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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:61-69

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:61.)
© 2000 American Heart Association, Inc.

Vascular Biology

Improvement of Nitric Oxide–Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation

Andreas H. Wagner; Thomas Köhler; Uwe Rückschloss; Ingo Just; Markus Hecker

From the Department of Cardiovascular Physiology, University of Göttingen (A.H.W., T.K., M.H.); the Institute of Pathophysiology, University of Halle (U.R.); and the Institute of Pharmacology and Toxicology, University of Freiburg (I.J.), Germany.

Correspondence to Dr Markus Hecker, Abteilung Herz- und Kreislaufphysiologie, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.


*    Abstract
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Abstract—Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester–stimulated superoxide anion (O2-) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 µmol/L). The HMG-CoA reductase product mevalonic acid (400 µmol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester–stimulated O2- formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced {approx}2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O2- formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O2- in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.


Key Words: HMG-CoA reductase inhibitor(s) • endothelial dysfunction • coronary heart disease • nitric oxide • superoxide anion • NADPH oxidase • p21 Rac


*    Introduction
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 *Introduction
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The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), collectively referred to as "statins," on coronary events have generally been attributed to the well-documented LDL cholesterol–lowering properties of these drugs. However, despite the strong association between drug concentration, blood lipid level, and patient benefit shown in the CARE, 4S, and WOSCOPS trials, HCRIs seem to exert at least part of their cardioprotective action by mechanisms other than simply lowering the lipid load of the vessel wall. Thus, HCRIs induce regression of atherosclerotic lesions in patients with coronary heart disease (CHD) with and without hypercholesterolemia1 and improve endothelial dysfunction, a hallmark of atherosclerotic blood vessels, in patients with moderately elevated total serum cholesterol within 1 month.2 At the cellular level, they modulate leukocyte activity and adhesiveness,3 4 inhibit vascular smooth muscle cell proliferation both in vitro and in vivo,5 6 and reduce the synthesis of chemokines (monocyte chemotactic protein-1) in mesangial cells7 and that of adhesion molecules such as E-selectin in hyperlipidemic patients.8 All of these effects do not seem to be mediated by the decrease in serum LDL cholesterol but nonetheless may be related to an inhibition of HMG-CoA reductase through prevention of the isoprenylation or farnesylation of members of the p21 Rac, Ras, or Rho families of low-molecular-weight GTP-binding proteins.9 10 These G proteins are essential for the intracellular signaling of seven transmembrane–spanning receptors and play a major role in cell proliferation and other important cell functions by mediating the activation of downstream effector molecules.11 12 13

An imbalance between the production of superoxide anions (O2-) and nitric oxide (NO) in the vessel wall has long been suspected to play an important role in the pathogenesis of CHD. Clinically manifested by an attenuated dilator response to endothelium-dependent mediators (acetylcholine, bradykinin, substance P) or a decrease in blood flow as well as by an overt constrictor response to stimulation of the sympathetic nervous system, endothelial dysfunction in coronary arteries is thought to be due to an increased formation or, alternatively, an insufficient removal of O2-. Because NO and O2- interact chemically to neutralize each other at a rate that is controlled only by the limits of diffusion, an increase in the local concentration of O2- must lead to a decrease in the concentration of biologically active NO. In addition to mediating endothelium-dependent vasodilatation, NO also limits the expression of proatherosclerotic gene products, such as adhesion molecules and chemokines,14 15 16 in endothelial and presumably also in smooth muscle cells by preventing the activation of nuclear factor {kappa}B. A shift in the NO/O2- balance toward O2- therefore not only causes an increase in tone but also promotes expression of these gene products, thus initiating or advancing the atherosclerotic process.

