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Coinduction of Endothelial Nitric Oxide Synthase and Arginine Recycling Enzymes in Aorta of Diabetic Rats
NITRIC OXIDE: Biology and Chemistry Vol. 5, No. 3, pp. 252–260 (2001) doi:10.1006/niox.2001.0344, available online at http://www.idealibrary.com on
Coinduction of Endothelial Nitric Oxide Synthase and Arginine Recycling Enzymes in Aorta of Diabetic Rats Seiichi Oyadomari,* ,† Tomomi Gotoh,* Kazumasa Aoyagi,‡ Eiichi Araki,† Motoaki Shichiri,† and Masataka Mori* ,1 *Department of Molecular Genetics and †Department of Metabolic Medicine, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan; and ‡Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan
Received January 30, 2001; published online May 9, 2001
Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline–NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1–2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1–2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-1 (TGF-1) mRNA in the aorta was also induced. TGF-1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-1. The results also suggest that TGF-1 works antiatherogenically 1
To whom correspondence should be addressed at Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. Fax: ⫹81-96-373-5145. E-mail: [email protected]. 252
at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-1 functions atherogenically by inhibiting endothelial cell growth. © 2001 Academic Press Key Words: hyperglycemia; arginine; endothelial nitric oxide synthase; argininosuccinate synthetase; transforming growth factor-1.
Accelerated atherosclerosis in diabetes is caused by numerous metabolic disorders related to extravascular and intravascular factors. Endothelial dysfunction, the first step in atherosclerosis, occurs in diabetes. Decreased endothelium-dependent relaxation is a characteristic feature of endothelium dysfunction (1). Endothelium-dependent relaxation is mediated by endothelium-derived nitric oxide (NO, 2 nitric oxide) (2). NO has a number of important functions beyond simple modulation of vasodilation (3), including inhibition of platelet aggregation (4), adhesion molecule expression (5), and prevention of smooth muscle proliferation (6). Longterm blockade of nitric oxide synthase (NOS) leads to coronary microvascular remodeling and hypertro2 Abbreviations used: NO, nitric oxide; NOS, NO synthase; eNOS, endothelial NOS; AS, argininosuccinate synthetase; AL, argininosuccinate lyase; CAT, cationic amino acid transporter; TGF-1, transforming growth factor-1; STZ, streptozotocin; HUVEC, human umbilical vein endothelial cells.
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
phy in rats (7). Therefore, the loss of NO production may be a leading cause of atherosclerosis. NO is synthesized from arginine by NOS, generating citrulline. The availability of arginine is one of the rate-limiting factors of cellular NO production. Cellular arginine can be obtained from exogenous sources via cationic amino acid transporter (CAT) or by endogenous synthesis. Citrulline, which is formed by the NOS reaction, can be recycled to arginine through the citrulline–NO cycle. This recycling is accomplished by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), which are expressed at high levels in the liver and kidney and at low levels in many other cell types, including vascular endothelial cells (8). Despite considerable evidence for the decreased NO-mediated endothelium-dependent relaxation in long-term diabetes, enhanced NO-mediated endothelium-dependent relaxation has been observed in early stages of diabetes (9, 10). Plasma arginine and tissue arginine contents in the aortas of diabetic rats are decreased (11), and arginine administration restores endothelium-dependent relaxation in early but not in later stages of the disease (12). Thus, decreased availability of arginine and impaired synthesis of NO seem to be important in the development of endothelial dysfunction. However, the mechanism underlying changes in endothelium-dependent relaxation during the course of diabetes has remained unclear. We therefore investigated participation of endothelial NOS (eNOS) and enzymes of arginine metabolism in the pathogenesis of diabetes-associated endothelial dysfunction. Here we report that eNOS and the enzymes of arginine recycling are coinduced in the aorta in an early stage of the STZ-induced diabetic rat and that plasma nitrate plus nitrite is increased. Coinduction of the enzymes by TGF-1 is suggested. MATERIALS AND METHODS
Animals and STZ Treatment Male Wistar rats (90 –100 g, 5 weeks) were randomized to receive STZ (100 mg/kg body wt, Sigma, St. Louis, MO) (diabetic) or citrate buffer alone (control). STZ or citrate buffer was administered intraperitonealy once daily on 2 consecutive days. The
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rats were killed at 1, 2, and 4 weeks after treatment following anesthetization with ether. Plasma glucose and insulin concentration were measured using ¨ ngelholm, Swea glucose analyzer (HemoCue AB, A den) and an Insulin ELISA kit (insulin ELISA kit, Shibayagi, Shibukawa, Japan), respectively. Nitrate plus nitrites in the plasma was assayed by a fluorometric method using NO 2⫺/NO 3⫺ Assay Kit-F (Dojindo Laboratories, Kumamoto, Japan), after conversion of nitrate to nitrite by nitrate reductase (13). All procedures were approved by the Animal Care and Use Committee of Kumamoto University.
Cell Culture and TGF-1 Treatment Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA) and cultured in medium 199 containing 10% FCS (Intergen, Purchase, NY) on collagen type I-coated dishes. The cells used in the present study were from the 3 to 4 passage. Cells were incubated for variable periods of time or with varying concentrations of TGF-1 (R&D Systems, Minneapolis, MN).
Northern Blot Analysis Total RNA (2 g per lane) was electrophoresed in formaldehyde–agarose gels and blotted onto nylon membranes. Northern blot analysis was performed as described (14). Digoxigenin-labeled antisense RNA probes were synthesized from cDNAs under the control of the T7 or SP6 promoter with a DIG RNA-labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) using as templates pcDNAIIreNOS (15), pcDNAII-rAS, (8), pcDNAII-rAL (8), pGEM-rCAT-1, pGEM-rCAT-2 (16), pGEM-mTGF1, pCS41 (pBluescriptII SK- with human eNOS cDNA, a gift from S. Kamitani, Kyoto University), and pGEM-hAS, respectively. cDNA for rat CAT-1 (nt 202– 804; GenBank Accession No. L10152), mouse TGF-1 (nt 693–1217; GenBank Accession No. M13177), and human AS (nt 28 –1482; GenBank Accession No. X01630) were isolated by PCR. Chemiluminescence signals were quantified using an Las-1000 plus chemiluminescence image analyzer (Fuji Photo Film Co., Tokyo, Japan).
