This Article Abstract Full Text (PDF) All Versions of this Article: 108/2/211    most recent 01.CIR.0000079311.39939.94v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maggi, A. Articles by Puglisi, L. Search for Related Content PubMed PubMed Citation Articles by Maggi, A. Articles by Puglisi, L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL NITRIC OXIDE STREPTOZOTOCIN Related Collections Angiogenesis Animal models of human disease Other Vascular biology

(Circulation. 2003;108:211.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Diabetes Undermines Estrogen Control of Inducible Nitric Oxide Synthase Function in Rat Aortic Smooth Muscle Cells Through Overexpression of Estrogen Receptor-ß

Adriana Maggi, PhD^*; Andrea Cignarella, PhD^*; Alessia Brusadelli, PhD; Chiara Bolego, PhD; Christian Pinna, PhD; Lina Puglisi, PhD

From the Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Disease (A.M., A.B.), University of Milan, Italy.

Correspondance to Prof Adriana Maggi, Center of Excellence on Neurodegenerative Disease, Via Balzaretti 9, 20133 Milan, Italy. E-mail adriana.maggi{at}unimi.it

Received January 30, 2003; revision received March 27, 2003; accepted March 31, 2003.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Previous reports from our group have shown that 17ß-estradiol reduces the synthesis and activity of inducible nitric oxide synthase (iNOS) in rat aortic smooth muscle cells (SMC) in response to inflammatory mediators. In this study, we investigated the effect of 17ß-estradiol on iNOS function in aortic SMC from streptozotocin-diabetic rats.

Methods and Results— Comparative analysis of NO release and of iNOS mRNA and protein content after 24-hour stimulation with a cytokine mixture revealed milder iNOS activation in diabetic than in control SMC. Furthermore, 17ß-estradiol dose-dependently blocked iNOS synthesis and activity in control but not in diabetic SMC. The defective estrogen response in diabetic SMC at 24 hours could not be attributed to reduced expression of estrogen receptors (ER). In fact, mRNA and protein levels of ER and, to a greater extent, of ERß, were increased in diabetic compared with nondiabetic SMC. Cytokines decreased ER and ERß expression in both groups. However, 17ß-estradiol dose-dependently restored the expression of ER but further downregulated that of ERß, indicating a differential regulation of ER isoforms.

Conclusions— Estrogenic control of iNOS was impaired in diabetic SMC. This was associated with a larger increase of ERß than of ER protein, whereas 17ß-estradiol regulated the two isoforms in an opposite fashion. Thus, modifications in the estrogen modulation of iNOS and in the expression pattern of ER may be involved in diabetic vascular dysfunction.

Key Words: myocytes • nitric oxide synthase • diabetes mellitus • hormones

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Diabetes is a cardiovascular disease.¹ Substantial evidence indicates that diabetic vascular dysfunction is associated with marked alterations of nitric oxide (NO) pathways.² In the presence of high blood glucose levels, the increased vascular generation of oxygen-derived free radicals, primarily superoxide anions, quenches the biological activity of NO.^2–4 This may result in a transient increase in NO synthesis through upregulation of endothelial NO synthase (eNOS) in endothelial cells, and even more so through activation of the inducible NOS (iNOS) isoform in smooth muscle cells (SMC) and macrophages. The sustained accumulation of high NO levels generated by iNOS can be toxic through reaction with superoxide to yield peroxynitrite, thereby playing a central role in the pathophysiology of inflammation and oxidant stress.⁵ Thus, the iNOS-mediated increase in NO formation may be a major mediator of diabetic vascular dysfunction. Accordingly, functional expression of iNOS has been reported in SMC from the superior mesenteric arteries of rats with chronic diabetes.⁶

We previously showed that diabetes impairs vascular function in female streptozotocin-diabetic rats by interfering with NO pathways.⁷ In women, diabetes abolishes the gender advantage in cardiovascular risk typical of premenopause.⁸ This protection is thought to be mediated, at least in part, by endogenous 17ß-estradiol (E₂) through mechanisms still under investigation.

