Asymmetric Dimethylarginine Produces Vascular Lesions in Endothelial Nitric Oxide Synthase–Deficient Mice
Involvement of Renin-Angiotensin System and Oxidative Stress
Objective— Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis.
Methods and Results— ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT1 receptor antagonist), which simultaneously suppressed vascular lesion formation.
Conclusions— These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo.
Endothelium-derived nitric oxide (NO), synthesized from l-arginine by endothelial NO synthase (eNOS), has several important antiatherogenic actions.1–5 Indeed, reduction of endothelial NO synthesis (endothelial dysfunction) predisposes the blood vessel to arteriosclerosis,1–5 and the eNOS-deficient (eNOS-KO) mice exhibit accelerated vascular lesion formation.6,7 As pharmacological tools to inhibit endothelial NO synthesis, synthetic l-arginine analogues have been used in vitro and in vivo. Among them, Nω-nitro-l-arginine methyl ester (l-NAME) is the most frequently used agent.1–5 Long-term treatment with l-NAME is known to cause arteriosclerotic coronary lesions, especially at microvascular levels, in experimental animals.8,9 This model with l-NAME is regarded as a useful animal model for examining the protective roles of endothelium-derived NO in the pathogenesis of arteriosclerosis.8,9
However, it is controversial whether these vascular effects of l-NAME are caused primarily by the inhibition of endothelial NO synthesis for the following reasons: first, the importance of endothelium-derived NO decreases as the vessel size becomes smaller,10 whereas l-NAME–induced vascular lesions are prominent at microvascular levels;8 second, long-term treatment with l-NAME does not reduce eNOS activity;11 third, multiple actions of l-NAME other than simple inhibition of NO synthesis have been reported.12,13 The most appropriate way to address this issue is to use mice that are deficient in the eNOS gene and to examine whether long-term treatment with l-NAME causes coronary vascular lesions in those mice. We have recently shown that treatment with l-NAME causes a comparable extent of coronary arteriosclerotic lesions in wild-type and eNOS-KO mice.14
Asymmetric dimethylarginine (ADMA) is a naturally occurring l-arginine analogue derived from the proteolysis of proteins containing methylated arginine residues.15–17 When administered acutely, ADMA inhibits purified NO synthase catalytic activity, endothelium-derived NO bioavailability, and endothelium-dependent NO-mediated vascular response, all of which are reversed by l-arginine (but not by d-arginine).5,18,19 Plasma concentrations of ADMA have been found to be elevated in patients with arteriosclerosis,20 as well as with coronary risk factors, such as hypertension21 or hypercholesterolemia,22 and elevated ADMA levels are associated with impaired endothelium-dependent NO-mediated vasodilation. On the basis of these findings, ADMA is thought to be an important endogenous NO synthase inhibitor, involving the pathogenesis of endothelial dysfunction and arteriosclerosis.23–26 In the present study, we tested our hypothesis that the mechanism(s) other than simple inhibition of endothelial NO synthesis is also involved in the long-term vascular effects of ADMA, as in the case of l-NAME.
The present study was reviewed and approved by the Ethics Committee of Animal Care and Experimentation, the University of Occupational and Environmental Health and was performed according to the Institutional Guidelines for Animal Experiments and the Law (No.105) and Notification (No.6) of the Japanese Government.
Male eNOS-KO and wild-type mice (8 to 10 weeks of age) were used. Disruption of the eNOS gene was confirmed by polymerase chain reaction (PCR) of genomic DNA.27 The eNOS-KO mice were derived from a cross between C57BL/6 and SV129J mice and were backcrossed to C57BL/6 mice over 10 generations. Thus, C57BL/6 mice (Charles River Japan Inc, Yokohama, Japan) were used as wild genotype control.
The following 17 groups were studied: control and eNOS-KO mice that received subcutaneous saline infusion, subcutaneous ADMA infusion (20, 40, and 60 mg/kg per day, Sigma, St. Louis, Mo), NG-monomethyl-l-arginine (l-NMMA) (1 mg/mL; Sigma) in drinking water, subcutaneous ADMA infusion (60 mg/kg per day) plus l-arginine (70 mg/mL; Sigma) in drinking water, subcutaneous ADMA infusion (60 mg/kg per day) plus temocapril (0.1 mg/mL; Sankyo Pharmaceutical Co, Tokyo, Japan) in drinking water, or subcutaneous ADMA infusion (60 mg/kg per day) plus olmesartan medoxomil (5 mg/kg; Sankyo) in chow, or wild-type mice that received subcutaneous ADMA infusion (60 mg/kg per day) plus hydralazine (0.05 mg/mL) in drinking water. In all of these groups, the treatments were performed for 4 weeks. Saline or ADMA was infused via an implanted osmotic minipump (Model 1002; Alzet, Palo Alto, Calif). The pumps were placed into the subcutaneous space of the mice anesthetized with ketamine (45 mg/kg intraperitoneally, Sankyo) through a small incision in the back of the neck. The actual daily intake of water containing drugs was 4 to 6 mL.
