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Am J Physiol Heart Circ Physiol 283: H2363-H2370, 2002. First published August 1, 2002; doi:10.1152/ajpheart.00151.2002
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Vol. 283, Issue 6, H2363-H2370, December 2002

Nucleoside reverse transcriptase inhibitors impair endothelium-dependent relaxation by increasing superoxide

Roy L. Sutliff1, Sergey Dikalov2, Daiana Weiss2, Jeremy Parker1, Scott Raidel1, Andrea K. Racine1, Rodney Russ1, Chad P. Haase1, W. Robert Taylor2, and William Lewis1

1 Department of Pathology and Laboratory Medicine and 2 Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nucleoside reverse transcriptase inhibitors (NRTIs) have been used successfully to reduce acquired immunodeficiency syndrome mortality. However, the use of these compounds is associated with numerous tissue toxicities, including cardiomyopathy. These studies address the effects of NRTIs on vascular function. Functional assays of contraction and relaxation were performed on isolated mouse aorta segments obtained from FVB/n mice exposed to zidovudine (AZT), stavudine, or water for 35 days. AZT and stavudine treatment impaired sensitivity to endothelium-dependent relaxation by acetylcholine. Dihydroethidium staining revealed that AZT treatment was associated with an increase in superoxide levels. Pretreatment of AZT-treated vessels with tiron (1 mM), a free radical scavenger, restored endothelium-dependent relaxation in mice. In cellular preparations, electron spin resonance measurements revealed elevated superoxide in cultured endothelial cells exposed to AZT; elevation was dependent on the length of exposure. These results indicate that NRTIs impair endothelium-dependent relaxation by increasing superoxide levels and suggest that NRTI therapy contributes to cardiovascular complications in acquired immunodeficiency syndrome.

vascular; reactive oxygen species; nitric oxide; zidovudine; stavudine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THERE IS AMPLE EVIDENCE of disturbed vascular function in human immunodeficiency virus (HIV)-1-infected patients (26). HIV-1 infection is associated with vasculitis in small blood vessels (3, 34) and with aneurysms in medium or large arteries (35). Serum levels of markers of endothelial activation, including von Willebrand factor antigen, soluble thrombomodulin, and E-selectin, correlate with disease severity in patients infected with HIV-1 (44, 45). A study examining the capacity of arteries from HIV-positive patients to vasodilate established that postischemic reactive hyperemia, a measure of endothelial function, is impaired in HIV-infected patients (40). Similar impaired endothelium-dependent relaxation has been consistently demonstrated in patients with hypercholesterolemia and coronary artery disease (6, 7, 49, 53). Free radical generation is widely implicated as a major cause for impaired endothelium-dependent relaxation (for reviews see Refs. 36 and 41).

Highly active antiretroviral therapy has significantly reduced morbidity and mortality of acquired immunodeficiency syndrome (AIDS) (42). Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT, 3'-azido-2',3'-dideoxythymidine) and stavudine (2',3'-didehydro-3'-dideoxythymidine, D4T), are integral to highly active antiretroviral therapy. Mitochondrial toxicity, recognized as a common side effect of NRTIs, is associated with the development of a number of tissue-specific toxicities, which may limit the long-term use of NRTIs in AIDS treatment (1, 8, 28). Early AZT research demonstrated skeletal and cardiac mitochondrial myopathies (4, 8, 9, 19, 23). Target tissues for toxicity may differ significantly among the NRTIs on the basis of specific tissue requirements for oxidative phosphorylation, cellular uptake, or phosphorylation state (28). The nature of selective tissue effects is not well understood. The first goal of this study was to determine whether NRTI treatment impacts vascular function.

