QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.111641v1 320/2/591    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyazaki-Akita, A. Articles by Iguchi, A. Search for Related Content PubMed PubMed Citation Articles by Miyazaki-Akita, A. Articles by Iguchi, A.

CARDIOVASCULAR

17-Estradiol Antagonizes the Down-Regulation of Endothelial Nitric-Oxide Synthase and GTP Cyclohydrolase I by High Glucose: Relevance to Postmenopausal Diabetic Cardiovascular Disease

Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan (A.M.-A., T.H., Q.F.D., A.I.); Department of Pharmacology, Fujita Health University School of Medicine, Toyoake, Japan (H.S., T.N.); and Department of Pharmacology, School of Medicine, University of Toyama, Toyama, Japan (Y.H.)

Received July 27, 2006; Revision received October 31, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
In postmenopausal women, the risk of diabetic cardiovascular disease drastically increases compared with that of men or premenopausal women. However, the mechanism of this phenomenon has not yet been clarified. We hypothesized that the beneficial effects of estrogen on endothelial function may be relevant to protection against hyperglycemia-induced vascular derangement. Bovine aortic endothelial cells were incubated for 72 h in the presence and absence of the physiological concentration of 17-estradiol (17-E2) under normal and high-glucose conditions. The presence of 17-E2 significantly counteracted the reduction in basal nitric oxide production under high-glucose conditions. This finding was associated with the recovery of endothelial nitric-oxide synthase (eNOS) protein expression, tetrahydrobiopterin (BH4) levels, and the activity and gene expression of GTP cyclohydrolase I (GTPCH-I), a rate-limiting enzyme for BH4 synthesis. Both the gene transfer of estrogen receptor using adenovirus and treatment with the protein kinase C inhibitor bisindolylmaleimide I significantly enhanced the effects of 17-E2 treatment under high-glucose conditions, whereas these effects were abolished by the estrogen receptor antagonist ICI 182,780 (faslodex). Transfection of small-interfering RNA targeting eNOS resulted in a marked reduction in GTPCH-I mRNA under both normal and high-glucose conditions, but this reduction was strongly reversed by 17-E2. These results suggest that the activation of ER with 17-E2 can counteract high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen deficiency may result in an exaggeration of hyperglycemia-induced endothelial dysfunction, leading to the development of cardiovascular disease in postmenopausal diabetic women.

Several lines of evidence have suggested the link of excess vascular oxidative stress with impaired NO activity in patients with diabetes (Tesfamariam and Cohen, 1992; Hayashi et al., 2005). It has been reported that supplementation with BH4 improves endothelial-dependent vasodilation by increasing NO activity in patients with type II diabetes mellitus (Heitzer et al., 2000). Thus, hyperglycemia and insulin resistance may be pathogenic factors that lead to decreased vascular relaxation via an imbalance of due to a relative deficiency of BH4 in endothelial cells (Shinozaki et al., 1999). Previously, we reported that a high-glucose concentration enhanced oxidative stress by eNOS dysfunction and activation of NADPH oxidase in BAECs (Ding et al., 2004).

Diabetes and abnormal glucose tolerance are associated with increased risk of cardiovascular disease (Kannel and McGee, 1979). The effect of diabetes on cardiovascular disease seems to be worse in women than in men (Barrett-Connor et al., 1991). Because the risk of cardiovascular disease dramatically increases with age, especially after menopause (Gordon et al., 1978), the influence of postmenopausal hormone use on cardiovascular disease in postmenopausal women has been a subject of interest. Recent reports from Heart and Estrogen/Progestin Replacement Study (Kanaya et al., 2003) and Women's Health Initiative Hormone (Margolis et al., 2004) randomized trials indicate that postmenopausal hormone replacement therapy (HRT) reduces the risk of diabetes. Therefore, it remains important to gain a better understanding of the molecular mechanisms that confer cardiovascular-protective effects of hormone replacement therapy in postmenopausal women with diabetes.

Laboratory evidence suggests that the vascular endothelium is an important target of estrogen. It has been shown that estrogen activates eNOS via genomic and nongenomic mechanisms, leading to an increase in NO (Hayashi et al., 1995b; Simoncini et al., 2000). Thus, we hypothesized that the profound effect of estrogen on eNOS-associated endothelial function may be relevant for obtaining protection against hyperglycemia-induced vascular impairment. In the present study, we demonstrate that treatment with 17-E2 at the physiological concentration can significantly counteract the down-regulation of eNOS and GTPCH-I in BAECs under high-glucose conditions. We found that 17-E2 increased GTPCH-I expression independently of eNOS expression, despite the finding that GTPCH-I expression was strongly regulated by the presence of eNOS.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. We used 17-estradiol (Sigma-Aldrich, St. Louis, MO), D-glucose, D-mannitol (Wako Pure Chemicals, Osaka, Japan), bisindolylmaleimide I (Calbiochem, Darmstadt, Germany), Takara One Step RNA PCR kit (Takara, Kyoto, Japan), eNOS monoclonal antibody (BD Biosciences, San Jose, CA), estrogen receptor antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), -actin antibody (Abcam, Cambridge, UK), siGENOME set of four duplexes (siRNAs targeting eNOS) (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and control (nonsil.) siRNA fluorescein (QIAGEN, Miami, FL). Lipofectamine 2000 and Opti-MEM were purchased from Invitrogen (Carlsbad, CA). ICI 182,780 was kindly provided by AstraZeneca Pharmaceuticals (Macclesfield, UK). GTPCH-I antibody was kindly provided from Prof. H. Ichinose (Tokyo Institute of Technology, Yokohama, Japan) who made it using recombinant full-length human GTPCH-I.

