Heme Oxygenase-

【摘要】  Objective- Nitrate tolerance is likely attributable to an increased production of reactive oxygen species (ROS) leading to an inhibition of the mitochondrial aldehyde dehydrogenase (ALDH-2), representing the nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN) bioactivating enzyme, and to impaired nitric oxide bioactivity and signaling. We tested whether differences in their capacity to induce heme oxygenase-1 (HO-1) might explain why PETN and not GTN therapy is devoid of nitrate and cross-tolerance.

Methods and Results- Wistar rats were treated with PETN or GTN (10.5 or 6.6 µg/kg/min for 4 days). In contrast to GTN, PETN did not induce nitrate tolerance or cross-tolerance as assessed by isometric tension recordings in isolated aortic rings. Vascular protein and mRNA expression of HO-1 and ferritin were increased in response to PETN but not GTN. In contrast to GTN therapy, NO signaling, ROS formation, and the activity of ALDH-2 (as assessed by an high-performance liquid chromatography-based method) were not significantly influenced by PETN. Inhibition of HO-1 expression by apigenin induced "tolerance" to PETN whereas HO-1 gene induction by hemin prevented tolerance in GTN treated rats.

Conclusions- HO-1 expression and activity appear to play a key role in the development of nitrate tolerance and might represent an intrinsic antioxidative mechanism of therapeutic interest.

Tolerance to nitroglycerin is likely attributable to an increased production of reactive oxygen species and an inhibition of its bioactivating enzyme. Pentaerythrityl tetranitrate (PETN) therapy is devoid of tolerance, potentially because of HO-1 induction. Inhibition of HO-1 expression induced "tolerance" to PETN, whereas HO-1 induction prevented tolerance in nitroglycerin-treated rats.

【关键词】  organic nitrates nitrate tolerance heme oxygenase reactive oxygen species

Introduction

When given acutely, organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) have potent antiischemic effects. 1 Their clinical usefulness during long-term treatment, however, is limited because of the development of tolerance and cross-tolerance to endothelium-dependent and independent vasodilators. 2 The phenomenon of nitrate tolerance was observed first in response to treatment with GTN, 3 but seems also to be shared by other organic nitrates such as isosorbide dinitrate (ISDN) 4 and mononitrate (ISMN), but interestingly not by pentaerithrityl tetranitrate (PETN). 5,6 See page 1673

Recently, a novel bioactivation pathway for GTN was reported, and the mitochondrial aldehyde dehydrogenase (ALDH-2) was identified as the bioactivating enzyme. 7 The link between GTN-induced oxidative stress and the development of nitrate tolerance was established by the demonstration of increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production within mitochondria, 8 which inactivate the ALDH-2 by oxidizing sulfhydryl (SH)-groups in the active center of the enzyme. 9

Further evidence for an important role of mitochondria in the development of nitrate tolerance was provided in studies with mice with partial manganese superoxide dismutase deficiency (Mn-SOD +/- mice). With these studies we could demonstrate that Mn-SOD +/- mice had a significantly higher susceptibility for the development of nitrate tolerance compared with wild-type mice. 10 Further support for a causal involvement of mitochondrial ROS in nitrate tolerance came from experiments where mitochondria-targeted antioxidants were able to prevent the development of tolerance. 11

Recently, we were able to demonstrate that PETN bioactivation is also mediated by the ALDH-2 and therefore that this organic nitrate shares the same biotransformation process like GTN. 12,13 When isolated aortic rings were pretreated with high doses of PETN or GTN to induce in vitro tolerance and subsequently challenged with low concentrations of PETN, the loss of vasodilator responsiveness (tolerance) was much more pronounced in GTN-pretreated vessels, indicating that GTN induced a marked degree of cross-tolerance to PETN. This finding is consistent with several animal 14,15 and human studies 5,6,16 showing that treatment with PETN failed to induce tolerance.

