CyA and OxLDL cause endothelial dysfunction in isolated arteries through endothelin-mediated stimulation of O2- formation

Jan Galle, Cordula Lehmann-Bodem, Ullrich Hübner, Alexandra Heinloth and Christoph Wanner

Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany

Correspondence and offprint requests to: Dr Jan Galle, Department of Medicine, Division of Nephrology, University Hospital Würzburg, Joseph-Schneider-Str. 2, D-97080 Würzburg, Germany.



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
Background. Cyclosporin A (CyA) and oxidized low-density lipoprotein (OxLDL) cause endothelial dysfunction, partly through stimulation of O2- formation (which can inactivate nitric oxide). We investigated whether CyA and OxLDL potentiate their influence on oxidative stress, whether endothelin (ET) is a mediator of CyA- and OxLDL-induced O2- formation, and whether enhanced oxidative stress results in further attenuation of endothelium-dependent vasodilation.

Methods and results. Human LDL was oxidized by Cu++. O2- formation of isolated rat aortic rings was measured using a chemiluminescence assay. Incubation (60 min) of aortic rings with CyA (10 ng–10 µg/ml) or with OxLDL (300 µg/ml) caused a significant, dose-dependent increase of the basal O2- formation. Pretreatment of the aortic rings with CyA (10 ng/ml) further enhanced the OxLDL-induced O2- formation by factor 1.9. The enhancement of the OxLDL-induced stimulation of O2- formation by CyA could be completely blocked by BQ123, a selective endothelin-1 (ET-1) receptor antagonist. Likewise, exogenously applied ET-1 (1 nM) potentiated the OxLDL-induced O2- formation by factor 1.8. Endothelium-dependent dilation was measured in isolated rings of rabbit aorta superfused with physiological salt solution in an organ bath. Incubation of the aortic rings with CyA (10 µg/ml, 60 min) or with OxLDL (300 µg/ml, 60 min) alone did not attenuate endothelium-dependent dilations. However, coincubation of the aortic rings with CyA+OxLDL in the presence of diethyl-dithio-carbamate, an inhibitor of the endogenous superoxide dismutase, caused a 60% inhibition of acetylcholine-induced dilator responses.

Conclusions. Coincubation of isolated aortic rings with CyA and OxLDL causes a potent enhancement of vascular O2- formation. ET-1 seems to be mediator of the CyA-induced O2- formation. Enhanced oxidative stress results in further attenuation of endothelium dependent vasodilation.

Keywords: atherosclerosis; endothelin; endothelium; oxidative stress; superoxide radical; transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
Cyclosporin (CyA) is a frequently used medication in patients undergoing organ transplantation or suffering from diseases which require immune suppression [1]. One important side effect of CyA treatment is systemic hypertension. CyA-induced arterial hypertension develops independently from the original disease and is likely to be caused by various mechanisms, including enhanced vascular constrictor responses to different agonists [2,3], release of endothelial constricting factors [4], and impaired release of endothelium-derived nitric oxide (NO) and dilator prostaglandins [58]. Recently, it has been suggested that CyA may stimulate formation of oxygen-derived radicals in vascular endothelial cells or mesangial cells [9,10]. Enhanced O2- formation could significantly contribute to CyA-induced arterial hypertension, since O2- inactivates the important endothelial vasodilator NO [11].

Another frequently observed feature of patients receiving CyA treatment are raised plasma levels of low density lipoprotein (LDL) [12,13]. Interestingly, LDL—particularly after oxidative modification—exerts effects on vascular tone similar to those observed under CyA-treatment: oxidized (Ox) LDL increases the vascular contractility [14,15] and inactivates NO [16,17]. In further analogy to CyA, some of the effects of OxLDL can be attributed to its potent stimulation of O2- formation in vascular cells [18,19].