Cell Culture. BAECs were obtained from a fetal calf as described previously (Hayashi et al., 1995b) and cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% (v/v) calf serum (CS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells were allowed to reach 80% confluence, and they were then stimulated with different concentrations of D-glucose (5.5, 10.5, and 30.5 mM) as well as other reagents in phenol-red free Dulbecco's modified Eagle's medium with 2% (v/v) CS. We defined the control as the status under 5.5 mM glucose concentration, which is the same concentration as normal human plasma. Mannitol was used as a control to rule out the effect of osmotic pressure. Subconfluent cell monolayers were studied at six to eight passages.

Determination of Intracellular BH4 Levels and GTPCH-I Activity. BAECs were harvested with trypsin, pelleted by centrifugation, and frozen at –80°C. BH4 measurements were performed following the high-performance liquid chromatography procedure described by Fukushima and Nixon (1980). GTPCH-I activity was assayed as described by Sawada et al. (1986), based on the quantification of D-erythro-neopterin by high-performance liquid chromatography after the conversion of enzymaticaly formed D-erythro-7,8-dihydroneopterin triphosphate into D-erythro-neopterin by sequential iodine oxidation and dephosphorylation.

Reverse Transcription-Polymerase Chain Reaction Analysis of GTPCH-I mRNA and eNOS mRNA. Total RNA was isolated from BAECs with TRIzol reagent (Invitrogen) following the manufacturer's protocol. One microgram of total RNA was reverse-transcribed to cDNA using 200 U/µl Moloney murine leukemia virus reverse transcriptase, and the cDNA samples were analyzed for GTPCH-I and eNOS by PCR. PCR was performed in 25 µl of reaction volume containing 80.2 mM each of Ex-Taq polymerase buffer and deoxynucleotide phosphate as well as mixture, 81.5 mM MgCl₂, and 2.5 U of Ex-TaqDNA polymerase, oligonucleotide primers (0.5 µM each), and the cDNA. The programmed cycles were as follows: one cycle of 50°C x 30 min and 94°C x 2 min followed by 40 cycles of 94°C x 30 s, 60°C x 30 s, and 72°C x 1 min. The PCR-amplified product was analyzed by 1% agarose gel electrophoresis. The bovine GTPCH-I primer sequences were 5'-CCGCCTACTCGTCCATCCTGA-3' (sense) and 3'-ACCTCGCATTACCATACACAT-5' (antisense). The bovine eNOS primer sequences were 5'-CCGTGTCCAACATGCTGCT-3' (sense) and 3'-ACCTCGCATTACCATACACAT-5' (antisense). The bovine -actin primer sequence were 5'-CGAGCATTCCCAAAGTTCTACAGTG-3' (sense) and 3'-CTACATACTTCCGAAAACCAGGGG-5' (antisense).

Western Blot Analysis of eNOS and Estrogen Receptor Protein. Total protein was extracted by adding lysis buffer (10 mM Tris, pH 7.4, 1% SDS, and 1 mM sodium carbonate). Protein was quantified, and 10 µg of protein was loaded into each lane of 7.5% (12.5% for -actin) polyacrylamide gel. The protein was electrophoresed and transferred to polyvinylidene difluoride membrane, blocked with 2% skimmed milk powder, and then incubated with primary antibody overnight at 4°C and horseradish peroxidase-conjugated anti-mouse IgG antibody for 1 h. The bands were developed in the dark on the film (Fuji Medical X-Ray film; Fuji Photo Film, Tokyo, Japan) and measured densitometrically by an NIH Image analyzer. Equal protein loading was confirmed by Coomassie Brilliant Blue and amido black staining of protein in each lane of the same blot.

Measurement of Nitrite. As described in our previous report (Ding et al., 2004), the concentration of nitrite:metabolites of NO in 10 µl of culture medium was determined with an automated NO detector high-performance liquid chromatography system (ENO10; EICOM, Kyoto, Japan), where nitrite was detected based on Griess reaction to form a purple azo dye, and then absorbance at 540 nm was detected. Values for nitrite level were normalized for cell protein. The incubated medium was not completely free from nitrite; therefore, an aliquot of medium underwent the same process as the medium obtained from the cultured cells. We usually used the nitrite value obtained with the medium alone as a blank, and it was subtracted from all the samples. Our preliminary study confirmed that coincubation with the NOS inhibitor N^G-nitro-L-arginine methyl ester at 1 mM decreased a nitrite level after subtraction to less than 5% of the previous subtracted value, indicating that nitrites in the medium where BAECs are present are mostly from NOS-mediated NO production.

Construction of an Adenovirus Vector Carrying ER and Transfer into Cultured Endothelial Cells. Human ER cDNA cloned into pBR322 was inserted into pAxCAwt. Recombinant adenoviruses were constructed by Takara (Kyoto, Japan). Adenoviruses carrying an Escherichia coli lacZ gene encoding a nucleus-localized variant of -galactosidase were also used. We grew 5 x 10⁵ BAECs in a six-well plate for 24 h and then incubated cells with adenoviruses at a multiplicity of infection of 20 for 24 h.