The development of nitrate tolerance has been demonstrated to be associated with increased vascular production of ROS. 17 Accordingly, cotreatment with antioxidants such as vitamin C 18 as well as folate 19 or substances that reduce oxidative stress indirectly such as ACE-inhibitors 20 were able to prevent the development of the nitrate tolerance.

In vitro studies with isolated cells have shown that PETN treatment upregulates the expression of heme oxygenase-1 (HO-1) and subsequently ferritin, which have been described to possess strong antioxidative properties. 21,22 Whether this may also occur in response to in vivo treatment with PETN remains to be established.

Based on these findings we sought to determine whether or not in vivo treatment with PETN will cause tolerance to PETN and cross-tolerance to GTN and the endothelium-dependent vasodilator acetylcholine (ACh), respectively, and whether a lack of induction of tolerance and endothelial dysfunction may be linked to stimulatory effects of this compound on HO-1. Furthermore, we wanted to clarify whether the modulation of HO-1 in general by inducers or inhibitors may lead to an aggravation or inhibition of tolerance in response to 4-day treatment with GTN or PETN.

Materials and Methods

Male Wistar rats (Charles River, Sulzfeld, Germany) were infused for 4 days with either GTN (6.6 µg/kg/min in ethanol or the solvent as a control) or PETN (10.5 µg/kg/min in DMSO or the solvent as a control). PETN and its metabolites were detected in whole blood using a high-performance liquid chromatography (HPLC)-based method with chemiluminescent nitrogen detection (CLND). Hemodynamic effects (blood pressure) were assessed telemetrically in freely moving animals. Endothelial and smooth muscle function were measured in organ chambers by isometric tension experiments-the vasodilation in response to endothelium-dependent acetylcholine or -independent GTN. Vascular NO/cGMP signaling was determined by phosphorylation of VASP (P-VASP on Western blot), protein expression by SDS-PAGE followed by Western blotting. Vascular and cardiac mRNA was assessed by real-time polymerase chain reaction (PCR). HO-1 activity was measured by serum bilirubin levels. Vascular and cardiac oxidative stress was determined using L-012 enhanced chemiluminescence. ALDH activity was measured by conversion of a benzaldehyde derivative to its benzoic acid product using an HPLC-based assay. Key experiments consisted of the induction of HO-1 by hemin (1 x 25 mg/kg i.p.) and suppression by apigenin (10 mg/kg/d) in GTN- or PETN-treated rats as well as measurement of the above described parameters in these animals. For detailed protocols please the supplemental materials, available online at http://atvb.ahajournals.org.

Results

PETN Metabolites in Whole Blood

The successful administration of PETN was verified by the levels of its metabolites in whole blood. The results are summarized in supplemental Figure I and supplemental Table I.

Hemodynamic Responses

On the first day of therapy, MAP was significantly decreased in both groups (GTN and PETN) ( P <0.05). After 6 days of continuous treatment, MAP was still decreased in PETN-treated animals, whereas in the GTN-group MAP was even higher than baseline compatible with hemodynamic tolerance to GTN, but not PETN ( Figure 1 A).

Figure 1. Mean arterial blood pressure (MAP, A), vascular NO/cGMP signaling (P-VASP, B), and endothelial/smooth muscle function (C and D) after in vivo treatment with PETN and GTN, respectively. A, MAP was measured telemetrically before (set to zero), 1 day and 6 days after nitrate treatment. Data are presented as mean±SEM obtained from 6 rats in a cross-over study. * P <0.05 vs first day GTN in vivo. B, The effects of nitrate treatment on P-VASP (phosphorylated VAsodilator Stimulated Phosphoprotein at serine 239) levels were measured by Western blotting. C and D, Vascular function was assessed by isometric tension experiments, and dose-response relationships of isolated rat aortic vessel segments were established for acute challenges with PETN, ACh, and GTN. Importantly, in vivo PETN treatment did not cause tolerance or cross-tolerance to GTN and ACh, respectively. Data are mean±SEM of n=8-20 (tension studies) and 18-27 (P-VASP) independent experiments. * P <0.05 vs solvent control.