Plasma concentrations of ADMA were determined by high-performance liquid chromatography (HPLC).28 Systolic blood pressure was measured by the tail-cuff method under conscious conditions.
The mice were euthanized by inhalation of overdose diethyl ether (Wako Pure Chemical Industries Ltd, Osaka, Japan). The aorta was cannulated and perfused with 4% paraformaldehyde solution under physiological pressure. Then, the heart was removed and embedded in paraffin, and the slices were stained with Masson trichrome solutions. The sections were scanned using a light microscope equipped with a 2-dimensional analysis system (IBAS; Carl Zeiss, Jena, Germany). The extent of medial thickening and the extent of perivascular fibrosis of the coronary arteries were evaluated by the ratio of medial thickness to internal diameter and the ratio of perivascular fibrosis area to total vascular area, respectively. In each heart, >10 large epicardial coronary arteries (internal diameter, 219±6 μm) and coronary microvessels (internal diameter, 31±4 μm) were examined, and average values were used.
Measurement of Nitrite Plus Nitrate and NO
Nitrite plus nitrate (NOx) concentrations in the plasma and urine were assessed by the Griess method after 15 hours of fasting.29 NO release in the coronary arteries was directly measured with an NO-sensitive electrode (Model 501; Inter Medical Co, Nagoya, Japan).14,30 The mouse hearts were isolated and a NO-sensitive electrode was stuck and placed perpendicularly into the left anterior descending coronary artery in oxygenated Krebs–Ringer bicarbonate solution at 37°C.
Sections of paraffin-embedded tissue were cut in thicknesses of 3 μm. Endogenous peroxidase was inhibited with 5 mmol/L hydrogen peroxide solution. The sections were incubated with protein-blocking serum to minimize spurious background staining. Then they were incubated with rabbit polyclonal antibody (Transduction Laboratories, Franklin Lakes, NJ) at a dilution of 1:500 for neuronal NOS (nNOS) or 1:100 for inducible NOS (iNOS) for 1 hour at room temperature, or with mouse monoclonal angiotensin-converting enzyme (ACE) antibody (Chemicon International Inc, Temecula, Calif) at a dilution of 1:200. An avidin biotin immunoperoxidase system was used to detect the antigen. The specificity of immunostaining was confirmed by omission of the primary antibody or by replacement of the primary antibody to an isotype-matched primary antibody (a nonimmune IgG).31
The heart, the aorta, or the brain was excised aseptically. They were gently flushed with cold phosphate-buffered saline (Gibco, Paisley, UK) and were homogenized in 500 μL of buffer containing 1.1 μmol/L leupeptin, 0.7 μmol/L aprotinin, 120 μmol/L phenylmethanesulfonyl fluoride (PMSF), 0.7 μmol/L pepstatin, 1 mmol/L iodoacetamide, and 1 mmol/L diisopropylfluorophosphate at 4°C. The homogenate was used for Western blot analysis as reported previously.31
Reverse-Transcriptase Polymerase Chain Reaction
mRNA was isolated from the heart or the aorta by guanidine hydrochloride extraction and oligo(dT) cellulose column separation. Reverse-transcriptase polymerase chain reaction (RT-PCR) for iNOS was performed with PC-701 thermocycler (Astec, Fukuoka, Japan) using a moloney murine leukemia virus (M-MLV) RT kit (Gibco) and a Takara Ex Tag kit (Takara, Tokyo, Japan).32 PCR products were subjected to electrophoresis in 1% agarose gels stained with ethidium bromide and analyzed using an FLA-2000 fluoroimage analyzer (Fujifilm, Tokyo, Japan).