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OYADOMARI ET AL. TABLE I
Characteristics of Study Rats
1W 2W 4W
Group
n
Body weight (g)
Plasma glucose (mM)
Plasma insulin (pM)
Plasma insulin (pM)
Cont STZ Cont STZ Cont STZ
6 6 6 6 6 6
158.6 ⫾ 0.7 106.8 ⫾ 3.2* 226.7 ⫾ 5.6 110.3 ⫾ 3.6* 282.2 ⫾ 5.1 117.1 ⫾ 4.9*
10.4 ⫾ 0.7 38.6 ⫾ 2.2* 11.8 ⫾ 0.8 41.9 ⫾ 1.7* 11.2 ⫾ 0.5 42.5 ⫾ 2.7*
164.1 ⫾ 16.0 48.7 ⫾ 3.6* 176.4 ⫾ 5.6 47.1 ⫾ 2.2* 170.9 ⫾ 10.2 41.0 ⫾ 5.6*
12.56 ⫾ 0.41 13.37 ⫾ 0.24** 11.87 ⫾ 0.46 13.26 ⫾ 0.23** 11.22 ⫾ 0.14 9.92 ⫾ 0.35**
Note. Plasma nitrite plus nitrate concentration was measured using a fluorometric assay. Data are shown as means ⫾ SE. Significant differences were evaluated by Student’s t test: *P ⬍ 0.001, **P ⬍ 0.05 vs control.
Western Blot Analysis The frozen samples were homogenized in lysis buffer (1% SDS, 300 mM NaCl, 1 mM PMSF, 10 mM Tris–HCl, pH 7.4). The homogenates were boiled for 5 min and centrifuged at 17,000g for 5 min at 4°C, and the supernatants served as tissue extracts. Tissue extracts were separated on SDS–PAGE and transferred to nitrocellulose membranes. Western blot analysis was done using polyclonal antibodies against rat eNOS (Transduction Laboratories, Lexington, KY) or antisera against rat AS protein (8). For immunodetection we used an ECL kit (Amersham Pharmacia Biotech). Chemiluminescence signals were quantified using the Las-1000 plus. Cell Proliferation Assay Cell growth was assayed by a colorimetric procedure using a Cell Counting Kit-8 (Dojindo Laboratories). This assay is modified from the method using MTT (17). HUVEC were seeded onto 96-well plates with a density of 5.0 ⫻ 10 3 cells per well. The cells were incubated with various concentrations of TGF-1 or vehicle. On day 3, the medium was replaced with fresh medium of the same content. On day 5, the medium was discarded and the cells were subjected to assay. Statistical Analysis Data were expressed as means ⫾ SE. Statistical significance of differences between groups was evaluated by using unpaired Student’s t test. When the
P value was less than 0.05, the difference was considered to be statistically significant.
RESULTS
Characteristics of Study Rats Diabetes was induced in male Wistar rats by using a selective -cell toxin STZ. Plasma glucose was markedly raised at each point in time after STZ treatment, compared with control (Table I). STZinduced diabetic rats did not gain weight. Plasma insulin in diabetic rats was lower than control at all time points. In plasma, nitrate and nitrite are the major degradation products of NO. Therefore, we measured plasma nitrite plus nitrate (Table I). The plasma nitrate plus nitrite levels in control were gradually decreased with age. The levels were modestly but significantly increased 1 and 2 weeks after STZ treatment and decreased at 4 weeks of disease compared with controls. These results suggest increased NO production in an early stage of diabetes. eNOS, AS, and AL mRNAs in Aortas of STZInduced Diabetic Rats The plasma nitrate plus nitrite levels reflect NO production but represent the sum of NO production by all tissue. Thus, we examined alterations of mRNAs for eNOS and related enzymes of arginine metabolism in the aortas of STZ-induced diabetic rats by using Northern blot analysis (Fig. 1). eNOS mRNA of 4.4 kb was increased 1 week after STZ
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
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whereas AL mRNA was induced more slowly and weakly. CAT-1 and CAT-2 mRNAs in Aortas of STZInduced Diabetic Rats Arginine can be imported into the cells via CAT-1 and CAT-2, which have amino acid transport properties consistent with those of system y ⫹. To identify alterations of CAT-1 and CAT-2 mRNAs, Northern blot analysis was performed (Fig. 2). CAT-1 mRNA of about 3.4 and 7.9 kb was detected in the aorta but remained little changed after STZ treatment. CAT-2 mRNA of about 4.5 and 8.0 kb, which was detected in the liver, was undetectable in the aorta of STZtreated rats at all points. eNOS and AS Proteins in Aortas of STZ-Induced Diabetic Rats The induction of eNOS and AS proteins in the aortas of STZ-induced diabetic rats was examined
FIG. 1. Northern blot analysis for eNOS, AS, and AL mRNAs in the aortas of STZ-induced diabetic rats. (A) Total RNAs (2.0 g) were subjected to blot analysis. The representative chemiluminograms for eNOS, AS, and AL mRNAs at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. The bottom panels show ethidium bromide staining of the 28S and 18S rRNAs. The chemiluminograms for eNOS (B), AS (C), and AL (D) mRNAs were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6) at indicated times.
treatment, reached a maximum (three- to fourfold) at 2 weeks, and decreased at 4 weeks. AS mRNA of about 1.5 kb began to increase 1 week after STZ treatment, reached a maximum (three- to fourfold) at 2 weeks, and decreased at 4 weeks. AL mRNA of about 2.0 kb showed little change at 1 week and was markedly increased at 2 weeks in one rat, but not in other animals. The AL mRNA was increased significantly at 4 weeks. The maximally induced levels of AS and AL mRNAs were much lower than those in the liver, an organ in which these enzymes are highly expressed. These results show that the increase in eNOS and AS mRNAs in the aortas of STZ-induced diabetic rats has a similar kinetics,
FIG. 2. Northern blot analysis for CAT-1 and CAT-2 mRNAs in the aortas of STZ-induced diabetic rats. Total RNAs (2.0 g) were subjected to blot analysis. The chemiluminograms for CAT-1 and CAT-2 mRNAs at the indicated weeks after treatment with vehicle (Cont) or STZ are shown: Pc, positive control; rat brain RNA for CAT-1 mRNA and rat liver RNA for CAT-2 mRNA.
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OYADOMARI ET AL.