As shown in Figure 1C through 1 E, PETN caused neither tolerance nor cross-tolerance to GTN and ACh. In contrast, GTN in vivo treatment caused tolerance to GTN, cross-tolerance to PETN, and endothelial dysfunction ( Figure 1C through 1 E and supplemental Table II). One should also note that the trinitrate metabolite of PETN, pentaerithrityl trinitrate (PETriN), showed similar vasodilator potency as compared with GTN, but did not induce tolerance or cross-tolerance (supplemental Figure V). This protective effect of PETN and PETriN was also not mimicked by NO-independent vasodilators of the Ca-antagonist group, amlodipine and nifedipine (supplemental Figure IX). Interestingly, GTN in vivo treatment induced no cross-tolerance to ISMN but mild cross-tolerance to ISDN (supplemental Table III).

PETN Treatment Increases and GTN Treatment Decreases the Activity of the cGMP-Dependent Kinase

Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at serine 239 reflects cGMP-dependent protein kinase I (cGK-I) activity and accordingly vascular NO bioavailability. P-VASP levels in aortic tissue from PETN-treated animals were significantly higher compared with controls ( Figure 1 B), whereas GTN treatment lead to a marked decrease in P-VASP levels as before 23 ( Figure 1 B).

Mitochondrial and Vascular Reactive Oxygen and Nitrogen Species Formation

In vivo treatment with PETN did not stimulate ROS (quantified by chemiluminescence) formation in isolated heart mitochondria nor in intact aortic vessel segments ( Table ). In contrast, as shown before, 24 in vivo treatment with GTN significantly increased mitochondrial and vascular ROS formation ( Table ).

Effect of PETN or GTN In Vivo Treatment on ROS Formation, ALDH, and Heme Oxygenase Activities

ALDH-2 Dehydrogenase and Esterase Activity

Mitochondrial ALDH-2 and total vascular ALDH dehydrogenase activity, as well as esterase activity, were not modified by in vivo PETN treatment ( Table ). In contrast, in vivo GTN therapy decreased mitochondrial ALDH-2 activity, vascular ALDH, as well as esterase activity ( Table ).

Effects of In Vivo Treatment With GTN or PETN on Expression of ALDH-2, HO-1, Endothelial NO Synthase (eNOS), Soluble Guanylyl Cyclase (sGC), and HO-1 Activity

The expression of the GTN and PETN bioactivating enzyme ALDH-2 was significantly decreased in vessels from in vivo GTN-treated rats, but not in vessels from PETN-treated animals ( Figure 2 A). In addition, cardiac ALDH-2 protein expression was decreased by GTN (supplemental Figure VI). PETN but not GTN increased HO-1 expression in the aorta ( Figure 2 A). Cardiac HO-1 protein expression was significantly decreased in GTN-treated rats (supplemental Figure VI). eNOS expression was increased in response to treatment with both organic nitrates ( Figure 2 B). The expression of the sGC subunit ß 1 was increased by GTN and not by PETN treatment ( Figure 2 B). PETN infusion increased plasma bilirubin levels compatible with increased HO-1 activity. In contrast, GTN treatment decreased bilirubin levels ( Table ).

Figure 2. Protein expression of ALDH-2 and HO-1 (A) as well as eNOS and sGCß 1 (B) in isolated aortic vessel segments of rats upon treatment with PETN or GTN. A, ALDH-2 expression decreased significantly by GTN but not by PETN treatment. HO-1 expression was not changed in response to GTN, but significantly increased in response to PETN in vivo treatment. B, eNOS expression was increased significantly in response to both organic nitrates whereas sGC expression was upregulated solely in GTN-treated rats. Below the densitometric data the representative original blots are shown. Data are mean±SEM of n=4-6 (ALDH-2), 15-28 (HO-1), 12-16 (eNOS), and 15-23 (sGC) independent experiments. * P <0.05 vs solvent control.

