane sulfonate, pH 4.35) (9, 34). The flow was 0.75 ml/min at 30°C. The optimum potential for detection of 3-NT was found to be +800 mV. To assess the selectivity of the system for 3-NT, two other electrodes were set to bracket the optimal detection potential: +700 and +900 mV, respectively. Confirmation of 3-NT elution was further established by an addition of 10 mM sodium hydrosulfite to putative 3-NT-containing samples. This treatment chemically reduces 3-NT to 3-aminotyrosine, silencing the otherwise observed 3-NT electrochemical signal at +800 mV.

Quantification of BH2 and BH4 in tissue extracts. Tissues (hearts, lungs, livers, kidneys, brains, and aortae) were rinsed of blood using ice-cold phosphate-buffered saline solution (pH 7.2–7.4), minced with scissors, and then homogenized in 1–5 ml/g wet wt of tissue in buffer containing (in mM) 50 Tris, 150 NaCl, 0.1 EDTA, and 20 mM CHAPS (pH 7.4). For aortae, we pooled tissues from three mice for each measurement. Ice-cold protein precipitation buffer (300 µl; 0.1 M phosphoric acid and 0.23 M TCA) was added to the tissue homogenate (100 µl aliquot), followed by immediate centrifugation (2 min at 12,000 g; 4°C). A portion of the resulting supernatant (100–150 µl) was placed in an HPLC autosampler vial, mixed with dithioerythritol (6 mM final concentration), and analyzed for biopterins by electrochemical and fluorometric detection.

Samples were injected onto an HPLC system configured with sequential multichannel electrochemical CoulArray (ESA) and fluorescence detection. Biopterins were resolved isocratically with a 100-mm C18 column (Microsorb-MV, Varian, CA) running mobile phase (50 mM sodium acetate, 5 mM citric acid, 48 µM EDTA, and 0.3 mM dithioerythritol, pH 5.2) at a flow rate of 0.75 ml/min and at room temperature or 30°C. The optimum potential for detection of BH4 was determined to be +125 mV. Two additional electrodes were set sequentially at –350 and +600 mV to restore initial BH4 levels (via reduction) and then fully oxidize biopterins within the sample, respectively, allowing for confirmation of BH4 presence and detection of all biopterins based on fluorescence, irrespective of redox state. Quantifications of BH4, BH2, and biopterin were done by comparison with external standards after normalizing for sample protein content.

Western blot analysis of tissue homogenates. Tissues were homogenized in a minimum volume (500 µl) of lysis buffer [containing 50 mM Tris·HCl (pH 8), 10 mM EDTA, 1% Tween 20, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride], sonicated, and centrifuged (13,000 rpm), as described previously (12). Proteins in the tissue supernatants (30–40 µg) were separated by SDS/PAGE (7.5% polyacrylamide gel). Proteins from the gel were transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore), which were rinsed and allowed to dry (2 h or overnight) before probing with specific antibodies. Membranes were blocked (5%; 1 h) and then incubated overnight with either a polyclonal rabbit iNOS (sc-650; Santa Cruz) in milk (1%) or monoclonal mouse eNOS (No. 610296; BD Transduction) or nNOS (No. 610308; BD Transduction) antibodies in BSA (1%). After primary incubation, membranes were washed and probed with goat-anti mouse secondary antibody (No. 170-6516; Bio-Rad) in milk (5%; 1 h). When using eNOS and nNOS antibodies, membranes were blocked (in 5% milk; 1 h), again following incubation with the primary antibody as directed by the manufacturer's instructions. Membranes were then washed and probed with a secondary antibody [iNOS: anti-rabbit secondary antibody, sc-2004 (Santa Cruz), in 1% milk for 1 h; eNOS and nNOS: goat-anti mouse secondary antibody, No. 170-6516 (Bio-Rad) 5% in milk for 1 h]. All blots were also probed for actin using a polyclonal goat actin antibody (No. sc-1615; Santa Cruz) in milk (1%; 90 min) and an anti-goat secondary antibody (No. sc-2922; Santa Cruz) in milk (1%; 1 h). Bands were visualized by enhanced chemiluminescence (RPN2132; ECL Plus Western Blotting Detection Kit, GE Healthcare) on film (Kodak BioMax MR film) and scanner (Typhoon Trio+ Variable Mode; GE Healthcare). Some membranes were stripped and reprobed. Toward this end, membranes were incubated with a stripping solution [containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, and 0.1 M 2-mecaptoethanol] in a shaking hot water bath (50°C for 30 min).

