Accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, attenuated dimethylarginine dimethylaminohydrolase activity and intimal hyperplasia in premenopausal human uterine arteries

Atherosclerosis 178 (2005) 231–239

Accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, attenuated dimethylarginine dimethylaminohydrolase activity and intimal hyperplasia in premenopausal human uterine arteries Renzo Y. Loyaga-Rendona , Shuichi Sakamotoa , Masashi Beppua , Takeshi Asoa , Mihoko Ishizakab , Ryoko Takahashib , Hiroshi Azumab,∗ a

b

Comprehensive Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113-8519, Japan Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-0062, Japan Received 16 October 2003; received in revised form 7 September 2004; accepted 22 September 2004 Available online 21 November 2004

Abstract The present study was designed to investigate the involvement of nitric oxide synthase (NOS), endogenous NOS inhibitors, arginase, which shares l-arginine as a common substrate with NOS, and dimethylarginine dimethylaminohydrolase (DDAH) as a metabolizing enzyme of NOS inhibitors in the occurrence of intimal hyperplasia in premenopausal human uterine arteries. Fifty-two uterine arteries were obtained from 52 patients undergoing total hysterectomy with an informed consent for the present study. All specimens were assessed histologically and the intima:media ratio (%) was evaluated as an index of intimal hyperplasia. Nineteen specimens were found to be histologically normal (intima:media ratio = 16.1 ± 0.8%), whereas remaining 33 specimens were categorized as intimal hyperplasia (intima:media ratio = 34.4 ± 1.5%). The intimal hyperplasia was associated with the impaired cyclic GMP production without change in endothelial NOS activity per se, accumulation of endogenous NOS inhibitors in endothelial cells, attenuated DDAH activity in endothelial cells and enhanced arginase activity in endothelial cells and smooth muscle layer. These findings suggest that the impaired cyclic GMP production as a marker of NO production is possibly due to the accumulated endogenous NOS inhibitors and enhanced arginase activity, which, in turn, closely relates to the occurrence of intimal hyperplasia, and that the impaired DDAH activity would result in the accumulation of endogenous NOS inhibitors in endothelial cells. Because of the enhanced arginase activity in endothelial cells and smooth muscle layer, the accelerated polyamine biosynthetic pathway may be implicated in the occurrence of intimal hyperplasia in premenopausal human uterine arteries. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Intimal hyperplasia; Impaired NO production; NOS; Endogenous NOS inhibitors; DDAH; Arginase

1. Introduction Despite the accumulated knowledge on the pathogenesis of atherosclerosis, mechanisms underlying the initiation and progression of the disorder are poorly understood. Recently, attention has been paid to the impaired NO production in endothelial cells as an important pathogenic factor of atherosclerosis. Since NO mediates endothelium-dependent ∗

Corresponding author. Tel.: +81 3 5280 8095; fax: +81 3 5280 8095. E-mail address: [email protected] (H. Azuma).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.09.006

vasodilatation [3], and inhibits platelet adhesion and aggregation [4], monocyte and leukocyte adhesion [18], smooth muscle cell growth [32], ornithine decarboxylase (ODC, which is a rate-limiting enzyme in the polyamine biosynthetic pathway) [33] and endothelin production as a potent mitogen [9], the impaired NO production is thought to be involved in the pathogenesis of atherosclerosis [34]. Recently, it has been reported that accumulation of endogenous NOS inhibitors such as NG -monomethyl-larginine (l-NMMA) and asymmetric NG ,NG -dimethyl-larginine (ADMA) in plasma [16] and tissues [36] might in

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part explain the mechanism decreasing NO production. Elevated endogenous NOS inhibitors are reportedly known to be involved in diseases such as hypertension [23], chronic renal failure [11], hypercholesterolemia [21], diabetes mellitus [38] and intimal hyperplasia [35]. l-NMMA and ADMA are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to l-citrulline and monomethylamine (or dimethylamine in the case of ADMA) [8]. Thus, the impaired DDAH activity results in the accumulated endogenous NOS inhibitors and, in turn, the impaired NO production [17]. Indeed, homocysteine inhibits DDAH activity, causing ADMA to accumulate and inhibit NO synthesis, suggesting the known effect of homocysteine to impair endothelium-mediated NO-dependent vasodilatation [30]. Furthermore, a glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in diabetes mellitus [37]. l-Arginine as a substrate for NOS is also consumed in pathways other than NO production [22]. Arginase metabolizes l-arginine to urea and l-ornithine through the urea cycle. It has been reported that the increased arginase activity decreases the l-arginine availability for NOS and, therefore, attenuates NO production [25]. It is also reportedly known that two products of the l-arginine–NO pathway interfere with cell growth by distinct mechanism. NG -hydroxy-larginine (NOHA) as an intermediate of NO production and NO itself appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively [32]. Ornithine is the principal precursor for production of polyamines, which are required for cell proliferation [24]. Cell proliferation is a main feature of atherosclerotic lesions. It might be possible that the NO and polyamine pathways could be important in the development of atherosclerosis. Therefore, the present experiments were designed to investigate the involvement of endothelial NOS, endogenous NOS inhibitors, arginase and DDAH in causing the intimal hyperplasia in premenopausal human uterine arteries.

