Endothelial Protection by Pentaerithrityl Trinitrate: Bilirubin and Carbon Monoxide as Possible Mediators

Abstract

Pentaerithrityl tetranitrate (PETN) is a long-acting donor of nitric oxide (NO) and has recently been characterized as an antianginal agent that, in contrast with other nitric acid esters, does not induce oxidative stress and is therefore free of tolerance. Moreover, animal experiments have revealed that PETN actively reduces oxygen radical formation in vivoand specifically prevents atherogenesis and endothelial dysfunction. Because heme oxygenase-1 (HO-1) has been described as an antiatherogenic and cytoprotective gene in the endothelium, our aim was to investigate the effect of the active PETN metabolite pentaerithrityl trinitrate (PETriN) on HO-1 expression and catalytic activity in endothelial cells. Endothelial cells derived from human umbilical vein were incubated with PETriN (0.01–1 mM) for 8 hr. PETriN increased HO-1 mRNA and protein levels in a concentration-dependent fashion up to 3-fold over basal levels. Elevation of HO-1 protein was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of both carbon monoxide and the endogenous antioxidant, bilirubin. Pretreatment of endothelial cells with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that the active PETN metabolite, PETriN, stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein, HO-1, in endothelial cells. Increased HO-1 expression and ensuing formation of bilirubin and carbon monoxide may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.

Nitric acid esters, such as glyceryl trinitrate, were introduced into therapy more than a century ago and are still widely used for the treatment of myocardial ischemia and its main symptom, angina pectoris. The basic mechanisms responsible for the vasodilatory and anti-ischemic action of organic nitrates involve bioactivation of, and nitric oxide (NO) release from, these compounds that have therefore been termed NO donors. The intracellular target of NO (donors) is the soluble isozyme of guanylyl cyclase that, upon activation by NO, increases its synthesis rate of the second messenger cGMP (1–3).

Sustained treatment of cardiovascular diseases with organic nitrates has long been known to induce tolerance to the hemodynamic and anti-ischemic effects of these drugs in humans and animals (4, 5). Despite its first description early this century, nitrate tolerance still poses an unsolved clinical problem and is one of the main limitations to the use of nitrates. Newer studies show that prolonged exposure of rabbits to organic nitrates is associated with enhanced superoxide production in the blood vessel wall (6, 7), suggesting that oxidative stress may cause vascular desensitization to nitrates.

Pentaerithrityl tetranitrate (PETN) is a long-acting NO donor and has recently been described as an organic nitrate ester that, in contrast to other nitric acid esters, does not induce oxidative stress and is therefore free of tolerance (8, 9). The lack of tolerance under PETN therapy was recently confirmed in a human in vivo study (10). Moreover, animal experiments revealed that PETN actively reduces oxygen radical formation in vivo (11) and specifically prevents atherogenesis and endothelial dysfunction in cholesterol-fed rabbits, possibly as a consequence of its radical scavenging properties (12). On the basis of these findings, clinical interest in this drug has been substantially renewed; however, the basic mechanisms underlying its particular antioxidant profile have remained unclear. Direct quenching or neutralization of superoxide radicals by NO (13) as the sole underlying cause appears unlikely because the antioxidant activity of PETN has a delayed onset and persists even after PETN treatment has ended (11, 12). Therefore, a plausible mode of action might be the induction of genes that protect cells from damage by reactive oxygen species. Heme oxygenase (HO)-1 has recently emerged as an NO/cGMP-inducible stress gene with cytoprotective and antiatherogenic functions (14–19). Increased HO-1 expression leads to degradation of heme and accumulation of iron, bilirubin, and carbon monoxide (CO) followed by reduced sensitivity of tissues to oxidant damage (14–19). Of these metabolites, bilirubin acts as a direct antioxidant (19, 20), whereas CO may exert tissue protective actions primarily through its vasodilator and antiplatelet effects (14, 21). Our aim, therefore, was to investigate the effect of the active PETN metabolite, pentaerithrityl trinitrate (PETriN), on HO-1 expression and catalytic activity in endothelial cells and to explore the protective role of bilirubin under these conditions.

Materials and Methods

Materials.

Fetal bovine serum (FBS), cell culture media and penicillin-streptomycin were obtained from Gibco (Eggenstein, FRG). The Chemiluminescence Western Blotting Kit and antirabbit peroxidase-conjugated secondary antibody were from Boehringer Mannheim (Mannheim, FRG). PETriN was provided by Alpharma-Isis (Langenfeld, FRG). S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was purchased from Alexis Deutschland GmbH (Grünberg, FRG). Isosorbide dinitrate (ISDN) was from Schwarz Pharma (Monheim, FRG). For HO-1 probes, the template was an EcoRI restriction fragment of the human HO-1 cDNA (clone 2/10), which was kindly provided by Dr. Rex Tyrrell, School of Pharmacy and Pharmacology, University of Bath, UK (22). The polyclonal antibody against human ferritin and all other chemicals were bought from Sigma (Deisenhofen, FRG).