TGF-1 mRNA in Aortas of STZ-Induced Diabetic Rats TGF-1 plays an important role in the pathogenesis of atherosclerosis (18) and regulates a wide variety of cellular activities through the transcription of various genes. Thus, the expression of TGF-1 mRNA was examined by using Northern blot analysis (Fig. 4). TGF-1 mRNA of 2.5 kb was weakly detected in control animals. The mRNA was increased 1 week after STZ treatment and further increased at 2 weeks (four- to fivefold). At 4 weeks, the mRNA remained highly increased, but was little increased in one animal. The mean value at 4 weeks was a little lower than that at 2 weeks. Effect of TGF-1 on eNOS and AS mRNAs in HUVEC FIG. 3. Western blot analysis of eNOS and AS in the aortas of STZ-induced diabetic rats. (A) The thoracic aorta extracts (40 g of protein) were subjected to blot analysis. The representative chemiluminograms for eNOS protein at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. (B) Aorta extracts (40 g of protein) or liver extracts (1.0 g of protein) were subjected to blot analysis. The representative chemiluminograms for AS protein are shown. (C) The chemiluminograms for eNOS protein were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6). (D) The chemiluminograms for AS protein were quantified, and the amount of protein relative to that in the liver was calculated. The results are shown as means ⫾ SE (n ⫽ 6). AS protein was barely detected in control rats (Cont).
by Western blot analysis (Fig. 3). eNOS protein of about 140 kDa was increased 1 week after STZ treatment, reached a maximum (four- to fivefold) at 2 weeks, and decreased at 4 weeks. Because AS protein was barely detectable in the aortas of the control rats, the amount of AS protein was calculated relative to that in the liver where this protein is highly and constitutively expressed as a urea cycle enzyme. AS protein of about 46 kDa increased 1 week after STZ treatment, reached a maximum (twofold compared to the level at 1 week) at 2 weeks, and decreased at 4 weeks. The concentration of AS protein in aorta of STZ-treated rats at 2 weeks was about 2.5% of that in the liver. These results support findings in the case of eNOS and AS mRNAs.
From the in vivo studies, we hypothesized that coinduction of eNOS and AS in the vasculature, in the presence of diabetes, is mediated by TGF-1. To test this hypothesis, the effect of TGF-1 on the expression of eNOS and AS mRNAs in HUVEC was examined by Northern blot analysis (Fig. 5). HUVEC were incubated with or without 1 ng/ml TGF-1 for 2 to 24 h. eNOS mRNA increased 2 h after treatment, reached a maximum at 6 –12 h, and decreased slightly at 24 h. On the other hand, AS
FIG. 4. Northern blot analysis for TGF-1 mRNA in the aortas of STZ-induced diabetic rats. (A) Total RNAs (2.0 g) were subjected to blot analysis. The representative chemiluminograms for TGF-1 mRNA at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. (B) The chemiluminograms were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6) at indicated times.
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
Effect of TGF-1 on Proliferation of HUVEC The effect of TGF-1 on the proliferation of HUVEC was also studied (Table II). When the cells were cultured for 5 days in the absence of TGF-1, the number of cells increased by about 3.6-fold. TGF-1 inhibited HUVEC proliferation in a dosedependent manner. Half-maximal inhibition of proliferation occurred at about 1 ng/ml TGF-1. Little proliferation of the cells was seen with 10 ng/ml. However, neither morphological changes nor apoptosis of cells was observed (data not shown). DISCUSSION
FIG. 5. Northern blot analysis for eNOS and AS mRNAs after TGF-1 treatment in HUVEC. Total RNAs (2.0 g) were subjected to blot analysis. (A) The representative chemiluminograms for eNOS and AS mRNAs at the indicated hours after treatment with 1 ng/ml TGF-1 are shown: Li, rat liver RNA. (B) The representative chemiluminograms for eNOS and AS mRNAs at 6 h after TGF-1 treatment at indicated concentrations are shown. (C and D) The chemiluminograms of time course (A) and dose response (B) were quantified, and the results are shown as means ⫾ SE (n ⫽ 3), relative to means for control rats (n ⫽ 3).
mRNA began to increase at 6 h, reached a maximum at 12 h, and decreased thereafter. These results show that both eNOS and AS are induced by TGF-1 in endothelial cells. Dose dependency of TGF-1 on eNOS and AS mRNAs is shown in Figs. 5B and 5D. eNOS mRNA was induced by TGF-1 nearly maximally at 0.1 ng/ml, whereas AS mRNA was little induced at this concentration and induced nearly maximally at 1.0 ng/ml.
Although long-term diabetes is characterized by impaired endothelium-dependent relaxation, enhanced NO-mediated endothelium-dependent relaxation at an early stage of diabetes was suggested by aortic ring experiments of experimental diabetes (9) and by agonist-stimulated blood flow responses in patients with early type 1 diabetes (10). Our finding in the present study revealed a novel mechanism of the altered NO production in diabetic endothelium by expression of eNOS and availability of the NOS substrate arginine. In addition to NOS activity, NO production depends on the availability of arginine, the substrate of NOS reaction. There is no de novo synthesis of arginine in vascular endothelial cells, where the first two enzymes of arginine synthesis, carbamylphosphate synthetase I and ornithine transcarbamylase, are absent. Arginine can be taken up from outside the cells by CAT-1 or CAT-2 or resynthesized from citrulline by AS and AL via the citrulline–NO cycle. Previous studies showed that vascular endothelial cells convert citrulline to arginine (19), and this conversion is increased when the cells are stimulated to produce NO (20). Coinduction
TABLE II
Effect of TGF-1 on Proliferation of HUVEC TGF-1 (ng/ml)
0
0.01
0.1
1
10
Cell number (⫻10 3)
4.29 ⫾ 0.16
4.70 ⫾ 0.26
3.03 ⫾ 0.30
3.31 ⫾ 0.10
2.29 ⫾ 0.13
Note. HUVEC (5 ⫻ 10 3 cells) were cultured with varying concentrations of TGF-1. After 5 days culture, cell proliferation was measured using a modified MTT assay. Results are shown as means ⫾ SE (n ⫽ 5). Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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of iNOS and AS was noted in murine macrophage cells (21–23), murine aortic smooth muscle cells (24), rat astroglioma cells (25), activated macrophages of rat and mouse tissues in vivo (22, 23), and rodent and human pancreatic -cells (26). The present study shows for the first time that eNOS, AS, and AL are coinduced. In contrast, CAT-1 and CAT-2 mRNAs remained little changed. These results suggest that NO production in the aorta is enhanced in an early stage of diabetes and that the citrulline–NO cycle is important in NO production. Induction of AL was less marked and later than that of eNOS and AS. AS is apparently rate-limiting in the citrulline recycling reaction in these cells, and the basal level of AL may be sufficient for recycling. These results are consistent with our previous findings in rat spleen and lung tissues, where induction of AL is much less marked than iNOS and AS after LPS treatment (22). TGF-1 has been implicated in the pathogenesis of atherosclerosis (18), vascular hypertrophy in hypertension (27), vascular response to injury after balloon angioplasty (28), and angiogenesis in wound healing (29). In the present study, we found that TGF-1 mRNA increases in the rat aorta in early stages of STZ-induced diabetes. In cases of type 2 diabetes, elevated plasma levels of TGF-1 were noted (30). Treatment of endothelial cells with a high concentration (25 mM) of glucose was reported to stimulate the release of active TGF-1 from 1.2 ng/ml at 5 mM glucose to 1.7 ng/ml (31). Here we demonstrated that physiological concentrations of TGF-1 induce eNOS and AS mRNAs in HUVEC. Therefore, induction of eNOS and AS in the diabetic aorta appears to be mediated by TGF-1. TGF-1 was reported to increase eNOS mRNA and protein mediated by NF-1 in bovine aortic endothelial cells (32). However, the promoter regions of the AS and AL genes have no NF-1 binding consensus sequence (33). AS mRNA was induced more slowly than eNOS mRNA in HUVEC after TGF-1 treatment. Therefore, the induction of AS mRNA by TGF-1 may be mediated by the proceeding induction of another transcription factor. Further examination of the transcriptional regulation of the eNOS, AS, and AL genes is needed. On the other hand, TGF-1 was also shown to inhibit the proliferation of HUVEC. Although eNOS and AS are still increased at 4
FIG. 6. Model for the regulation of the citrulline–NO cycle by high glucose via TGF-1 in aortic endothelial cells. High glucose induces TGF-1 expression, and increased TGF-1 stimulates expression of eNOS and AS in endothelial cells via autocrine signaling. On the other hand, elevation of TGF-1 also results in inhibition of endothelial cell growth, which may be partially responsible for the decrease in NO production in diabetes of long duration.