HCRIs seem to be capable of interfering with this NO/O2- balance; the question, however, is at what level. Thus, it was recently shown that simvastatin and lovastatin upregulate NO synthase (NOS) expression in cultured endothelial cells by increasing the stability of its mRNA.17 18 The functional consequences of this increased NOS expression, however, are difficult to extrapolate from the cell culture model and therefore remain to be determined. Moreover, it is not clear whether this is a class effect of the HCRIs. Therefore, we compared the effects of three different HCRIs, atorvastatin, cerivastatin, and pravastatin, on the formation of O2- in endothelium-intact segments of the rat aorta as well as that of atorvastatin on the NO-mediated dilator response of these segments to acetylcholine. In addition, we have attempted to elucidate at what level HCRIs affect the NO/O2- balance in these segments.


*    Methods
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Materials
Acetylcholine chloride, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), bis-N-methyl-acridinium nitrate (lucigenin), mevalonic acid lactone, NG-monomethyl-L-arginine (L-NMMA), phenylarsine oxide (PAO), phenylephrine hydrochloride, phorbol dibutyrate (PDB), polymyxin B sulfate, xanthine oxidase from buttermilk, and xanthine were obtained from Sigma, and exoenzyme (C3) from Clostridium botulinum from List Biological Laboratories (via Quadratech). NG-Nitro-L-arginine (L-NNA) was from Bachem. Recombinant bovine superoxide dismutase (SOD, Peroxinorm) was generously provided by Grünenthal, atorvastatin by Goedecke/Parke-Davis, cerivastatin by Bayer, and pravastatin by Bristol-Myers Squibb.

Mevalonic acid lactone was converted to sodium mevalonic acid by solubilization in 0.1 mol/L sodium hydroxide, heating at 50°C for 2 hours, and adjustment of the pH to 7.4 with 0.1 mol/L hydrochloric acid.19 Clostridium sordellii lethal toxin was prepared from strain 6018 as described.20

Preparation and Incubation of Ring Segments of Rat Thoracic Aorta
Aortas (1.5-mm ID) were isolated from pentobarbitone-anesthetized male Wistar rats (250 to 300 g body weight), cleaned under sterile conditions of adherent fat and connective tissue, and cut into ring segments {approx}5 to 7 mm long. Segments were placed in 2 mL of Waymouth medium (Life Technologies) containing 10% FBS in the absence (solvent control, 0.05% dimethylsulfoxide, vol/vol) or presence of the HCRIs at different concentrations and incubated for 1 to 22 hours at 37°C in an incubator gassed with 5% CO2 (vol/vol). In some experiments, the lumen of the segments was carefully denuded with the aid of a roughened steel cannula (2.0-mm OD), and the absence of endothelium was confirmed by the lack of a relaxant response of these segments to acetylcholine (1 to 10 pmol) after preconstriction with phenylephrine (10 µmol/L) in a superfusion bioassay system (see below). For vascular reactivity studies, incubations took place in serum-free Waymouth medium to which polymyxin B sulfate was added at a concentration of 100 U/mL.

Vascular Reactivity Studies
Four ring segments were tested simultaneously by mounting them between force transducers and a rigid support for measurement of isometric force (TSE). The rings were superfused at 1 mL/min with warmed (37°C), oxygenated (95% O2, 5% CO2) Krebs-Henseleit solution, pH 7.4 (composition in mmol/L: Na+ 144.0, K+ 5.9, Cl- 126.9, Ca2+ 1.6, Mg2+ 1.2, H3PO4- 1.2, SO42- 1.2, HCO3- 25.0, D-glucose 11.1, and diclofenac 0.001). Passive tension was adjusted over a 30-minute equilibration period to 2.7±0.1 g (n=40). Thereafter, the rings were preconstricted to 4.3±0.1 g of tension (n=40) with 10 µmol/L phenylephrine. After a stable plateau of constriction had been reached, increasing doses of acetylcholine (1 to 30 nmol, {approx}1 to 30 µmol/L) were administered as a bolus injection into the superfusate, and the ensuing relaxant response was monitored with the aid of a digital PC-operated analysis system (Biosys, TSE). In some experiments, SOD (100 U/mL) was continuously infused into the superfusate.