Measurement of serum lipid profiles. Blood was removed from euthanized mice from the right ventricle and atrium following a surgical opening of the thoracic cavity. The blood was placed on ice (10 min), centrifuged (3,300 g for 5 min), and stored at 4°C. Glucose was measured using a FreeStyle Blood Glucose Meter (Therasense, Alameda, CA). Cholesterol, LDL, and triglycerides were measured using Cholesterol E, L-Type LDL-C and L-Type TG H assay kits (Wako Chemicals USA, Richmond, VA), respectively. The kits were used according to protocols supplied by the manufacturer.

Statistical analyses. Data are presented as means ± SE with significant differences determined by a two-tailed nonparametric Mann-Whitney t-Test. P < 0.05 was defined as being statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Western diet-evoked increases in protein 3-NT accumulation and BH4 oxidation in ApoE–/– mouse tissues. Hearts, lungs, livers, kidneys, and brains were harvested from 3- and 27-wk-old ApoE–/– mice for comparison of protein (3-NT) and BH4/BH2 levels. The elder mice were fed a Western (atherogenic) diet during the 24 wk immediately before being euthanized (see MATERIALS AND METHODS for details).

As shown in Fig. 1A, protein 3-NT levels were relatively low in all examined tissues of 3-wk-old ApoE–/– mice (i.e., 5–20 pmol/mg), except in brain, where levels were somewhat greater ({approx}50 pmol/mg protein), and in heart, where levels were below the limit of detection (<1 pmol/mg protein). Following 24 wk on a Western diet, 3-NT levels increased significantly in kidney, lung, and heart to a level that was 10-fold to >280-fold over prediet levels measured in 3-wk-old ApoE–/– mice. A 24-wk Western diet also failed to elicit a significant increase in protein 3-NT levels in the brain, beyond the relatively high prediet brain levels found in 3-wk-old mice.


Figure 1
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  		Fig. 1. Protein 3-nitrotyrosine (3-NT; A), tetrahydrobiopterin (BH4; B), and dihydrobiopterin (BH2; C) in tissues from apolipoprotein E-deficient (ApoE–/–) mice before and after feeding a Western diet for 24 wk. Hearts, lungs, livers, kidneys, and brains were removed from ApoE–/– mice at 3 wk of age (n = 4) and after feeding a Western diet for an additional 24 wk (n = 9). Organs were analyzed for protein 3-NT and biopterin levels, as described in MATERIALS AND METHODS. *P < 0.05, significantly different values for protein 3-NT, BH4, and BH2 in tissues from mice fed a Western diet for 24 wk compared with tissues from 3-wk-old mice.