weeks in the aortas of STZ-induced diabetic rats, plasma nitrite plus nitrate levels were decreased. This disparity may be explained at least partially by the inhibition of endothelial cell growth by TGF-1 and/or the decreased availability of tetrahydrobiopterin, a critical cofactor for the NOS reaction. Recent studies suggest that tetrahydrobiopterin is decreased in the course of diabetes (34). Future studies of tetrahydrobiopterin metabolism in diabetes are needed. Thus, TGF-1 may have double-edged roles in the development of atherosclerotic lesions. Namely, the induction of TGF-1 in the early stages of diabetes may work antiatherogenically by upregulating the citrulline–NO cycle and increasing NO production, whereas prolonged elevation of TGF-1 may function atherogenically by inhibiting endothelial cell growth and reducing NO production (Fig. 6). Plasma and vascular tissue arginine in diabetic rats is decreased (11). Many studies have shown that arginine supplementation can improve endothelial dysfunction and NO production in diabetics (35, 36). Importantly, arginine administration restores endothelium-dependent relaxation in early but not in later stages of the disease (12). Overexpression of eNOS can partially improve impaired endothelium-dependent relaxation (37).
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
Furthermore, endothelial dysfunction coinciding with an increased eNOS expression was observed in aortic banding-induced hypertensive rats (38) and in Watanabe heritable hyperlipidemic rabbits (39). Thus, increased eNOS expression related to impaired endothelium-dependent relaxation is apparently not specific to diabetic vasculature but may be a common feature in the progression of atherosclerosis. However, arginine administration in some patients with coronary artery disease cannot increase NO production (40). While many possibilities remain to be examined, one possible explanation for the lack of effect of arginine supplementation is that transport or intracellular concentrations of arginine are limiting. Based on all these findings, we propose that sustained induction of AS and AL might be a therapeutic target for improving endothelium dysfunction, in addition to activation or induction of eNOS or transfer of the eNOS gene. ACKNOWLEDGMENTS We thank S. Kamitani (Kyoto University) for the human eNOS cDNA. We also thank R. Shindo for technical assistance and M. Ohara for comments on the manuscript. This work was supported in part by Grant-in-Aid 10557020 to M. Mori from the Ministry of Education, Science, Sports, and Culture of Japan.
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laxing factor: Cultured endothelial cells recycle L-citrulline to L-arginine. Proc. Natl. Acad. Sci. USA 87, 8612– 8616. 20. Mitchell, J. A., Hecker, M., and Vane, J. R. (1990). The generation of L-arginine in endothelial cells is linked to the release of endothelium-derived relaxing factor. Eur. J. Pharmacol. 176, 253–254. 21. Nussler, A. K., Billiar, T. R., Liu, Z. Z., and Morris, S. M., Jr. (1994). Coinduction of nitric oxide synthase and argininosuccinate synthetase in a murine macrophage cell line. Implications for regulation of nitric oxide production. J. Biol. Chem. 269, 1257–1261. 22. Nagasaki, A., Gotoh, T., Takeya, M., Yu, Y., Takiguchi, M., Matsuzaki, H., Takatsuki, K., and Mori, M. (1996). Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats. RNA blot, immunoblot, and immunohistochemical analyses. J. Biol. Chem. 271, 2658 –2662. 23. Salimuddin, Nagasaki, A., Gotoh, T., Isobe, H., and Mori, M. (1999). Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide. Am. J. Physiol. 277, E110 –E117. 24. Hattori, Y., Campbell, E. B., and Gross, S. S. (1994). Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis. J. Biol. Chem. 269, 9405–9408. 25. Zhang, W. Y., Takiguchi, M., Koshiyama, Y., Gotoh, T., Nagasaki, A., Iwase, K., Yamamoto, K., Takeshima, H., Negi, A., and Mori, M. (1999). Expression of citrulline–nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells. Brain Res. 849, 78 – 84. 26. Flo¨dstrom, M., Morris, S. M., Jr., and Eizirik, D. L. (1996). Role of the citrulline–nitric oxide cycle in the functional response of adult human and rodent pancreatic islets to cytokines. Cytokine 8, 642– 650. 27. Sarzani, R., Brecher, P., and Chobanian, A. V. (1989). Growth factor expression in aorta of normotensive and hypertensive rats. J. Clin. Invest. 83, 1404 –1408. 28. Nikol, S., Isner, J. M., Pickering, J. G., Kearney, M., Leclerc, G., and Weir, L. (1992). Expression of transforming growth factor-1 is increased in human vascular restenosis lesions. J. Clin. Invest. 90, 1582–1592. 29. Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H., and Fauci, A. S. (1986). Transforming growth factor type : Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 83, 4167– 4171.