In view of these pathophysiological similarities of CyA and OxLDL, we aimed to study their stimulatory effect on O2- formation in more detail. We hypothesized that their influence on O2- formation is potentiated by each other, and that increased O2- formation leads to a more pronounced attenuation of endothelium-dependent, NO-mediated dilation. Furthermore, it has been shown that CyA and OxLDL induce the release of endothelin (ET) from vascular cells [4,20,21] and that ET receptor antagonists play a protective role in acute CyA toxicity [22] and prevent CyA increased vasoconstriction [23]. We therefore analysed the role of ET in the stimulation of O2- formation. For this purpose we used isolated rabbit and rat aortic rings and measured O2- formation using a chemiluminescence method. Attenuation of endothelium-dependent dilations was analysed in the isolated arteries in an organ bath under isometric conditions.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
Reagents
CyA (Sandoz, Basel, Switzerland) was dissolved in dimethyl sulfoxide (DMSO) (Serva, Heidelberg, FRG) and further diluted in Krebs-HEPES buffer of the following composition (in mM): CaClx2 H2O 1.8, KCl 2.6, MgCl2x6 H2O 0.5, NaCl 137, N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) 9.1, glucose 2.8, NaH2PO4 0.35, pH 7.3. Endothelin-1 (ET-1) and cyclo(D-asp-pro-D-val-leu-D-trp) (BQ-123) were from Alexis (Grünberg, FRG). Pentobarbital was from Byk Gulden (Konstanz, FRG). Phenylephrine (PE) was from Hoechst (Frankfurt, FRG). Sodium nitroprusside (SNP), indomethacin, acetylcholine (ACh), superoxide dismutase (SOD), catalase, xanthin, xanthine oxidase, ethylene-diamine-tetraacetic acid (EDTA), diethyldithiocarbamate (DDC), 4,5 dihydroxy-1,3-benzene disulfonic acid salt (TIRON), NG-nitro-L-arginine, bis-N-methylacridinium (lucigenin), phorbol-12-myristate- 13-acetate (PMA), and butylated-hydroxy-toluene (BHT) were from Sigma (Munich, FRG). All drugs if not indicated otherwise were further diluted in Krebs-HEPES buffer. Indomethacin was dissolved in ethanol-0.1 mol/l NaHCO3 (1:3) v/v.

Isolation and oxidation of LDL
Pooled human LDL was obtained from healthy subjects from the local blood bank and was isolated, as described recently, by ultracentrifugation [24]. Protein content of lipoproteins was measured using a commercially available kit (Sigma protein kit) which is based on a modification of the method as initially described by Lowry [25]. Lipoprotein concentrations are always given as microgram protein per ml solution. Homogeneity of lipoproteins was tested by agarose gel electrophoresis (REP-HDL-plus cholesterol electrophoresis, Helena Diagnostika, Hartheim, FRG). Lipoproteins were prepared fresh every 2 weeks. During this period apolipoprotein B remained intact and not degraded.

Preparation of OxLDL was carried out as described recently [24]. Briefly, antioxidant-free LDL (300 µg protein/ml) was incubated with CuSO4 (5 µmol/l) in PBS at 20°C for 30 h. The degree of oxidation was quantified by the increase in relative mobility on agarose gel (lipidophor electrophoresis kits, IMMUNO, Heidelberg, FRG), indicating an enhanced negative charge of OxLDL [26]. The relative mobility of OxLDL on agarose gel electrophoresis as an index for lipoprotein oxidation was 1.7–2.1 compared with native LDL.

Blood vessel preparation and measurement of O2- formation by lucigenin-chemiluminescence
In order to investigate O2- formation from isolated rat aorta, the thoracic aorta was removed from female rats (6 weeks old, 200–300 g) killed by exsanguination after an intraperitoneal dose of pentobarbital (300–400 mg/kg). All procedures were carried out in accordance with the guidelines of the German Ministry of Agriculture for the use and care of laboratory animals. The blood vessels were placed in Krebs-HEPES buffer, cleaned of fat and connective tissue, and cut into ring segments (5 mm long). To measure O2- formation from these rings, we utilized a chemiluminescence assay with lucigenin that reacts specifically with O2-, resulting in the release of photons, which can be detected in a photomultiplier tube. This method is highly sensitive and allows the study of time kinetics. Detection of chemiluminescence with lucigenin was carried out as described recently [27] in a scintillation counter with a single photomultiplier tube (LUMAT LB 9501/16, Berthold-Instruments, Wildbad, FRG). The aortic ring segments were incubated for 60 min at 37°C in Krebs-HEPES buffer containing 10 ng–10 µg/ml CyA, or 300 µg/ml OxLDL, or their respective buffers as control. The influence of PMA, of ET-1, and of BQ123 was studied by adding the respective substances to the incubation medium. Control experiments were performed to prove that the respective solvents of the substances had no influence on the chemiluminescence signal. Incubations were carried out in the presence of 10 mM DDC because preliminary experiments revealed that the signals obtained in the absence of an SOD-inhibitor were below the detection limit. Thereafter the segments were transferred into the scintillation vials containing 0.25 mM lucigenin in a final volume of 2 ml. Counts were obtained at 1-min intervals at room temperature. To correct for background, counts obtained from vials containing all components with the exception of the aortic rings were subtracted from these signals. At the end of the experiments, the aortic segments were dried, and the dry weight of each segment was determined. The time course of the chemiluminescence signal of aortic segments is always shown as scintillation counts per second and per mg tissue.

