High Glucose Increases Nitric Oxide Synthase Expression and Superoxide Anion Generation in Human Aortic Endothelial Cells
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.
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 hormones12 13 increase eNOS mRNA and protein, whereas tumor necrosis factor-α 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.
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.
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−
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.
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).
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 (5×10−4 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⇓).
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|
|PCR||=||polymerase chain reaction|
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.
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.
- Copyright © 1997 by American Heart Association
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