30. Pfeiffer, A., Middelberg-Bisping, K., Drewes, C., and Schatz, H. (1996). Elevated plasma levels of transforming growth factor- 1 in NIDDM. Diabetes Care 19, 1113–1117. 31. Morishita, R., Nakamura, S., Nakamura, Y., Aoki, M., Moriguchi, A., Kida, I., Yo, Y., Matsumoto, K., Nakamura, T., Higaki, J., and Ogihara, T. (1997). Potential role of an endothelium-specific growth factor, hepatocyte growth factor, on endothelial damage in diabetes. Diabetes 46, 138 –142. 32. Inoue, N., Venema, R. C., Sayegh, H. S., Ohara, Y., Murphy, T. J., and Harrison, D. G. (1995). Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor- 1. Arterioscler. Thromb. Vasc. Biol. 15, 1255– 1261. 33. Takiguchi, M., and Mori, M. (1995). Transcriptional regulation of genes for ornithine cycle enzymes. Biochem. J. 312, 649 – 659. 34. Pieper, G. M. (1997). Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J. Cardiovasc. Pharmacol. 29, 8 –15. 35. Pieper, G. M., Siebeneich, W., Moore-Hilton, G., and Roza, A. M. (1997). Reversal by L-arginine of a dysfunctional arginine/nitric oxide pathway in the endothelium of the genetic diabetic BB rat. Diabetologia 40, 910 –915. 36. Matsunaga, T., Okumura, K., Ishizaka, H., Tsunoda, R., Tayama, S., Tabuchi, T., and Yasue, H. (1996). Impairment of coronary blood flow regulation by endothelium-derived nitric oxide in dogs with alloxan-induced diabetes. J. Cardiovasc. Pharmacol. 28, 60 – 67. 37. Lund, D. D., Faraci, F. M., Miller, F. J., Jr., and Heistad, D. D. (2000). Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation 101, 1027–1033. 38. Bouloumie, A., Bauersachs, J., Linz, W., Scholkens, B. A., Wiemer, G., Fleming, I., and Busse, R. (1997). Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production. Hypertension 30, 934 –941. 39. Kanazawa, K., Kawashima, S., Mikami, S., Miwa, Y., Hirata, K., Suematsu, M., Hayashi, Y., Itoh, H., and Yokoyama, M. (1996). Endothelial constitutive nitric oxide synthase protein and mRNA increased in rabbit atherosclerotic aorta despite impaired endothelium-dependent vascular relaxation. Am. J. Pathol. 148, 1949 –1956. 40. Blum, A., Hathaway, L., Mincemoyer, R., Schenke, W. H., Kirby, M., Csako, G., Waclawiw, M. A., Panza, J. A., and Cannon, R. O., III. (2000). Oral L-arginine in patients with coronary artery disease on medical management. Circulation 101, 2160 –2164.
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Coinduction of Endothelial Nitric Oxide Synthase and Arginine Recycling Enzymes in Aorta of Diabetic Rats Seiichi Oyadomari,* ,† Tomomi Gotoh,* Kazumasa Aoyagi,‡ Eiichi Araki,† Motoaki Shichiri,† and Masataka Mori* ,1 *Department of Molecular Genetics and †Department of Metabolic Medicine, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan; and ‡Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan
Received January 30, 2001; published online May 9, 2001
Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline–NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1–2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1–2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-1 (TGF-1) mRNA in the aorta was also induced. TGF-1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-1. The results also suggest that TGF-1 works antiatherogenically 1
To whom correspondence should be addressed at Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. Fax: ⫹81-96-373-5145. E-mail: [email protected]. 252
at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-1 functions atherogenically by inhibiting endothelial cell growth. © 2001 Academic Press Key Words: hyperglycemia; arginine; endothelial nitric oxide synthase; argininosuccinate synthetase; transforming growth factor-1.
Accelerated atherosclerosis in diabetes is caused by numerous metabolic disorders related to extravascular and intravascular factors. Endothelial dysfunction, the first step in atherosclerosis, occurs in diabetes. Decreased endothelium-dependent relaxation is a characteristic feature of endothelium dysfunction (1). Endothelium-dependent relaxation is mediated by endothelium-derived nitric oxide (NO, 2 nitric oxide) (2). NO has a number of important functions beyond simple modulation of vasodilation (3), including inhibition of platelet aggregation (4), adhesion molecule expression (5), and prevention of smooth muscle proliferation (6). Longterm blockade of nitric oxide synthase (NOS) leads to coronary microvascular remodeling and hypertro2 Abbreviations used: NO, nitric oxide; NOS, NO synthase; eNOS, endothelial NOS; AS, argininosuccinate synthetase; AL, argininosuccinate lyase; CAT, cationic amino acid transporter; TGF-1, transforming growth factor-1; STZ, streptozotocin; HUVEC, human umbilical vein endothelial cells.
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
phy in rats (7). Therefore, the loss of NO production may be a leading cause of atherosclerosis. NO is synthesized from arginine by NOS, generating citrulline. The availability of arginine is one of the rate-limiting factors of cellular NO production. Cellular arginine can be obtained from exogenous sources via cationic amino acid transporter (CAT) or by endogenous synthesis. Citrulline, which is formed by the NOS reaction, can be recycled to arginine through the citrulline–NO cycle. This recycling is accomplished by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), which are expressed at high levels in the liver and kidney and at low levels in many other cell types, including vascular endothelial cells (8). Despite considerable evidence for the decreased NO-mediated endothelium-dependent relaxation in long-term diabetes, enhanced NO-mediated endothelium-dependent relaxation has been observed in early stages of diabetes (9, 10). Plasma arginine and tissue arginine contents in the aortas of diabetic rats are decreased (11), and arginine administration restores endothelium-dependent relaxation in early but not in later stages of the disease (12). Thus, decreased availability of arginine and impaired synthesis of NO seem to be important in the development of endothelial dysfunction. However, the mechanism underlying changes in endothelium-dependent relaxation during the course of diabetes has remained unclear. We therefore investigated participation of endothelial NOS (eNOS) and enzymes of arginine metabolism in the pathogenesis of diabetes-associated endothelial dysfunction. Here we report that eNOS and the enzymes of arginine recycling are coinduced in the aorta in an early stage of the STZ-induced diabetic rat and that plasma nitrate plus nitrite is increased. Coinduction of the enzymes by TGF-1 is suggested. MATERIALS AND METHODS
Animals and STZ Treatment Male Wistar rats (90 –100 g, 5 weeks) were randomized to receive STZ (100 mg/kg body wt, Sigma, St. Louis, MO) (diabetic) or citrate buffer alone (control). STZ or citrate buffer was administered intraperitonealy once daily on 2 consecutive days. The
253
rats were killed at 1, 2, and 4 weeks after treatment following anesthetization with ether. Plasma glucose and insulin concentration were measured using ¨ ngelholm, Swea glucose analyzer (HemoCue AB, A den) and an Insulin ELISA kit (insulin ELISA kit, Shibayagi, Shibukawa, Japan), respectively. Nitrate plus nitrites in the plasma was assayed by a fluorometric method using NO 2⫺/NO 3⫺ Assay Kit-F (Dojindo Laboratories, Kumamoto, Japan), after conversion of nitrate to nitrite by nitrate reductase (13). All procedures were approved by the Animal Care and Use Committee of Kumamoto University.
