High Glucose via Peroxynitrite Causes Tyrosine Nitration and Inactivation of Prostacyclin Synthase That Is Associated With Thromboxane/Prostaglandin H₂ Receptor–Mediated Apoptosis and Adhesion Molecule Expression in Cultured Human Aortic Endothelial Cells

Both poly- and monoclonal antibodies against 3-nitrotyrosine were bought from Upstate Biotechnologies (Lake Placid, NY). Dialyzed pronase E was from Boehringer Mannheim (Indianapolis, IN). l-Glucose, mannitol, calcium ionophore A23187, cytochrome c, superoxide dismutase (SOD), catalase, tyrosine, 3-nitrotyrosine, nitrate reductase, and pig gelatin were obtained from Sigma (St. Louis, MO). Monoclonal antibodies against PGIS and PGH₂ as well as enzyme-linked immunosorbent assay (ELISA) kits to measure 6-keto-PGF1α, and cyclic guanosine monophosphate (GMP) were obtained from Cayman Chemicals (Ann Arbor, MI). Human soluble intercellular adhesion molecules (sICAMs) ELISA kits were bought from R&D systems (Minneapolis, MN). Cell culture media were obtained from Gibco BRL (Grand Island, NY). Other chemicals and organic solvents of highest grade were obtained from local suppliers.

HAECs (American Type Culture Collection, Rockville, MD) were cultured in Ham’s F12 medium, 10% fetal bovine serum, 100 μg/ml heparin, and 30 μg/ml endothelial cell growth supplement. Cells were seeded in 60 × 15-mm culture dishes precoated with 0.1% pig gelatin. When they reached confluence, the cells were maintained in 1% fetal calf serum and exposed to normal glucose (5.5 mmol/l) or high glucose (30 or 44 mmol/l) for 7 days, during which the medium was changed every 2 days. Control groups (to account for media hyperosmolarity) were exposed to mannitol (24.5 or 39.5 mmol/l) in normal medium containing glucose (5.5 mmol/l). After incubation, the media were collected and assays were conducted as described below. For measurements of O₂·⁻ and cyclic GMP, the growth media was replaced by phosphate-buffered saline (PBS) containing no glucose. Unless otherwise noted, these measurements were performed on cells or the PBS bathing cells, which were stimulated by the calcium ionophore A23187 (10⁻⁵ mol/l, 2 h).

The release of O₂·⁻ was assayed by the SOD-inhibitable cytochrome c reduction assay, as described previously (24,25). Cytochrome c was present throughout the 2-h incubation of cells stimulated with A23187.

The level of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the culture medium exposed to cells during the last 2 days of the exposure period were measured by using the Griess reaction. For total NO_x, NO₃⁻ was first converted to NO₂⁻ by adding nitrate reductase.

After incubation in the presence of A23187 for 2 h in PBS, cells were quickly scraped into PBS and sonicated twice on ice. The supernatants were immediately separated by centrifugation at 10,000g for 5 min at 4°C and assayed for cyclic GMP by using ELISA kits according to the instructions provided by the supplier.

PGIS activity was assayed by incubating cells with PGH₂ (10⁻⁵ mol/l for 3 min), and PGIS activity was assessed by levels of 6-keto-PGF1α in the cell supernatant. In addition, for assessment of endogenous PGI₂ formation, levels of 6-keto-PGF1α were measured in cells stimulated with the calcium ionophore 23187 (10⁻⁵ mol/l for 2 h) as described previously (10,15,16,17,18,19).

Nitration of PGIS in cell extracts (0.5 mg protein) was identified by immunoprecipitation and Western blotting, as previously described (16,17,18,19). The corresponding bands in Western blots were calculated by using a phospho-imaging system (Bio-Rad) as the integrated values from the area × density. The value obtained for protein from cells exposed to 5 mmol/l glucose were taken as 100%.

After incubation, the cells were scraped into ice-cold 0.1 mol/l sodium acetate (pH 7.2) and sonicated twice on ice after being washed twice with ice-cold PBS (pH 7.4). The samples were hydrolyzed with dialyzed pronase E in the presence of 10 mmol/l CaCl₂ for 60–72 h at 55°C, as described previously (26).