Preparation of siRNAs and Transfection of siRNAs into Endothelial Cells. Four siRNAs targeting human eNOS with the following sense and antisense sequences were used: numbers 1, 5'-UGAAGCACCUGGAGAAUGAUU-3' (sense) and 5'-PUCAUUCUCCAGGUGCUUCAUU-3' (antisense); 2, 5'-CGGAACAGCACAAGAGUUAUU-3' (sense) and 5'-PUAACUCUUGUGCUGUUCCGUU-3' (antisense); 3, 5'-CGAGGAGACUUCCGAAUCUUU-3' (sense) and 5'-PAGAUUCGGAAGUCUCCUCGUU-3' (antisense); and 4, 5'-AGGAGAUGGUCAACUAUUUUU-3' (sense) and 5'-PAAAUAGUUGACCAUCUCCUUU-3' (antisense). Control (nonsil.) siRNA and fluorescein (20 µM) were used for negative control. The siRNAs were dissolved in siRNA buffer (20 mM KCl, 6.0 mM HEPES, pH 7.5, and 0.2 mM MgCl₂) to prepare a 20 µM stock solution. We grew 3 x 10⁵ BAECs in a six-well plate for 24 h and then transfected with siRNAs in 1.5 ml of serum and antibiotics-free medium using Lipofectamine 2000. Five microliters of Lipofectamine 2000 in 245 µl of Opti-MEM was incubated at room temperature for 5 min and then added in 5 µl of siRNA stock solution in 245 µl of Opti-MEM. After 20-min incubation at room temperature, siRNAs-lipid complex solution was added to the cells in serum. After incubation for 6 h at 37°C, the medium was replaced with 2% CS and 1% penicillin (100 U/ml), streptomycin (100 µg/ml) contained medium and cultured for 48 h.

Statistical Analysis. Data were obtained from three or four different experiments. Results are expressed as the mean of independent experiments (mean ± S.D.). Overall differences between groups were analyzed using a two-way ANOVA followed by the Fisher's post hoc test for determining differences between means when more than two groups were compared. An independent t test was used when only two groups were compared. In all tests, a probability level of P < 0.05 was used as the decision rule for significance testing.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effects of a High-Glucose Levels and 17-E2 on BH4 Levels and GTPCH-I Activity and Expression. Abnormally low levels of cofactor BH4 may be a factor involved in eNOS dysfunction; therefore, we measured BH4 levels in BAECs cultured for 72 h under high-glucose conditions. Under the two high-glucose conditions (10.5 and 30.5 mM), BH4 levels were significantly decreased by 20% compared with those under normal glucose conditions (5.5 mM) (Fig. 1A). In contrast, no change was observed in the number of cells with exposure to high glucose [95.3 ± 4.8 and 92.2 ± 6.1% of the control (5.5 mM glucose) with 10.5 and 30.5 mM glucose, respectively]. To evaluate whether 17-E2 is effective at ameliorating eNOS dysfunction, BAECs were treated with 17-E2. The addition of 17-E2 (1 nM) significantly increased BH4 levels not only under normal but also under high-glucose conditions (Fig. 1A). Even under hyperglycemic conditions, almost the same amount of BH4 was obtained as under normal glucose conditions. To rule out an osmotic effect, we added 25 mM mannitol to 5.5 mM glucose and 20 mM mannitol to 10.5 mM glucose. It was confirmed that mannitol did not affect BH4 levels [102.3 ± 2.4 and 107.5 ± 6.2% of control (5.5 mM glucose) with and without 17-E2, respectively] or GTPCH-I activity (98.3 ± 2.9 and 103.5 ± 6.1% of the control 5.5 mM glucose with and without 17-E2, respectively).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Effect of 17-E2 on BH4 levels (A), GTPCH-I activity (B), and GTPCH-I mRNA expression (C) in BAECs under different glucose levels. Cells were incubated for 72 h with different concentrations of D-glucose (5.5, 10.5, or 30.5 mM) in the absence or presence of 1 nM 17-E2. Mannitol was added to rule out an osmotic pressure effect. It was confirmed that mannitol exerted no effect on either BH4 levels or GTPCH-I activity. In C, representative reverse transcription-PCR data are shown in the top image. Note that there is no apparent difference in -actin mRNA, which was used as an internal control. Each result is expressed as the percentage of the value compared with that of the band obtained under normal (5.5 mM) glucose conditions without 17-E2. *, P < 0.05 compared with normal (5.5 mM) glucose in the absence of 17-E2. , P < 0.05 compared with samples treated with 10.5 mM glucose in the absence of 17-E2. #, P < 0.05 compared with samples treated with 30.5 mM glucose in the absence of 17-E2 (A and B) or with those treated at a normal glucose concentration in the presence of 17-E2 (C), as determined by ANOVA followed by Fisher's post hoc test. HG, high (30.5 mM) glucose; Man, mannitol (25 mM); NG, normal glucose.

Effects of High Glucose and 17-E2 on eNOS Protein Expression. To determine whether a longer period of exposure to high glucose can affect the levels of expression of eNOS, we analyzed the expression levels of eNOS protein in BAECs cultured for 72 h under high-glucose conditions. In this study, a high-glucose concentration was found to reduce the level of expression of eNOS protein, with a decrease of 19% at 10.5 mM and of 36% at 30.5 mM (Fig. 2A) after 72 h. We previously reported that 17-E2 (10^–1010^–8 M) increases eNOS expression in BAECs (Hayashi et al., 1995b). In this study, 1 nM 17-E2 caused 1.4- and 2.1-fold increases in eNOS protein expression under normal and high-glucose conditions, respectively (Fig. 2B). Interestingly, an even more marked effect of 17-E2 treatment on eNOS protein expression was observed under high-glucose conditions; this effect was similar to that of 17-E2 treatment on the expression of GTPCH-I, which is involved in the synthesis of cofactor BH4.