Blood vessel preparation and measurement of tension
Isolated rabbit aortic rings were superfused with Krebs-HEPES buffer under isometric conditions, and force development was measured as described previously [19]. Briefly, the thoracic aorta was removed from New Zealand white rabbits of either sex (4–5 months old, 2.5–3.5 kg) killed by exsanguination after an intraperitoneal dose of pentobarbital (300–400 mg/ml). Further procedures were carried out as described for rat aortas. The ring segments (4–5 mm long) were mounted into small organ baths (volume 2 ml) and connected to a strain gauge transducer (Hugo Sachs, Hugstetten, FRG) to record changes in isometric tension. The aortic rings were superfused at a constant rate (3 ml/min) with Krebs-HEPES buffer (37°C, bubbled with a 95% O2 and 5% CO2 gas mixture). At the start of each experiment, the rings were stretched to a resting tension of 2.5 g to optimize vasoconstrictor responses and then contracted with PE (1 µM). Once the contractions had reached a plateau, the endothelial integrity of the preparations was verified by adding 1 µM ACh to the superfusate. Only arteries with a vasodilator response of >70% inhibition of preconstriction was considered endothelium-intact. To confirm that the ACh-induced vasodilation was mediated by endothelium-derived NO, in parallel experiments the inhibitory effect of NG-nitro-L-arginine on the vasodilator responses to ACh was investigated as described [28]. The preparations were then washed and allowed to equilibrate with Krebs-HEPES buffer for 45 min before being contracted a second time with PE. When a stable vasoconstriction to PE of 3–5 g was reached, concentration–response curves to ACh (3 nM–3 µM) were constructed. The effects of CyA (10 µg/ml) and of OxLDL (300 µg/ml) on the vasodilator responses to ACh were investigated by pretreating aortic rings with CyA or OxLDL for 90 min before the ACh concentration–response curve was determined. This was usually about 60 min, and CyA or OxLDL were still present. In each experiment, three aortic rings obtained from the same animal were studied simultaneously, with one ring serving as time-matched solvent control for CyA- or OxLDL-treated arteries. All experiments were performed in the presence of indomethacin (10 µmol/l) added to the intraluminal perfusate to exclude the influence of prostanoid factors.

Statistics
Data are presented as means±SEM of number of experiments (n). Statistical difference of data in Figures 1 and 2GoGo was determined by comparing the area under the curve with a computed software program (SigmaStat, Jandel Scientific, Erkrath, FRG). Data in Figures 3–7GoGoGoGoGo represent means of 20 repetitive measurements of counts per second over a 20 min detection period, and data are compared by Students paired t-test. For multiple comparisons of data, Bonferroni's correction was applied. Vasodilator responses are expressed in per cent of the initial steady state constriction induced by PE. Drug effects in Figure 8Go were compared by two-way ANOVA (one factor being repeated measures of drug concentrations and the other factor being the particular intervention), followed by all pairwise multiple comparison procedures (Tukey test). Differences were considered significant at an error probability of P<0.05.



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Fig. 1. CyA dose-dependently induces O2- formation in isolated rat aorta. Time course of lucigenin-mediated chemiluminescence as a parameter for O2- formation in control and in CyA-treated rat arteries. Treatment with CyA (10 ng–10 µg/ml) for 60 min significantly and dose-dependently increased the basal chemiluminescence signal of the arteries (n=8). The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown).

 


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Fig. 2. Comparison of the effect of CyA, OxLDL and PMA on O2- formation in isolated rat aorta. Time course of lucigenin-mediated chemiluminescence as a parameter for O2- formation in control, CyA-, OxLDL-, and phorbol-ester (PMA)-treated rat arteries. Treatment with CyA (10 ng/ml), OxLDL (300 µg/ml), or PMA (100 µM) significantly increased the basal chemiluminescence signal of the arteries (n=7). The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown).