Cell Culture
Porcine aortic endothelial cells (PAECs) were detached by treatment with dispase (1.6 U/mL for 7 minutes at 37°C). They were seeded in either gelatin-coated 96-well multiwell plates (Berthold) or 100-mm Petri dishes and grown to confluence in DMEM/Ham’s F12 medium (1:1, vol/vol) containing 20% FBS, as described previously.21 RAW 264.7 macrophages were grown in DMEM containing 20% FBS, as described before.22

Detection of O2- Formation
Formation of O2- by xanthine oxidase or the rat aortic segments was determined photometrically as described in detail elsewhere23 by monitoring of the SOD-sensitive reduction of ferricytochrome c at 550 nm as a function of time. Alternatively, O2- formation was determined by monitoring of the lucigenin-enhanced chemiluminescence in a Lumat LB 9501 luminometer (Berthold). Briefly, lucigenin (final concentration of 250 µmol/L) was added to a plastic cuvette containing Krebs-HEPES buffer (composition in mmol/L: NaCl 135.3, KCl 4.7, CaCl2 1.8, MgSO4 1.2, K2HPO4 1.2, Na-EDTA 0.026, Na-HEPES 10, and D-glucose 11.1), pH 7.4 (final volume 0.6 mL). O2- production was stimulated by the addition of PDB (5 µmol/L) after the background and basal O2- formation had been measured. The assay was calibrated by monitoring of the chemiluminescence signal of known amounts of O2- generated by xanthine oxidase (0.05 U) and xanthine (10 to 50 µmol/L), which had previously been determined spectrophotometrically by monitoring of the reduction of ferricytochrome c. The chemiluminescence assay was specific for O2-; no light emission was recorded in the presence of authentic NO or H2O2.

When PAECs were used for determination of O2- formation, they were grown in 96-well multiwell plates, and chemiluminescence was monitored by lucigenin-enhanced chemiluminescence (final concentration of 250 µmol/L) in a Microlumat LB 99P microplate luminometer (Berthold). For this purpose, the medium was removed and replaced by Krebs-HEPES buffer (final volume 250 µL). O2- production was stimulated by the addition of 5 µmol/L PDB after lucigenin had been injected and basal O2- formation measured.

NADPH Oxidase Assay
NADPH oxidase activity was measured by a luminescence assay with 250 µmol/L lucigenin as electron acceptor and either 100 µmol/L NADH or 100 µmol/L NADPH as the substrate (final volume 0.9 mL). Aortic segments with and without endothelium were homogenized in Krebs-HEPES buffer (pH 7.0) and worked up as described above. No activity could be measured in the absence of NADH or NADPH. The reaction was started by the addition of 30 to 50 µg protein, and the ensuing chemiluminescence signal was monitored as described above for the aortic segments.

Determination of NOS Activity
NOS activity was determined by monitoring of the conversion of L-[3H]arginine to L-[3H]citrulline. The assay was performed essentially as described previously.21 In brief, aortic rings ({approx}8 mm in length) were placed in 90 µL ice-cold 50 mmol/L Tris HCl buffer (pH 7.4) containing 1.15% (wt/vol) KCl, 1 mmol/L EDTA, 5 mmol/L D-glucose, 0.1 mmol/L DL-dithiothreitol, 2 mg/L leupeptin, 2 mg/L pepstatin A, and 70 mg/L Pefabloc (Biomol) and homogenized with a glass-glass homogenizer. The crude homogenate was centrifuged twice at 750g for 5 minutes to remove bigger fragments. The protein content of the supernatant was measured according to the method of Bradford,24 and aliquots (50 µg total protein) were incubated (double determination) for 10 to 60 minutes with 1 µmol/L L-[3H]arginine (total activity 1 µCi).