In Situ Hybridization
The frozen heart was cut in thicknesses of 12 μm using a cryostat and mounted onto gelatin/chrome alum-coated slides. The sections were fixed in 4% paraformaldehyde for 5 minutes and incubated in saline containing 0.25% acetic anhydride and 0.1 mol/L triethanolamine (TEA) for 10 minutes. They were then dehydrated and delipidated in chloroform. In situ hybridization histochemistry for nNOS was performed as we reported previously.33
Measurements were performed immediately after the mice were euthanized. To assess the extent of oxidative stress, superoxide anion production in the isolated heart was measured by the lucigenin-enhanced chemiluminescence method.14 Superoxide anion production was expressed as relative light units per second per mg wet weight.
Plasma Homocysteine Levels
Plasma homocysteine concentrations were determined by HPLC and electrochemical detection, as described previously.34
Results are expressed as mean±SEM. Statistical analyses were performed by 2-way ANOVA followed by Scheffe post-hoc test for multiple comparisons. A value of P<0.05 was considered to be statistically significant.
Plasma ADMA concentrations
Basal plasma ADMA concentrations in wild-type (0.6±0.1 μmol/L, n=5) and eNOS-KO (0.7±0.1 μmol/L, n=5) mice were almost identical to the previously reported plasma ADMA concentrations in healthy human subjects (0.4 to 1.0 μmol/L).18,35 Long-term treatment with ADMA (20, 40, and 60 mg/kg per day) significantly increased plasma ADMA concentrations in both wild-type (2.0±0.2, 2.5±0.3, and 3.1±0.2 μmol/L, respectively, n=5 each) and eNOS-KO (1.9±0.2, 2.3±0.2, and 2.8±0.3 μmol/L, respectively, n=5 each) mice in a dose-dependent manner. These increased levels were similar to the reported plasma ADMA concentrations in patients with peripheral arterial occlusive disease (2.5 to 3.5 μmol/L).20 Because these levels were within the pathological levels seen in humans, we used the dose of 60 mg/kg per day of ADMA in the following experiments.
Arterial systolic blood pressure was slightly but significantly higher in eNOS-KO mice (127±3 mm Hg) than in wild-type mice (98±5 mm Hg; P<0.05, n=10 each). Long-term treatment with ADMA significantly increased blood pressure in both wild-type (120±3 mm Hg) and eNOS-KO mice (138±4 mm Hg; P<0.05, n=10 each).
Formation of Coronary Vascular Lesions
In wild-type mice, long-term treatment with ADMA caused significant medial thickening (expressed as wall to lumen ratio) and perivascular fibrosis in coronary microvessels, but not in large coronary arteries (Figure 1A and 1C). Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of medial thickening and perivascular fibrosis in coronary microvessels (but not in large coronary arteries) that was comparable to that in wild-type mice (Figure 1B and 1D). These vascular effects of ADMA were not reversed by simultaneous treatment with l-arginine (70 mg/mL) in both strains (Figure 2A through 2D). Another endogenous l-arginine analogue, l-NMMA (1 mg/mL),36 similarly induced a comparable extent of medial thickening and perivascular fibrosis in coronary microvessels in wild-type and eNOS-KO mice (Figure I, available online at http://atvb.ahajournals.org).
In wild-type mice, antihypertensive treatment with hydralazine (0.05 mg/mL) normalized systolic blood pressure (120±3 to 95±4 mm Hg) but failed to inhibit the formation of medial thickening (0.2±0.1 to 0.8±0.1) or perivascular fibrosis (0.2±0.1 to 0.7±0.1) in coronary microvessels caused by ADMA treatment (n=5).
Long-term treatment with ADMA did not significantly reduce either plasma (74±4 to 106±3 μmol/L) or urine (1.6±0.5 to 1.8±0.6 μmol/L) NOx concentrations, markers of systemic NO production in wild-type mice (n=5 each). In addition, long-term treatment with ADMA did not significantly affect either basal vascular NO release (assessed by the reduction in NO levels in coronary arteries of isolated heart induced by acute administration of 10−3 M Nω-nitro-l-arginine, l-NNA [0.29±0.09 μmol/L without and 0.38±0.02 μmol/L with ADMA treatment, respectively]), or acetylcholine (10−5 M)-stimulated vascular NO release (0.63±0.12 μmol/L without and 0.52±0.19 μmol/L with ADMA treatment, respectively), markers of local NO production, in wild-type mice (n=5 each).