Thus far, two estrogen receptors (ER) have been identified: ER and ERß. These proteins are encoded by two separate genes, are distinct structurally and functionally, and are expressed in endothelial cells,⁹ SMC,^10,11 and macrophages.¹² In endothelial cells, E₂ increases the expression of eNOS, thereby controlling the tone of underlying SMC,¹³ and may also rapidly activate eNOS through ER-mediated mechanisms not involving gene expression.^14,15 Conversely, E₂ blocks the synthesis of iNOS induced by inflammatory stimuli in SMC¹¹ and macrophages,¹² suggesting that the beneficial effect of the hormone arises from a combined action on different vascular cell types.

The aim of the present study was to further investigate the effects of E₂ on the expression and activity of iNOS in cultured aortic SMC from diabetic rats. In this study, we show that iNOS response to inflammatory events in SMC from diabetic rats is less sensitive to estrogen, probably on account of altered ER/ERß ratio. These findings indicate possible mechanisms underlying the increased risk of cardiovascular disease in diabetic premenopausal women.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
E₂, L-arginine, sepiapterin, ascorbic acid, L-nitroarginine-methyl ester (L-NAME), S-ethylisothiourea (EIT), and streptozotocin (STZ) were obtained from Sigma. The anti-rabbit iNOS polyclonal antibody was purchased from Transduction Laboratories (Lexington, Ky). The anti-ER and anti-ERß antibodies were kindly provided by Dr Geoffrey L. Greene (University of Chicago). Oligodeoxynucleotides were synthesized by Amersham Biosciences.

Diabetes Induction
Diabetes was induced by intravenous injection of STZ (65 mg/kg) in male Sprague-Dawley rats weighing 200 to 225 g (Charles River, Calco, Italy). STZ was freshly dissolved in citrate buffer (pH 4.5); control animals were injected with vehicle. Diabetes induction was considered successful when glycosuria was detectable 1 week after treatment. When the animals were killed, they had plasma glucose levels >25 mmol/L. The procedures followed were in accordance with institutional guidelines of the University of Milan.

Cell Culture
SMC were obtained from aortic intimal-medial layers of nondiabetic and 28-day diabetic rats according to Ross.¹⁶ Cells were grown in medium 199 (M199) as previously described.¹¹ The medium was replaced with phenol red–free M199 with 10% FCS for 5 days whenever the effects of E₂ were tested. Twenty-four hours before starting experiments, cells were synchronized in medium containing 0.4% FCS. SMC were then incubated for the indicated time with a cytokine mixture comprising 10 ng/mL interleukin-1ß (IL-1ß), 10 ng/mL interferon- (IFN-), 25 ng/mL tumor necrosis factor (TNF)-, and 10 µg/mL lipopolysaccharide (LPS). We previously demonstrated that this mixture consistently stimulated iNOS protein synthesis in SMC.¹¹ E₂ and/or other compounds were added along with cytokines.

Gene Expression Analysis
Total RNA was extracted from SMC (2x10⁵ cells/well) and rat uterus (as a positive control for ER mRNA expression) with the Bio/RNA-X Cell kit (Bio/Gene, Kimbolton, UK). RNA was reverse transcribed using MMLV reverse transcriptase H(-) (Promega). Amplification of ER and ERß cDNAs was carried out with the use of published primer sequences.¹¹ Amplification of iNOS cDNA was achieved by using the primer pair 5'-ATGGCCGACCTGAT-GTTGCC-3' (reverse) and 5'-TTTGACCAGAGGACCCAGAG-3' (forward). The intensity of PCR product bands was normalized to that of the housekeeping gene GADPH by densitometric analysis with the use of the Quantity-One software (Bio-Rad).