View larger version (26K): [in this window] [in a new window]   Fig. 2. A, changes in eNOS protein expression in BAECs under different glucose levels. B, effects of 17-E2 on the high-glucose-induced decrease in eNOS protein expression in BAECs. Cells were incubated for 72 h with different concentrations of D-glucose (5.5, 10.5, or 30.5 mM) in the absence or presence of 1 nM 17-E2. Mannitol (25 mM) was added to the samples to rule out an osmotic pressure effect. Each densitometric result is expressed as the relative percentage of the band obtained under abnormal glucose conditions compared with the band obtained under normal (5.5 mM) glucose conditions in the absence of 17-E2 taken as 100%. Representative immunoblots are shown in the top trace of each panel. *, P < 0.05 compared with the sample exposed to a normal glucose concentration in the absence of 17-E2. #, P < 0.05 compared with the sample exposed to a high (30.5 mM) glucose concentration and in the absence of 17-E2, as determined by ANOVA, followed by Fisher's post hoc test.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effect of 17-E2 on the nitrite concentrations in the cultured medium of BAECs under normal and high-glucose conditions. Cells were incubated for 72 h with 5.5 or 30.5 mM glucose in the absence (open bar) or presence (solid bar) of 1 nM 17-E2. The nitrite concentrations are expressed as percentage of the value obtained under normal glucose conditions in the absence of 17-E2. *, P < 0.05 compared with normal glucose in the absence of 17-E2. #, P < 0.05 versus high glucose in the absence of 17-E2, by ANOVA followed by the Fisher's post hoc test. Each result is expressed as percentage of the value under normal (5.5 mM) glucose conditions. Man, 25 mM.

Roles of ER and Protein Kinase C in the Effects of 17-E2 on eNOS under High-Glucose Conditions. To determine the mechanism underlying the effects of 17-E2 under high-glucose conditions, we investigated the role of estrogen receptors and PKC. Two types of ER have been identified to date (ER and ER) (Walter et al., 1985; Kuiper et al., 1996). ER is known to have a protective effect on vascular tissues, and a deficiency in the expression of ER in endothelial cells is known to accelerate atherosclerosis (Rubanyi et al., 1997; Sudhir et al., 1997). In contrast, ER is more widely present in tissues, and in females it is abundantly expressed in smooth muscle cells (Mendelsohn, 2002). In this study, we examined the role of ER in eNOS expression in BAECs under high-glucose conditions. Although ER protein levels tended to become lower under high-glucose conditions compared with those under normal glucose conditions, gene transfer of ER resulted in a successful increase in ER under both conditions (Fig. 4A). The reduction in eNOS protein expression by exposure to high levels of glucose was counteracted by gene transfer of ER using adenovirus as well as by the administration of 17-E2 (Fig. 4, A and B). The up-regulating effect of 17-E2 was abolished by incubation of cells with ICI 182,780, a specific antagonist of ER, for 72 h (Fig. 4C). Furthermore, ICI 182,780 significantly decreased eNOS expression in the presence of 17-E2 under normal glucose conditions. It is thought that PKC might be activated by hyperglycemia and thereby reduce eNOS expression (Srinivasan et al., 2004). The PKC inhibitor bisindolylmaleimide I significantly enhanced the protein expression of eNOS in the presence of 17-E2 under high-glucose conditions without affecting that under normal glucose conditions (Fig. 4C).

View larger version (33K): [in this window] [in a new window]   Fig. 4. A, effects of ER overexpression by gene transfer of adenovirus vector (Ad ER) on ER protein expression in BAECs under normal and high-glucose conditions. Where indicated, cells were treated with 1 nM 17-E2. B, effects of ER overexpression by gene transfer of Ad ER on eNOS protein expression in BAECs under normal and high-glucose conditions. The cells transfected with adenovirus carrying an E. coli lacZ gene (Ad LacZ) were served as control. Where indicated, cells were treated with 1 nM 17-E2. *, P < 0.05 compared with normal glucose, Ad LacZ transfection. #, P < 0.05 compared with normal glucose, Ad LacZ transfection in the presence of 17-E2. , P < 0.05 compared with high-glucose, Ad LacZ transfection, by ANOVA followed by the Fisher's post hoc test. C, effects of ICI 182,780 and bisindolylmaleimide I on eNOS protein expression in BAECs treated with 17-E2 under normal and high-glucose conditions. Cells were incubated for 72 h with 1 µM ICI 182,780 and 3 µM bisindolylmaleimide I. ***, P < 0.001 compared with normal glucose in the presence of 17-E2. #, P < 0.05; ##, P < 0.01 compared with high glucose in the presence of 17-E2. , P < 0.05 compared with normal glucose in the presence of 17-E2 and bisindolylmaleimide I, by ANOVA followed by the Fisher's post hoc test. In both A and B, each result is expressed as percentage of the control value under normal glucose conditions. Representative immunoblots are shown in the top trace of each panel. Note that ER is usually detected as a very faint band. HG, 30.5 mM; NG, 5.5 mM.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Effect of siRNAs targeting eNOS on GTPCH-I mRNA expression in BAECs. A, representative reverse transcription-PCR data showing that two of four eNOS siRNAs (numbers 2 and 3) successfully eliminated eNOS mRNA 48 h after transfection and reduced GTPCH-I mRNA. Neg, negative control. B, representative immunoblot data showing no detection of eNOS protein 48 h after transfection of successful eNOS siRNA (numbers 2 and 3). C, representative immunoblot data showing no detection of eNOS protein and GTPCH-1 protein 48 h after transfection of successful eNOS siRNA. D, modulation by 1 nM 17-E2 of the effect of successful eNOS siRNA (2) on the mRNA expression level of GTPCH-I in BAECs under normal (5.5 mM) and high (30.5 mM)-glucose conditions. *, P < 0.05 compared with control. #, P < 0.05 compared with transfection of eNOS siRNA in the absence of 17-E2, by ANOVA followed by the Fisher's post hoc test.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, the impairment of endothelial function under hyperglycemic conditions was detected as a decrease in NO production resulting from the down-regulation of eNOS expression, BH4 levels, and the GTPCH-I expression and activity in BAECs. This decrease in NO production could lead to increased oxidative stress and impaired vascular dilation. Thus, it is most likely that an enhancement of BH4 synthesis and NO bioavailability may be important in preventing diabetic cardiovascular complications. Previously, we reported that under high-glucose conditions, the activation of NADPH oxidase and the uncoupling of eNOS due to a BH4 deficiency increased ROS (Ding et al., 2004).