 
The total biopterin content (sum of BH4 and BH2) in organs of 3-wk-old ApoE–/– mice ranged from a low of 2 pmol/mg protein in heart to a high of 25 pmol/mg protein in liver; intermediary levels were found in lung, brain, and kidney, in that order (see Fig. 1, B and C). Notably, total biopterin was found exclusively in its fully reduced form, BH4, in hearts, lungs, kidneys, and brains of 3-wk-old mice (Fig. 1B), i.e., BH2 was essentially undetectable (Fig. 1C). In liver, a minor but significant fraction of total biopterin was found in 3-wk-old mice as BH2 ({approx}13%) (Fig. 1C), with the remainder being BH4 (Fig. 1B). We were unable to detect the further-oxidized species, biopterin, in any tissue examined. The relatively high liver levels of BH4 and BH2 found are in accord with a hepatic role of BH4 as a releasable redox-active cofactor for support of aromatic amino acid hydroxylases; this is in contrast to the predominant function of lower BH4 levels in NOS-rich tissues as a tightly bound cofactor. Following 24 wk on a Western diet, the total biopterin content of some organs increased significantly, up to twofold (in heart, lung, and liver), whereas in others it did not significantly change (kidney and brain); nonetheless, in all organs, a significant accumulation of BH2 was observed (Fig. 1C). The extent of BH2 accumulation ranged from 34–50% of the total biopterin in the organs studied. Notably, the atherogenic diet-evoked accumulation of BH2 on a percent basis was low in brain, where protein 3-NT accumulation was also among the least.

iNOS gene deletion attenuates the accumulation of protein 3-NT and BH2 in tissues of Western diet-fed ApoE–/– mice. Experiments were performed to assess the extent to which iNOS expression is required for Western diet-evoked protein 3-NT accumulation and BH4 oxidation in ApoE–/– mouse organs. ApoE–/– and ApoE–/–-iNOS–/– mice were used for this purpose. Similar experiments were conducted to investigate the effects of feeding these mice a regular chow diet for 24 wk.

Figure 2 shows the influence of iNOS gene expression on protein 3-NT accumulation in hearts, lungs, livers, kidneys, and brains of ApoE–/– mice fed a regular chow diet (Fig. 2, left) and a Western diet (Fig. 2, right) for 24 wk. With respect to mice fed a regular chow diet, there were no differences in 3-NT levels measured in organs from ApoE–/– mice with and without the presence of iNOS, apart from the heart (5.80 ± 2.9 pmol/mg protein in iNOS–/– mice vs. 39.3 ± 12.2 pmol/mg protein in iNOS+/+ mice). In mice fed a Western diet, the results show that the majority of diet-evoked 3-NT accumulation in most organs is dependent on iNOS gene expression. Indeed, protein 3-NT accumulation was suppressed by iNOS gene deletion in heart by 76% (70.0 ± 30.7 pmol/mg protein in NOS–/– vs. 286.1 ± 75.0 pmol/mg protein iNOS+/+ mice), lung by 97% (7.1 ± 3.9 pmol/mg protein in iNOS–/– vs. 231.6 ± 70.0 pmol/mg protein in iNOS+/+ mice) and liver by 86% (8.7 ± 4.3 pmol/mg protein in iNOS–/– vs. 62.6 ± 16.8 pmol/mg protein in iNOS+/+ mice). Lesser suppressive, but not significant, effects of iNOS gene expression were observed on protein 3-NT accumulation in kidney (39%; 96.5 ± 24.2 pmol/mg protein in iNOS–/– vs. 158.5 ± 46.3 pmol/mg protein in iNOS+/+ mice) and brain (59%; 27.8 ± 14.9 pmol/mg protein in iNOS–/– mice vs. 68.3 ± 35.4 pmol/mg protein in iNOS+/+ mice), suggesting a contribution of other NOS isoforms to protein nitration in these organs. Even in heart, where a 76% decrease in protein 3-NT accumulation was observed in iNOS–/– mice, the residual 24% of 3-NT accumulation suggests that another NOS isoform importantly contributes to atherogenesis-associated protein nitration.


Figure 2
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  		Fig. 2. Protein 3-NT in tissues from ApoE–/– and ApoE–/–-iNOS–/– mice fed either regular chow or Western diets for 24 wk. Hearts, lungs, livers, kidneys, and brains were removed from ApoE–/– (n = 5, regular chow diet; and n = 9, Western diet) or ApoE–/–-iNOS–/– (n = 5, regular chow diet; and n = 20, Western diet) mice. Organs were analyzed for protein 3-NT levels as described in MATERIALS AND METHODS. *P < 0.05, significantly different values from mice with the ability to express iNOS compared with iNOS-null mice. #P < 0.05, significantly different values between mice on a Western diet compared with the same genotype on a regular chow diet.

