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(Circulation. 1997;96:25-28.)
© 1997 American Heart Association, Inc.

Articles

High Glucose Increases Nitric Oxide Synthase Expression and Superoxide Anion Generation in Human Aortic Endothelial Cells

Francesco Cosentino, MD, PhD; Keiichi Hishikawa, MD, PhD; Zvonimir S. Katusic, MD, PhD; ; Thomas F. Lüscher, MD

From the Departments of Cardiology and Cardiovascular Research, University Hospitals, Bern and Zürich, Switzerland, and Departments of Anesthesiology and Pharmacology (Z.S.K.), Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Thomas F. Lüscher, MD, FACC, FESC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. E-mail100771.1237{at}compuserve.com

	Abstract

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Background Hyperglycemia is a primary cause of premature vascular disease. Endothelial cell dysfunction characterized by diminished endothelium-dependent relaxations is likely to be involved. Little is known about the molecular mechanisms of hyperglycemia-induced endothelial dysfunction.

Methods and Results This study was designed to determine the effect of hyperglycemia on the L-arginine/nitric oxide (NO) pathway. Expression of endothelial nitric oxide synthase (eNOS) mRNA and production of NO were studied in human aortic endothelial cells exposed to control levels (5.5 mmol/L) and high levels (22.2 mmol/L) of glucose for 5 days. We examined the effect of glucose on NO release by measuring changes in nitrite (NO₂^-) levels by Griess reaction. Superoxide anion (O₂^-) production was also examined by the ferrocytochrome c assay. NOS mRNA and protein expression, which were evaluated by reverse transcription–polymerase chain reaction and Western blotting, were approximately twofold greater in endothelial cells exposed to high glucose. Elevated glucose levels increased NO₂^- production by only 40% but increased the release of O₂^- by more than threefold.

Conclusions The present study demonstrates that prolonged exposure to high glucose increases eNOS gene expression, protein expression, and NO release. However, upregulation of eNOS and NO release is associated with a marked concomitant increase of O₂^- production. These results provide the molecular basis for understanding how chronic exposure to elevated glucose leads to an imbalance between NO and O₂^-. This may explain impaired endothelial function and be important for diabetic vascular disease.

Key Words: diabetes mellitus • endothelium-derived factors • free radicals

	Introduction

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The relation between diabetes and premature vascular disease is well established.^{1 2} One of the defects involves endothelial dysfunction characterized by impaired endothelium-dependent responses.^{3 4} In isolated blood vessels, exposure to elevated glucose causes endothelial dysfunction.^{5 6} However, the molecular mechanisms of hyperglycemia-induced endothelial dysfunction remain unknown. The endothelium acts as a transducer of humoral signals and physical forces. Many signals modify eNOS activity and NO release. Human eNOS has been cloned.⁷ The promoter region of the human eNOS gene contains tentative regulatory sequences, including phorbol esters, cAMP, and acute-phase, shear stress, and sterol-responsive elements.⁸ Exercise,⁹ mechanical stimuli,^{10 11} and sex hormones^{12 13} increase eNOS mRNA and protein, whereas tumor necrosis factor- decreases eNOS mRNA posttranscriptionally.¹⁴ This suggests that arterial tone is modulated by changes in expression of eNOS and NO production. Because little is known about the effects of hyperglycemia on the NO pathway, we studied the effect of an elevated concentration of glucose on eNOS mRNA and protein expression and production of NO and superoxide anion (O₂^-) in cultured human aortic endothelial cells.

	Methods

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Cell Culture
Human aortic endothelial cells were obtained from Clonetics. Cells were first grown to confluence in humidified air (5% CO₂ at 37°C). Then control (5.5 mmol/L) or elevated glucose concentrations (22.2 mmol/L) were administered for 5 days with concomitant lowering of serum concentration in the medium to 2% to keep the cells in the quiescent state. Cells up to passage 6 were used.

Amplification of eNOS mRNA by RT-PCR
The relative expression of eNOS mRNA in control and high-glucose–treated endothelial cells was evaluated by RT-PCR. Cellular RNA was reverse transcribed and first-strand cDNA was used as a template in PCR. cDNA aliquots were amplified with primers specific for eNOS and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a Perkin-Elmer GeneAmp 9600 cycler.