Western Blot Analysis
After quantification by Lowry’s method,¹⁷ 28 µg of cell protein was loaded onto discontinuous gradient SDS-PAGE (10% to 5%) gels. After electrophoresis, proteins were transferred to nitrocellulose membrane and incubated first with the primary antibodies (all 1:1000) overnight, then with the peroxidase-conjugated secondary antibody (Bio-Rad) for 1 hour. Proteins were detected by chemiluminescence (Amersham Biosciences).

Nitrite Assay
For nitrite measurement by Griess’ reaction,¹¹ 200 µL/well of medium was placed in a 96-well plate, and 20 µL of 6.5 mol/L HCl and 20 µL of 37.5 mmol/L sulfanilic acid was added. After incubation for 10 minutes, 20 µL of 12.5 mmol/L N-(1-naphthyl)-ethylenediamine was added. Optical density was read after 20 minutes at 550 nm (nitrite values are expressed as µmol nitrite/mg cell protein).

Statistical Analysis
Data were obtained from at least 3 independent experiments, each value representing mean±SEM of duplicate or triplicate determinations. Differences between mean values of multiple groups were analyzed by 1-way ANOVA with Fisher’s analysis. Values of P<0.05 were considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
E₂ Prevents Cytokine-Induced Secretion of Nitrite in SMC From Control But Not From Diabetic Rats
In cultured control SMC exposed to a cytokine mixture for 24 hours, nitrite levels in the medium increased 4.5-fold (Figure 1), ¹¹ but this was significantly opposed by E₂ (10^-11 to 10^-9 mol/L). Basal nitrite levels were not affected by E₂ in either group. In SMC from diabetic rats, cytokines increased nitrite production only 2.4-fold, and E₂ did not block cytokine effects but led to a slight dose-dependent increase in nitrite levels (Figure 1). This suggests that diabetes induced alterations in SMC metabolism that persisted in the absence of high glucose levels and affected the estrogenic control of nitrite production.

View larger version (31K): [in this window] [in a new window]   Figure 1. Effects of E2 on cytokine-induced nitrite accumulation in culture medium of aortic SMC from control and diabetic rats. SMC were grown in phenol red–free M199 medium for 5 days, synchronized in 0.4% FCS for 24 hours, and stimulated with cytokine cocktail (10 ng/mL IL-1ß, 10 ng/mL IFN-, 25 ng/mL TNF-, and 10 µg/mL LPS) for 24 hours. Increasing concentrations of E2 (10-11 to 10-9 mol/L) were added at the same time as cytokines where indicated. Results are mean±SEM of 6 to 7 independent experiments performed in duplicate. *P<0.01 vs untreated; P<0.05 vs untreated diabetic and cytokine-treated control; **P<0.01 vs cytokine-treated control; P<0.05 vs cytokine-treated diabetic (ANOVA).

Some vascular dysfunction can be reversed by increasing NOS substrate or adding antioxidants.² We then evaluated whether the altered response to cytokine stimulation in diabetic SMC was related to shortage of iNOS substrate or cofactors or to increased oxidant activities. As shown in the Table, addition of increasing concentrations of the iNOS substrate L-arginine or cofactor sepiapterin did not augment nitrite accumulation in the medium of diabetic SMC, suggesting that those were not limiting steps for iNOS activity. Similarly, addition of either ascorbic acid or lercanidipine (a vascular-selective calcium channel blocker with marked antioxidant properties) failed to affect nitrite production, suggesting that no increased oxidant burden impaired iNOS activity.