 
Figure 3 shows the influence of iNOS gene expression on the biopterin content (BH4 and BH2, individually) of hearts, lungs, livers, kidneys, and brains of ApoE–/– mice fed both a regular chow diet and a 24-wk Western diet. With a regular chow diet, iNOS did not contribute to any differences in either BH4 (Fig. 3A, left) or BH2 (Fig. 3B, left) in any of the tissues examined. Figure 3C, left, expresses these measurements as a BH2-to-BH4 ratio and further highlights the fact that iNOS does not play a role in BH4 and BH2 levels when animals are fed a regular chow diet. However, as shown previously in Fig. 1, B and C, the administration of an atherogenic diet was associated with a marked oxidation of tissue BH4, as evidenced by the accumulation of 34–50% of total biopterin as BH2 in each of the study tissues (Fig. 3, A and B, right). Furthermore, with the expression of these data as a BH2-to-BH4 ratio, it is evident that the atherogenic diet induced BH2 levels in all tissues of animals bearing the iNOS gene, apart from the liver (Fig. 3C, right) compared with a regular chow diet (Fig. 3C, left). This atherogenic diet-induced accumulation of BH2 was significantly and near-completely prevented by iNOS gene deletion in heart (0.12 ± 0.10 pmol/mg protein in iNOS–/– vs. 1.3 ± 0.3 pmol/mg protein in iNOS+/+ mice), lung (0.38 ± 0.11 pmol/mg protein in iNOS–/– vs. 5.4 ± 1.7 pmol/mg protein in iNOS+/+ mice), and kidney (1.2 ± 0.3 pmol/mg protein in iNOS–/– vs. 5.5 ± 1.5 pmol/mg protein in iNOS+/+ mice) (Fig. 3B, right). This protection against BH4 oxidation afforded by iNOS gene deletion was concomitant with a diminished total biopterin content in the lung, kidney, and brain (73%, 50%, and 28% reduction, respectively, compared with that of iNOS+/+ mice). Modest nonsignificant protection was afforded by iNOS gene deletion against atherogenic diet-induced BH2 accumulation in brain (1.0 ± 0.3 pmol/mg protein in iNOS–/– vs. 2.0 ± 0.5 pmol/mg protein in iNOS+/+ mice), and protection was not evident in liver (22.0 ± 4.9 pmol/mg protein in iNOS–/– vs. 15.2 ± 4.4 pmol/mg protein in iNOS+/+ mice) (Fig. 3B, right). Whereas iNOS expression affected BH4 levels in the heart (2.6 ± 0.2 pmol/mg protein in iNOS–/– vs. 1.4 ± 0.3 pmol/mg protein in iNOS+/+) and lung (2.6 ± 0.5 pmol/mg protein in iNOS–/– vs. 5.3 ± 2.1 pmol/mg protein in iNOS+/+), it had no effect on absolute levels of BH4 in liver, kidney, and brain (Fig. 3A, right).


Figure 3
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  		Fig. 3. BH4 (A) and BH2 (B) levels in tissues from ApoE–/– and ApoE–/–-iNOS–/– mice fed either regular chow or Western diets for 24 wk. Hearts, lungs, livers, kidneys, and brains were removed from ApoE–/– (n = 5, regular chow diet; and n = 9, Western diet) or ApoE–/–-iNOS–/– (n = 5, regular chow diet; and n = 20, Western diet) mice. Organs were analyzed for BH4 and BH2 levels, as described in MATERIALS AND METHODS. *P < 0.05, significantly different values for BH4 and BH2 in tissues from mice with the ability to express iNOS compared with iNOS-null mice. #P < 0.05, significantly different values between mice on a Western diet compared with the same genotype on a regular chow diet. C: results in A and B expressed as a ratio of BH2 to BH4.