Western Blot
eNOS protein was analyzed by Western blot using an anti-human eNOS antibody (Transduction Laboratories) as previously described.¹³

Measurement of NO
We evaluated NO production by measuring levels of nitrite (NO₂^-), the oxidized product of NO, by Griess reaction as previously described.^{13 15} Briefly, basal and ionomycin-stimulated production were measured by subtracting NO₂^- values at time 0 from cumulative concentrations obtained after 3 hours' incubation and 60 minutes' exposure to ionomycin (1 µmol/L), respectively.

Measurement of O₂^-
Production of O₂^- was measured as the superoxide dismutase–inhibitable reduction of cytochrome c, as previously described.^{16 17}

Statistical Analysis
Results are expressed as mean±SEM; n indicates number of experiments. Statistical evaluation of the data was performed by use of unpaired Student's t test and ANOVA followed by Fisher's test. A value of P<.05 was considered statistically significant.

	Results

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Effect of Glucose on eNOS mRNA and Protein Expression
The exposure to a high level of glucose (22.2 mmol/L) for 5 days significantly increased the expression of eNOS mRNA compared with control (5.5 mmol/L; Fig 1). By contrast, the expression of GAPDH mRNA did not change in endothelial cells exposed to high levels of glucose (PCR product: 8.6±1.8 and 10±1.8 ng for control and high-glucose–treated cells, respectively; n=9). When control and high-glucose–treated cells were compared, expression of eNOS, as detected by Western blotting, was also greater in cells exposed to high glucose. Densitometric analysis showed an almost twofold increase in eNOS protein expression (Fig 1). However, when endothelial cells were exposed to a similar concentration of mannitol, eNOS protein expression was not affected (OD: 100±17 and 108±20 arbitrary units for control and mannitol-treated cells, respectively; n=3).

View larger version (33K): [in this window] [in a new window]   Figure 1. Effect of glucose on eNOS mRNA and protein expression assessed by RT-PCR and Western blotting, respectively, in human aortic endothelial cells exposed to control levels (5.5 mmol/L) or high levels of glucose (22.2 mmol/L). A, Representative ethidium bromide agarose gel and PCR product. B, Western blot and densitometric quantification of eNOS in protein homogenates. Data are mean±SEM (n=7 and 3, respectively). *P<.05 vs corresponding control.

Effect of Glucose on NO and O₂^- Production
Both basal and stimulated NO₂^- production by ionomycin (1 µmol/L) were increased in endothelial cells exposed to high levels of glucose (Fig 2). The stimulatory effect of ionomycin was inhibited by L-NMMA (5x10^-4 mol/L, 410±33 and 63±15 pmol per well per hour in the absence and in the presence of L-NMMA, respectively; n=4). However, O₂^- production was more than 300% higher in high-glucose–treated cells (Fig 2).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effect of glucose on (A) basal and ionomycin-stimulated release of nitrite (NO2-) and (B) production of superoxide anion (O2-). Data are mean±SEM (n=6). *P<.05 vs corresponding control.

	Discussion

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This study demonstrates for the first time that in human aortic endothelial cells, prolonged exposure to high glucose concentrations increases eNOS gene expression, protein expression, NO₂^- release, and production of O₂^-. Diabetes is an important cardiovascular risk factor and is associated with impaired endothelium-dependent relaxations. Hence, we would have expected glucose-induced downregulation of the eNOS gene. However, eNOS mRNA and protein expression were approximately twofold higher in endothelial cells exposed to elevated glucose. Accordingly, NO production, which we assessed by measuring changes in NO₂^- levels, was increased by 40%. However, the production of O₂^- was increased more than 300% in high-glucose–treated cells. The interaction of O₂^- with NO is very rapid and leads to inactivation of NO and production of the potent oxidant peroxynitrite.^{18 19} This may contribute to impaired endothelial function by stimulating arachidonic acid metabolism, lipid peroxidation, and prostanoid production.^{20 21} In isolated arteries, prolonged exposure to elevated glucose concentrations impairs endothelium-dependent relaxations.^{5 6} Hyperglycemia-induced endothelial dysfunction may result from decreased production of NO, inactivation of NO by oxygen-derived free radicals, or increased production of contracting factors.²² Our results clearly indicate that the synthesis and release of NO are not diminished after exposure to high concentrations of glucose. On the contrary, basal and ionomycin-induced production of NO₂^- were increased in high-glucose–treated cells. Selective upregulation of eNOS mRNA and protein expression together with the inhibitory effect of L-NMMA on NO₂^- production are consistent with this conclusion.