Determination of Total Aortic SOD Activity
Tissues were homogenized as described above for determination of NOS activity. SOD activity of the prepared homogenates was assayed with the Microlumat LB 99P luminometer by direct monitoring of the inhibition of O2- formation with the xanthine (50 µmol/L)/xanthine oxidase (0.01 U) system as the source of O2- and lucigenin-enhanced chemiluminescence (final concentration of 250 µmol/L) in Krebs-HEPES buffer (250 µL final volume, pH 7.4). One unit of SOD activity was defined as the amount of protein that inhibited by 50% the rate of O2- formation under these conditions. Final protein concentrations in samples used for total SOD assay were 24 µg/mL. Recombinant bovine SOD was used as a control.

Protein Kinase C Activity Measurements and Western Blot Analyses
The PepTag assay (Promega) was used for nonradioactive detection of protein kinase C (PKC) activity in partially purified homogenates. Homogenates from rat aortic segments (750g supernatant) and microsomal fractions from PAECs (100 000g pellet) were prepared as described previously21 after 18 hours of incubation in the absence and presence of 10 µmol/L atorvastatin. PKC isolated from rat brain (Promega) was used as control enzyme; the purified enzyme consists primarily of {alpha}, ß, and {gamma} isoforms with lesser amounts of {delta} and {zeta} isoforms.

Immunoblotting analysis of specific PKC isoform activation in cultured PAECs after incubation with atorvastatin was also performed. The resuspended 100 000g pellets of the microsomal fraction and the cytosolic fraction (40 to 50 µg protein per lane) were separated by denaturing 10% polyacrylamide gel electrophoresis in the presence of SDS according to standard protocols and then transferred to nitrocellulose membranes (Amersham-Pharmacia Biotech). Transferred proteins were probed by isotype-specific anti-PKC antibodies (Life Technologies). To visualize protein bands, anti-rabbit immunoglobulin conjugated to horseradish peroxidase (1:7500 dilution; Sigma-Aldrich) and the chemiluminescence detection method (SuperSignal Blaze chemiluminescent substrate; Pierce Chemical) were used.

Reverse Transcription–Polymerase Chain Reaction Analysis
The frozen aortic segments were minced under liquid nitrogen with the aid of a mortar and pestle, and total RNA was isolated by solid-phase extraction with the RNeasy kit from Qiagen. For reverse transcription (RT), the following RT components were added to the reaction vials: 1 to 5 µg of total RNA, 4 µL 5x first-strand buffer; 1 µL dNTP mixture (10 mmol/L each dATP, dGTP, dCTP, and dTTP at neutral pH), 1 µL oligo(dT) (500 µg/mL), 2 µL DL-dithiothreitol (0.1 mol/L), and 1 µL Superscript II reverse transcriptase (Life Technologies) in a total volume of 20 µL. The vials were incubated for 45 minutes at 40°C; thereafter, the RT reaction was terminated by heating to 72°C for 10 minutes. For polymerase chain reaction (PCR), the expression of endothelial constitutive NOS (ecNOS), Cu2+/Zn2+-SOD, Mn2+-SOD, and elongation factor 2 (EF-2) mRNA as an internal standard was assessed by semiquantitative PCR as follows: A unique 2-minute period for complete denaturation at 94°C in the beginning, followed by a primer-specific number of cycles of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 58°C, and 1 minute of primer extension at 72°C, with an additional 5 minutes at 72°C for final extension at the end. The expression of NADPH oxidase (gp91-phox subunit) was assessed by PCR as follows: A unique 1-minute period for complete denaturation at 95°C in the beginning, followed by 40 cycles of 30 seconds of denaturation at 95°C, 30 seconds of annealing at 58°C, and 30 seconds of primer extension at 72°C, with an additional 3 minutes at 72°C for final extension at the end. For quantitative measurement of gp91-phox, a DNA standard with a 192-bp deletion was produced, allowing the development of a competitive-PCR method.25