HO-1 and Ferritin mRNA Expression

HO-1 mRNA expression was slightly but significantly increased by GTN treatment (supplemental Figure II), whereas in vivo PETN administration markedly upregulated HO-1 mRNA levels in accordance with the protein expression data (supplemental Figure II). A similar observation was made for mRNA levels of ferritin. GTN in vivo increased ferritin expression significantly in the heart, whereas no change was observed in the aorta (supplemental Figure II). In response to PETN treatment, the increase in ferritin expression was markedly stronger (supplemental Figure II).

Induction and Inhibition of HO-1 Protein Expression by Hemin and Apigenin

Single i.p. injection of GTN-treated rats with the HO-1 inducer hemin increased vascular HO-1 mRNA expression and protein ( Figure 3C and 3 D). Subsequently, tolerance was prevented and the cGK-I activity was increased ( Figure 3A and 3 B). In addition, oxidative stress within mitochondria was reduced and ALDH-2 activity improved significantly ( Figure 3E and 3 F). Coinfusion of PETN-treated rats with the inhibitor of HO-1 expression apigenin induced a "tolerance-like" state (supplemental Figure IIIa), indicating that PETN-treated rats developed tolerance in the absence of the protective effects of HO-1. Accordingly, apigenin cotreatment increased mitochondrial ROS formation (supplemental Figure IIIb). The decrease of mitochondrial oxidative stress by HO-1 induction by hemin and the increase of ROS formation induced by HO-1 suppression with apigenin could be attributed at least in part to increased or decreased bilirubin levels. Indeed, we could demonstrate the potent antioxidant properties of bilirubin, by demonstrating a concentration-dependent inhibitory effect on mitochondrial ROS formation in isolated mitochondria from rats treated in vivo with GTN (supplemental Figure IV). Finally, the HO-1 inducer hemin significantly stimulated the expression of eNOS protein in aortas from GTN-treated rats, whereas apigenin, an HO-1 inhibitor, had no effect on eNOS protein levels in PETN-treated rats at all (supplemental Figure VII).

Figure 3. Effect of coadministration of the HO-1 inducer hemin on GTN-induced tolerance. Hemin (25 mg/kg) was administrated by single i.p. injection on day 3 of GTN treatment. Hemin cotreatment markedly improved vascular GTN responsiveness as demonstrated by isometric tension studies (A) and increased the activity of the cGK-I as indicated by increased vascular P-VASP formation (B). Hemin treatment lead to a significant increase in HO-1 protein levels in aorta (C), HO-1 mRNA expression in heart (D), to a significant decrease in mitochondrial ROS formation (E) and improvement of mitochondrial ALDH-2 activity (F). Data are mean±SEM of n=8-12 (A), 5 (B), 4 (C), 5 (D), 40 (E), and 6-18 (F) independent experiments. In panels B and C, 2 original blots are shown for each group. * P <0.05 vs GTN treatment.

Discussion

The results of the present study demonstrate that 2 different organic nitrates such as GTN and PETN, both being bioactivated by the enzyme ALDH-2, have marked differing effects on the development of tolerance, oxidative stress, ALDH-2 expression, and desensitization to the endothelium-dependent vasodilator ACh (so-called cross-tolerance). PETN and GTN treatment upregulated eNOS protein while GTN in addition upregulated sGC expression. The vascular levels of phosphorylated VASP, however, indicated that cGK-I activity was increased solely in response to long-term PETN treatment. Upregulation of HO-1 by hemin prevented tolerance to GTN whereas inhibition of HO-1 activity by apigenin induced a "tolerance-like" state in PETN-treated rats, pointing to a crucial role of this enzyme in the modulation of the degree of tolerance in response to the use of organic nitrates. This is the first description of PETN effects on these parameters in rats whereas GTN was used for in-study comparison. The suppression of GTN-induced tolerance by HO-1 upregulation is a new observation.