Superoxide anions are attractive candidates as mediators of endothelial dysfunction in diabetes.²³ In agreement with our results, O₂^- formation is involved in glucose-induced changes of endothelial Ca²⁺/endothelium-derived relaxing factor signaling.²⁴ Indeed, superoxide dismutase, a scavenger of O₂^-, prevents the impaired endothelium-dependent relaxations caused by elevated glucose.²⁵ In diabetic arteries, O₂^- may produce contractile effects not only by inactivation of NO but also via formation of hydrogen peroxide and hydroxyl radical, which stimulate the production of contractile prostanoids.^{23 24 25 26} Our findings support the hypothesis of an increased NO inactivation by O₂^- as an important mechanism for the impairment of endothelium-dependent relaxations in arteries exposed to high levels of glucose. The mechanisms by which high glucose levels simultaneously increase eNOS expression and production of O₂^- are not known. The experiments with mannitol certainly rule out an effect of osmolarity. One potential mechanism is the synthesis of diacylglycerol and protein kinase C activation.²⁷ Indeed, protein kinase C is chronically activated in diabetic tissues.²⁸ The promoter region of the human eNOS gene contains a phorbol ester–responsive element.⁸ In normal blood vessels, activation of protein kinase C by phorbol esters reduces endothelium-dependent relaxations as in diabetes.⁶ Hence, the release of O₂^- and prostaglandins by protein kinase C may explain the impaired endothelium-dependent relaxations.²⁹

An increased production of O₂^- may also occur via auto-oxidation of glucose and/or nonenzymatic protein glycation.³⁰ Further studies are needed to elucidate the signal-transduction pathway involved.

In summary, this study demonstrates that elevated concentrations of glucose increase eNOS gene and protein expression as well as NO release. However, upregulation of eNOS and increased NO release are associated with a marked concomitant increase of O₂^- production. These findings may explain the impaired endothelial function and be important in the development of diabetic vascular disease.

	Selected Abbreviations and Acronyms

 

eNOS = endothelial nitric oxide synthase L-NMMA = NG-monomethyl-L-arginine NO = nitric oxide PCR = polymerase chain reaction RT = reverse transcription

	Acknowledgments

 
This work was supported in part by the Swiss National Research Foundation grant 32-35541.91 (Dr Lüscher), National Heart, Lung, and Blood Institute grant HL-535427 (Dr Katusic), and the Mayo Foundation (Dr Katusic). Dr Cosentino was supported by a grant from Bristol-Myers Squibb, Italy, for cardiovascular research.

	Footnotes

 
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996, and previously published in abstract form (Circulation. 1996;94 [suppl I]:I-1093).

Received March 26, 1997; revision received May 8, 1997; accepted May 13, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kannel WB, McGee DL. Diabetes and cardiovascular disease: the Framingham Study. JAMA. 1978;241:2035-2038.
   
2. Cohen RA, Tesfamariam B. Diabetes mellitus and the vascular endothelium. In Ruderman N, ed. Hyperglycemia, Diabetes and Vascular Disease. New York, NY: Oxford University Press; 1992:44-49.
   
3. Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA. Impaired neurogenic and endothelium-dependent relaxation of human penile smooth muscle: the pathophysiological basis for impotence in diabetes mellitus. N Engl J Med. 1989;320:1025-1030.[Abstract]
   
4. Tesfamariam B, Jakubowski JA, Cohen RA. Contraction of diabetic rabbit aorta due to endothelium-derived PGH₂/TXA₂. Am J Physiol. 1989;257:H13272-H13277.
   
5. Tesfamariam B, Brown ML, Deykin D, Cohen RA. Elevated glucose promotes generation of endothelium-derived vasoconstrictor prostanoids in rabbit aorta. J Clin Invest. 1990;85:929-932.
   
6. Tesfamariam B, Brown ML, Cohen RA. Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. J Clin Invest. 1991;87:1643-1648.
   