 


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Fig. 3. O2- formation in rat aorta is further enhanced by coincubation with OxLDL and CyA. Mean O2- formation in terms of lucigenin-mediated chemiluminescence over a 20 min period in control, OxLDL (300 µg/ml)-, CyA (10 ng/ml)-, and OxLDL+CyA-treated rat arteries. Treatment with CyA or OxLDL significantly increased the basal chemiluminescence signal of the arteries (n=8). The combination of CyA and OxLDL further enhanced the chemiluminescence signal. The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown). *Indicates P<0.05 of OxLDL- or Cya-treated arteries vs controls. #Indicates P<0.05 of OxLDL+CyA-treated arteries vs aortic rings treated with OxLDL or CyA alone.

 


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Fig. 4. Stimulation of O2- formation by CyA in rat aorta can be blocked by the ET-receptor antagonist BQ123. Mean O2- formation in terms of lucigenin-mediated chemiluminescence over a 20 min period in control, CyA (10 ng/ml)-, and CyA+BQ123 (1 µM)-treated rat arteries. Treatment with CyA significantly increased the basal chemiluminescence signal of the arteries (n=7). Additional treatment with the ETA receptor antagonist BQ123 prevented the increase of the chemiluminescence signal. The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown). *Indicates P<0.05 of CyA-treated arteries vs controls. #Indicates P<0.05 of CyA-treated arteries vs CyA+BQ123.

 


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Fig. 5. Stimulation of O2- formation by OxLDL+CyA in rat aorta can be blocked by the ETA-receptor antagonist BQ123. Mean O2- formation in terms of lucigenin-mediated chemiluminescence over a 20 min period in control, OxLDL (300 µg/ml)+CyA (10 ng/ml)-, and OxLDL+CyA+BQ123 (1 µM)-treated rat arteries. Treatment with OxLDL+CyA significantly increased the basal chemiluminescence signal of the arteries (n=7). Additional treatment with the ETA-receptor antagonist BQ123 prevented the increase of the chemiluminescence signal. The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown). *Indicates P<0.05 of OxLDL+CyA-treated arteries vs controls. #Indicates P<0.05 of OxLDL+CyA-treated arteries vs OxLDL+CyA+BQ123.

 


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Fig. 6. ET-1 induces O2- formation in isolated rat aorta and potentiates the OxLDL stimulated O2- formation. Time course of lucigenin-mediated chemiluminescence as a parameter for O2- formation in control, ET-1 (1 nM)-, OxLDL (300 µg/ml)-, and OxLDL+ET-treated rat arteries. Treatment with OxLDL or ET alone significantly increased the basal chemiluminescence signal of the arteries (n=8). The combination of OxLDL and ET further enhanced the chemiluminescence signal. The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown). *Indicates P<0.05 of OxLDL- or ET-treated arteries vs controls, and #indicates P<0.05 of OxLDL+ET vs OxLDL alone.

 


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Fig. 7. Stimulation of O2- formation by OxLDL+ET-1 in rat aorta can be blocked by the ETA-receptor antagonist BQ123. Mean O2- formation in terms of lucigenin-mediated chemiluminescence over a 20 min period in control, OxLDL (300 µg/ml)-, OxLDL+ET (1 nM), and OxLDL+ET+BQ123 (1 µM)-treated rat arteries. Treatment with OxLDL significantly increased the basal chemiluminescence signal of the arteries (n=8), which was significantly further enhanced by ET. Additional treatment with the ETA receptor antagonist BQ123 prevented the increase of the chemiluminescence signal. The O2- scavenger TIRON completely blunted the chemiluminescence signal in all treatment groups (not shown). *Indicates P<0.05 of OxLDL+ET-treated arteries vs OxLDL. #Indicates P<0.05 OxLDL+ET-treated arteries vs OxLDL+ET+ BQ123-treated arteries.

 


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Fig. 8. OxLDL and CyA potentiate their damaging influence on ET-dependent vasodilation. Effect of 90 min incubation with OxLDL (300 µg/ml) and CyA (10 µg/ml) in the presence of DDC (1 mM) on the ACh-induced, ET-dependent dilation of isolated rabbit aortic rings. Incubation of PE-precontracted aortic rings with OxLDL and CyA significantly attenuated dilator responses in the presence of the SOD-inhibitor DDC. In the absence of DDC, or in arteries treated with OxLDL or CyA alone, vasodilations were not attenuated (data not shown). Dilator responses are expressed as percent of precontraction (4.8 g in the OxLDL/CyA group and 4.4 g in the control group). Data are means±SEM of seven experiments. *Indicates P<0.05.