A Varian Prostar pump (Walnut Greek, CA) equipped with a reverse-phase column (25 × 0.46 cm i.d., Ultrasphere 5-μm octyldecylsulfate column) was used under isocratic conditions with 50 mmol/l aqueous sodium acetate (pH 3.1) containing 10% methanol at a flow rate of 1 ml/min, which was continually sparged with helium. After optimization of the electrochemical detector (Varian Star 9080) conditions by hydrodynamic voltametry, the oxidation potential for 3-nitrotyrosine was set at 0.85 mV, and the absorption was measured at 365 nm with a photodiode array detector (Varian). 3-Nitrotyrosine was quantified by its absorption at 365 nm, by its electrochemical detector response, and by co-elution with 3-nitrotyrosine standard. 3-Nitrotyrosine in samples was completely eliminated by reduction with sodium dithionite, as expected.

After incubation, the culture media from the last 2 days of the exposure period were collected. The levels of sICAMs in culture media were measured by using ELISA kits, as instructed by the provider.

Endothelial cell DNA fragmentation was assayed with the diphenylamine assay, as reported elsewhere (27). The percentage of DNA fragmentation was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.

Results are expressed per milligram protein, as measured by the Bio-Rad protein assay kits in cells scraped from the culture dishes after each experiment. One-way analysis of variance and Student’s t tests were used where appropriate, and P < 0.05 was regarded as significant.

As shown in Fig. 1A, exposure of HAECs to high glucose (30 mmol/l) significantly increased the release of O₂·⁻ in the cells stimulated with the calcium ionophore A23187. Mannitol (30 mmol/l) did not mimic the effects of high glucose.

As shown in Fig. 1B, the release of total NO_x (NO₂⁻ and NO₃⁻) increased modestly in the culture media collected from the last 2 days from cells exposed to high glucose. However, the increased release of NO_x was largely caused by an increased formation of NO₃⁻, a breakdown product of ONOO⁻ (7,8). As a result, the NO₃⁻/NO₂⁻ ratios were significantly increased in high glucose–exposed cells, suggesting an increased formation ONOO⁻ in cells exposed to high glucose.

Compared with NO, ONOO⁻ is a much less potent activator of guanylyl cyclase (28), thus the formation of ONOO⁻ decreases cyclic GMP. As expected, cells cultured in high glucose and then exposed to PBS containing A23187 showed a significant decrease in cyclic GMP formation compared with cells exposed to control glucose or to high mannitol, consistent with reduced NO bioactivity in cells exposed to high glucose (Fig. 1C).

Further studies were conducted to determine whether the increased production of ONOO⁻ in HAECs exposed to high glucose inhibited PGIS activity. As shown in Fig. 1D, high glucose significantly inhibited calcium ionophore-stimulated endogenous PGI₂ release (as measured by levels of its degradation product 6-keto-PGF_1α) as well as PGIS activity (as measured by the conversion of exogenous PGH₂ into 6-keto-PGF_1α). In contrast, mannitol did not affect A23187-stimulated PGI₂ release or cellular PGIS activity.

As shown in Fig. 2A, growth in high glucose increased PGIS immunoprecipitates obtained with antibodies against 3-nitrotyrosine (Fig. 2A). In addition, increased 3-nitrotyrosine was also observed in Western blots of immunoprecipitates obtained with PGIS antibodies (Fig. 2B). Because high glucose did not affect the expression of PGIS (data not shown), these results can only be explained by increased tyrosine nitration of PGIS in cells exposed to high glucose. Exposure of HAECs to mannitol was not associated with nitration of PGIS (Figs. 2A and B).

We further used HPLC-electrochemical/UV detection to confirm the identity of and quantify 3-nitrotyrosine. Homogenates of HAECs or immunoprecipitates obtained with anti-PGIS antibody were digested with pronase E, and 3-nitrotyrosine was identified by co-elution with standard 3-nitrotyrosine and its sensitivity to reduction by sodium dithionite. As shown in Fig. 2C, exposure to high glucose (44 mmol/l) significantly increased 3-nitrotyrosine content in both HAEC homogenates and immunoprecipitates obtained with PGIS antibodies. The data in Fig. 2C also indicate that 3-nitrotyrosine detected in cell homogenates was largely derived from nitrated PGIS, indicating that PGIS is the main target of ONOO⁻ in cells exposed to high glucose.