Expression of nNOS or iNOS
No expression of nNOS or iNOS was noted in coronary microvessels of eNOS-KO mice by immunostaining, Western blotting, in situ hybridization, or RT-PCR before ADMA treatment (Figure II, available online at http://atvb.ahajournals.org). With our methods, we were able to detect the expression of nNOS protein and mRNA in the brains of eNOS-KO mice, and the expression of iNOS protein and mRNA in the hearts and the aortas taken from eNOS-KO mice 12 hours after intraperitoneal injection of lipopolysaccharide (20 mg/kg), confirming the accuracy of our methods (Figure II).
Tissue ACE Expression
After treatment with ADMA, ACE immunoreactivity increased markedly in both wild-type and eNOS-KO mice to a comparable extent, mainly in the adventitia (Figure 3A). No such increase in ACE immunoreactivity was noted in mice cotreated with temocapril (0.1 mg/mL), which is an ACE inhibitor, or olmesartan (5 mg/kg), which is an angiotensin II type 1 (AT1) receptor antagonist (Figure 3A).
Treatment with ADMA for 3 days significantly increased cardiac lucigenin chemiluminescence in both mice to a comparable extent (Figure 3B). The ADMA-induced increase in lucigenin chemiluminescence was again normalized by cotreatment with temocapril or olmesartan in both strains (Figure 3B).
Prevention of Coronary Vascular Lesion Formation
Medial thickening and perivascular fibrosis induced by ADMA were also abolished by simultaneous treatment with temocapril or olmesartan in both strains (Figure 4A and 4B).
Plasma Homocysteine Levels
Long-term treatment with ADMA did not significantly affect plasma homocysteine concentrations in either wild-type mice (5.2±0.2 μmol/L without and 6.0±0.3 μmol/L with ADMA treatment, respectively, n=5 each) or in eNOS-KO mice (6.2±0.7 μmol/L without and 6.4±0.4 μmol/L with ADMA treatment, respectively, n=5 each).
The novel findings of this study were as follows: (1) long-term treatment with ADMA induced coronary microvascular lesions in wild-type and eNOS-KO mice to a comparable extent; (2) the treatment also caused an upregulation of vascular ACE and an increase in superoxide production in both strains to a comparable extent; (3) these changes were not a consequence of the developed hypertension; (4) the effects of ADMA were not antagonized by administration of l-arginine; and (5) neither systemic nor vascular NO production was attenuated by treatment with ADMA. These results provide the first direct evidence that mechanisms other than simple inhibition of endothelial NO synthesis are involved in the long-term vascular effects of ADMA in vivo.
Plasma ADMA Concentrations
Basal levels of plasma ADMA concentrations in mice were similar to those in healthy humans.18,35 Increased plasma ADMA concentrations in mice induced by treatment with ADMA were equivalent to plasma ADMA concentrations in patients with peripheral arterial occlusive disease.20 Thus, the mouse plasma levels of ADMA under our experimental conditions were within the human pathophysiological range.20
No Effect of Blood Pressure
Arterial blood pressure was significantly elevated after long-term treatment with ADMA in both wild-type and eNOS-KO mice. Treatment with hydralazine prevented the ADMA-induced hypertension but failed to suppress the ADMA-induced coronary vascular lesion formation. These results indicate that the long-term vascular effects of ADMA were not caused by an elevation of arterial blood pressure.
No Effect of ADMA on NO Production
Plasma or urine NOx concentrations, markers of systemic NO production, were unaffected by long-term treatment with ADMA in wild-type mice. Moreover, basal or acetylcholine-stimulated vascular NO release, markers of local NO production, were also unaltered by treatment with ADMA. Therefore, it is possible that long-term treatment with ADMA does not reduce either systemic or local vascular NO production.
Involvement of NO-Independent Mechanisms in Long-Term Vascular Effects of ADMA
ADMA inhibits NO synthases by competing with l-arginine. Because the plasma levels of ADMA attained by treatment with ADMA were far less compared with that of l-arginine,37 it may be reasonable that ADMA treatment does not affect NO production. The following lines of evidence further support the involvement of NO-independent mechanisms in the long-term vascular effects of ADMA. First, the effects of ADMA could not be reversed by l-arginine. Second, ADMA treatment did not decrease NO production. Third, ADMA treatment caused a comparable extent of vascular lesion formation irrespective of the presence or absence of eNOS. Fourth, ADMA treatment also caused a comparable extent of direct ACE upregulation and oxidative stress irrespective of the presence or absence of eNOS. Fifth, the vascular lesions were induced in coronary microvessels of eNOS-KO mice, on which expression of other NO synthases was absent.