NRTI therapy has been linked to the generation of free radicals in a number of tissue types. NRTI-mediated oxidative damage has been reported in the heart, skeletal muscle, and liver (10, 22, 46, 51). Furthermore, treatment with antioxidants significantly reduced the development of NRTI-mediated skeletal and cardiac myopathies (10, 51). Generation of reactive oxygen species in vascular tissues may have significant effects on the availability of nitric oxide (NO) for endothelium-dependent relaxation and, therefore, may result in impaired endothelium-dependent relaxation (25). Thus a second goal of the present study was to examine the hypothesis that NRTI-mediated free radical generation contributes to impaired endothelium-dependent relaxation and that NRTI effects can be ameliorated by treatment with a free radical scavenger.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NRTI treatment. For this study, 5-wk-old FVB/n male mice (20-25 g; Taconic Farms, Germantown, NY) were treated with D4T or AZT for 35 days according to methodologies previously described (30). Each mouse received drinking water (vehicle) alone, water containing AZT (courtesy of Glaxo Wellcome, Research Triangle Park, NC; 0.7 mg/ml), or water containing D4T (Clinical Pharmacology Unit, Center for AIDS Research, Atlanta VA Medical Center, Decatur, GA; 0.036 mg/ml) ad libitum. Volume consumed was determined in individually housed mice, and consumption (mg · kg-1 · day-1) was calculated. Fresh water was replenished on alternate days. AZT consumption averaged 105 mg · kg-1 · day-1, and D4T consumption averaged 10.3 ± 0.6 mg · kg-1 · day-1. No mortality occurred from AZT or D4T administration. All mice gained or maintained weight over the 35-day protocol. Studies were performed according to approved Institutional Animal Care and Use Committee guidelines.

Aorta preparation. Mice were killed by CO2 asphyxiation. Aortas were rapidly excised and rinsed in cold bicarbonate-buffered physiological saline solution (PSS), and loose fat and connective tissue were removed. Aortas were maintained in PSS (118 mM NaCl, 4.73 mM KCl, 1.2 mM MgSO4, 0.025 mM EDTA, 1.2 mM KH2PO4, 2.5 mM CaCl2, 11 mM glucose, and 25 mM NaH2CO3, pH 7.4, in 95% O2-5% CO2 at 37°C) for the remainder of the experiment.

Aorta force measurements. Aorta contractile and relaxation properties were analyzed as described previously (50). Briefly, 5-mm endothelium-intact aortic rings were isometrically mounted. Concentration-isometric force curves were generated in response to the depolarizing contractile agent KCl (0-50 mM) and receptor-mediated agonist phenylephrine (PE, 0.1 nM-10 µM). Developed forces were expressed as a percentage of the maximal force generated in response to KCl or PE. Relaxation responses to acetylcholine (ACh, 1 nM-100 µM) and the NO donor sodium nitroprusside (SNP, 0.1 nM-1 µM) were examined after precontraction with 300 nM PE, which yields 80-90% maximum contraction. In a separate series of experiments, ACh relaxation was determined after 45 min of incubation with 1 mM tiron, a free radical scavenger. Percent relaxation was calculated by determining the difference between the force generated in response to PE and the force after addition of vasorelaxant. Data are expressed as percentage of force generated in response to PE.

Blood pressure. Systolic blood pressures in mice were determined using an automated tail-cuff system (17). Briefly, mice were acclimated to the procedure for seven consecutive days, and basal pressure was measured. After basal determinations, water or AZT was administered. Systolic pressure measurements were recorded three times per week over the 5-wk treatment period to maintain acclimatization to the procedure. Averaged data were plotted for the baseline and at 7-day increments. On each day of blood pressure determination, 2 sets of 10 measurements were taken. The first set of 10 was discarded to reduce variability.

In situ measurement of superoxide. Dihydroethidium (DHE) staining for superoxide was carried out as described previously (39). Aortas were harvested, placed in cold PBS, and embedded for cryosectioning in optimal cutting temperature compound (Tissue-Tek). Sections (30 µm) were cut on a microtome, and 10 µM DHE was added to the sections. A coverslip was placed over the sections, and the sections were incubated at 37°C for 30-40 min. Ethidium staining was visualized using confocal micrsocopy. Superoxide signal specificity was confirmed by incubating sections with polyethylene glycol (PEG)-conjugated superoxide dismutase (SOD, 50 U/ml) for 30 min at 37°C.