Cell Culture.

Human endothelial cells derived from the umbilical cord were obtained as a cell line (ECV304) from the European Collection of Cell Cultures (23). ECV304 endothelial cells were grown in M199 containing 10% FBS, streptomycin (100 μg/ml) and penicillin (100 U/ml) in a humidified incubator at 37°C (95% room air, 5% CO₂). ECV304 cells were used for all experiments except those looking at cell viability. Cell viability analysis was performed using porcine aortic endothelial cells, which are known to be particularly sensitive to damage inflicted by oxygen free radicals (24). These cells were isolated from porcine aorta and characterized as described previously (24). They were maintained and subcultured (up to passage 3) in Dulbecco’s modified Eagle medium supplemented with 15% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were grown in a humidified incubator at 37°C (95% room air, 5% CO₂).

HO-1 mRNA Analysis.

Subconfluent endothelial cells in 150-mm culture dishes were incubated for 8 hr in the presence of control media or NO donors. Total RNA was extracted using Trizol reagent (Gibco, Eggenstein, FRG). Northern blot analysis was performed as previously described (25). Briefly, samples containing equal amounts of RNA (20–30 μg) were separated in a 1% denaturing formaldehyde gel. Separated RNA was transferred onto a positively charged nylon membrane by vacuum transfer (500 mbar). The transferred RNA was fixed by baking at 120°C for 30 min. After that, the membranes were hybridized with a random primed digoxigenin-labeled human HO-1 cDNA probe (22) overnight at 50°C. The detection was conducted using the DIG detection kit from Boehringer Mannheim according to the instructions of the supplier. Equal loading was assessed by comparing the 28s ribosomal RNA bands after ethidium bromide staining of gels (26, 27).

HO-1 Protein Analysis.

Endothelial cells were cultured in 150-mm dishes as described above. After an 8-hr incubation with control media or PETriN, cells were washed and extracted as described previously (18). Protein (150 μg) was applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, protein was transferred to a nitrocellulose membrane, and a polyclonal antibody to rat HO-1 (StressGen, Victoria, BC, Canada) was used to identify HO-1 protein content. Antigen antibody complexes were visualized with the horseradish peroxidase chemiluminescence system according to the manufacturer’s instructions (Boehringer, Mannheim). Quantitation of HO-1 protein content was performed using computer-assisted videodensitometry (Eagle Eye II-system, Stratagene, La Jolla, CA).

Bilirubin Formation.

Confluent endothelial cells in 150-mm culture dishes were incubated for 8 hr in the presence of control media, PETriN, or ISDN. The method used for determination of HO activity via bilirubin formation follows the protocol published by Motterlini and co-workers (28). Briefly, after the incubation, cells were washed twice with phosphate-buffered saline, gently scraped off the dish and centrifuged (1000g, 10 min, 4°C). The cell pellet was suspended in MgCl₂ (2 mM) phosphate (100 mM) buffer (pH 7.4), frozen at −70°C, thawed three times, and finally sonicated on ice before centrifugation at 18,000g for 10 min at 4°C. The supernatant (400 μl) was added to a NADPH-generating system containing 0.8 mM NADPH, 2 mM glucose-6-phosphate, 0.2 U glucose-6-phosphate-1-dehydrogenase, and 2 mg protein of rat liver cytosol prepared from the 105,000g supernatant fraction as a source of biliverdin reductase, potassium phosphate buffer (100 mM, pH 7.4), and hemin (10 μM) in a final volume of 200 μl. The reaction was conducted for 1 hr at 37°C in the dark and terminated by addition of 1 ml of chloroform. The extracted bilirubin was calculated by the difference in absorption between 464 and 530 nm using a quartz cuvette (extinction coefficient, 40 mM⁻¹ × cm⁻¹ for bilirubin). HO activity was measured as picomoles of bilirubin formed per milligram of endothelial cell protein per hour. Basal HO activity was in a range between 200 and 600 pmol bilirubin/mg protein/hr.

CO Formation.