2. Methods 2.1. Collection of human uterine arteries Informed consent was obtained from patients, complying with the Ethics Committee Guides of Tokyo Medical and Dental University. Fifty-two human uterine arteries were collected from 52 patients undergoing abdominal total hysterectomy. Premenopausal women were enrolled in sequential manner. Patients with malignant disease except for intracervical neoplasia, as a reason of surgery, were excluded from the present study. Immediately after obtaining the uterus, the ascending branches of the uterine arteries in the parametrium were carefully dissected in the ice-cold modified Krebs solution. The composition of the modified Krebs solution (mM) was as follows: NaCl, 118.0; KCl, 4.7; MgSO4 ·7H2 O, 1.2;

CaCl2 ·2H2 O, 2.5; KH2 PO4 , 1.2; NaHCO3 , 25.0; and glucose, 10.0, which was bubbled with 95% O2 and 5% CO2 (pH 7.4). Surrounding connective tissue and fat were trimmed off. Arteries were stored at −80 ◦ C until the measurements except the ones for measurement of cyclic GMP production. Since the amount of uterine artery specimens was limited, it was impossible to perform all determinations in specimens from one patient. Determinations were therefore carried out in a sequential manner. The first 11 uterine arteries were used for the measurement of cyclic GMP production, the following 16 for determinations of NOS and arginase activities, and the following 12 for measurement of concentrations of l-arginine, ADMA, l-NMMA and SDMA in endothelial cells. The last 13 specimens were used for determination of DDAH activity in endothelial cells. 2.2. Histological assessment of intimal hyperplasia Light microscopic examination was performed according to the method described previously [31] in a blinded manner for all uterine artery specimens. Because uterine arteries are meandering, three specimens were excised from different parts (distal, middle and proximal part) of the artery to make the assessment impartially. The magnitude of intimal hyperplasia was given as the ratio of intimal area to medial area (intima:media ratio in percentage), which had been assessed histologically (Elastica-Van Gieson, ×40). The areas of intima and media were calculated by computed image analysis (Java Image Software, Bethesda, Maryland, USA) according to the method described previously [31,35]. Results were given as a mean intima:media ratio of three parts. Arteries were classified into two groups according to the histological examination, that is, histologically normal arteries as group I and arteries with intimal hyperplasia as group II. The classification was made taking 22% limit of intima:media ratio as normal, which was validated in the previous reports [31,35]. 2.3. Cyclic guanosine monophosphate (cyclic GMP) measurement Eleven uterine arteries, collected in order, were used within 2 h after the isolation for determining cyclic GMP production. Ring preparations, weighing approximately 10 mg with intact endothelium, were cut off with razor blade. Cyclic GMP levels were measured as described previously [31,35]. Briefly, preparations, which had been processed without damaging endothelium, were preincubated in the modified Krebs solution for 20 min at 37 ◦ C, transferred into a fresh modified Krebs solution and followed by a further 30 min incubation. Preparations were then rapidly transferred into 10% trichloroacetic acid (TCA) with liquid nitrogen in order to stop the reaction. Norepinephrine (NE, 10−6 M) and acetylcholine (ACh, 10−5 M) were added immediately and 15 min after transferring the specimens into the fresh modified Krebs solution, respectively. All experiments were performed in the presence of 10−5 M 3-isobutyl-1-methylxanthine (IBMX) as