View this table: [in this window] [in a new window]   Effects of Different Supplementations on Nitrite Content in Medium of Aortic SMC From Diabetic Rats

In both SMC groups, the cytokine-induced nitrite increase was reversed by the nonselective NOS inhibitor L-NAME and the relatively selective iNOS inhibitor EIT (Figure 2), suggesting that it was secondary to iNOS induction. After 24-hour cytokine challenge, nitrite levels were again greater in the media of control SMC than in those of diabetic SMC. However, at 48 hours, nitrite content in the two groups was comparable. Further investigation showed that such differences reflected a distinct temporal pattern of iNOS protein induction. In fact, we consistently measured the highest iNOS protein levels after 48-hour cytokine treatment in diabetic SMC and after 24 hours in control SMC (Figure 3). After 72 hours, iNOS protein levels decreased, yet were significantly higher than in untreated cells. This delayed response to cytokine stimulation in diabetes was not restricted to isolated SMC but was also observed in whole aortic rings in culture under identical conditions (not shown).

View larger version (30K): [in this window] [in a new window]   Figure 2. Nitrite production by control and diabetic SMC after 24-hour and 48-hour incubation with cytokines. SMC were grown in M199 medium+10% FCS until confluence, synchronized in 0.4% FCS for 24 hours, and stimulated as described in the legend to Figure 1 for the indicated time in the presence of the NOS inhibitors L-NAME (0.3 mmol/L) and EIT (10 µmol/L). Nitrite production was measured in medium aliquots by Griess’ reaction. Data are expressed as mean±SEM of 5 to 7 independent experiments, each performed in duplicate. *P<0.01 vs basal; P<0.01 vs cytokines.

View larger version (35K): [in this window] [in a new window]   Figure 3. Time course of iNOS protein synthesis in control and diabetic SMC. SMC were grown and stimulated as described in the legend to Figure 2. Amount of iNOS in cell lysates was quantified by scanning densitometry. Representative Western blot of iNOS is shown. Data are expressed as mean±SEM of 3 independent experiments. *P<0.01 vs control.

E₂ Exerts Opposite Modulation of iNOS Synthesis in Control and Diabetic SMC
We then evaluated the content of iNOS mRNA and protein after E₂ treatment. As expected, iNOS mRNA was undetectable in untreated SMC from either group, as shown by semiquantitative RT-PCR. In control SMC, cytokine stimulation for 24 hours increased iNOS mRNA content; a modest induction was observed in diabetic SMC as well. However, the overall effect of E₂ was opposite in the two groups. In fact, E₂ (10^-11 to 10^-9 mol/L) dose-dependently reduced iNOS mRNA levels in control SMC but increased iNOS mRNA in diabetic SMC severalfold compared with cytokine-treated diabetic and 2-fold compared with cytokine-treated control SMC (data not shown).

Western blot experiments showed that E₂ reduced in a dose-dependent manner iNOS protein content in cytokine-treated control SMC (Figure 4A). By contrast, treatment with E₂ did not affect iNOS protein levels in diabetic SMC despite increased mRNA levels. As shown in Figure 4B, after 48-hour cytokine stimulation, E₂ was about as effective in control as in diabetic SMC at reducing iNOS protein content, indicating that the early difference in response to E₂ between control and diabetic SMC leveled off at later time points. Similar results were obtained for iNOS activity (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 4. Effects of E2 (10-11 to 10-9 mol/L) on cytokine-induced iNOS protein synthesis in control and diabetic SMC. SMC were grown and incubated for 24 hours (A) and 48 hours (B), as described in the legend to Figure 1. Amount of iNOS in cell lysates was quantified by scanning densitometry. Representative Western blots for iNOS are shown. Data are expressed as mean±SEM of 3 to 5 independent experiments. *P<0.01 vs cytokines (ANOVA).

Loss of E₂-Inhibitory Control on iNOS Gene Expression Correlates With Decreased ER and Increased ERß Expression
The antithetical control of E₂ on iNOS expression in SMC from diabetic and nondiabetic rats after 24-hour cytokine incubation could not be ascribed to deficiencies in iNOS system. This led us to investigate whether diabetes had altered the expression pattern of ERs. It was apparent that (1) the mRNA of ER was more abundant than that of ERß in untreated diabetic and nondiabetic SMC and (2) both ER and ERß mRNA levels were consistently greater in diabetic than in control SMC under any condition tested (Figure 5). Cytokine incubation for 24 hours markedly reduced ER mRNA levels, whereas E₂ (10^-11 to 10^-9 mol/L) dose-dependently restored ER expression in both groups. On cytokine treatment, ERß mRNA decreased by 50% in diabetic SMC and fell to barely detectable levels in control SMC (Figure 5). In sharp contrast to ER, E₂ dose-dependently reduced ERß mRNA in diabetic SMC but had no detectable effect in control SMC.