The sequence of the rat ecNOS-specific primers (courtesy of Dr Patrick Diel, Department of Morphology and Tumor Research, German Sport University of Cologne, Germany) was 5'-AGTGGAAGTGGTTCCGCTG-3' (sense) and 5'-GAGATGG-TCAAGTTGGGAGC-3' (antisense), and the predominant cDNA amplification product was predicted to be 450 bp in length. The following primers with the respective GenBank library accession number and position of the PCR product in the coding sequence and predicted size were used for amplification: rat Cu2+/Zn2+-SOD (X05634, position 58 to 505; 448 bp) 5'-GCAGAAGGC-AAGCGGTGAAC-3' (sense) and 5'-TAGCAGGACAGCAGAT-GAGT-3' (antisense); rat Mn2+-SOD (Y00497, position 329 to 945; 617 bp) 5'-CCCTAAGGGTGGTGGAGAAC-3' (sense) and 5'-GGCCTTATGATGACAGTGAC-3' (antisense); gp91-phox (X04011, position 814 to 1350, 537 bp) 5'-CCTATGACTTGGAAATGGAT-3' (sense) and 5'-CAGAGCCAGTAGAAGTAGAT-3' (antisense); L-selectin (CD62L) (S79523, position 156 to 717, 562 bp); 5'-CCTGAAGCTGTGGATCTGGAC-3' (sense) and 5'-CTCAGGG-GCCTTCAAAGGCTC-3' (antisense); and EF-2 (Z11692, position 1990 to 2207, 218 bp) 5'-GACATCACCAAGGGTGTGCAG-3' (sense) and 5'-GCGGTCAGCACACTGGCATA-3' (antisense).

To ensure that the PCR amplification was indeed semiquantitative, ie, in the linear phase of the exponential amplification curve, each PCR protocol was established for different numbers of cycles and amounts of cDNA. Furthermore, for each newly synthesized cDNA, the abundance of EF-2 cDNA was measured first, calculated, and used as a reference point to adjust the amount of cDNA from each sample for PCR amplification.

Data Analysis
Unless indicated otherwise, results are expressed as mean±SEM of n observations. Student’s unpaired t test was used to determine the statistical significance of differences between the means. A value of P<0.05 was considered to be statistically significant.


*    Results
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Phorbol Ester–Stimulated O2- Formation
Incubation of endothelium-intact segments of the rat aorta with the PKC activator PDB resulted in a prominent concentration-dependent increase in O2- formation (EC50 {approx}1 µmol/L), which was preceded by a lag phase of 2 to 3 minutes duration and reached a maximum after 9 to 16 minutes, depending on the concentration used. A PDB concentration of 5 µmol/L was chosen for all further experiments, because this gave a near-maximum response (range 1 to 12 pmol · mg-1 · min-1). PDB-stimulated O2- formation occurred almost exclusively in the endothelium of the rat aorta, because the response to PDB in denuded segments was reduced by >90% (Figure 1aDown). In this context, it did not matter whether the segments were denuded mechanically or the endothelial cells were removed enzymatically by perfusion with dispase (not shown). In the presence of SOD (100 U/mL), the PDB-stimulated endothelium-dependent increase in O2- formation was inhibited by >90%, whereas basal O2- formation appeared to be unaffected (Figure 1bDown). Idebenone (1 µmol/L), a potent antioxidant,26 inhibited both basal and PDB-stimulated O2- formation by 60% and >90%, respectively, whereas the specific PKC inhibitor Ro 31-8220 (1 µmol/L) inhibited stimulated but not basal O2- formation by >90% (n=3, P<0.05). AEBSF (5 mmol/L)27 and PAO (1 µmol/L),28 two inhibitors of the assembly of NADPH oxidase in phagocytes, also attenuated PDB-stimulated O2- formation by 75% and 78%, respectively (Figure 1cDown). Of note was that two different NOS inhibitors, L-NNA and L-NMMA (100 µmol/L), did not significantly affect PDB-stimulated O2- formation when they were incubated with the endothelium-intact segments for 1 or 18 hours (not shown). Moreover, all three HCRIs (10 µmol/L) had no direct effect on O2- formation catalyzed by xanthine/xanthine oxidase (not shown). Finally, to verify that PDB-stimulated O2- formation indeed originated in the endothelium and was not due to adherent or infiltrated leukocytes, the mRNA level of CD62L, a specific marker for these cells,29 was monitored in both endothelium-intact (Figure 2Down) and denuded (not shown) segments. In contrast to the positive control (rat spleen), RT-PCR analysis failed to detect CD62L mRNA in these segments, even at a very high degree of amplification (37 cycles).