Superoxide levels. Bovine aortic endothelial cells (BAECs) were maintained as described elsewhere, and electron spin resonance (ESR) was used to measure the production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· from endothelial cells after exposure to AZT for 7 and 14 days (11-13). Briefly, cells were cultured in a 10-cm dish, washed with PBS, scraped from the dish, and suspended in 250 µl of incubation buffer containing (in mM) 5.5 glucose, 1.4 CaCl2, 0.015 diethylentriaminepentaacetic acid, 2.6 NaCl, and 5.0 KCl in sodium phosphate buffer (19.5 mM NaH2PO4 and 53.5 mM Na2HPO4, pH 7.4). Suspensions of endothelial cells were kept on ice until used. The sample containing 1 mM 1-hydroxy-3-carboxypyrrolidine (CPH) was transferred to a 100-µl capillary that was inserted in a Bruker superhigh Q microwave cavity. ESR spectra were recorded at room temperature for 11 min. The effect of AZT treatment on formation of superoxide radicals was determined by monitoring the oxidation of CPH to paramagnetic 3-carboxyproxyl (CP) using ESR spectroscopy. PEG-SOD (25 U/ml) was added to the samples to verify that the observed signal was O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. The rates of CP formation were measured from the ESR kinetics by monitoring the amplitude of the low-field component of the ESR spectrum. The concentration of CP was determined from a calibration curve for intensity of the ESR signal at various concentrations of CP nitroxide.

Statistics. Data from concentration-response curves were compared using one-way ANOVA after determination of EC50 values for individual curves. Significance was defined as P < 0.05 for all tests.

Chemicals. All chemicals were analytic grade I and were obtained from Sigma (St. Louis, MO).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of D4T and AZT treatment on aorta contractility. To determine the effects of AZT and D4T on smooth muscle contraction, isometric force development was measured in aortas from untreated, AZT-treated, and D4T-treated FVB/n mice (Fig. 1). Neither AZT nor D4T treatment significantly affected contractile sensitivity to KCl (a depolarizing agent) or PE (a receptor-dependent agonist).


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  		Fig. 1.   Contractility of aortas from mice treated with water (), zidovudine (AZT, triangle ), and stavudine (D4T, down-triangle). Aortas were isometrically mounted, and concentration-isometric force curves were generated in response to KCl (A) and phenylephrine (B). Neither AZT nor D4T treatment significantly affected sensitivity of aortas to KCl or phenylephrine. Values are means ± SE (n = 6).

Effects of NRTIs on endothelium-dependent relaxation. To assess NRTI effects on endothelium-dependent relaxation, isolated mouse aortas were stimulated with 3 µM PE, which approximates the ED80, and then treated with ACh. Concentration-relaxation relationships indicate that AZT and D4T treatment decreased ACh sensitivity of aortas. EC50 values are presented in Table 1. AZT and D4T treatment also decreased the maximum relaxation produced by ACh (Table 1). AZT treatment also caused a significant decrease in the maximal percent relaxation to the endothelium-dependent vasorelaxant vasoactive intestinal peptide. Maximal percent relaxation values were 54.6 ± 3.6 and 34.4 ± 3.5 for aortas from water- and AZT-treated mice, respectively.

                              
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  		Table 1.   EC50 and maximum relaxation for ACh and SNP

SNP-mediated relaxation. Recent studies established that NO mediates ACh relaxation in mouse aorta (5, 33). To determine whether differences in ACh-mediated relaxation were attributable to differential responses of the vascular smooth muscle to NO, concentration-relaxation relationships were generated in response to the NO donor SNP (Fig. 2B, Table 1). Neither D4T nor AZT treatment significantly affected SNP-mediated relaxation.


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  		Fig. 2.   Acetylcholine (A) and sodium nitroprusside (B) concentration-relaxation relationship of aortas from mice treated with water (), AZT (triangle ), and D4T (down-triangle). Aortas were precontracted with 300 nM phenylephrine and exposed to increasing concentrations of acetylcholine or sodium nitroprusside. Nucleoside reverse transcriptase inhibitor (NRTI) treatment reduced endothelium-dependent relaxation but not endothelium-independent relaxation of mouse aorta. Statistical comparisons are summarized in Table 1. Values are means ± SE (n = 6).

DHE staining. DHE is converted to ethidium bromide in the presence of superoxide. Ethidium bromide intercalates into DNA and has been used as an indirect measure of superoxide generation (39). Figure 3 shows DHE staining in representative untreated and AZT-treated aortas (n = 3). Significantly more DHE staining was present in the endothelium and the smooth muscle layers of aorta from AZT-treated mice. Consistent with previous studies (38, 48), pretreatment with membrane-permeable PEG-conjugated SOD eliminated DHE staining (data not shown).