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a non-selective inhibitor of phosphodiesterases. The net production of cyclic GMP was expressed as the difference between the production with NE plus ACh and that with NE plus ACh plus 10−4 M NG -nitro-l-arginine (NOARG) as an inhibitor of NOS. The basal cyclic GMP level was taken as the value without any agonist and antagonist except for IBMX. The protein concentration of the samples was determined by use of the protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). 2.4. Nitric oxide synthase (NOS) activity in endothelial cells The NOS activity was determined according to the method described previously with minor modifications [36]. In brief, endothelial cells were collected from uterine arteries weighting approximately 150 mg by the method described above. The buffer used for this purpose was composed of 50 mM Tris–HCl, 10 mM CHAPS, 2 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 ␮M pepstatin A and 2 ␮M leupeptin (pH 7.4). The collected endothelial cells were sonicated at 50 W, 28 kHz (GSD-50, SMT Co., Tokyo, Japan) for 15 s (three times at 1 min intervals), and the homogenate was centrifuged at 10,000 × g, for 20 min at 4 ◦ C to separate the supernatant, in which protein concentration was determined using BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Incubation mixtures consisted of 100 ␮l of the supernatant and 20 ␮l of the buffer described above containing 1 mM NADPH, 4 ␮M flavin adenine dinucleotide (FAD), 4 ␮M flavin mononucleotide (FMN), 10 ␮M tetrahydrobiopterin (BH4 ), 1 mg/l calmodulin, 2.5 mM CaCl2 , and 0.1 ␮Ci/ml [14 C(U)]-l-arginine (specific activity: 11,581 MBq/mmol). The reaction mixture was incubated at 37 ◦ C for 1 h in a shaking water bath. The incubation was terminated by keeping the tubes on ice for 5 min. Samples were then applied to a 1 ml column of Dowex AG50W-X8 (Na+ form) to remove unmetabolized [14 C]-larginine. The columns were then washed with 1.5 ml distilled water, and [14 C]-l-citrulline was quantified in the flowthrough fraction using a liquid scintillation counter (TRICARB 2750TR/LL, Packard Instrument Co., Meriden, CT, USA). NOS activity was expressed as pmol l-citrulline/(mg protein per 60 min). The activity was also measured in the presence of 100 ␮M l-NOARG or 30 ␮M aminoguanidine as an inhibitor of inducible NOS [29]. Calcium-independent NOS activity was measured in the incubation mixture removing CaCl2 and containing 20 mM EDTA. The net activity was expressed as the difference between activities in the absence and presence of l-NOARG. 2.5. Arginase activity in endothelial cells and smooth muscle layer The arginase activity in endothelial cells was measured in the same preparations as those obtained for the determination of NOS activity. Remaining smooth muscle layer was ho-

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mogenized in a Polytron (Kinematica, Lucerne, Switzerland), three times at maximum speed for 20 s each, to a 25% homogenate in the buffer consisting of 50 mM Tris–HCl, 10 mM CHAPS, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 ␮M pepstatin A and 2 ␮M leupeptin, pH 7.4. The homogenate was centrifuged at 10,000 × g for 20 min at 4 ◦ C, and the supernatant was decanted from the pellet. Arginase activity was measured by determining the conversion of l-[guanido-14 C]arginine to [14 C]-urea according to the method described previously with minor modifications [2]. Aliquots of tissue extracts (10 ␮l) were incubated in a final volume of 100 ␮l buffer containing 9 mM Tris–HCl, 0.08 ␮Ci/ml of l[guanido-14 C]-arginine (specific activity: 1776 MBq/mmol) and 1 mM MnCl2 , pH 9.6, for 2 h at 37 ◦ C. Reactions were terminated by the addition of 400 ␮l of ice-cold stop buffer containing 250 mM sodium acetate and 100 mM urea, pH 4.5. Arginase exerts essentially no activity at the low pH of the stop solution. Samples were passed through a column containing 1.5 ml Dowex 50W-X8 resin to remove unmetabolized l-[guanido-14 C]-arginine. The columns were then washed with 1.5 ml of distilled water, and [14 C]-urea was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB2750TR/LL, Packard Instrument Co., Meriden, CT, USA). The enzyme activity was determined in the presence and absence of 20 ␮M NG -hydroxy-larginine as an arginase inhibitor [32]. Results were given as the net activity calculated from the difference of the activities in the presence and absence of NOHA. 2.6. Endogenous methylarginines and l-arginine Contents of NG -monomethyl-l-arginine, asymmetric symmetric NG ,NG -dimethyll-arginine (SDMA) and l-arginine in endothelial cells were determined by means of high-performance liquid chromatography (HPLC) as reported previously [35,36]. Endothelial cells were collected from the uterine artery specimens by gently rubbing of the luminal surface according to the method described previously [35]. The collected endothelial cells were sonicated at 50 W, 28 kHz (GSD-50, SMT Co., Tokyo, Japan) for 15 s (three times at 1 min intervals) in 5 mM icecold N-(2-hydroxyethyl)-piperazine-N -2-ethanesulphonic acid (HEPES) buffer (pH 7.4). The homogenate was centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernatant was separated and lyophilized with the aid of the centrifugal vaporizer (CVE-100D, Eyela, Tokyo, Japan). Twentymicroliters aliquots of the reconstituted solution with HEPES buffer were assayed for DNA by a fluorometric method [1]. After addition of TCA in a final concentration of 5%, the centrifugation at 1600 × g for 15 min was followed to obtain the supernatant, of which 100 ␮l aliquot was applied for HPLC to determine l-arginine and methylarginines. The minimum concentration of methylarginines that can be detected was 5 pM. To estimate intracellular concentration of l-NMMA, ADMA, SDMA and l-arginine, we obtained the equation between the number of endothelial cells (y) and DNA concen-