Confluent endothelial cells were grown in 75-cm² flasks and incubated for 8 hr with PETriN, ISDN, or vehicle. At the end of the incubation period, the culture media was aspirated and the cells were washed with phosphate-buffered saline, scraped with a rubber policeman into 10 ml of phosphate-buffered saline and centrifuged (3000g, 4°C, 10 min). Pellets were resuspended in potassium phosphate buffer (100 mM, pH 7.4) and sonicated for 15 sec. Cell debris was sedimented at 1000g for 5 min and the cytosolic fraction was used for the CO release assay. Assays were conducted in subdued lighting. Twenty-microliter aliquots of cell sonicates were reacted with 20 μl of hemin (150 μM) and 20 μl NADPH (4.5 mM) in a septum-sealed vial at 37°C. Blanks consisted of cell sonicates reacted with hemin only. Vials were purged with CO-free air and allowed to incubate for 15 min. The reaction was stopped with dry ice, and CO release into the vial headspace was analyzed by gas chromatography using a reduction gas analyzer (29).

Cell Viability Analysis.

Endothelial cells were seeded at 2 × 10⁴ cells/well in 96-well microtiter plates in 100 μl of media containing 15% FBS. After a 24-hr incubation at 37°C, cells reached confluence and were incubated for 8 hr in the presence of PETriN, ISDN, or bilirubin. Then, hydrogen peroxide was given to the cells and incubation at 37°C was continued for 24 hr, followed by a cytotoxicity assay. Cell viability was measured by staining with crystal violet as previously described (18, 30). This colorimetric test allows assessment of the remaining viable cells after the incubation procedure. Cells were washed with phosphate-buffered saline, fixed with methanol for 5 min and then stained for 10 min with a 0.1% crystal violet solution. After three washes with tap water, the dye was eluted with 0.1 M trisodium citrate in 50% ethanol for 10 min. Optical density at 630 nm was measured using a microtiter plate reader (Biotek EL 311s).

Results

In endothelial cells, an 8-hr incubation with PETriN (10–500 μM) increased HO-1 mRNA levels in a concentration-dependent fashion (Fig. 1). The NO donor SNAP served as control and likewise stimulated HO-1 mRNA expression (Fig. 1 ). HO-1 induction by PETriN also occurred at the protein level (Fig. 2 ). These effects on HO-1 expression were accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of CO (Fig. 3 ) and bilirubin (Fig. 4 ) in lysates from cells treated with PETriN. ISDN, another long-acting nitrate, was without effect under these conditions (Figs. 3 and 4 ). An 8-hr pretreatment with PETriN protected endothelial cells from hydrogen peroxide-mediated toxicity, whereas ISDN failed to exert a cytoprotective action (Fig. 5 ). Endothelial protection by PETriN was mimicked by exogenous bilirubin, which led to an almost complete reversal of hydrogen peroxide-induced toxicity (Fig. 6 ). PETriN, ISDN, or bilirubin alone had no significant effect on cell viability under these conditions.

Discussion

The organic nitrate, PETN, is known to possess antioxidant properties that are thought to be the underlying cause for its specific pharmacological profile. In contrast to other long-acting nitrates, PETN induces tolerance-free vasodilation in humans and was reported to prevent endothelial dysfunction as well as atherogenesis in cholesterol-fed rabbits (8–12, 31). However, the exact nature of the vasoprotective signaling pathways triggered by PETN has remained obscure.

The present study demonstrates that the active PETN metabolite, PETriN, stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein, HO-1, in endothelial cells. In addition, pretreatment with PETriN was followed by increased resistance of endothelial cells to oxidant injury. The capacity to protect the endothelium in vitro may translate into and explain the previously observed antiatherogenic actions of PETN in vivo (8–12, 31). PETriN is a dinitrated Phase I metabolite of PETN and a highly potent donor of NO (32). PETriN undergoes hepatic circulation, tends to accumulate, and is therefore held responsible for the sustained vasodilatory and anti-ischemic effects of PETN (32–34). The parent compound, PETN, is immediately metabolized after oral administration, not detectable in plasma and is therefore considered to merely function as a prodrug for PETriN (34). In our study, increased HO-1 expression and endothelial protection occurred at micromolar concentrations of PETRiN, which are well within the range of plasma or tissue levels that can be expected during oral therapy (34). Higher concentrations, between 0.5 and 1 mM PETriN, were required to confirm increased HO activity in cell-free assays of bilirubin and CO formation. This inevitable loss of sensitivity during the ex vivo measurement of specific enzyme activity in a broken cell system has been reported previously and is due to induced HO-1 not being fully recoverable during the complex preparation of lysate from intact cells that were pre-exposed to NO donors (18, 28).