View larger version (32K): [in this window] [in a new window]   Figure 5. Effects of E2 (10-11 to 10-9 mol/L) on mRNA levels of ER in control and diabetic SMC. SMC were grown and stimulated as described in the legend to Figure 1. Total RNA was extracted and cDNAs of ER and ERß amplified as detailed in the Methods section. Levels of mRNA for ERs were normalized to GADPH mRNA by densitometry analysis. In control SMC, cytokine treatment with or without E2 led to virtually undetectable ERß mRNA levels. Representative RT-PCR assays are shown. Data are expressed as mean of 3 independent experiments.

In contrast to the strong accumulation of ER mRNA in diabetic with respect to nondiabetic SMC, the levels of ER protein were increased by 40% to 50% only (Figure 6). In both SMC groups, cytokines strongly decreased ER levels, and this was dose-dependently reversed by E₂. Experimental diabetes, on the other hand, had a marked impact on ERß protein level, which was >than 2-fold higher than in nondiabetic SMC. ERß content was not affected by cytokine treatment but was significantly diminished by E₂, particularly in diabetic SMC (Figure 6).

View larger version (32K): [in this window] [in a new window]   Figure 6. Effects of E2 (10-11 to 10-9 mol/L) on ER and ERß protein synthesis in control and diabetic SMC. SMC were grown and incubated as described in the legend to Figure 1. Amounts of ER and ERß in cell lysates were quantified by scanning densitometry. Representative Western blots for both ERs are shown. Data are expressed as mean±SEM of 3 independent experiments. *P<0.05 vs cytokine-treated (ANOVA).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study we show that the cytokine-mediated iNOS activation and the estrogen response were very different in aortic SMC from diabetic compared with control rats and that this effect was associated with a significant change in ER and ERß relative content. These differences may be relevant to diabetic vascular dysfunction.

Data from our group and other groups indicate that the E₂ antagonism to the synthesis of inflammatory proteins is mediated by ER because it occurs in cell cultures at hormone concentrations compatible with ER transcriptional activation, is blocked by specific ER antagonists, and is linked to ER expression.^11,12,23,24 In vitro transfection experiments show that ER is indispensable to E₂ regulation of iNOS transcription. There is little evidence, however, for a direct interaction of estrogen with the iNOS promoter,²⁵ which lacks canonical estrogen responsive elements. As proposed for the effect of estrogens on other molecules of the inflammatory cascade, it is conceivable that iNOS mRNA synthesis is controlled by ERs by interaction with transcription factors such as NF-B, AP-1, or STATs.^26,27 Paech et al²⁸ reported that E₂ has opposite influence on NF-B and AP-1 transcription activity, depending on its association with ER or ERß. Considering a differential effect of the two ER subtypes on iNOS expression, we propose that the altered ER/ ERß ratio reported here for diabetic SMC, and in particular the remarkable upregulation of ERß, underlies the loss of E₂ negative control on iNOS activity. ER and ERß expression was increased in diabetic compared with control SMC, with a relative increase in ER and ERß protein levels of 1.4- and 2.5-fold, respectively. Although no experiments were performed in this study to distinguish functionally between ER and ERß, our findings suggest that the increased ERß expression was associated with the defective response to E₂ and to cytokines observed in diabetes. Supporting this hypothesis are recent studies in ERß-deficient mice describing an important role for ERß in regulating vascular function and blood pressure.²⁹ The authors showed that ERß is involved in estrogen-induced accumulation of iNOS protein in endothelium-denuded aortic rings as well as iNOS gene transcription in normal vessels. This lends further support to the view that the altered estrogenic control of iNOS function in diabetic SMC was mediated mainly by ERß.