NG ,NG -dimethyl-l-arginine,

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tration (x) by the least square method: y = (14.3x + 6) × 104 (r = 0.9840) [19]. From a determination of DNA concentration in endothelial cell supernatant, the number of endothelial cells was calculated. If the intracellular water space is assumed to be 2 pl/endothelial cell [10], intracellular concentrations of l-arginine and methylarginines can be calculated. To evaluate the inhibitory effects of methylarginines (l-NMMA and ADMA) on the NOS activity, we harvested endothelial cells from thoracic aorta of male adult rabbits. The NOS activity was determined as described above in the presence (1, 3, 10, 30 and 100 ␮M) or absence of the methylarginines. Since the plasma levels of methylarginines were not determined in the patients in the current experiment, the plasma concentrations of l-arginine, l-NMMA, ADMA and SDMA were measured in 14 healthy volunteers (23–54 years old) and compared to the concentrations in endothelial cells of histologically normal groups I and II with intimal hyperplasia in order to understand the differences between plasma and endothelial cell levels.

Fig. 1. Intima:media ratio in groups I and II. Nineteen uterine artery specimens out of 52 obtained from 52 patients were histologically normal. The mean intima:media ratio of these 19 specimens was estimated to be 16.1 ± 0.8% (range: 9.5–21.6%). These specimens were classified as group I. Various degree of intimal hyperplasia was observed in the remaining 33 specimens, of which intima:media ratio was estimated to be 34.4 ± 1.5% (range: 23.3–57.0%). These specimens were classified as group II. ***Significant difference at p < 0.005.

Meriden, CT, USA). DDAH activity was expressed as pmol l-citrulline/(mg protein per 2 h). 2.8. Statistical analysis

2.7. Dimethylarginine dimethylaminohydrolase activity in endothelial cells DDAH activity was measured by determining the conversion of [3 H]-l-NMMA to [3 H]-l-citrulline. Endothelial cells were collected from uterine arteries weighting approximately 150 mg by the method described previously [35]. The buffer used for this purpose was composed of 100 mM sodium phosphate buffer (pH 6.5), 1 mM PMSF, 2 mM 2mercaptoethanol, 1 ␮M pepstatin A and 2 ␮M leupeptin. The collected endothelial cells were sonicated at 50 W, 28 kHz (GSD-50, SMT Co., Tokyo, Japan) for 15 s (three times at 1 min intervals), and the homogenate was centrifuged at 10,000 × g, for 20 min at 4 ◦ C to separate the supernatant, in which protein concentration was determined using BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Incubation mixtures consisted of 90 ␮l of the supernatant and 10 ␮l of the buffer described above containing 20 mM EDTA, 0.1 ␮M l-NMMA and 0.01 ␮Ci/ml of l-[3 H]-NMMA. [3 H]l-NMMA [NG -monomethyl-l-arginine(2,3,4-3 H)] (specific activity: 2.00 TBq/mmol) was manufactured by Daiichi Pure Chemicals Co. (Ibaragi, Japan). Radiochemical purity of [3 H]-l-NMMA which had been determined by Dupon/NEN Boston Analytical Service (Boston, MS, USA) with the aid of high-performance liquid chromatography was 97.6%. The reaction mixture was incubated at 37 ◦ C for 2 h in a shaking water bath. The incubation was terminated by keeping the tubes on ice for 5 min. Samples were applied to a 1 ml column of Dowex AG50W-X8 (Na+ form) to remove unmetabolized [3 H]-l-NMMA. The columns were then washed with 1.5 ml distilled water, and [3 H]-l-citrulline was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB 2750TR/LL, Packard Instrument Co.,

Data are shown as mean ± standard error of mean. For comparison between two groups, unpaired t-test was used. Significant difference was accepted if p < 0.05.

3. Results 3.1. Morphological findings Nineteen specimens out of 52 uterine arteries isolated from 52 patients were histologically normal. The mean intima:media ratio of these 19 specimens was determined to be 16.1 ± 0.8% (range: 9.5–21.6%). These specimens were classified as group I. In the remaining 33 specimens, various degrees of intimal hyperplasia were observed, of which intima:media ratio was estimated to be 34.4 ± 1.5% (range: 23.3–57.0%). These specimens were classified as group II. The value in group II was significantly (p < 0.005) greater than that in group I (Fig. 1). The medial layer was histologically normal in all specimens. The histologically normal group I was compared with the hyperplasic group II in the following parameters. 3.2. Cyclic GMP production Fresh 11 uterine artery specimens (within 2 h after the isolation) obtained from 11 patients were subjected to the measurement of cyclic GMP production. Basal and stimulated productions of the nucleotide with acetylcholine were abolished in the presence of 100 ␮M NOARG and in specimens from which endothelium had been removed. Cyclic GMP productions varied considerably among 11 speci-