 


   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
Measurement of O2- formation OxLDL and CyA induce O2- formation in isolated rat aorta
We first assessed the effect of OxLDL and of CyA alone on O2- formation in isolated rat aortic rings using the chemiluminescence method with lucigenin as indicator for O2-. Aortic rings were incubated for 60 min with CyA (10 ng–10 µg/ml), or for 60 min with OxLDL (300 µg/ml). CyA dose-dependently increased the basal O2- formation of rat aortic rings and induced its maximal effect at a concentration of 10 µg/ml (Figure 1Go). At a concentration of 10 ng/ml, CyA reached a similar potency as the reference stimulation with the phorbol ester PMA (100 µM), whereas 300 µg/ml OxLDL was even more potent (Figure 2Go). The concentration for OxLDL was chosen according to results of previous studies on OxLDL-stimulated O2- formation [18,19], and because preliminary experiments revealed that OxLDL at a lower concentration (100 µg/ml) had no significant effect on endothelium-dependent dilations. However, at a concentration of 100 µg/ml, a small, but significant enhancement of O2- formation could be observed (data not shown). The following experiments investigated the influence of CyA on the OxLDL-induced O2- formation and were performed with 10 ng/ml CyA and 300 µg/ml OxLDL.

O2- formation in rat aorta is further enhanced by coincubation with OxLDL and CyA
Next we studied whether coincubation of rat aortic rings with OxLDL and CyA further enhances the stimulation of O2- formation. Mean O2- formation in terms of lucigenin-mediated chemiluminescence over a 20 min period in control, OxLDL (300 µg/ml)-, CyA (10 ng/ml)- and OxLDL+CyA-treated rat arteries is shown in Figure 3Go. Treatment with CyA or OxLDL significantly increased the basal chemiluminescence signal of the arteries (compare with Figure 2Go). The combination of CyA and OxLDL significantly further enhanced the chemiluminescence signal.

It has been suggested that some of the vascular effects of CyA and of OxLDL are mediated by release of ET-1 from the arterial endothelium [4,21,23,29]. In order to analyse whether stimulation of O2- formation by CyA alone as well as the enhancement of the OxLDL-induced O2- formation by CyA can be linked to increased ET-1 activity, we performed (i) a series of experiments with the ETA receptor antagonist BQ123, and (ii) a series of experiments with exogenously applied ET-1 to prove that the CyA effect can be mimicked by ET-1.

Stimulation of O2- formation by CyA and by OxLDL/CyA can be blocked by the ETA-receptor antagonist BQ123
Incubation of rat aortic rings with the ETA-receptor antagonist BQ123 (1 µM) completely prevented the stimulation of O2- formation induced by CyA (10 ng/ml) alone (Figure 4Go). BQ123 also significantly reduced stimulation of O2- formation induced by the combination of CyA (10 ng/ml) and OxLDL (300 µg/ml) (Figure 5Go) and by OxLDL alone (by ~50%, data not shown). Control experiments with the phorbol ester PMA (100 µM) revealed that BQ123 had no unspecific effect on basal or PMA-stimulated O2- formation (data not shown). Thus, ET-1 is a likely mediator of the CyA+OxLDL effect on O2- formation.

ET-1 induces O2- formation in isolated rat aorta and potentiates the OxLDL stimulated O2- formation
We then investigated whether exogenously applied ET-1 stimulated O2- formation in rat aortic rings, and whether ET-1 enhanced the OxLDL-induced O2- formation in a fashion similar to CyA. ET-1 (0.1–10 nM) dose-dependently increased basal O2- formation with a maximal effect at 1 nM. Figure 6Go shows the effect of 1 nM ET-1 alone and its enhancement of the OxLDL(300 µg/ml)-induced O2- formation. Thus, ET-1 exerted similar effects as CyA.

Stimulation of O2- formation by OxLDL+ET-1 in rat aorta can be blocked by BQ123
Finally, we tested whether the effect of ET-1 on the OxLDL-induced stimulation of O2- formation can be blocked by BQ123. Figure 7Go shows O2- formation in control, OxLDL(300 µg/ml)-, OxLDL+ET-1(1 nM)- and OxLDL+ET-1+BQ123(1 µM)-treated rat arteries. Treatment with the ETA receptor antagonist BQ123 prevented the increase of the chemiluminescence signal induced by ET-1.