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  		Fig. 3.   Detection of superoxide in aortas from mice treated with vehicle or AZT. Fluorescent photomicrographs [magnification ×10 (A and B) and ×20 (C and D)] show aortas labeled with dihydroethidium (DHE) using confocal microscopy. Superoxide staining was considerably less intense in aortas from mice treated with vehicle (A and C) than in aortas from mice treated with AZT (B and D). E and F: vessels stained with hematoxylin and eosin (magnification ×5).

Tiron pretreatment. Free radicals, such as superoxide, cause the development of endothelial dysfunction in several murine models (20, 47). To confirm that the effects of AZT on endothelium-dependent relaxation were due to the production of free radicals, ACh relaxation relations were generated in aortas from AZT- and water-treated mice pretreated with the free radical scavenger tiron (1 mM) for 45 min.

Similar to the study described above, aortas from mice exposed to AZT were less sensitive than controls to ACh-mediated relaxation (EC50 = 99.4 ± 4.5 and 22.3 ± 1.7 nM for AZT-treated and untreated aortas, respectively, P < 0.001). Tiron pretreatment did not significantly affect ACh sensitivity of untreated vessels. Importantly, tiron pretreatment restored the EC50 of AZT-treated aortas to levels observed in untreated vessels (27.4 + 5.5 nM; Fig. 4).


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  		Fig. 4.   Acetylcholine concentration-relaxation relationship in aortas from water- and AZT-treated mice in the absence and presence of tiron. Isometrically mounted water- and AZT-treated aortas were pretreated with the free radical scavenger tiron for 45 min and contracted with 300 nM phenylephrine. Concentration-relaxation relationships for acetylcholine were generated. Tiron pretreatment abrogated effects of AZT treatment on acetylcholine relaxation. Values are means ± SE (n = 6).

Blood pressure. To determine whether AZT alters arterial tone in vivo, systolic blood pressure was measured in mice before and after AZT treatment. Despite the observed effects of AZT on endothelium-dependent relaxation ex vivo, concomitant alterations in systolic blood pressure were absent (Fig. 5).


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  		Fig. 5.   Systolic blood pressure during 5 wk of AZT treatment. Animals were acclimated to an automated tail-cuff system for 1 wk, and baseline systolic pressure was determined for 3 consecutive days. AZT treatment (200 mg · kg-1 · day-1) was initiated, and blood pressure was monitored 3 times per week during treatment. Averaged data are plotted for baseline and at 7-day increments. Values are means ± SE (n = 8).

Superoxide after AZT treatment of BAECs. The effects of AZT treatment on formation of superoxide radicals were determined by monitoring the oxidation of CPH using ESR spectroscopy. BAECs were treated with 1 µM AZT for 7 and 14 days, and PEG-conjugated SOD-inhibitable superoxide production was determined (Fig. 6). After 7 days, no effect of AZT on superoxide production was observed. However, after 14 days of AZT treatment, superoxide production was increased by >30% relative to control BAECs.


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  		Fig. 6.   Superoxide production after exposure of bovine aortic endothelial cells (BAECs) to 1 µM AZT for 7 and 14 days. A: representative traces of electron spin resonance (ESR) time scans of superoxide dismutase-inhibitable 3-carboxyproxyl formation in control BAECs and BAECs exposed to AZT for 14 days. Inset: ESR spectrum of 3-carboxyproxyl nitroxide depicting position of time scan. B: summary of superoxide production in control BAECs and BAECs treated with AZT for 7 and 14 days. Values are means ± SE (n = 4). *P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that D4T and AZT profoundly impair endothelium-dependent relaxation in murine aortas. Increased superoxide levels mediate these adverse functional effects of D4T and AZT. Arterial smooth muscle cell function is not affected by D4T or AZT treatment, because the contractile response to KCl and PE is unchanged. Relaxation responses to SNP are also unaffected, indicating that the smooth muscle component of cGMP pathways is unchanged. Surprisingly, although no functional evidence suggests smooth muscle toxicity, DHE staining indicates that superoxide production is increased in medial smooth muscle. These apparent paradoxical results agree with a number of studies demonstrating normal relaxation to SNP in the presence of increased superoxide (14, 15, 21). Mechanisms for the dissociation between elevated superoxide and unaffected relaxation to SNP remain unclear.