 
Comparative expression of eNOS and nNOS in tissues of Western diet-fed ApoE–/– and ApoE–/–-iNOS–/– mice. Figure 4 compares eNOS and nNOS protein expression in the heart, lung, liver, kidney, and brain of ApoE–/– and ApoE–/–-iNOS–/– mice fed a Western diet for 24 wk. Notably, eNOS protein was detected in all tissues examined from both ApoE–/– and ApoE–/–-iNOS–/– mice. In contrast, whereas nNOS protein was robustly detected in the brains and hearts of ApoE–/– and ApoE–/–-iNOS–/– mice, very little or no nNOS was observed in the lung, liver, or kidney. These findings provide no evidence of compensation for iNOS gene deletion by the other NOS isoforms, eNOS and nNOS.


Figure 4
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  		Fig. 4. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein levels in ApoE–/– and ApoE–/–-iNOS–/– mice fed a Western diet for 24 wk. Hearts, lungs, livers, kidneys, and brains were removed from ApoE–/– mice containing iNOS (n = 3) or without iNOS (ApoE–/–-iNOS–/–; n = 3) and analyzed for protein eNOS or nNOS expression, as described in MATERIALS AND METHODS. Each lane represents tissue from a separate mouse. Results are representative of measurements repeated twice.

 
Figure 5 depicts 3-NT, BH4 and BH2, and NOS protein levels in aortae from ApoE–/– and ApoE–/–-iNOS–/– mice fed a regular chow or Western diet. Administration of a Western diet significantly increased 3-NT levels in aortae from ApoE–/– mice compared with ApoE–/–-iNOS–/– mice (Fig. 5A). Notably, lower 3-NT levels were found in mice fed a regular chow diet. With respect to BH4 and BH2, very low levels were measured in each case which were unaffected by either iNOS gene deletion or diet (Fig. 5B). Since these levels are close to the detection limit of our instrumentation, it is notable that small differences would be difficult to discern. An examination of NOS protein levels in lesions removed from aortae of Western diet-fed mice revealed the presence of iNOS protein in Western diet-fed ApoE–/– mice (Fig. 5C), which was missing, as expected, in lesions from ApoE–/–-iNOS–/– mice. Furthermore, iNOS was not detected in the surrounding aortic tissue of these Western diet-fed mice (data not shown). It is noteworthy that aortae from regular chow-fed mice do not contain the volume of lesions observed in Western diet-fed mice, which prevented us from conducting a similar study in these animals. Figure 5C also shows that eNOS and low levels of nNOS were present at indistinguishable levels in ApoE–/– and ApoE–/–-iNOS–/– lesions and thus do not appear to compensate for the absence of iNOS.


Figure 5
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  		Fig. 5. Protein 3-NT (A), BH4 and BH2 levels (B), and nitric oxide synthase protein expression (C) in aortae from ApoE–/– and ApoE–/–-iNOS–/– mice. 3-NT, BH4, and BH2 levels were measured in aortae from ApoE–/– mice with and without iNOS fed a regular chow or Western diet (n = 10 for each category). For each separate measurement, it was necessary to pool aortae from 2 to 3 mice. *P < 0.05, significantly different values. C: lesions were dissected from aortae removed from ApoE–/– mice with and without iNOS that had been fed a Western diet for 24 wk and probed for iNOS, eNOS, and nNOS. Each lane represents a measurement using lesions from a separate mouse.