The observation that ER and ERß protein content was regulated by E₂ in an opposite fashion in aortic SMC (Figures 5 and 6 ) provides further evidence for the potential divergent effects of the two receptors.^28,30 By upregulating ER, E₂ may enhance the protective antiinflammatory effects mediated by this receptor subtype in these cells while limiting potential proinflammatory effects with ERß downregulation. Interestingly, enhanced transcription of both ER subtypes occurred in diabetic cells, although the increase in protein level was more evident for ERß. It is conceivable that ER protein underwent translational regulation, as was the case for iNOS; this phenomenon has been reported to occur in diabetes.^31,32 Accordingly, the cytokine-dependent synthesis of iNOS was delayed and significantly blunted in diabetic cells possibly due to altered levels of transcription factors or of intracellular signaling molecules such as intracellular Ca²⁺, as suggested by previous studies in SMC.³³ The cytokine response itself was blunted but not abolished in diabetic SMC at the 24-hour time point. Therefore, the altered time course of iNOS response to cytokines and the corresponding loss of estrogenic effect on this phenomenon, along with the changes in the ER expression pattern, appeared to be part of diabetic vascular dysfunction in aortic SMC.

Activation of iNOS in SMC leads to rapid production of high amounts of NO, which causes pathological vasodilation. In addition, excess NO may interact with superoxide to generate peroxynitrite, which in turn modifies the function of several proteins, contributing to the tissue damage occurring in vascular sepsis and inflammation.¹⁸ Our results may partially explain why diabetes abolishes the gender-specific vascular protection afforded by estrogen reported in epidemiological studies.⁸ Thus, understanding the mechanism for the vascular alterations in estrogen response and ER levels in diabetes would be particularly relevant from a clinical perspective. Estrogen inhibits the iNOS activation associated with ovariectomy,¹⁹ uraemia,²⁰ transplantation,²¹ and inflammatory stimuli.^11,22 Estrogen also appears to be effective in clinical conditions sharing inflammatory features such as multiple sclerosis, Alzheimer’s disease, osteoporosis, and periodontitis, indicating that the beneficial effects of estrogen reflect its antiinflammatory action. In view of the possible role of ERß in the development of diabetic vascular dysfunction, it would be interesting to assess the role of ERß in other inflammatory disorders in which the hormone exerts protective effects. These studies might eventually lead to the development of novel drugs for prevention and treatment of inflammatory disease affecting the aging woman.

	Acknowledgments

 
This work was supported by grants from the Italian Association for Cancer Research (A.M.), Ministry of Education and University (II National Research Program on Drugs and COFIN), European Union (QLRT-2001-02221), and CARIPLO Foundation (A.M. and A.C.).

	Footnotes

 
*Drs Maggi and Cignarella contributed equally to this study.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation. 1999; 100: 1134–1146.[Free Full Text]

2. De Vriese AS, Verbeuren TJ, Van de Voorde J, et al. Endothelial dysfunction in diabetes. Br J Pharmacol. 2000; 130: 963–974.[CrossRef][Medline] [Order article via Infotrieve]

3. Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986; 250: H822–H827.[Medline] [Order article via Infotrieve]

4. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care. 1996; 19: 257–267.[Abstract]

5. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996; 271: C1424–C1437.[Medline] [Order article via Infotrieve]

6. Bardell AL, MacLeod KM. Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats. J Pharmacol Exp Ther. 2001; 296: 252–259.[Abstract/Free Full Text]

7. Bolego C, Cignarella A, Zancan V, et al. Diabetes abolishes the vascular protective effects of estrogen in female rats. Life Sci. 1999; 64: 741–749.[CrossRef][Medline] [Order article via Infotrieve]