   Measurement of endothelium-dependent dilations
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
Endothelium-dependent dilations of isolated arteries can be attenuated by OxLDL [16,17] or by CyA [6,7] alone, depending on the concentration and the duration of the exposure to the substances. However, at concentrations similar to those used in this study, OxLDL or CyA have no major impact on ACh-induced, endothelium-dependent dilations [30]. But, OxLDL and CyA increase O2- formation and O2- can inactivate NO [11], potentially resulting in attenuation of endothelium-dependent dilations. In view of the strong enhancement of the OxLDL-induced O2- formation by CyA, we wondered whether coincubation of isolated arteries with OxLDL and CyA results in impaired endothelial function.

OxLDL and CyA potentiate their damaging influence on endothelium-dependent vasodilation
The effect of 90 min incubation with OxLDL (300 µg/ml) and CyA (10 µg/ml) in the presence of DDC (1 mM, inhibitor of the endogenous endothelial SOD) on the ACh-induced, endothelium-dependent dilation of isolated rabbit aortic rings is shown in Figure 8Go. Incubation of PE-precontracted aortic rings with OxLDL and CyA together, significantly attenuated dilator responses. In arteries treated with OxLDL or CyA alone and in the absence of DDC (at the concentration given and at a 90 min incubation period), vasodilations were not attenuated (data not shown).

Thus, enhanced oxidative stress by OxLDL and CyA may result in severe attenuation of endothelial function in terms of impaired endothelium-dependent dilations.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
In this study, we confirmed that OxLDL induces oxidative stress in vascular tissue and showed for the first time directly that CyA stimulates O2- formation in isolated rat aortic rings, confirming indirect evidence from previous studies. The salient new findings of this investigation are: (i) that concomitant treatment with CyA and OxLDL further enhances their influence on O2- formation and (ii) that ET-1 is a mediator of CyA- and OxLDL-induced O2- formation. As expected, enhanced oxidative stress resulted in attenuation of endothelium-dependent vasodilation.

The regulation of vascular tone is of major importance for the maintenance of physiological organ perfusion and for the control of arterial blood pressure. It is well known that the arterial endothelium plays an active part in this regulation [31,32], and that many drugs or other pathological agents affect regulation of vascular tone via disturbance of endothelial function [33]. Both CyA and OxLDL are such pathological agents that are considered to cause endothelial dysfunction [9,34,35]. In the case of CyA, endothelial dysfunction is believed to be a mechanism that importantly contributes to one of its major side effects, namely arterial hypertension [36,37]. Among the many factors that attenuate endothelial function, reactive oxygen species seem to play an important role [38]. Virtually all cells of the vascular wall are a potential source for formation of reactive oxygen species. For example O2- can be produced by endothelial cells and by smooth muscle cells via a NAD(P)H oxidase [39,40], but also by macrophage-derived foam cells [41]. Low concentrations of O2- are continuously formed, however, once there is an imbalance established, excess O2- will inactivate endothelial NO [11], resulting in formation of peroxynitrite [42], and eventually in formation of the highly toxic hydroxyl radical [38]. This cascade of events will finally cause attenuation of the NO-mediated endothelium-dependent dilation. Thus, all agents that enhance vascular O2- formation may significantly contribute to attenuation of endothelial function. CyA and OxLDL are agents that can enhance oxidative stress [9,10,18,19], and it has been suggested that this might be the underlying mechanism for their negative impact on endothelial function in terms of impaired endothelium-dependent dilations.

While the stimulation of O2- formation in vascular preparations by OxLDL alone has been described before [18,19,43], evidence from previous studies for enhanced oxidative stress induced by CyA was only indirect, coming from the preventive effect of the antioxidative enzymes SOD and catalase [9,10]. Here, using the sensitive chemiluminescence assay, we show directly that CyA dose-dependently stimulates O2- formation in isolated rat aortic rings. The stimulatory effect was potent, in as much as the stimulation with CyA at a concentration of 10 ng/ml reached a similar potency as the reference stimulation with the phorbol ester PMA, a strong stimulator of O2- formation [44]. The combined effect of OxLDL and CyA had not been investigated before. The rationale to study such a combined effect bases on the fact that lipid disorders are common in patients under CyA treatment [12,13], which implies that a modulation of the individual effect of CyA or OxLDL by each other may be of relevance for patients in vivo. In our in vitro system, the OxLDL-induced O2- formation was further enhanced by concomitant treatment with CyA by a factor of 1.9. Thus, concomitant treatment resulted in a stimulation of O2- formation that was superadditive to the individual effects of CyA or OxLDL. As could be expected, this strong stimulation of O2- formation resulted in a strong attenuation of endothelium-dependent dilations in rabbit aorta. Further evidence for the role of O2- in this attenuation of endothelium-dependent dilations can be derived from the observation that inhibition of the endogenous endothelial SOD with DDC increased the effect of CyA and OxLDL.