 
iNOS gene deletion does not influence serum lipid profiles and glucose in either regular chow or Western diet-fed ApoE–/– and ApoE–/–-iNOS–/– mice. As shown in Fig. 6, serum cholesterol, glucose, triglycerides, and LDL profiles were indistinguishable in mouse cohorts on ApoE–/– and ApoE–/–-iNOS–/– genetic backgrounds within the two diet groups. These results indicate that the protection afforded by iNOS gene deletion against protein nitration and BH4 oxidation was not due to a correction of lipid profile defects. A further conclusion that can be drawn from Fig. 6 is that the administration of a Western diet resulted in significant increases in serum cholesterol and LDL (ApoE–/–: 875 ± 70 and 812 ± 68 mg/dl cholesterol and LDL, respectively; and ApoE–/–-iNOS–/–: 789 ± 66 and 772 ± 113 mg/dl cholesterol and LDL, respectively) compared with mice fed a regular chow diet (ApoE–/–: 517 ± 79 and 475 ± 51 mg/dl cholesterol and LDL, respectively; ApoE–/–-iNOS–/–: 501 ± 35 and 360 ± 21 mg/dl cholesterol and LDL, respectively). For comparison, C57BL/6J control wild-type mice on a regular chow diet for 28 wk exhibited much lower serum cholesterol (87 ± 8 mg/dl), triglycerides (50 ± 3 mg/dl), and LDL (32 ± 15 mg/dl) values. Thus the mice on the Western diet in our study exhibited serum cholesterol, triglyceride, and LDL levels that were, respectively, 10-, 3-, and 25-fold over values measured for control wild-type mice on a regular diet. In contrast, glucose levels in nonfasted control wild-type mice (187 ± 10 mg/dl) were comparable with those reported for ApoE–/– (regular diet 232 ± 14 mg/dl; Western diet 213 ± 22 mg/dl) and ApoE–/–-iNOS–/– mice (regular diet 206 ± 13 mg/dl; Western diet 175 ± 13 mg/dl) regardless of diet.


Figure 6
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  		Fig. 6. iNOS gene deletion does not influence serum lipid profiles and glucose levels in either regular chow or Western diet-fed ApoE–/– mice. Serum levels of cholesterol, glucose, triglyceride, and LDL were measured in wild-type mice fed a regular chow diet for 28 wk (left) and in ApoE–/– and ApoE–/–-iNOS–/– mice fed a regular chow (middle) or Western diet (right) for 24 wk, as described in MATERIALS AND METHODS. Numbers represent averages from using serum from 10 or more mice. *P << 0.05 compared with wild-type mice. #P << 0.05 compared with counterparts on a regular chow diet.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings establish an essential and specific role for iNOS-derived reactive nitrogen species in the profound accumulation of protein 3-NT and BH2 in peripheral organs of a murine genetic model of diet-induced atherosclerosis.

Atherosclerosis is a chronic vasoinflammatory condition (42), characterized by endothelial dysfunction (16), defined by insufficient NO bioactivity (6). Paradoxically, immunohistochemistry and Western blot analysis experiments have shown an increase in NOS enzyme expression in atherosclerotic lesions from animal disease models (4) and human patients (7). NO insufficiency, despite abundant NOS in atherosclerotic vessels, has been reconciled by two additional disease features: 1) accelerated consumption of NO bioactivity, via the near diffusion-limited reaction of NO with O2bullet– that emanates from multiple cell sources [including NADPH oxidase, xanthine oxidase, inefficiencies of mitochondrial electron transport, and uncoupled NOSs (31)] and 2) diminished NO biosynthetic activity of NOSs, owing to ONOO–-mediated oxidation of the essential NOS cofactor, BH4 (29). Since BH4 oxidation results in NOS uncoupling and a consequent increase in the rate of NOS-derived O2bullet– production (50), atherosclerotic blood vessels may be prone to a feed-forward acceleration in the rate of ONOO– generation, at the expense of NO bioactivity. In addition to failing endothelium-mediated vasodilatation, the resulting loss of NO bioactivity can promote vascular inflammation (17) and thereby accelerate lesion progression. BH4 oxidation by reactive nitrogen species provides a unifying mechanistic link between oxidative stress and endothelial dysfunction in chronic vascular conditions, including atherogenesis.