We inferred that 17-E2 may reverse hyperglycemia-induced endothelial dysfunction, because premenopausal women have a lower incidence of cardiovascular disease than do postmenopausal women and men of a similar age (Gordon et al., 1978; Fujishima et al., 1996). Our present results showed that treatment with the physiological concentration of 17-E2 (1 nM) in endothelial cells simultaneously exposed to high levels of glucose increased not only the eNOS expression level but also intracellular BH4 as well as GTPCH-I activity and gene expression levels. Interestingly, 17-E2 was found to be more effective under high-glucose conditions. The characteristic effects of 17-E2 on hyperglycemia have been noted in a previous clinical study (Colacurci et al., 1998), although the underlying mechanisms have not yet been investigated. It has been reported that 17-E2 elevates mRNA levels of GTPCH-I and intracellular BH4 levels in microvascular endothelial cells in the brain (Serova et al., 2004). However, the overall pattern of effects of 17-E2 on BH4 and GTPCH-I observed in that study seems to differ somewhat from the pattern observed in BAECs in the present study, based on differences between microvessels and muscular and elastic arteries. Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and insulin have also been shown to increase both BH4 levels and GTPCH-I activity in endothelial cells (Ishii et al., 2001; Hattori et al., 2003). However, to the best of our knowledge, the present study is the first to indicate that the beneficial effect of 17-E2 on eNOS under high-glucose conditions may be associated with a recovery of GTPCH-I in endothelial cells.

There are several reports showing the profound effect of high levels of glucose on eNOS expression. Shrinivasan et al. (2004) have documented that, in human aortic endothelial cells, acute elevated glucose (4 h) led to the up-regulation of eNOS expression; however, chronic elevated glucose (17 days) was associated with decreases in eNOS expression and nitrite production as well as with increases in the mitochondrial production of ROS. Our previous study demonstrated that high-glucose exposure for 24 h significantly increased eNOS mRNA expression (Ding et al., 2004). Noyman et al. (2002) have found reduced levels of eNOS expression and activity with a decrease in insulin sensitivity in BAECs cultured under high-glucose conditions for 2 weeks. In contrast, one previous study revealed that eNOS expression and NO production are increased in human aortic endothelial cells cultured under high-glucose conditions for 2 weeks. Possible reasons for this apparent discrepancy may include the use of different types of endothelial cells and different culture conditions. In agreement with recent results (Noyman et al., 2002; Srinivasan et al., 2004), the present results revealed decreases in eNOS protein expression and nitrite production in BAECs cultured under high-glucose conditions for 13 days.

This study supports our previous reports showing a gender difference in NO production in the aortas of animals and humans (Hayashi et al., 1992, 1995a, 2002). We have reported that atherosclerosis developed more slowly in female rabbits than in male rabbits fed a high-cholesterol diet, which may have been caused by a difference in the basal release as well as the stimulated release of NO (Hayashi et al., 1995a,b, 2000, 2002). The results of the present study also suggest that estrogen contributes to the improvement of endothelial function and provides protection against the progression of atherosclerosis. We have also shown that basal eNOS can be activated by 17-E2 at physiological concentrations (10^–1210^–8 M) in young cells (passaged fewer than six times), whereas ER is decreased in old cells (passaged more than 12 times) (Hayashi et al., 1995b). We preliminarily observed that the expression of ER at passages 6 to 8 tended to be lower under high-glucose than under normal glucose conditions (Fig. 4A). Accordingly, the gene transfer of ER may be important for allowing estrogen to act effectively against the type of endothelial dysfunction caused by aging and hyperglycemia. Treatment with 17-E2 following the gene transfer of ER using adenovirus resulted in a large increase in eNOS expression in BAECs. This effect was abolished by the addition of ICI 182,780, an ER antagonist, which confirmed the involvement of genomic activity.