The potential mediator role of ET-1 in the individual or combined effect of CyA or OxLDL on O2- formation had also not been investigated before. ET-1, however, may be of particular importance because both CyA and OxLDL may exert part of their pathological effects via increased release or formation of ET-1 [4,20,21,45], and ET-1 has been shown to increase O2- formation in lung tissue, brain vessels and in macrophages [4648]. It was therefore one major scope of this study to analyse whether ET-1 was involved in the stimulation of O2- formation in vascular tissue. The evidence for a role of ET-1 in stimulation of O2- formation provided by our data is 2-fold. First, the ET-1 antagonist BQ123 which binds to the ETA-receptor prevented stimulation of O2- formation by CyA and OxLDL. Secondly, exogenously applied ET-1 dose-dependently stimulated O2- formation in rat aortic rings, and 1 nM ET-1 enhanced the OxLDL-induced O2- formation in a fashion similar to 10 ng/ml CyA. Thus, ET-1 exerted similar effects as CyA, and all these effects could be blocked by BQ123. These findings imply that CyA and OxLDL stimulated O2- formation in aortic rings through the release of ET-1. As shown in Figures 4 and 5GoGo, BQ123 almost completely blocked the increase in O2- formation induced by CyA alone or induced by CyA+OxLDL, while—as shown in Figure 7Go—BQ123 only blocked the ET-1 sensitive portion when OxLDL and ET-1 were given in combination. The reason for the different potency of BQ123 is unclear. However, the combination of OxLDL+ET-1 was the most potent stimulus during this investigation, which might explain why it was only incompletely blocked by 1 µM BQ123. Recently, it has been demonstrated that blockade of the ETA receptor improved NO-mediated, endothelium-dependent dilations in apolipoprotein E-deficient mice [49]. Thus, an alternative explanation for the action of BQ123 in our study is that it acted via increased NO release, which would also result in a reduced chemiluminescence signal due to the rapid reaction between NO and O2- [38]. It was beyond the scope of our study to analyse the cellular origin of O2- produced from aortic rings. However, from other in vitro investigations we know that the endothelial or smooth muscle cell NAD(P)H oxidase is an important O2- generating system [39,40]. It has recently been shown that stimulation of the NAD(P)H oxidase in rat smooth muscle cells involves a protein kinase C (PKC)-dependent step [50], and that ET-1 is a potent stimulator of PKC in vascular smooth muscle cells [51]. Thus, PKC-mediated stimulation of NAD(P)H oxidase via ET-1 may be responsible for the OxLDL- and CyA-induced stimulation of O2- formation. Another potential source for O2- is the endothelial NO synthase (eNOS), e.g. when an important cofactor of the eNOS, tetrahydrobiopterin, is not available in sufficient concentration [5254]. Since acute administration of CyA also enhances NO activity [55], one could speculate that CyA increases vascular O2- formation via eNOS under certain conditions.



   Conclusions
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 
We demonstrate in this study that coincubation of isolated aortic rings with CyA and OxLDL causes a potent enhancement of vascular O2- formation. ET-1 seems to be mediator of the CyA-induced O2- formation. Enhanced oxidative stress results in further attenuation of endothelium dependent vasodilation. These findings may be of relevance for patients with lipid disorders under immune suppression with CyA.



   Acknowledgments
 
The skilful technical assistance of Mrs Susanne Clemens-Richter, Mrs Elke Baumeister, Mrs Traudel Baier and Mrs Margarete Röder is gratefully acknowledged. Grant support from the Deutsche Forschungsgemeinschaft to J.G. (Ga 431/1–4 and Ga 431/2–1).



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Measurement of endothelium...
 Discussion
 Conclusions
 References
 

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Received for publication: 13. 4.99
Accepted in revised form: 4.11.99