This study employed a genetic approach to elucidate, organ by organ, the specific role of iNOS-derived reactive species in hallmark oxidative reactions of atherogenesis: protein 3-NT accumulation and BH4 oxidation. Notably, each of these oxidative reactions has been attributed to increased ONOO– production in atherosclerotic blood vessels, arising from an accelerated production of O2bullet– and reaction with NOS-derived NO. Since the kinetics of BH4 oxidation by ONOO– far exceed the rate of oxidation by O2bullet– (29), the formation of ONOO– is likely to be the key determinant of the BH4 oxidation status in the endothelium of atherosclerotic blood vessels. Multiple sources of NO can contribute to ONOO– overproduction in atherogenic lesions. However, a growing body of literature has implicated uncoupled eNOS as a predominant contributor to ONOO– formation in advanced lesions (41). The present findings reveal an essential, permissive role of iNOS in atherogenic-associated reactions of ONOO– in diverse tissues.

We observed a marked accumulation of protein 3-NT and oxidized BH4 (i.e., BH2) in organs of ApoE–/– mice, after 24 wk of feeding a Western diet. The extent of 3-NT accumulation varied dramatically in the various organs studied. Whereas heart, lung, liver, and kidney exhibited the greatest 3-NT accumulation (ranging from a 280-fold to 6-fold increase), brain proteins showed only a small and nonsignificant increase in 3-NT (0.3-fold). This differential tissue distribution of protein 3-NT accumulation is likely to reflect the extent of ONOO– generated within each organ, which, in turn, can be related to the extent of antioxidant reserves and tissue propensity for atherogenic diet-evoked tissue inflammation. It is important to consider that 3-NT formation may also arise from nitrite metabolism by a peroxidase enzyme in the presence of hydrogen peroxide (36, 39). The failure of a Western diet to increase protein 3-NT in brain is a predictable consequence of the protection afforded by the blood-brain barrier against central nervous system entry of proinflammatory cytokines. Although brain 3-NT was not increased by a 24-wk atherogenic diet, the brain is an apparent site of marked overproduction of NO and NO-derived species even in healthy young mice (3-wk-old ApoE–/–). This is indicated by a fourfold or greater increase in brain protein 3-NT versus 3-NT in every other organ studied herein.

In contrast to the organ-to-organ variability observed for protein 3-NT accumulation, we observed a uniform atherogenic diet-induced oxidation of BH4. Indeed, oxidation of BH4 to BH2 ranged from 30% to 50% of total biopterin in all organs (apart from aortae) analyzed from Western diet-fed ApoE–/– mice. Before commencement of the atherogenic diet, it is notable that BH2 was undetectable in any tissue but liver, where BH2 levels were only {approx}12% of total biopterin. Furthermore, BH2 levels were low in tissues from ApoE–/– mice administered a regular chow diet. Thus accumulation of tissue BH2 may provide a telling biomarker for advanced atherosclerotic disease. It has previously been reported that BH2 levels remain unchanged in aortae of ApoE–/– mice on a Western diet compared with wild-type mice (10). In our study, we also observed that BH2 did not accumulate in aortae from Western diet-fed mice compared with regular chow-fed mice. However, our results concerning BH4 levels are at variance with the previous study, in which an accumulation of BH4 was noted in mice on an atherogenic diet (10). Our results indicate that BH4 levels are similar in mice fed either a regular chow or Western diet. A difference is that hypercholesterolemia was more severe in our atherosclerotic model (cholesterol = 22 ± 2 mmol/l) compared with that in the earlier report (5.6 ± 0.3 mmol/l) (10). We measured circulating levels of cholesterol that were ~10 times higher than in wild-type mice. Indeed, in atherosclerotic rabbits with cholesterol levels 28 times higher than in control animals, markedly diminished BH4 levels were found (50a). Thus BH4 availability may depend on the severity of hypercholesterolemia.