PKC may be yet another important factor in eNOS regulation in hyperglycemia. Ohara et al. (1995) have shown that PKC inhibits eNOS expression in BAECs. It has been suggested that the decrease in eNOS expression observed in hyperglycemia is mediated by PKC and mitochondrial ROS via the activation of oxidative stress transcription factor AP-1 (Srinivasan et al., 2004). eNOS activated by the phosphorylation of Ser1177 through the phosphatidylinositol 3-kinase/Akt pathway is inactivated by the phosphorylation of Thr495 and dephosphorylation of Ser1177 through PKC (Matsubara et al., 2003). Hyperglycemia seems to alter the signaling pathway activating eNOS via modification at the Akt site (Du et al., 2001) and the activation of PKC (Ishii et al., 2001; Naruse et al., 2006). In the present study, the PKC inhibitor bisindolylmaleimide I enhanced the effects of 17-E2 on eNOS expression under high-glucose conditions, suggesting that the recovery effect of 17-E2 on high-glucose-induced eNOS down-regulation is regulated by PKC.

Furthermore, this study is the first to demonstrate that eNOS expression levels affect GTPCH-I expression levels in endothelial cells. We thus found that transfection with siRNA-targeting eNOS reduced the expression of GTPCH-I mRNA. This suggests that the decrease in eNOS observed under pathological conditions such as inflammation, atherosclerosis, or diabetes accelerates endothelial dysfunction by decreasing levels of the cofactor BH4. However, even when eNOS siRNA was transfected, 17-E2 was capable of increasing GTPCH-I expression levels. Thus, this effect of 17-E2 was more clearly demonstrated in an experiment using eNOS siRNA, in which 17-E2 enhanced the level of GTPCH-I mRNA expression, regardless of whether GTPCH-I was down-regulated by high-glucose conditions.

In diabetes, elevated blood glucose, altered insulin signaling, increased ROS, enhanced PKC, and inflammation can together or separately lead to a decrease in NO bioavailability (Endemann and Schiffrin, 2004). High-glucose levels reduced eNOS expression, GTPCH-I expression, and BH4 levels in BAECs. Finally, accelerated NO degradation by ROS might result in a decline in NO bioavailability, although we failed to confirm the possible involvement of oxidative stress in the high-glucose-induced decrease in eNOS expression in BAECs. Importantly, 17-E2 treatment ameliorated these endothelial cell abnormalities under high-glucose conditions, which seemed to be related to the activation of ER and masking by PKC.

The results of the present study may contribute to an understanding of the anti-atherogenic effects of estrogen in preventing endothelial dysfunction in diabetes mellitus (Fig. 6). If estrogen also prevents endothelial NO dysfunction under high-glucose conditions in humans, premenopausal diabetic women may be protected against the progression of atherosclerosis. Moreover, the prevalence of coronary heart disease in premenopausal women is much lower than that in the same generation of males or that of postmenopausal women. HRT in postmenopausal diabetic women may provide the benefit of reduced risk of developing cardiovascular disease. However, it should be noted that WHI (Pradhan et al., 2002) and Women's Health Initiative Memory Study (Brinton, 2005) indicated the risk associated with HRT (0.625 mg of conjugated equine estrogen and 2.5 mg of medroxyprogesterone acetate per day) for stroke, coronary heart disease, and Alzheimer's disease, even in diabetic patients. However, most of the patients in the study had another coronary risk factor such as obesity (66%), smoking habit (50%), and hypertension (35%). Moreover, the data from the WHI from patients without a uterus who had been prescribed only conjugated equine estrogen showed no increase in coronary heart disease. It is generally thought that estrogen may be atheroprotective (Hayashi et al., 2002); however, in most clinical cases, concomitant administration of progesterone, which masks the beneficial effects of estrogen, is necessary to prevent uterine cancer. Therefore, we cannot conclude that estrogen has a negative effect, simply based on the results of the WHI study. The present study conclusively demonstrates that the activation of ER with 17-E2 can counteract the high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen may be of potential use in the reversal of hyperglycemia-induced endothelial dysfunction, in turn retarding the development of cardiovascular disease in postmenopausal diabetic women. The clinical implications of the present findings will need to be addressed in future studies.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Diagram of the possible mechanism underlying the effect of 17-E2 on hyperglycemia-induced endothelial dysfunction. Left, hyperglycemia would cause endothelial dysfunction by decreasing eNOS expression and BH4 synthesis and by activating PKC. The decrease in eNOS expression could lead to a decrease in GTPCH-I expression. PKC may inactivate eNOS possibly due to phosphorylation of Thr495 and dephosphorylation of Ser1177. Right, 17-E2 via activation of ER could increase eNOS expression and BH4 synthesis even in hyperglycemia, and activate eNOS by phosphorylating Ser1177 through the phosphatidylinositol 3 (PI3)-kinase/Akt pathway. The increase in eNOS expression by 17-E2 may be masked by PKC.

	Footnotes

 
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.111641.

ABBREVIATIONS: NO, nitric oxide; BH4, tetrahydrobiopterin; eNOS, endothelial nitric-oxide synthase; GTPCH-I, GTP cyclohydrolase I; BAEC, bovine aortic endothelial cell; HRT, hormone replacement therapy; 17-E2, 17-estradiol; siRNA, small-interfering RNA; CS, calf serum; PCR, polymerase chain reaction; ER, estrogen receptor; NOS, nitric-oxide synthase; ANOVA, analysis of variance; ROS, reactive oxygen species; PKC, protein kinase C; ICI 182,780, 7-[9-[(4,4,5,5,5-pentafluoropentylsulphinyl)-nonyl]-estra-1,3,5(10)-triene-3,17-diol, faslodex; HG, high glucose; NG, normal glucose; Man, mannitol.