Cell Culture and TGF-1 Treatment Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA) and cultured in medium 199 containing 10% FCS (Intergen, Purchase, NY) on collagen type I-coated dishes. The cells used in the present study were from the 3 to 4 passage. Cells were incubated for variable periods of time or with varying concentrations of TGF-1 (R&D Systems, Minneapolis, MN).
Northern Blot Analysis Total RNA (2 g per lane) was electrophoresed in formaldehyde–agarose gels and blotted onto nylon membranes. Northern blot analysis was performed as described (14). Digoxigenin-labeled antisense RNA probes were synthesized from cDNAs under the control of the T7 or SP6 promoter with a DIG RNA-labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) using as templates pcDNAIIreNOS (15), pcDNAII-rAS, (8), pcDNAII-rAL (8), pGEM-rCAT-1, pGEM-rCAT-2 (16), pGEM-mTGF1, pCS41 (pBluescriptII SK- with human eNOS cDNA, a gift from S. Kamitani, Kyoto University), and pGEM-hAS, respectively. cDNA for rat CAT-1 (nt 202– 804; GenBank Accession No. L10152), mouse TGF-1 (nt 693–1217; GenBank Accession No. M13177), and human AS (nt 28 –1482; GenBank Accession No. X01630) were isolated by PCR. Chemiluminescence signals were quantified using an Las-1000 plus chemiluminescence image analyzer (Fuji Photo Film Co., Tokyo, Japan).
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OYADOMARI ET AL. TABLE I
Characteristics of Study Rats
1W 2W 4W
Group
n
Body weight (g)
Plasma glucose (mM)
Plasma insulin (pM)
Plasma insulin (pM)
Cont STZ Cont STZ Cont STZ
6 6 6 6 6 6
158.6 ⫾ 0.7 106.8 ⫾ 3.2* 226.7 ⫾ 5.6 110.3 ⫾ 3.6* 282.2 ⫾ 5.1 117.1 ⫾ 4.9*
10.4 ⫾ 0.7 38.6 ⫾ 2.2* 11.8 ⫾ 0.8 41.9 ⫾ 1.7* 11.2 ⫾ 0.5 42.5 ⫾ 2.7*
164.1 ⫾ 16.0 48.7 ⫾ 3.6* 176.4 ⫾ 5.6 47.1 ⫾ 2.2* 170.9 ⫾ 10.2 41.0 ⫾ 5.6*
12.56 ⫾ 0.41 13.37 ⫾ 0.24** 11.87 ⫾ 0.46 13.26 ⫾ 0.23** 11.22 ⫾ 0.14 9.92 ⫾ 0.35**
Note. Plasma nitrite plus nitrate concentration was measured using a fluorometric assay. Data are shown as means ⫾ SE. Significant differences were evaluated by Student’s t test: *P ⬍ 0.001, **P ⬍ 0.05 vs control.
Western Blot Analysis The frozen samples were homogenized in lysis buffer (1% SDS, 300 mM NaCl, 1 mM PMSF, 10 mM Tris–HCl, pH 7.4). The homogenates were boiled for 5 min and centrifuged at 17,000g for 5 min at 4°C, and the supernatants served as tissue extracts. Tissue extracts were separated on SDS–PAGE and transferred to nitrocellulose membranes. Western blot analysis was done using polyclonal antibodies against rat eNOS (Transduction Laboratories, Lexington, KY) or antisera against rat AS protein (8). For immunodetection we used an ECL kit (Amersham Pharmacia Biotech). Chemiluminescence signals were quantified using the Las-1000 plus. Cell Proliferation Assay Cell growth was assayed by a colorimetric procedure using a Cell Counting Kit-8 (Dojindo Laboratories). This assay is modified from the method using MTT (17). HUVEC were seeded onto 96-well plates with a density of 5.0 ⫻ 10 3 cells per well. The cells were incubated with various concentrations of TGF-1 or vehicle. On day 3, the medium was replaced with fresh medium of the same content. On day 5, the medium was discarded and the cells were subjected to assay. Statistical Analysis Data were expressed as means ⫾ SE. Statistical significance of differences between groups was evaluated by using unpaired Student’s t test. When the
P value was less than 0.05, the difference was considered to be statistically significant.
RESULTS
Characteristics of Study Rats Diabetes was induced in male Wistar rats by using a selective -cell toxin STZ. Plasma glucose was markedly raised at each point in time after STZ treatment, compared with control (Table I). STZinduced diabetic rats did not gain weight. Plasma insulin in diabetic rats was lower than control at all time points. In plasma, nitrate and nitrite are the major degradation products of NO. Therefore, we measured plasma nitrite plus nitrate (Table I). The plasma nitrate plus nitrite levels in control were gradually decreased with age. The levels were modestly but significantly increased 1 and 2 weeks after STZ treatment and decreased at 4 weeks of disease compared with controls. These results suggest increased NO production in an early stage of diabetes. eNOS, AS, and AL mRNAs in Aortas of STZInduced Diabetic Rats The plasma nitrate plus nitrite levels reflect NO production but represent the sum of NO production by all tissue. Thus, we examined alterations of mRNAs for eNOS and related enzymes of arginine metabolism in the aortas of STZ-induced diabetic rats by using Northern blot analysis (Fig. 1). eNOS mRNA of 4.4 kb was increased 1 week after STZ
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
255
whereas AL mRNA was induced more slowly and weakly. CAT-1 and CAT-2 mRNAs in Aortas of STZInduced Diabetic Rats Arginine can be imported into the cells via CAT-1 and CAT-2, which have amino acid transport properties consistent with those of system y ⫹. To identify alterations of CAT-1 and CAT-2 mRNAs, Northern blot analysis was performed (Fig. 2). CAT-1 mRNA of about 3.4 and 7.9 kb was detected in the aorta but remained little changed after STZ treatment. CAT-2 mRNA of about 4.5 and 8.0 kb, which was detected in the liver, was undetectable in the aorta of STZtreated rats at all points. eNOS and AS Proteins in Aortas of STZ-Induced Diabetic Rats The induction of eNOS and AS proteins in the aortas of STZ-induced diabetic rats was examined
FIG. 1. Northern blot analysis for eNOS, AS, and AL mRNAs in the aortas of STZ-induced diabetic rats. (A) Total RNAs (2.0 g) were subjected to blot analysis. The representative chemiluminograms for eNOS, AS, and AL mRNAs at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. The bottom panels show ethidium bromide staining of the 28S and 18S rRNAs. The chemiluminograms for eNOS (B), AS (C), and AL (D) mRNAs were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6) at indicated times.