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Fig. 2. Basal and net productions of cyclic GMP in histologically normal specimens and specimens with intimal hyperplasia. Fresh 11 uterine artery specimens (within 2 h after the isolation) obtained from 11 patients were subjected to the measurement of cyclic GMP production. The mean basal and net productions were calculated to be 4.2 ± 0.7 and 8.7 ± 1.1 fmol/mg protein for the histologically normal specimens (n = 4), and 0.3 ± 0.1 (p < 0.005) and 1.3 ± 0.6 (p < 0.005) fmol/mg protein for specimens with intimal hyperplasia (n = 7), respectively. The intima:media ratio (%) was estimated to be 13.6 ± 2.2 in the former (n = 4) and 36.4 ± 4.0 in the latter (n = 7, p < 0.005). ***Significant difference vs. corresponding value in the basal and net productions at p < 0.005.

mens (0–4.5 fmol/mg protein for the basal production and 0–11.8 fmol/mg protein for the net production). However, if these values were arranged according to the histological findings, the mean basal and net productions were calculated to be 4.2 ± 0.7 and 8.7 ± 1.1 fmol/mg protein for the histologically normal specimens (n = 4), and 0.3 ± 0.1 (p < 0.005) and 1.3 ± 0.6 (p < 0.005) fmol/mg protein for specimens with intimal hyperplasia (n = 7), respectively. These results are shown in Fig. 2. The intima:media ratio (%) was estimated to be 13.6 ± 2.2 in the former (group I; n = 4) and 36.4 ± 4.0 in the latter (group II) (n = 7, p < 0.005), of which values were evaluated according to the criteria described previously [31]. 3.3. NO synthase (NOS) activity The NOS activity was greatly inhibited in the presence of 20 mM EDTA or 100 ␮M NOARG as a nonselective inhibitor of NOS, but remained unaffected by 30 ␮M aminoguanidine as an inhibitor of inducible NOS [29], indicating that the NOS activity determined in the present experiment was due dominantly to the constitutive isozyme [12]. Mean NOS activities were determined to be 153 ± 18 pmol l-citrulline/(mg protein per 1 h) for group I (n = 5) and 154 ± 10 pmol l-citrulline/(mg protein h−1 ) for group II (n = 11). These values were not significantly different from each other. 3.4. Arginase activity The net arginase activity was given as the difference between total activity and the activity in the presence of 20 ␮M NOHA as a selective arginase inhibitor [32]. The

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Fig. 3. Arginase activities in endothelial cells (A) and smooth muscle layer (B) of group I with normal intima and group II with intimal hyperplasia. The arginase activity in endothelial cells was determined to be 957 ± 132 pmol urea/(mg protein per 2 h) for group I (n = 5) and 1780 ± 85 pmol urea/(mg protein per 2 h) for group II (n = 11). The enzyme activity in endothelial cells was relatively higher (p < 0.005) than that in the smooth muscle layer in both groups. ***Significant difference vs. corresponding value in endothelial cells and smooth muscle layer at p < 0.005.

arginase activity in endothelial cells was determined to be 957 ± 132 pmol urea/(mg protein per 2 h) for group I (n = 5) and 1780 ± 85 pmol urea/(mg protein per 2 h) for group II (n = 11). The enzyme activity was significantly (p < 0.005) higher in group II with intimal hyperplasia (Fig. 3A). The arginase activity in the smooth muscle layer of which endothelial cells had been removed was also significantly (p < 0.005) higher in group II (792 ± 74 pmol urea/(mg protein per 2 h), n = 11) than the value in group I (363 ± 53 pmol urea/(mg protein per 2 h), n = 5) (Fig. 3B). Arginase activity in endothelial cells was relatively higher (p < 0.005) than that in the smooth muscle layer in both groups. 3.5. Concentrations of l-arginine and methylarginines in endothelial cells The mean concentrations of l-NMMA and ADMA in endothelial cells were determined to be 0.7 ± 0.1 and 0.6 ± 0.1 ␮M in group I (n = 5) and 3.0 ± 0.3 and 2.9 ± 0.8 ␮M in group II (n = 7), respectively. The concentrations were significantly (p < 0.005) higher in group II than those in group I. The relative figures of l-NMMA:ADMA were approximately 1:1 in all cases. Changes in the concentrations of l-NMMA plus ADMA are shown in Fig. 4A. l-Arginine concentration tended to decrease in group II, but not significantly different (324 ± 43 ␮M in group I and 206 ± 36 ␮M in group II). Concentrations of SDMA remained unchanged in both groups (0.3 ± 0.1 ␮M in group I and 0.4 ± 0.1 ␮M in group II). The IC50 values of l-NMMA and ADMA for NOS were determined to be 2.7 ± 0.2 ␮M (n = 5) and 15.4 ± 1.0 ␮M (n = 5) in the rabbit thoracic aortic endothelial cells, respectively. The inhibitory effect of 3 ␮M l-NMMA and ADMA (the mean concentration determined in the hyperplasic arteries) on the NOS activity was 54.8 ± 2.0% (n = 5) and 16.8 ± 1.4% (n = 5), respectively. On the other hand, the