As shown in Fig. 3A, U46619, a TP-receptor agonist, significantly increased endothelial cell DNA fragmentation and sICAM shedding. The effect of U46619 was antagonized by SQ29548, a selective TP receptor antagonist. Figure 3B shows that increased endothelial cell apoptosis and sICAM shedding occurred in cells exposed to high glucose. SQ29548 partially blocked these effects of high glucose. These results indicate that growth in high glucose, likely via inhibition of PGIS and consequent shunting to its substrate, PGH₂, or other eicosanoids activates TP receptors and causes endothelial cell apoptosis and sICAM expression.

If tyrosine nitration is responsible for inactivation of PGIS, and this in turn is responsible for TP receptor stimulation, then preventing PGIS nitration should also prevent TP receptor stimulation. Accordingly, studies were done to determine the effects of scavenging O₂·⁻ or inhibiting the synthesis of NO, either of which should decrease ONOO⁻ formation. Figure 4A shows that treatment of HAECs with polyethythene glycol–conjugated SOD (300 units/ml) throughout the exposure to high glucose decreased the nitrotyrosine immunoreactive protein in immunoprecipitates obtained with anti-PGIS antibodies. N^G-monomethyl-l-arginine methyl ester (l-NAME; 0.5 mmol/l) also decreased tyrosine nitration of PGIS, but there was no apparent effect of the xanthine oxidase inhibitor, oxypurinol (0.01 mmol/l).

Figure 4B shows that either SOD or l-NAME treatment also largely prevented the inhibition of PGIS activity in high glucose media, as reflected by 6-keto-PGF_1α levels measured in the media incubated with the cells during the last 2 days of culture. In addition, sICAM levels obtained in the same media from cells incubated in high glucose and treated with SOD or l-NAME increased significantly less than those incubated with high glucose alone. Note that treatment of cells with the TP antagonist SQ29548 (10⁻⁵ mol/l) had no significant effect on the inhibition of PGIS caused by high glucose, but it prevented the increase in sICAMs in HAECs exposed to high glucose.

Vascular cells are capable of generating ONOO⁻ because of their capacity to simultaneously release O₂·⁻ and NO. Previous studies have demonstrated that high glucose triggers O₂·⁻ formation in vascular cells (4,24,25,29). Potential sources of this O₂·⁻ could be NAD(P)H oxidases (29), xanthine oxidase (2), mitochondria (4), or NO synthase (30). In the present study, exposure of HAECs to high glucose significantly increased the release of O₂·⁻, as previously reported (24,25). The reaction of NO and O₂·⁻ results in ONOO⁻, which is much less potent than NO in stimulating guanylyl cyclase (28). Indeed, cyclic GMP content was significantly decreased in HAECs exposed to high glucose, suggesting increased inactivation of NO by O₂·⁻. Levels of NO₃⁻, a stable breakdown product of ONOO⁻ (7,8,9), were significantly increased, whereas those of NO₂⁻, a product of the reaction of NO with molecular oxygen, declined. This indicated that a major change in the fate of NO occurred in HAECs exposed to high glucose, characterized by its rapid reaction with O₂·⁻ to produce ONOO⁻. These results suggest that an increase of O₂·⁻ and ONOO⁻ explains the decrease in NO bioactivity in cells exposed to high glucose.

Convincing evidence for ONOO⁻ formation in cells exposed to high glucose comes from the identification of tyrosine-nitrated PGIS. Although other pathways of tyrosine nitration have been also suggested (31), 3-nitrotyrosine is regarded as the “footprint” of ONOO⁻ formation in vivo. Here, we examined whether tyrosine nitration of PGIS occurred because this enzyme is tyrosine nitrated by low concentrations of ONOO⁻ (15,16,17). Increased staining with an antibody against 3-nitrotyrosine was found in the immunoprecipitates obtained with antibodies against PGIS in cells exposed to high glucose, although the levels of PGIS expression were not changed. Tyrosine nitration of PGIS was further established by finding that PGIS-positive staining was concomitantly present in the immunoprecipitates obtained with antibody against 3-nitrotyrosine. Moreover, a substantially increased 3-nitrotyrosine content measured by HPLC was found both in cell homogenates and in immunoprecipitates obtained with PGIS antibodies from cells grown in high-glucose media. Remarkably, 3-nitrotyrosine in PGIS was the main component of 3-nitrotyrosine detected in cells exposed to high glucose. Indeed, Western blots with a polyclonal antibody against 3-nitrotyrosine of the immunoprecipitates obtained with a monoclonal antibody against 3-nitrotyrosine yielded a single major band with the molecular weight of PGIS (data not shown), further indicating that 3-nitrotyrosine mainly originated from PGIS.