Address correspondence to: Dr. Toshio Hayashi, Department of Geriatrics, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: hayashi{at}med.nagoya-u.ac.jp

	References

Top Abstract Materials and Methods Results Discussion References

 

Albrecht EW, Stegeman CA, Herringa P, Henning RH, and Goor HV (2003) Protective role of endothelial nitric oxide synthase. J Pathol 199: 8–17.[CrossRef][Medline]

Brinton RD (2005) Investigative models for determining hormone therapy-induced outcomes in brain: evidence in support of a healthy cell bias of estrogen action. Ann NY Acad Sci 1052: 57–74.[Abstract/Free Full Text]

Barrett-Connor EL, Cohn BA, Wingard DL, and Edelstein SL (1991) Why is diabetes mellitus a stronger risk factor for fatal ischemic heart disease in women than in men? The Rancho Bernardo Study. J Am Med Assoc 265: 627–631.[Abstract]

Cai H and Harrison DG (2000) Endothelial dysfunction in cardiovascular disease: the role of oxidant stress. Circ Res 87: 840–844.[Abstract/Free Full Text]

Calles-Escandon J and Cipolla M (2001) Diabetes and endothelial dysfunction: a clinical perspective. Endocr Rev 22: 36–52.[Abstract/Free Full Text]

Colacurci N, Zarcone R, Mollo A, Russo G, Passaro M, de Seta L, and de Franciscis P (1998) Effects of hormone replacement therapy on glucose metabolism. Phanminerva Med 40: 18–21.

Creager MA, Luscher TF, Beckman JA, and Cosentino F (2003) Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I. Circulation 108: 1527–1532.

Ding QF, Hayashi T, Packiasamy AR, Miyazaki A, Fukatsu A, Shiraishi H, Nomura T, and Iguchi A (2004) The effects of high glucose on NO and through endothelial GTPCH1 and NADPH oxidase. Life Sci 75: 3185–3194.[CrossRef][Medline]

Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, and Brownlee M (2001) Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Investig 108: 1341–1348.[CrossRef][Medline]

Endemann DH and Schiffrin EL (2004) Nitric oxide, oxidative excess, and vascular complications of diabetes mellitus. Curr Hypertens Rep 6: 85–89.[Medline]

Fujishima M, Kiyohara Y, Kato I, Ohmura T, Iwamoto H, Nakayama K, Ohmori S, and Yoshitake T (1996) Diabetes and cardiovascular disease in a prospective population survey in Japan. The Hisayama Study. Diabetes 45: S14–S16.

Fukushima T and Nixon JC (1980) Analysis of reduced forms of biopterin in biological tissue and fluids. Anal Biochem 102: 176–188.[CrossRef][Medline]

Gesierich A, Niroomand F, and Tiefenbacher CP (2003) Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism. Basic Res Cardiol 98: 69–75.[CrossRef][Medline]

Gordon T, Kannel WB, Hjortland MC, and McNamara PM (1978) Menopause and coronary heart disease. The Framingham Study. Ann Intern Med 89: 157–161.[Medline]

Hattori Y, Nakanishi N, Akimoto K, Yoshida M, and Kasai K (2003) HMG-CoA reductase inhibitor increases GTP cyclohydrolase I mRNA and tetrahydrobiopterin in vascular endothelial cells. Arterioscler Thromb Vasc Biol 23: 176–182.[Abstract/Free Full Text]

Hayashi T, Fukuto JM, Ignarro LJ, and Chaudhuri G (1992) Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci USA 89: 11259–11263.[Abstract/Free Full Text]

Hayashi T, Fukuto JM, Ignarro LJ, and Chaudhuri G (1995a) Gender differences in atherosclerosis formation. Possible role of nitric oxide. J Cardiovasc Pharmacol 26: 792–802.[Medline]

Hayashi T, Jayachandran M, Sumi D, Thakur NK, Esaki T, Muto E, Kano H, Asai Y, and Iguchi A (2000) Physiological concentration of 17beta-estradiol retards the progression of severe atherosclerosis induced by a high-cholesterol diet plus balloon catheter injury: role of NO. Arterioscler Thromb Vasc Biol 20: 1613–1621.[Abstract/Free Full Text]

Hayashi T, Juliet PA, Kano-Hayashi H, Tsunekawa T, Dingqunfang D, Sumi D, Matsui-Hirai H, Fukatsu A, and Iguchi A (2005) NADPH oxidase inhibitor, apocynin, restores the impaired endothelial-dependent and –independent responses and scavenges superoxide anion in rats with type 2 diabetes complicated by NO dysfunction. Diabetes Obes Metab 7: 334–343.[CrossRef][Medline]

Hayashi T, Kano H, Sumi D, Iguchi A, Ito I, and Endo H (2002) The long-term effect of estriol on endothelial function and bone mineral density in octogenarian women. J Am Geriatr Soc 50: 777–778.[CrossRef][Medline]

Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, Hidaka H, and Iguchi A (1995b) Estrogen increases endothelial nitric oxide by a receptor-mediated system. Biochem Biophys Res Commun 214: 847–855.[CrossRef][Medline]

Heitzer T, Krohn K, Albers S, and Meinnertz T (2000) Tetrahydrobiopterin improves endothelin-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia 43: 1435–1438.[CrossRef][Medline]