7. Marsden PA, Schappert KT, Chen HS, Flowers M, Sundell CL, Wilcox JM, Lamas S, Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett. 1992;307:287-293.[Medline] [Order article via Infotrieve]
   
8. Marsden PA, Heng HHQ, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem. 1993;268:17478-17488.[Abstract/Free Full Text]
   
9. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze T. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res. 1994;74:349-353.[Abstract/Free Full Text]
   
10. Nishida K, Harrison DG, Navas JP, Fisher AA, Dockrey SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ. Molecular cloning and characterization of the constitutive bovine endothelial cell nitric oxide synthase. J Clin Invest. 1992;90:2092-2093.
    
11. Awolesi MA, Sessa WC, Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. J Clin Invest. 1995;96:1449-1454.
    
12. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S. Induction of calcium-dependent nitric oxide synthase by sex hormones. Proc Natl Acad Sci U S A. 1994;91:5212-5216.[Abstract/Free Full Text]
    
13. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T. Upregulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett. 1995;360:291-293.[Medline] [Order article via Infotrieve]
    
14. Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase messenger RNA by shortening its half-life. Circ Res. 1993;73:295-309.
    
15. Hishikawa K, Nakaki T, Suzuki H, Saruta T, Kato R. Transmural pressure inhibits nitric oxide release from human endothelial cells. Eur J Pharmacol. 1992;215:329-331.[Medline] [Order article via Infotrieve]
    
16. Massey V. The microestimation of succinate and extinction coefficient of cytochrome c. Biochem Biophys Acta. 1959;34:255-257.[Medline] [Order article via Infotrieve]
    
17. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510-518.[Abstract/Free Full Text]
    
18. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620-1624.[Abstract/Free Full Text]
    
19. Squadrito GL, Pryor WA. The formation of peroxynitrite in vivo from nitric oxide and superoxide. Chem Biol Interact. 1995;96:203-206.[Medline] [Order article via Infotrieve]
    
20. Gurtner GH, Knoblauch A, Smith PL, Sies H, Adkinson NF. Oxidant- and lipid-induced pulmonary vasoconstriction mediated by arachidonic acid metabolites. J Appl Physiol. 1983;61:584-591.
    
21. Tate RM, Morris HG, Schroeder WR, Repine JE. Oxygen metabolites stimulate thromboxane production and vasoconstriction in isolated saline-perfused rabbit lungs. J Clin Invest. 1984;74:608-613.
    
22. Cohen RA. Dysfunction of vascular endothelium. Circulation. 1993;87(suppl V):V-67-V-76.
    
23. Tesfamariam B. Free radicals in diabetic endothelial cell dysfunction. Free Radic Biol Med. 1994;16:383-391.[Medline] [Order article via Infotrieve]
    
24. Graier WF, Simecek S, Kukowetz WR, Kostner GM. High D-glucose induced changes in endothelial Ca²⁺/EDRF signalling is due to generation of superoxide anions. Diabetes. 1996;45:1386-1395.[Abstract]
    
25. Tesfamariam B, Cohen RA. Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol. 1992;263:H321-H326.[Abstract/Free Full Text]
    
26. Katusic ZS, Schugel J, Cosentino F, Vanhoutte PM. Endothelium-dependent contractions to oxygen-derived free radicals in canine basilar artery. Am J Physiol. 1993;264:H859-H864.[Abstract/Free Full Text]
    
27. Lee TS, Saltsman KA, Ohashi H, King GL. Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications. Proc Natl Acad Sci U S A. 1989;86:5141-5145.[Abstract/Free Full Text]
    
28. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform ßII and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci U S A. 1992;89:11059-11063.[Abstract/Free Full Text]
    
29. Wu KK, Hatsakis H, Lo SS, Seong DC, Sanduja SK, Tai HH. Stimulation of de novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol esters. J Biol Chem. 1988;263:19043-19047.[Abstract/Free Full Text]
    
30. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405-412.[Abstract]
    

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				N. J. Alp and K. M. Channon Regulation of Endothelial Nitric Oxide Synthase by Tetrahydrobiopterin in Vascular Disease Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 413 - 420. [Abstract] [Full Text]

M. Raitakari, T. Ilvonen, M. Ahotupa, T. Lehtimaki, A. Harmoinen,