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Fig. 4. Concentrations of l-NMMA and ADMA as endogenous NOS inhibitors (A) and DDAH activity (B) in group I with normal intima and group II with intimal hyperplasia. The mean concentrations of l-NMMA and ADMA in endothelial cells were determined to be 0.7 ± 0.1 and 0.6 ± 0.1 ␮M in group I (n = 5) and 3.0 ± 0.3 and 2.9 ± 0.8 ␮M in group II (n = 7), respectively. DDAH activity was determined to be 6.2 ± 0.8 pmol l-citrulline/(mg protein per 2 h) (n = 8) in group II, of which value was significantly lower than that of 13.8 ± 0.8 pmol l-citrulline/(mg protein per 2 h) in group I (n = 5). ***Significant difference vs. corresponding value at p < 0.005. l-NMMA, NG -monomethyl-l-arginine; ADMA, asymmetric NG ,NG -dimethyl-l-arginine; DDAH, dimethylarginine dimethylaminohydrolase.

plasma concentrations of l-arginine, l-NMMA, ADMA and SDMA in healthy volunteers were determined to be 67 ± 5, 0.002 ± 0.001, 0.25 ± 0.03 and 0.25 ± 0.02 ␮M (n = 14), respectively. (Table 1). 3.6. Dimethylarginine dimethylaminohydrolase activity DDAH is an enzyme that metabolizes l-NMMA and ADMA to l-citrulline and monomethylamine (or dimethylamine in the case of ADMA) [8]. Thus, the decreased DDAH activity brings about an accumulation of l-NMMA and ADMA in endothelial cells [17,30,37]. We compared DDAH activity in endothelial cells. The enzyme activity was determined to be 6.2 ± 0.8 pmol l-citrulline/(mg protein per 2 h) (n = 8) in group II, which value was significantly (p < 0.005) lower than 13.8 ± 0.8 pmol l-citrulline/(mg protein per 2 h) of group I (n = 5) (Fig. 4B). 3.7. Baseline data of patients Age, systolic and diastolic blood pressure, total cholesterol and triglyceride were compared between groups I and II. As shown in Table 2, these values were not significantly different from each other. Systolic and diastolic blood pressure, total cholesterol and triglyceride were within normal range. Normal values in our hospital are 128–249 mg/dl for total cholesterol and 55–159 mg/dl for triglyceride. Cholesterol fractions and basal glucose levels were not determined in the present experiments.

Table 2 Baseline data of patients Group I (n = 19) Age (years) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mg/dl) Triglyceride (mg/dl)

45.2 123.3 76.2 192.7 110.9

± ± ± ± ±

1.1 2.9 2.9 7.2 12.8

Group II (n = 33) 45.9 119.4 72.5 191.4 97.1

± ± ± ± ±

0.7 2.4 2.1 5.8 6.4

Reproductive history, smoking history, cause for hysterectomy and cardiovascular health are shown in Table 3. These baseline data were not different between both groups except for the rate of smokers to the number of patients, which was approximately double in group I (36.8% versus 15.2% in group II). Intima:media ratios in group I were determined to be 15.5 ± 1.6% for smokers (n = 7) and 16.5 ± 1.0% for nonsmokers (n = 12). These values were not significantly different from each other. The intima:media ratio of 36.0 ± 5.8% for smokers (n = 5) in group II was again not significantly different from 34.1 ± 1.4% for non-smokers (n = 28) in the same group.

4. Discussion In spite of the limited number of specimens in each experiment, we demonstrated that the intimal hyperplasia in premenopausal human uterine arteries was associated with the decrease in endothelium- and NOS-dependent

Table 1 Concentrations of l-arginine and methylarginines in plasma of healthy volunteers, and in endothelial cells of patients with normal uterine arteries (group I) and arteries with intimal hyperplasia

Plasma ECs (group I) ECs (group II)

l-Arginine (␮M)

l-NMMA (␮M)

ADMA (␮M)

SDMA (␮M)

67 ± 5 324 ± 43 206 ± 36

0.002 ± 0.001 0.7 ± 0.1 3.0 ± 0.3

0.25 ± 0.03 0.6 ± 0.1 2.9 ± 0.8

0.25 ± 0.02 0.3 ± 0.1 0.4 ± 0.1

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Table 3 Reproductive history, smoking history, causes of hysterectomy and cardiovascular health in patients of two groups Reproductive history