However, we cannot deny the possibility that the vascular effects of ADMA might be mediated by inhibition of NO delivery from circulating sources, such as S-nitrosohemoglobin or other nitrosothiols, which might be derived from nNOS and/or iNOS. It remains to be fully elucidated whether the vascular effects of ADMA observed in eNOS-KO mice are caused by inhibition of other NOS. For this purpose, mice deficient in all 3 NOS isoforms would need to be developed.
To investigate the inter-relation between ADMA and homocysteine, we examined the influence of ADMA treatment on plasma homocysteine concentrations. However, plasma homocysteine levels were unaffected by the treatment, suggesting no association of homocysteine in the present study.
Involvement of Renin-Angiotensin System in Long-Term Vascular Effects of ADMA
Treatment with ADMA significantly increased tissue ACE expression in the perivascular area and cardiac superoxide production, which were normalized by ACE inhibition or AT1 receptor blockade along with the suppression of vascular lesion formation. Long-term administration of angiotensin II causes perivascular fibrosis in rats,38 which resembles the histopathologic changes seen in ADMA-induced vascular lesions. Furthermore, AT1 receptor stimulation elicits vascular production of reactive oxygen species, including superoxide, which, in turn, induces arteriosclerotic vascular lesions.39 Thus, enhanced tissue ACE expression and oxidative stress through AT1 receptor may contribute, at least in part, to the long-term vascular effects of ADMA, irrespective of the presence or absence of eNOS.
Multiple Nonspecific Effects of l-Arginine Analogues
l-NMMA, another endogenous l-arginine analogue, also induced a comparable extent of coronary microvascular lesions in both strains, as did ADMA. Our recent study demonstrated that synthetic l-arginine analogues, such as l-NAME or l-NNA, similarly elicited a comparable extent of coronary microvascular structural changes in both strains.14 It is therefore possible that nonspecific vascular effects may not be limited to ADMA in particular but may also be extended to l-arginine analogues in general. Multiple actions of l-arginine analogues have been reported, which include inhibition of cytochrome c reduction,13 endothelial generation of superoxide anions,40 antagonism of muscarinic acetylcholine receptors,12 impairment of urea cycle in which l-arginine is a substrate,41 and inhibition of the endothelium-independent relaxation induced by amiloride (an inhibitor of Na+-H+ exchange) and dibutyryl cAMP (a membrane-permeable cAMP analogue).42 Thus, multiple mechanisms other than simple inhibition of endothelial NO synthesis appear to be involved in the long-term vascular effects of l-arginine analogues.
Difference Between Short-Term and Long-Term Effects of ADMA
In contrast to its long-term effects, ADMA acutely inhibits endothelial NO synthesis both in vitro and in vivo.5,18,19 This notion is supported by the fact that the short-term effects of ADMA can be reversed by cotreatment with l-arginine (but not with d-arginine).5,18,19 Although the precise mechanisms for the difference between the short-term and long-term vascular effects of ADMA remains to be elucidated, it is conceivable that metabolites of ADMA may be accumulated at higher concentrations in blood vessels, exerting various unknown effects other than simple inhibition of endothelial NO synthesis, and that other compensatory mechanisms for NO bioavailability (eg, endothelial superoxide dismutase system or cofactors for eNOS) may be upregulated.
In summary, we were able to demonstrate a novel mechanism by which ADMA causes arteriosclerotic vascular lesion formation in an eNOS-independent manner. Direct upregulation of local ACE and increased oxidative stress via AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo. Our findings should contribute to a better understanding of the pathophysiological role of ADMA in arteriosclerosis.
We thank R. Tsunawaki, T. Ishida, and R. Maekado for their excellent technical assistance. This work was supported in part by grants-in-aid for Scientific Research (14570096) and for Exploratory Research (16650097 and 16659209) from the Ministry of Education, Culture, Sports, Science, and Technology, Tokyo, Japan; a research grant from the Japan Foundation of Cardiovascular Research, Tokyo, Japan; a research grant from the Sankyo Pharmaceutical Co, Tokyo, Japan; and a research grant from the University of Occupational and Environmental Health, Kitakyushu, Japan.
O. Suda and M. Tsutsui contributed equally to this work.
- Received March 16, 2004.
- Accepted May 30, 2004.
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