treatment, reached a maximum (three- to fourfold) at 2 weeks, and decreased at 4 weeks. AS mRNA of about 1.5 kb began to increase 1 week after STZ treatment, reached a maximum (three- to fourfold) at 2 weeks, and decreased at 4 weeks. AL mRNA of about 2.0 kb showed little change at 1 week and was markedly increased at 2 weeks in one rat, but not in other animals. The AL mRNA was increased significantly at 4 weeks. The maximally induced levels of AS and AL mRNAs were much lower than those in the liver, an organ in which these enzymes are highly expressed. These results show that the increase in eNOS and AS mRNAs in the aortas of STZ-induced diabetic rats has a similar kinetics,
FIG. 2. Northern blot analysis for CAT-1 and CAT-2 mRNAs in the aortas of STZ-induced diabetic rats. Total RNAs (2.0 g) were subjected to blot analysis. The chemiluminograms for CAT-1 and CAT-2 mRNAs at the indicated weeks after treatment with vehicle (Cont) or STZ are shown: Pc, positive control; rat brain RNA for CAT-1 mRNA and rat liver RNA for CAT-2 mRNA.
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TGF-1 mRNA in Aortas of STZ-Induced Diabetic Rats TGF-1 plays an important role in the pathogenesis of atherosclerosis (18) and regulates a wide variety of cellular activities through the transcription of various genes. Thus, the expression of TGF-1 mRNA was examined by using Northern blot analysis (Fig. 4). TGF-1 mRNA of 2.5 kb was weakly detected in control animals. The mRNA was increased 1 week after STZ treatment and further increased at 2 weeks (four- to fivefold). At 4 weeks, the mRNA remained highly increased, but was little increased in one animal. The mean value at 4 weeks was a little lower than that at 2 weeks. Effect of TGF-1 on eNOS and AS mRNAs in HUVEC FIG. 3. Western blot analysis of eNOS and AS in the aortas of STZ-induced diabetic rats. (A) The thoracic aorta extracts (40 g of protein) were subjected to blot analysis. The representative chemiluminograms for eNOS protein at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. (B) Aorta extracts (40 g of protein) or liver extracts (1.0 g of protein) were subjected to blot analysis. The representative chemiluminograms for AS protein are shown. (C) The chemiluminograms for eNOS protein were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6). (D) The chemiluminograms for AS protein were quantified, and the amount of protein relative to that in the liver was calculated. The results are shown as means ⫾ SE (n ⫽ 6). AS protein was barely detected in control rats (Cont).
by Western blot analysis (Fig. 3). eNOS protein of about 140 kDa was increased 1 week after STZ treatment, reached a maximum (four- to fivefold) at 2 weeks, and decreased at 4 weeks. Because AS protein was barely detectable in the aortas of the control rats, the amount of AS protein was calculated relative to that in the liver where this protein is highly and constitutively expressed as a urea cycle enzyme. AS protein of about 46 kDa increased 1 week after STZ treatment, reached a maximum (twofold compared to the level at 1 week) at 2 weeks, and decreased at 4 weeks. The concentration of AS protein in aorta of STZ-treated rats at 2 weeks was about 2.5% of that in the liver. These results support findings in the case of eNOS and AS mRNAs.
From the in vivo studies, we hypothesized that coinduction of eNOS and AS in the vasculature, in the presence of diabetes, is mediated by TGF-1. To test this hypothesis, the effect of TGF-1 on the expression of eNOS and AS mRNAs in HUVEC was examined by Northern blot analysis (Fig. 5). HUVEC were incubated with or without 1 ng/ml TGF-1 for 2 to 24 h. eNOS mRNA increased 2 h after treatment, reached a maximum at 6 –12 h, and decreased slightly at 24 h. On the other hand, AS
FIG. 4. Northern blot analysis for TGF-1 mRNA in the aortas of STZ-induced diabetic rats. (A) Total RNAs (2.0 g) were subjected to blot analysis. The representative chemiluminograms for TGF-1 mRNA at the indicated weeks after treatment with vehicle (Cont) or STZ are shown. (B) The chemiluminograms were quantified, and the results are shown as means ⫾ SE (n ⫽ 6), relative to means for control rats (n ⫽ 6) at indicated times.
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257
ARGININE METABOLISM AND NO IN DIABETIC AORTA
Effect of TGF-1 on Proliferation of HUVEC The effect of TGF-1 on the proliferation of HUVEC was also studied (Table II). When the cells were cultured for 5 days in the absence of TGF-1, the number of cells increased by about 3.6-fold. TGF-1 inhibited HUVEC proliferation in a dosedependent manner. Half-maximal inhibition of proliferation occurred at about 1 ng/ml TGF-1. Little proliferation of the cells was seen with 10 ng/ml. However, neither morphological changes nor apoptosis of cells was observed (data not shown). DISCUSSION
FIG. 5. Northern blot analysis for eNOS and AS mRNAs after TGF-1 treatment in HUVEC. Total RNAs (2.0 g) were subjected to blot analysis. (A) The representative chemiluminograms for eNOS and AS mRNAs at the indicated hours after treatment with 1 ng/ml TGF-1 are shown: Li, rat liver RNA. (B) The representative chemiluminograms for eNOS and AS mRNAs at 6 h after TGF-1 treatment at indicated concentrations are shown. (C and D) The chemiluminograms of time course (A) and dose response (B) were quantified, and the results are shown as means ⫾ SE (n ⫽ 3), relative to means for control rats (n ⫽ 3).
mRNA began to increase at 6 h, reached a maximum at 12 h, and decreased thereafter. These results show that both eNOS and AS are induced by TGF-1 in endothelial cells. Dose dependency of TGF-1 on eNOS and AS mRNAs is shown in Figs. 5B and 5D. eNOS mRNA was induced by TGF-1 nearly maximally at 0.1 ng/ml, whereas AS mRNA was little induced at this concentration and induced nearly maximally at 1.0 ng/ml.