Studies utilizing pharmacological approaches and NO synthase-deficient mice have clearly demonstrated that ACh relaxes mouse aortas by activating endothelial NO synthase (5, 24, 33). AZT and D4T markedly attenuate sensitivity and maximal relaxation elicited by ACh, indicating that NO-mediated relaxation is impaired after NRTI treatment. Additionally, this effect is not limited to the muscarinic agonist ACh, because endothelium-dependent relaxation to vasoactive intestinal peptide is also impaired after AZT treatment. Smooth muscle responses to NO were unaffected by NRTI treatment, as demonstrated by normal relaxation responses to the NO donor SNP.

Numerous studies have linked NRTI toxicity to increased free radical levels (10, 46, 51). Our results clearly demonstrate increased superoxide levels after exposure to AZT in situ, in vitro, and ex vivo. In situ results with DHE staining demonstrate that AZT treatment results in increased superoxide in aortas. Exposure of BAECs to 1 µM AZT increased the SOD-inhibitable superoxide. Furthermore, pretreatment of aortas from AZT-treated mice with tiron, a superoxide scavenger, restores endothelium-dependent relaxation. These results indicate that AZT-mediated effects on superoxide result in altered endothelium-dependent relaxation.

The effects of AZT on superoxide appear to depend on length of exposure to AZT. Treatment of BAECs with 1 µM AZT for 7 days had no effect on SOD-inhibitable superoxide production. Treatment of cells with 1 µM AZT for 14 days, on the other hand, resulted in increased superoxide production. Furthermore, exposure of isolated aortas in organ baths to 1 µM AZT for 1 h had no effect on endothelium-dependent relaxation (data not shown). Therefore, it appears that the effects of AZT on superoxide- and endothelium-dependent relaxation require prolonged exposure, as has been described for other tissue toxicities (8, 10, 27, 30, 31).

Mechanisms underlying NRTI-mediated endothelial dysfunction are poorly understood. Similarly, the potential role of mitochondrial toxicity in the development of altered endothelium-dependent relaxation remains to be clarified if analogies to other tissue effects are to be made. Mitochondrial dysfunction is thought to be the result of two interrelated pathophysiological mechanisms that result from defective oxidative phosphorylation: 1) increased production of mitochondrial reactive oxygen species and 2) reduced energy production (16, 27). Both have been reported after NRTI therapy and are associated with the development of NRTI-mediated cardiomyopathy. Furthermore, the production of free radicals has been implicated in mitochondrial DNA damage and dysfunction in endothelial cells (2).

Clinical implications of these findings are intriguing but not entirely clear. As reported elsewhere, doses of AZT consumed by mice in this study were higher on a milligram per kilogram per day basis than doses used in humans. Reductions in ACh sensitivity occurred in a concentration-dependent manner at 100 mg · kg-1 · day-1 AZT and 10 mg · kg-1 · day-1 D4T. These doses are higher than those employed in clinical use on a milligram per kilogram per day basis. Nonetheless, higher doses of AZT were used in these experiments on the basis of experience with this system and the fact that the plasma half-life of AZT in mice is five times lower than that observed in humans (52). AZT doses used in this study resemble those of other studies in rodent species including mice (30, 43) and rats (29, 32, 37). The doses of AZT resembled those for which toxic effects could easily be determined in as little as 35 days of treatment. Similar and lower doses have been reported to result in NRTI-mediated cardiotoxicity (18, 30, 37). Recent studies suggest that doses of ~50 mg mg · kg-1 · day-1 result in structural alterations in mitochondria in the same amount of time.

In summary, the present study demonstrates that NRTI treatment impairs endothelium-dependent relaxation by increasing superoxide. These endothelial effects of NRTIs may become increasingly important as the cardiovascular effects of AIDS are explored further and as NRTI therapy continues.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-59798 (to W. Lewis), the University Research Committee of Emory University, and American Heart Association Grant 003024N (to R. L. Sutliff).


    FOOTNOTES

Address for reprint requests and other correspondence: R. L. Sutliff, Dept. of Pathology, Emory University, 1639 Pierce Dr., Rm. 7120 WMRB, Atlanta, GA 30322 (E-mail: rsutlif{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 1, 2002;10.1152/ajpheart.00151.2002

Received 16 July 2002; accepted in final form 24 July 2002.


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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 283(6):H2363-H2370
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