Smoking history

Causes for hysterectomy

Cardiovascular health

Group I (n = 19) 0–4 Gravida (mean = 1.6)

Smokers (7)

Myoma (17) Adenomyosis (3) Ovarian tumor (1)

Within normal limits (17) Hypertension (2) Diabetes mellitus (1)

Non-smokers (12)

Carcinoma In situ, cervix (1)

Smokers (5)

Myoma (23) Adenomyosis (8) Endometriosis (3) Ovarian tumor (3)

Within normal limits (29) Hypertension (2) Premature ventricular Contraction (1)

Non-smokers (28)

Carcinoma In situ, cervix (4)

Left branch block (1) Diabetes mellitus (1)

0–3 Para (mean = 1.4)

Group II (n = 33) 0–7 Gravida (mean = 1.8)

0–3 Para (mean = 1.4)

cyclic GMP production, the accumulation of endogenous NOS inhibitors in endothelial cells, the enhanced arginase activities in endothelial cells and smooth muscle layer, and the impaired DDAH activity in endothelial cells, whereas the endothelial NOS activity per se remained unaltered. There are several questions that should be answered. It is important to demonstrate whether the accumulated concentrations of l-NMMA and ADMA in endothelial cells of specimens with intimal hyperplasia are sufficient to inhibit NOS. Endothelial concentrations of l-NMMA and ADMA were determined to be 3.0 ± 0.3 and 2.9 ± 0.8 ␮M in specimens with intimal hyperplasia (group II), respectively. The IC50 values of l-NMMA and ADMA for NOS, which had been prepared from endothelial cells of rabbit thoracic aorta, were determined to be 2.7 ± 0.2 and 15.4 ± 1.0 ␮M, respectively. l-NMMA and ADMA at 3 ␮M showed inhibitory effects on the NOS activity (54.8 ± 2.0%, n = 5, and 16.8 ± 1.4%, n = 5, respectively). Thus, the determined concentrations of the two methylarginines seem to be sufficient to inhibit endothelial NOS activity. l-NMMA and ADMA are endogenously produced inhibitors of NOS. In the plasma, ADMA and SDMA are the major circulating forms of methylarginines, whereas l-NMMA level is considerably low. An elevated level of ADMA has been found in multiple disorders where NOS dysfunction has been implicated, such as hypercholesterolemia [21], renal failure [11] and hypertension [23] among others [27]. Many researchers pay little attention to l-NMMA as an endogenous NOS inhibitor. However, we have reported that the content of l-NMMA is approximately equal to that of ADMA in the corpus cavernosum [36] and endothelial cells [35]. Masuda et al. [36] demonstrated that the content of lNMMA as well as that of ADMA increased in the corpus caverosum after ischemia without changing plasma methylarginines, and that the inhibitory effect of l-NMMA on NOS was more potent than that of ADMA. Furthermore, concordantly with the data shown in this report, it has been shown

that the intracellular ADMA levels are ∼10-fold higher than the reported range for plasma values. The above findings suggest that ratio and concentration of methylarginines differ between plasma and cells. Herein, we provide evidence that l-NMMA might also play an important role for the development of atherosclerosis through modulation of NO production. Thus, l-NMMA as an endogenous NOS inhibitor should be investigated in the regulation of NOS in the cells and tissues. There is a report describing that the l-arginine– methylarginine ratio in plasma contributes to systemic NO production in the rabbit [16]. Thus, it seems plausible that the ratio of intracellular l-arginine to methylarginines also contributes to NO production in endothelial cells. In the present experiments, the significantly decreased larginine:methylarginines ratio in endothelial cells (slightly decreased l-arginine and significantly increased methylarginines in group II) was associated with the impaired cyclic GMP production as a marker of NO production [7], suggesting that the accumulation of endogenous NOS inhibitors in endothelial cells would, at least partly, result in the impaired NO production. This suggestion seems to be partly supported by the finding that the NOS activity per se remained unaltered in the group II specimens. It could be argued that the decreased cyclic GMP production is due to an impaired guanylate cyclase activity rather than an impaired NO production. However, we showed in previous publications that the ability of sodium nitroprusside (SNP) as a NO donor to produce cyclic GMP was not different between histologically normal human uterine artery specimens and specimens with intimal hyperplasia [31] and that the relaxation in response to SNP remained unchanged even in hyperplasic specimens of human uterine arteries [13]. Therefore, we ruled out the possibility of the impaired guanylate cyclase activity to decrease cyclic GMP production and concluded that the decreased cyclic GMP production in specimens with intimal hyperplasia would be due to the impaired