In vivo and in vitro studies have demonstrated that induction of HO-1 causes anti-inflammatory, antiatherogenic, and cytoprotective effects (14–19). Moreover, the first human case of HO-1 deficiency, which has been reported as the result of a genetic disorder, showed severe, persistent endothelial damage and increased tissue vulnerability to oxidant injury besides growth retardation and anemia (35). Therefore, it is plausible to assume that the HO-1 induction observed in this study contributes to the specific antioxidant profile of PETN, including the lack of tolerance and prevention of atherogenesis. In agreement with this, the HO metabolite bilirubin, when added exogenously to the cells, profoundly increased cellular resistance to hydrogen peroxide toxicity with the surviving cell fraction nearing that of untreated cells (96%). This effect was seen at low micromolar concentrations of bilirubin, which are in the upper range of the reference interval for plasma levels. High-normal serum levels of bilirubin were reported to be inversely related to atherogenic risk and to provide protection against endothelial damage (35–37). Our findings lend support to the concept of bilirubin as a biologically important antioxidant (20) and to the role of HO-1 in PETriN-dependent endothelial protection. The other HO metabolite formed under the influence of PETriN, CO, has long been considered as being tissue protective solely by its anti-platelet and vasodilatory effects, the latter being of potential benefit also in antagonizing vascular tolerance (21). However, recent evidence points to direct anti-inflammatory properties of CO (38, 39), which may complement and support the cytoprotective and antioxidant actions of bilirubin. A third pathway, besides CO and bilirubin formation through which HO-1 induction leads to tissue protection, is the induction of secondary antioxidant proteins such as ferritin (15). Accordingly, ferritin has recently been characterized as NO inducible and, among the group of organic nitrates, specifically sensitive to PETN/PETriN (40).

In this study, another long-acting nitrate, ISDN, did not protect endothelial cells from oxidant damage. The absence of significant cytoprotection in the presence of ISDN was paralleled by a lack of HO-1 stimulatory capacity. ISDN had no significant effect on CO release or bilirubin formation. These observations are in agreement with previous reports demonstrating small or nondetectable amounts of NO released from ISDN in various cell types (41, 42). Interestingly, and in contrast to PETN, isosorbide nitrates are known to induce tolerance to their cardiovascular effects, presumably via oxidant stress (7, 43). Moreover, in earlier investigations aimed at assessing the antiatherogenic potential of nitrates, PETN, but not isosorbide nitrates, prevented plaque formation and endothelial dysfunction in animal models of atherosclerosis (12, 31). Thus, the ability to activate HO-1 induction and associated antioxidant pathways apparently distinguishes PETNs from other long-acting nitrates and may explain their different patterns of action in vivo.

Together, we have demonstrated that the active PETN metabolite, PETriN, stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and the ensuing formation of bilirubin and CO may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.

Figure 1.

Effect of PETriN and SNAP on HO-1 mRNA expression in endothelial cells. Incubations, mRNA isolation and Northern blot analysis were performed as described under Materials and Methods. The data shown are representative of three experiments with similar results.

Figure 2.

Effect of PETriN on HO-1 protein expression in endothelial cells. Incubations, protein isolation, and Western blot analysis were performed as described under Materials and Methods. The densitometric data (lower panel) are mean ± SEM of n = 3 separate experiments. *P < 0.05, treatment versus control (CON), one-way analysis of variance (ANOVA), and Bonferroni’s multiple comparison test. A representative Western blot analysis is shown in the upper panel.

Figure 3.

Effect of PETriN and ISDN on HO activity (measured as formation of CO) in endothelial cells. Incubations and assessment of CO formation were performed as described under Materials and Methods. All data shown are mean ± SEM of n = 3 separate experiments. *P < 0.05, treatment versus control (CON), one-way ANOVA, and Bonferroni’s multiple comparison test.

Figure 4.

Effect of PETriN and ISDN on HO activity (measured as formation of bilirubin) in endothelial cells. Incubations and assessment of bilirubin formation were performed as described under Materials and Methods. All data shown are mean ± SEM of n = 3 separate experiments. *P < 0.05, treatment versus control (CON), one-way ANOVA, and Bonferroni’s multiple comparison test.

Figure 5.

Effect of PETriN and ISDN on hydrogen peroxide-mediated toxicity in endothelial cells. Incubations were performed as described under Materials and Methods. All data shown are mean ± SEM of n = 3 separate experiments. *P < 0.05, treatment versus control (CON), one-way ANOVA, and Bonferroni’s multiple comparison test.

Figure 6.

Effect of birirubin on hydrogen peroxide-mediated toxicity in endothelial cells. Incubations were performed as described under Materials and Methods. All data shown are mean ± SEM of n = 3 separate experiments. *P < 0.05, treatment vs. control (CON), one-way ANOVA, and Bonferroni’s multiple comparison test.

Footnotes

1

To whom requests for reprints should be addressed at School of Pharmacy, Martin Luther University, Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany. E-mail:

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (Schr 298/8-3).

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