As we have demonstrated recently, the development of tolerance in response to organic nitrates correlates strongly with their capacity to stimulate mitochondrial ROS/RNS formation and to inhibit ALDH-2 activity. 12 Importantly, GTN, PETN, and its metabolite PETriN are bioactivated by ALDH-2, whereas ISDN, ISMN, pentaerithrityl dinitrate (PEDN), and pentaerithrityl mononitrate (PEMN) are likely to be bioactivated by other pathways, such as cytochrome P450 systems. 13,25 Interestingly, in contrast to GTN, PETN induces markedly less tolerance "in vitro", mitochondrial ROS/RNS formation, and inhibition of ALDH-2 activity, when being challenged acutely with high concentrations of this compound, 12 suggesting that "antioxidant" properties may come into play. 26

Indeed, in contrast to GTN, PETN treatment did not stimulate vascular ROS production ( Table ) and in addition did not inhibit the activity of the bioactivating enzyme ALDH-2 ( Table ). Likewise, GTN but not PETN treatment resulted in a significant downregulation of this nitrate reductase ( Figure 2 A). This explains, as shown before, why treatment with GTN induces cross-tolerance to PETN, whereas PETN treatment does not desensitize the vasculature to GTN. 12 These findings also indicate that, in addition to an inhibition of the activity of the ALDH-2, 8 a downregulation of the bioactivating enzyme also contributes to the tolerance phenomenon in response to chronic GTN treatment.

One candidate responsible for the antioxidant properties of PETN is the HO-1. Previous in vitro studies have indicated that incubation of cultured endothelial cells with PETN upregulates HO-1 and subsequently increases intracellular bilirubin, ferritin, and carbon monoxide levels, all of which may explain at least in part the antioxidative effects of this organic nitrate. 22 To address whether this may occur also in response to in vivo treatment with organic nitrates, we treated Wistar rats with GTN and PETN, respectively. In the present study, male Wistar rats were treated for 4 days with PETN using the infusion method which was adopted from the previously established protocol in our laboratory to induce tolerance in Wistar rats via GTN infusion (6.6 µg/kg/min). 8,24 The infusion rate of PETN (10.5 µg/kg/min) was similar to that of GTN.

Treatment of Wistar rats with both organic nitrates resulted in marked differences with respect to their effects on the antioxidant enzyme HO-1. Whereas GTN slightly modified HO-1 expression at the mRNA and protein level, a marked increase was established in response to PETN treatment in the vascular tissue, but also in the heart ( Figure 2 A and supplemental Figure II). HO-1 is the rate limiting enzyme in heme degradation to generate equimolar amounts of biliverdin, ferrous free iron, and carbon monoxide. Subsequently biliverdin is converted to bilirubin by the biliverdin reductase, and free iron is sequestered by ferritin. Thus, the upregulation of HO-1 was, as expected, paralleled by an increase in the expression of ferritin (supplemental Figure II). Indirect evidence for an upregulation of this antioxidant system was provided by the observed increase in plasma bilirubin levels in response to PETN but not GTN, reflecting increased HO-1 activity ( Table ).

The data summarized in supplemental Figure IV clearly indicate that bilirubin is a powerful inhibitor of ROS formation in mitochondria of animals treated in vivo with GTN. Bilirubin is more efficient than vitamin E in preventing lipid peroxidation in vitro. 27 In addition, higher serum levels are inversely related to the incidence of coronary artery disease. 28 Bilirubin prevents the activation of the vascular NADPH oxidase 29 and inhibits protein kinase C activity, 30 mechanisms that have been proposed to be mechanistically involved in the development of nitrate tolerance. 31,32

The induction of ferritin expression has been shown to provide marked antioxidant cellular protection by rapidly sequestering free cytosolic iron, the crucial catalyst of oxygen-centered radical formation via the Fenton reaction in biological systems. 33 Furthermore, the increase in expression of intracellular ferritin has been shown to reduce the cytotoxic effects of hydrogen peroxide in vascular endothelial cells. 34 Thus, it is conceivable to conclude that, although the metabolism of both PETN and GTN lead to increased ROS/RNS production, 12 the consequences for the metabolism of PETN can be neglected because of its simultaneous stimulatory effects on the activity and expression of HO-1, ferritin, and increased bilirubin levels.