In previous studies, we demonstrated that nitration of PGIS with exogenous ONOO⁻ inactivates the enzyme. In the present study, PGIS nitration in HAECs exposed to high glucose was accompanied by inactivation of the enzyme. The tyrosine nitration of PGIS and the decrease in its activity was in large part prevented by treating the cells with either SOD or l-NAME, a finding consistent with the proposal that ONOO⁻, produced in HAECs exposed to high glucose from O₂·⁻ and NO, tyrosine-nitrates and inactivates PGIS.

Inactivation of PGIS results in the accumulation of PGH₂ within vascular tissues and vasoconstriction via the TP receptor, a finding common to blood vessels in diabetes (2,10). Activation of TP receptors can trigger platelet aggregation, thrombus, vasospasm, cell apoptosis, and expression of adhesion molecules. We further addressed whether activation of TP receptors occurred as a consequence of PGIS inactivation and contributed to endothelial cell apoptosis and adhesion molecule expression in HAECs exposed to high glucose. In line with the results obtained with TP receptor agonists (12,14), activation of TP receptors by U46619 significantly increased endothelial apoptosis and shedding of sICAMs from cells into the media. The effect of U46619 on apoptosis and sICAMs was antagonized by SQ29548, indicating that the TP receptor mediated these effects. Furthermore, SQ29548 significantly inhibited the increased sICAM and endothelial apoptosis found to occur in HAECs exposed to high glucose, indicating that activation of TP receptors was at least partially responsible. Whereas the role of TP receptors can be adequately explained by increased levels of the PGI₂ precursor PGH₂ as a result of PGIS inactivation, further studies will be necessary to determine whether increased formation of other eicosanoids that are reported to act upon TP receptors, such as 8-isoprostanes or 15-hydroxyeicosatetraenoic acid, might contribute to high glucose–induced apoptosis and expression of adhesion molecules (32).

Nitration of PGIS by ONOO⁻ likely represents an important mechanism for diabetic vascular complications. Recent evidence demonstrates intense 3-nitrotyrosine staining in vascular endothelium and villous stroma of placentas from diabetic patients when compared with normal control subjects (33). The intimal localization of 3-nitrotyrosine staining indicates that endothelium-derived NO is inactivated by O₂·⁻ to produce the cytotoxic oxidant ONOO⁻. Although the identity of nitrated proteins in these tissues remains to be investigated, our preliminary results in diabetic apolipoprotein E–deficient mice suggest increased tyrosine nitration of PGIS in vivo (M.-H.Z., unpublished data).

In summary, exposure of HAECs to high glucose results in increased O₂·⁻, ONOO⁻, and tyrosine nitration of PGI₂ synthase. Associated activation of TP receptors increases endothelial cell apoptosis and adhesion molecule expression. Oxidant stress and nitration of PGIS could contribute to the initiation and progression of vascular complications of diabetes by several mechanisms, including decreased protective actions of both NO and PGI₂, and because the increased TP receptor stimulation favors endothelial apoptosis and expression of adhesion molecules. The evidence presented here that nitration of PGIS via ONOO⁻ occurs in endothelial cells exposed to high glucose helps to unify several previously proposed pathogenic vascular abnormalities in diabetes, including 1) decreased activity of NO, 2) decreased PGI₂, 3) vasoconstriction mediated by TP receptors, and 4) increased oxygen-derived free radicals.

This work was supported by a grant from the American Heart Association and the Juvenile Diabetes Foundation International (JDF) and by a National Institutes of Health-JDF sponsored grant (PO1-HL96005-05). We are grateful to Dr. T. Klein for kindly providing an antibody against human PGIS.