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NO production. Another possible explanation for the decreased cyclic GMP production found in the hyperplasic arteries would be an increased phosphodiesterase activity; the involvement of the latter mechanism remains to be investigated. l-Arginine as a substrate for NOS is also consumed in pathways other than NO production [22]. Arginase, which exists as the isoforms of constitutive arginase (arginase I) and inducible arginase (arginase II) in smooth muscle cells and endothelial cells [15,28], metabolizes l-arginine to urea and l-ornithine through the urea cycle. It is also reportedly known that the increased arginase activity decreases the larginine availability to NOS and, therefore, attenuates the NO production [25]. The present experiments demonstrated that the arginase activity in endothelial cells was significantly increased in group II with intimal hyperplasia. Thus, it is suggested that the impaired NO production in group II would be partly due to the decreased l-arginine availability resulting from the enhanced arginase activity. Since the arginase activity in endothelial cells was significantly increased, it might be expected that there will be decreased intracellular l-arginine levels in the hyperplasic arteries (group II). However, the l-arginine level decreased slightly but not significantly in group II. Although the intracellular l-arginine concentration exceeds by far the Km value of NOS, the exogenous application of l-arginine increases the NO production [27], which is known as “arginine paradox”. The proposed explanation of this phenomenon has been the existence of arginine pools, and colocalization of arginine transporters and NOS in caveolae [20]. Thus, if arginase colocalizes with NOS in caveolae, an increased arginase activity might deprive the adjacent NOS of l-arginine without decreasing the total levels of l-arginine. It has been reported that other enzymes involved in the arginine–citrulline metabolism, such as argininosuccinate synthase and argininosuccinate lyase, colocalize with NOS in caveolae [26], suggesting close spatial relationship between the enzymes necessary for arginine metabolism. However, further experiments should be performed to analyze the colocalization of arginase and NOS. DDAH is an enzyme that metabolizes l-NMMA and ADMA to l-citrulline and monomethylamine (and dimethylamine in the case of ADMA) [8]. Thus, the impaired DDAH activity results in the accumulated endogenous NOS inhibitors [17,30,37]. The DDAH activity was significantly lower in group II with intimal hyperplasia than that in the histologically normal group I (basal levels), leading us to assume that the accumulation of endogenous NOS inhibitors in endothelial cells would be due to the decreased metabolism of these inhibitors by DDAH. This possibility is partly supported by the findings that the intracellular concentration of SDMA, which is not a substrate for the metabolizing enzyme [8], remained unchanged in group II. On the other hand, it is reportedly known that l-arginine as well as methylarginines enters cells through the cationic amino acid transporter known collectively as system y+ [14]. The increased

transport would result in the increased intracellular concentrations of l-arginine, l-NMMA, ADMA and SDMA. However, the accumulated l-NMMA and ADMA were associated with unchanged SDMA and slightly decreased l-arginine in the present experiments. Therefore, the increased transmembrane transport is possibly not implicated in the accumulation of endogenous NOS inhibitors in endothelial cells. NO inhibits platelet adhesion and aggregation [4], which are considered as initiating events for the intimal hyperplasia [5]. It has also been demonstrated that NO inhibits vascular smooth muscle cell proliferation [6]. We have demonstrated that intimal hyperplasia is at least partly caused through the impaired ability of endothelial cells to produce NO, and that the accumulation of endogenous l-NMMA and ADMA is accompanied by the decreased NO production and occurrence of the intimal hyperplasia [35]. On the other hand, two products of the l-arginine–NO pathway interfere with cell growth by distinct mechanism. NG -hydroxy-l-arginine as an intermediate of NO production and NO itself appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively [32]. Ornithine decarboxylase is a rate-limiting enzyme in the polyamine biosynthetic pathway [33]. Polyamines are required for cell proliferation [24]. If these findings are considered together, the decreased productions of NO and probably NOHA, which are possibly brought about by the accumulation of endogenous NOS inhibitors and the increased arginase activity, would result in the cancellation of cell growth inhibition by NO and the acceleration of polyamine biosynthetic pathway and, in turn, closely relate to the occurrence of the intimal hyperplasia in premenopausal human uterine arteries. However, further investigations should be performed for a better understanding of the detailed mechanisms.

5. Conclusion In the human uterine arteries with intimal hyperplasia, the decreased cGMP production as a marker of NO production is possibly due to the accumulated endogenous NOS inhibitors and enhanced arginase activity. The accumulation of endogenous NOS inhibitors would be brought about by a decreased DDAH activity in endothelial cells. In addition, because of the enhanced arginase activity in endothelial and smooth muscle layer, the acceleration of the polyamine biosynthetic pathway might be implicated in the occurrence of intimal hyperplasia in premenopausal human uterine arteries.

Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research (14572152 to H.A.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Smoking Research Foundation, Japan (to H.A.), and the New Drug Research (NDR) Foundation, Japan (to H.A.).

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