8. Kannel WB, Wilson PW. Risk factors that attenuate the female coronary disease advantage. Arch Intern Med. 1995; 155: 57–61.[Abstract/Free Full Text]

9. Lindner V, Kim SK, Karas RH, et al. Increased expression of estrogen receptor-ß mRNA in male blood vessels after vascular injury. Circ Res. 1998; 83: 224–229.[Abstract/Free Full Text]

10. Karas RH, Patterson BL, Mendelsohn ME. Human vascular smooth muscle cells contain functional estrogen receptor. Circulation. 1994; 89: 1943–1950.[Abstract/Free Full Text]

11. Zancan V, Santagati S, Bolego C, et al. 17ß-Estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology. 1999; 140: 2004–2009.[Abstract/Free Full Text]

12. Vegeto E, Pollio G, Pellicciari C, et al. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-induced apoptosis. FASEB J. 1999; 13: 793–803.[Abstract/Free Full Text]

13. Furchgott RF. Endothelium-derived relaxing factor: discovery, early studies, and identification as nitric oxide. Biosci Rep. 1999; 19: 235–251.[CrossRef][Medline] [Order article via Infotrieve]

14. Mendelsohn ME. Nongenomic E. R-mediated activation of endothelial nitric oxide synthase: how does it work? What does it mean? Circ Res. 2000; 87: 956–960.[Abstract/Free Full Text]

15. Chen Z, Yuhanna IS, Galcheva-Gargova Z, et al. Estrogen receptor mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest. 1999; 103: 401–406.[Medline] [Order article via Infotrieve]

16. Ross R. The smooth muscle cell, II: growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol. 1971; 103: 2787–2796.[CrossRef]

17. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265–275.[Free Full Text]

18. Marshall HE, Merchant K, Stamler JS. Nitrosation and oxidation in the regulation of gene expression. FASEB J. 2000; 14: 1889–1900.[Abstract/Free Full Text]

19. Tamura K, Yamaguchi K, Kogo H. 17ß-Estradiol inhibits ovariectomy-induced expression of inducible nitric oxide synthase in rat aorta in vivo. Life Sci. 2000; 66: 259–264.

20. Noris M, Todeschini M, Zappella S, et al. 17ß-Estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases. Am J Physiol Renal Physiol. 2000; 279: F626–F635.[Abstract/Free Full Text]

21. Saito S, Aras RS, Lou H, et al. Effects of estrogen on nitric oxide synthase expression in rat aorta allograft and smooth muscle cells. J Heart Lung Transplant. 1999; 18: 937–945.[CrossRef][Medline] [Order article via Infotrieve]

22. Kauser K, Sonnenberg D, Diel P, et al. Effect of 17ß-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta. Br J Pharmacol. 1998; 123: 1089–1096.[CrossRef][Medline] [Order article via Infotrieve]

23. Cuzzocrea S, Santagati S, Sautebin L, et al. 17ß-Estradiol anti-inflammatory activity in carrageenan-induced pleurisy. Endocrinology. 2000; 141: 1455–1463.[Abstract/Free Full Text]

24. Vegeto E, Bonincontro C, Pollio G, et al. Estrogen prevents the lipopolysaccharide-induced inflammatory response in microglia. J Neurosci. 2001; 21: 1809–1818.[Abstract/Free Full Text]

25. Miller L, Alley EW, Murphy WJ, et al. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol. 1996; 59: 442–450.[Abstract]

26. Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994; 269: 4705–4708.[Abstract/Free Full Text]

27. Faulds MH, Pettersson K, Gustafsson JÅ, et al. Cross-talk between ERs and signal transducers and activators of transcription 5 is E₂ dependent and involves two functionally separate mechanisms. Mol Endocrinol. 2001; 15: 1929–1940.[Abstract/Free Full Text]

28. Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science. 1997; 277: 1508–1510.[Abstract/Free Full Text]

29. Zhu Y, Bian Z, Lu P, et al. Abnormal vascular function and hypertension in mice deficient in estrogen receptor ß. Science. 2002; 295: 505–508.[Abstract/Free Full Text]

30. Weatherman RV, Scanlan TS. Unique protein determinants of the subtype-selective ligand responses of the estrogen receptors (ERalpha and ERbeta) at AP-1 sites. J Biol Chem. 2001; 276: 3827–3832.[Abstract/Free Full Text]

31. Yu WJ, Juang SW, Chin WT, et al. Decrease of nitric oxide synthase in the cerebrocortex of streptozotocin-induced diabetic rats. Neurosci Lett. 1999; 272: 99–102.[CrossRef][Medline] [Order article via Infotrieve]

32. Perreault M, Dombrowski L, Marette A. Mechanism of impaired nitric oxide synthase activity in skeletal muscle of streptozotocin-induced diabetic rats. Diabetologia. 2000; 43: 427–437.[CrossRef][Medline] [Order article via Infotrieve]

33. Muniyappa R, Srinivas PR, Ram JL, et al. Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells. Hypertension. 1998; 31: 289–295.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Cignarella, C. Bolego, V. Pelosi, C. Meda, A. Krust, C. Pinna, R. M. Gaion, E. Vegeto, and A. Maggi Distinct Roles of Estrogen Receptor-{alpha} and {beta} in the Modulation of Vascular Inducible Nitric-Oxide Synthase in Diabetes J. Pharmacol. Exp. Ther., January 1, 2009; 328(1): 174 - 182. [Abstract] [Full Text] [PDF]

				C. Pinna, C. Bolego, P. Sanvito, V. Pelosi, R. Baetta, A. Corsini, R. M. Gaion, and A. Cignarella Raloxifene Elicits Combined Rapid Vasorelaxation and Long-Term Anti-Inflammatory Actions in Rat Aorta J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1444 - 1451. [Abstract] [Full Text] [PDF]

				C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2192 - 2199. [Abstract] [Full Text] [PDF]

				R. C. Christian, P. Y. Liu, S. Harrington, M. Ruan, V. M. Miller, and L. A. Fitzpatrick Intimal Estrogen Receptor (ER){beta}, But Not ER{alpha} Expression, Is Correlated with Coronary Calcification and Atherosclerosis in Pre- and Postmenopausal Women J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2713 - 2720. [Abstract] [Full Text] [PDF]

				C. Bolego, A. Cignarella, P. Sanvito, V. Pelosi, F. Pellegatta, L. Puglisi, and C. Pinna The Acute Estrogenic Dilation of Rat Aorta Is Mediated Solely by Selective Estrogen Receptor-{alpha} Agonists and Is Abolished by Estrogen Deprivation J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1203 - 1208. [Abstract] [Full Text] [PDF]

				P. Y. Liu, R. C. Christian, M. Ruan, V. M. Miller, and L. A. Fitzpatrick Correlating Androgen and Estrogen Steroid Receptor Expression with Coronary Calcification and Atherosclerosis in Men without Known Coronary Artery Disease J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1041 - 1046. [Abstract] [Full Text] [PDF]

				F. Barchiesi, E. K. Jackson, B. Imthurn, J. Fingerle, D. G. Gillespie, and R. K. Dubey Differential Regulation of Estrogen Receptor Subtypes {alpha} and {beta} in Human Aortic Smooth Muscle Cells by Oligonucleotides and Estradiol J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2373 - 2381. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 108/2/211    most recent 01.CIR.0000079311.39939.94v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maggi, A. Articles by Puglisi, L. Search for Related Content PubMed PubMed Citation Articles by Maggi, A. Articles by Puglisi, L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL NITRIC OXIDE STREPTOZOTOCIN Related Collections Angiogenesis Animal models of human disease Other Vascular biology