Ishii M, Shimizu S, Nagai T, Shiota K, Kiuchi Y, and Yamamoto T (2001) Stimulation of tetrahydrobiopterin synthesis induced by insulin: possible involvement of phosphatidylinositol 3-kinase. Int J Biochem Cell Biol 33: 65–73.[CrossRef][Medline]

Kanaya AM, Herrington D, Vittinghoff E, Lin F, Grady D, Bittner V, Cayley JA, and Barrett-Connor E (2003) Glycemic effects of postmenopausal hormone therapy: the Heart and Estrogen/Progestin Replacement Study. A randomized double-blind, placebo-controlled trial. Ann Intern Med 138: 1–9.[Abstract/Free Full Text]

Kannel WB and McGee DL (1979) Diabetes and glucose tolerance as risk factors for cardiovascular disease: the Framingham study. Diabetes Care 2: 120–126.[Abstract]

Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, and Gustafsson JA (1996) Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93: 5925–5930.[Abstract/Free Full Text]

Margolis KL, Bonds DE, Rodabough RJ, Tinker L, Phillips LS, Allen C, Bassford T, Burke G, Torrens J, Howard BV, et al. (2004) Effect of oestrogen plus progestin on the incidence of diabetes in postmenopausal women: results from the Women's Health Initiative Hormone Trial. Diabetologia 47: 1175–1187.[Medline]

Matsubara M, Hayashi N, Jing T, and Titani K (2003) Regulation of endothelial nitric oxide synthase by protein kinase C. J Biochem 133: 773–781.[Abstract/Free Full Text]

Mendelsohn ME (2002) Genomic and nongenomic effects of estrogen in the vasculature. Am J Cardiol 90: 3F–6F.[CrossRef][Medline]

Naruse K, Rask-Madesen C, Takahara N, Ha SW, Suzuma K, Way KJ, Jacobs JR, Clermont AC, Ueki K, Ohshiro Y, et al. (2006) Activation of vascular protein kinase C- inhibits Akt-dependent endothelial nitric oxide synthase function in obesity-associated insulin resistance. Diabetes 55: 691–698.[Abstract/Free Full Text]

Noyman I, Marikovsky M, Sasson S, Stark AH, Bernath K, Seger R, and Madar Z (2002) Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells. Nitric Oxide 7: 187–193.[CrossRef][Medline]

Ohara Y, Sayegh HS, Yami JJ, and Harrison DG (1995) Regulation of endothelial constitutive nitric oxide synthase by protein kinase C. Hypertension 25: 415–420.[Abstract/Free Full Text]

Pradhan AD, Manson JE, Rossouw JE, Siscovick DS, Mouton CP, Rifai N, Wallace RB, Jackson RD, Pettinger MB, and Ridker PM (2002) Inflammatory biomarkers, hormone replacement therapy, and incident coronary heart disease: prospective analysis from the Women's Health Initiative observational study. J Am Med Assoc. 288: 980–987.[Abstract/Free Full Text]

Rubanyi GM, Freay AD, Kauser K, Sukovich D, Burton G, Lubahn DB, Couse JF, Curtis SW, and Korach KS (1997) Vascular estrogen receptor and endothelium-derived nitric oxide production in mouse aorta. Gender difference and effect of estrogen receptor gene disruption. J Clin Investig 99: 2429–2437.[Medline]

Sawada M, Horikoshi T, Masada M, Akino M, Sugimoto T, Matsuura S, and Nagatsu T (1986) A sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin. Anal Biochem 154: 361–366.[CrossRef][Medline]

Serova LI, Maharjan S, Huang A, Sun D, Kaley G, and Sabban EL (2004) Response of tyrosine hydroxylase and GTP cyclohydrolase I gene expression to estrogen in brain catecholaminergic regions varies with mode of administration. Brain Res 1015: 1–8.[CrossRef][Medline]

Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, and Kikkawa R (1999) Abnormal biopterin metabolism is a major cause of impaired endothelial dependent relaxation through nitric oxide/ imbalance in insulin–resistant rat aorta. Diabetes 48: 2437–2445.[Abstract]

Simoncini T, Hafezu-Moghadam A, Brazil DP, Ley K, Chin WW, and Liao JW (2000) Interaction of oeestrogen receptor with the regulatory subunit of phosphatidylinositol-OH kinase. Nature (Lond) 47: 538–541.

Srinivasan S, Hatley ME, Bolick DT, Palmer LA, Edelstein D, Brownlee M, and Hedrick CC (2004) Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia 47: 1727–1734.[CrossRef][Medline]

Sudhir K, Chou TM, Chatterjee K, Smith EP, Williams TC, Kane JP, Malloy MJ, Korach KS, and Rubanyi GM (1997) Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation 96: 3774–3777.

Tesfamariam B and Cohen RA (1992) Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol 263: H321–H326.

Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E, Scrace G, Waterfield M, et al. (1985) Cloning of the human estrogen receptor cDNA. Proc Natl Acad Sci USA 82: 7889–7893.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. J. Lee, D. G. Kang, J. S. Kim, and H. S. Lee Effect of Buddleja officinalis on High-Glucose-Induced Vascular Inflammation in Human Umbilical Vein Endothelial Cells Experimental Biology and Medicine, June 1, 2008; 233(6): 694 - 700. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.111641v1 320/2/591    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyazaki-Akita, A. Articles by Iguchi, A. Search for Related Content PubMed PubMed Citation Articles by Miyazaki-Akita, A. Articles by Iguchi, A.