Although long-term diabetes is characterized by impaired endothelium-dependent relaxation, enhanced NO-mediated endothelium-dependent relaxation at an early stage of diabetes was suggested by aortic ring experiments of experimental diabetes (9) and by agonist-stimulated blood flow responses in patients with early type 1 diabetes (10). Our finding in the present study revealed a novel mechanism of the altered NO production in diabetic endothelium by expression of eNOS and availability of the NOS substrate arginine. In addition to NOS activity, NO production depends on the availability of arginine, the substrate of NOS reaction. There is no de novo synthesis of arginine in vascular endothelial cells, where the first two enzymes of arginine synthesis, carbamylphosphate synthetase I and ornithine transcarbamylase, are absent. Arginine can be taken up from outside the cells by CAT-1 or CAT-2 or resynthesized from citrulline by AS and AL via the citrulline–NO cycle. Previous studies showed that vascular endothelial cells convert citrulline to arginine (19), and this conversion is increased when the cells are stimulated to produce NO (20). Coinduction
TABLE II
Effect of TGF-1 on Proliferation of HUVEC TGF-1 (ng/ml)
0
0.01
0.1
1
10
Cell number (⫻10 3)
4.29 ⫾ 0.16
4.70 ⫾ 0.26
3.03 ⫾ 0.30
3.31 ⫾ 0.10
2.29 ⫾ 0.13
Note. HUVEC (5 ⫻ 10 3 cells) were cultured with varying concentrations of TGF-1. After 5 days culture, cell proliferation was measured using a modified MTT assay. Results are shown as means ⫾ SE (n ⫽ 5). Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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of iNOS and AS was noted in murine macrophage cells (21–23), murine aortic smooth muscle cells (24), rat astroglioma cells (25), activated macrophages of rat and mouse tissues in vivo (22, 23), and rodent and human pancreatic -cells (26). The present study shows for the first time that eNOS, AS, and AL are coinduced. In contrast, CAT-1 and CAT-2 mRNAs remained little changed. These results suggest that NO production in the aorta is enhanced in an early stage of diabetes and that the citrulline–NO cycle is important in NO production. Induction of AL was less marked and later than that of eNOS and AS. AS is apparently rate-limiting in the citrulline recycling reaction in these cells, and the basal level of AL may be sufficient for recycling. These results are consistent with our previous findings in rat spleen and lung tissues, where induction of AL is much less marked than iNOS and AS after LPS treatment (22). TGF-1 has been implicated in the pathogenesis of atherosclerosis (18), vascular hypertrophy in hypertension (27), vascular response to injury after balloon angioplasty (28), and angiogenesis in wound healing (29). In the present study, we found that TGF-1 mRNA increases in the rat aorta in early stages of STZ-induced diabetes. In cases of type 2 diabetes, elevated plasma levels of TGF-1 were noted (30). Treatment of endothelial cells with a high concentration (25 mM) of glucose was reported to stimulate the release of active TGF-1 from 1.2 ng/ml at 5 mM glucose to 1.7 ng/ml (31). Here we demonstrated that physiological concentrations of TGF-1 induce eNOS and AS mRNAs in HUVEC. Therefore, induction of eNOS and AS in the diabetic aorta appears to be mediated by TGF-1. TGF-1 was reported to increase eNOS mRNA and protein mediated by NF-1 in bovine aortic endothelial cells (32). However, the promoter regions of the AS and AL genes have no NF-1 binding consensus sequence (33). AS mRNA was induced more slowly than eNOS mRNA in HUVEC after TGF-1 treatment. Therefore, the induction of AS mRNA by TGF-1 may be mediated by the proceeding induction of another transcription factor. Further examination of the transcriptional regulation of the eNOS, AS, and AL genes is needed. On the other hand, TGF-1 was also shown to inhibit the proliferation of HUVEC. Although eNOS and AS are still increased at 4
FIG. 6. Model for the regulation of the citrulline–NO cycle by high glucose via TGF-1 in aortic endothelial cells. High glucose induces TGF-1 expression, and increased TGF-1 stimulates expression of eNOS and AS in endothelial cells via autocrine signaling. On the other hand, elevation of TGF-1 also results in inhibition of endothelial cell growth, which may be partially responsible for the decrease in NO production in diabetes of long duration.
weeks in the aortas of STZ-induced diabetic rats, plasma nitrite plus nitrate levels were decreased. This disparity may be explained at least partially by the inhibition of endothelial cell growth by TGF-1 and/or the decreased availability of tetrahydrobiopterin, a critical cofactor for the NOS reaction. Recent studies suggest that tetrahydrobiopterin is decreased in the course of diabetes (34). Future studies of tetrahydrobiopterin metabolism in diabetes are needed. Thus, TGF-1 may have double-edged roles in the development of atherosclerotic lesions. Namely, the induction of TGF-1 in the early stages of diabetes may work antiatherogenically by upregulating the citrulline–NO cycle and increasing NO production, whereas prolonged elevation of TGF-1 may function atherogenically by inhibiting endothelial cell growth and reducing NO production (Fig. 6). Plasma and vascular tissue arginine in diabetic rats is decreased (11). Many studies have shown that arginine supplementation can improve endothelial dysfunction and NO production in diabetics (35, 36). Importantly, arginine administration restores endothelium-dependent relaxation in early but not in later stages of the disease (12). Overexpression of eNOS can partially improve impaired endothelium-dependent relaxation (37).
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ARGININE METABOLISM AND NO IN DIABETIC AORTA
Furthermore, endothelial dysfunction coinciding with an increased eNOS expression was observed in aortic banding-induced hypertensive rats (38) and in Watanabe heritable hyperlipidemic rabbits (39). Thus, increased eNOS expression related to impaired endothelium-dependent relaxation is apparently not specific to diabetic vasculature but may be a common feature in the progression of atherosclerosis. However, arginine administration in some patients with coronary artery disease cannot increase NO production (40). While many possibilities remain to be examined, one possible explanation for the lack of effect of arginine supplementation is that transport or intracellular concentrations of arginine are limiting. Based on all these findings, we propose that sustained induction of AS and AL might be a therapeutic target for improving endothelium dysfunction, in addition to activation or induction of eNOS or transfer of the eNOS gene. ACKNOWLEDGMENTS We thank S. Kamitani (Kyoto University) for the human eNOS cDNA. We also thank R. Shindo for technical assistance and M. Ohara for comments on the manuscript. This work was supported in part by Grant-in-Aid 10557020 to M. Mori from the Ministry of Education, Science, Sports, and Culture of Japan.
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