Despite the substantial Western diet-elicited oxidation of BH4 in organs, it is notable that absolute BH4 levels were relatively undiminished; BH4 preservation was due to an increase in the total biopterin content in heart, liver, lung, or brain. When compared with mice on a regular chow diet, BH4 levels were slightly diminished. An increase in total biopterin is explained by a Western diet-induced and tissue-selective upregulation of the rate-limiting enzyme in the de novo BH4 biosynthesis, GTP cyclohydrolase 1 (GTPCH1). GTPCH1 is a well-established protein biomarker of tissue immunoactivation and inflammation (52) and was recently shown to be upregulated in aortae of ApoE–/– mice, but only after a minimum of 20 wk on a Western diet (10). We now broadly extend this prior finding of atherogenic diet-induced BH4 synthesis upregulation, from lesion-laden conductance vessels to diverse tissues with predominantly microvascular lesions [e.g., kidney (51)].

Prior studies using ApoE–/– mice have established that each of the three NOS gene products are expressed in atherosclerotic blood vessels (54) and can play a disease-modulatory role (13, 23, 26, 38). Inasmuch as atherogenesis is a vasoinflammatory condition, which is associated with proinflammatory cytokine release (42), it is reasonable that cytokine-induced iNOS could contribute prominently to the generation of reactive nitrogen species that mediate protein nitration, BH4 oxidation, and endothelial dysfunction. Studies reported herein using iNOS-null mice clearly establish the organ-specific role of iNOS-derived NO for BH4 oxidation and protein nitration in Western diet-fed ApoE–/– mice. Among the organs studied (heart, lung, liver, kidney, and brain), iNOS gene deletion was associated with an essentially complete protection against BH4 oxidation in all tissues but liver, and a >75% attenuation in protein 3-NT accumulation was found in all tissues but brain and lung. The differential contribution of iNOS-derived NO for protein nitration versus BH4 oxidation in any given tissue was unanticipated. This may be a consequence of multiple NOS isoforms and antioxidant defense mechanisms that are present in each tissue but nonuniformly distributed. Failure of iNOS gene deletion to suppress significantly protein nitration in kidney and brain can be explained by a major role of other NOS isoforms, likely eNOS and nNOS, respectively. Significantly, we found that eNOS and nNOS did not compensate for the absence of iNOS in any of the tissues examined.

In summary, our results demonstrate that iNOS-derived species are required for robust atherosclerosis-associated ONOO– production in peripheral organs. The observed organ-selective contribution of iNOS to ONOO– formation, deduced from protein 3-NT incorporation and BH4 oxidation, additionally suggests a variable contribution of constitutive NOS isoforms to the formation of proatherogenic reactive nitrogen intermediates in some organs. We hypothesize that although proinflammatory cytokine-induced iNOS provides the key source of NO for an initial acceleration of vascular ONOO– production during early stage atherogenesis, the consequent oxidation of BH4 would allow cofactor-depleted ("uncoupled") eNOS to serve as a sustaining source of ONOO– precursors in later-stage disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institutes of Health (NIH) Grants HL-46403 and HL-07423 (to D. P. Hajjar); HL-80702, HL-46403, and RR-19355 (to S. S. Gross); and HL-42630 (to N. Maeda) and a NIH T32 training grant in Cardiovascular Biology (to D. P. Hajjar). Additional support was provided by a Philip Morris USA and Philip Morris International grant (to R. K. Upmacis).


   ACKNOWLEDGMENTS
 
We are indebted to Cynthia Cheung and Albert Morrishow for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. K. Upmacis, Dept. of Pathology and Center of Vascular Biology, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (e-mail: rupmacis{at}med.cornell.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* R. K. Upmacis and M. J. Crabtree contributed equally to this work. Back


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