Next we studied the effects of treatment with organic nitrates on the expression of the eNOS and the sGC and the activity of the cGK-I. As shown before, GTN treatment upregulated eNOS and sGC expression associated, however, with a reduction of the vascular levels of phosphorylated VASP compatible with an inhibition of cGK-I activity ( Figures 2B and 1 B). In contrast, PETN treatment upregulated the expression of eNOS, which was paralleled by increase in P-VASP levels, which nicely fits with our observation that in contrast to GTN, PETN treatment beneficially influences NO, sGC, cGK-I signaling and therefore does not induce endothelial dysfunction ( Figure 1 C and supplemental Table II). However, one should note that PETN-dependent HO-1 induction leads also to increased CO formation, which itself, although it is a weak activator of sGC, could, at least in part, be responsible for the increased P-VASP formation in the PETN treated group.

The proof of concept was provided by experiments using the HO-1 inducer hemin in GTN-treated animals and the suppressor of HO-1, apigenin, in PETN-treated rats, respectively. Hemin treatment increased HO-1 expression and simultaneously prevented the development of GTN tolerance, reduced mitochondrial ROS formation, and increased mitochondrial ALDH-2 activity ( Figure 3 ). In contrast, the suppressor of HO-1 apigenin was able to significantly reduce vascular responsiveness to PETN and simultaneously increased mitochondrial ROS formation (supplemental Figure III).

We here provide an explanation why in vivo treatment with PETN is devoid of tolerance and cross-tolerance induction in response to prolonged in vivo treatment. In contrast to GTN, PETN does not increase vascular oxidative stress and therefore does not interfere with its bioactivation by ALDH-2 (supplemental Figure X). A likely explanation for this beneficial property of PETN is the induction of the antioxidant enzyme HO-1 and subsequent increases in the expression of ferritin in vascular tissue but also in the heart. This favorable characteristics of PETN may also explain why therapy with GTN but not PETN causes tolerance and stimulates ROS production in human subjects. 5

Future Implications

The exciting new observation is the capacity of inducers or inhibitors of HO-1 to modulate the development of tolerance and endothelial dysfunction in response to organic nitrates. The powerful inhibitory effects, eg, of the HO-1 inducer hemin on the development of tolerance in response to GTN may help to develop new agents, allowing to treat patients with GTN chronically without side effects like tolerance and endothelial dysfunction. The pharmacological profile of PETN, increasing vascular NO and decreasing superoxide levels, markedly resembles that of statins and ACE inhibitors. 35 It remains to be established, however, whether PETN may indeed represent the first organic nitrate, which will be able to beneficially influence prognosis in patients with coronary artery disease.

Acknowledgments

The expert technical assistance of Jörg Schreiner and Claudia Kuper is gratefully acknowledged. This article contains results that are part of the doctoral thesis of Meike Coldewey.

Sources of Funding

The present work was supported by a vascular biology grant from Actavis Deutschland GmbH (T.M.) as well as continuous funding by the German Research Foundation (DFG) (SFB 553-C17 to A.D. and T.M. and SFB 553-A1 to H.L. and U.F.).

Disclosures

D.S. is employee of Actavis Deutschland GmbH. T.M. received a vascular research grant from Actavis Deutschland GmbH.

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作者单位：Philip Wenzel; Matthias Oelze; Meike Coldewey; Marcus Hortmann; Andreas Seeling; Ulrich Hink; Hanke Mollnau; Dirk Stalleicken; Henry Weiner; Jochen Lehmann; Huige Li; Ulrich Förstermann; Thomas Münzel; Andreas DaiberFrom the II. Medizinische Klinik (P.W., M.O., M.C., U.H., H.M., T.M., A.D.