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  		Figure 1. a, PDB (5 µmol/L)–stimulated O2- formation in rat aortic segments with (intact) or without (denuded) endothelium estimated by lucigenin-enhanced chemiluminescence (n=5). *P<0.05 vs intact. b, PDB-stimulated and basal O2- formation by rat aortic segments in the presence of SOD (100 U/mL) estimated by lucigenin-enhanced chemiluminescence (n=3). *P<0.05 vs control. c, Inhibitory effects of AEBSF (5 mmol/L) and PAO (1 µmol/L) on PDB (5 µmol/L)–stimulated O2- formation in endothelium-intact segments (n=4). *P<0.05 vs control.



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  		Figure 2. Lack of detection of CD62L mRNA in endothelium-intact segments of the rat aorta incubated in the absence or presence of atorvastatin (atorva, 10 µmol/L). Representative RT-PCR analysis. Identical findings were obtained with >=4 segments derived from other animals.

HCRI Effects on PDB-Stimulated O2- Formation
Exposure of the endothelium-intact segments to atorvastatin (concentration range 0.01 to 10 µmol/L) for up to 1 hour increased rather than decreased PDB-stimulated O2- formation. However, prolonged exposure to the HCRI resulted in a progressive inhibition of basal and PDB-stimulated O2- formation (Figure 3Down) that was both time- and concentration-dependent (t0.5 {approx}8 hours, EC50 {approx}1 µmol/L). The maximum inhibitory effect of atorvastatin on PDB-stimulated O2- formation was observed at a concentration of 10 µmol/L after an 18-hour incubation period. Because lucigenin is suspected to redox-cycle at the concentration used and hence interfere with the chemiluminescence detection of O2-, experiments with atorvastatin (18-hour incubation) were performed again, but O2- formation determined via the SOD-sensitive reduction of ferricytochrome c. As shown in Figure 4aDown, both ways of monitoring O2- formation yielded essentially the same results.



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  		Figure 3. A, Concentration-dependent effect of atorvastatin on PDB (5 µmol/L)–stimulated O2- formation in rat aortic rings with intact endothelium (representative experiment). B, Time course of O2- production in the presence of 10 µmol/L atorvastatin. Representative experiment. Values for 1 hour (n=4) and 18 hours (n=9) represent the mean±SEM. *P<0.05 vs time zero.



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  		Figure 4. a, Effects of atorvastatin (atorva, 10 µmol/L) on PDB (5 µmol/L)–stimulated O2- formation, estimated by lucigenin-enhanced chemiluminescence (luc CL) or SOD-sensitive reduction of ferricytochrome c (cyt c) in rat aortic rings with intact endothelium (n=4 to 5). *P<0.05 vs control. b, Effects of atorvastatin (10 µmol/L), cerivastatin (ceriva, 1 µmol/L), or pravastatin (prava, 10 µmol/L) on basal and PDB-stimulated O2- formation after 18 hours of incubation (n=5). *P<0.05 vs control. c, Effect of atorvastatin (10 µmol/L, 18 hours) alone (-atorva) and in combination with 400 µmol/L mevalonic acid (+atorva) on PDB-stimulated O2- formation (n=7). *P<0.05 vs control; {dagger}P<0.05 vs atorvastatin alone.

Exposure of the segments to cerivastatin and pravastatin for the same period of time also resulted in a significant inhibition of PDB-stimulated O2- formation, with a maximum inhibitory effect of cerivastatin (>60%) and pravastatin (>50%) at concentrations of 1 and 10 µmol/L, respectively (Figure 4bUp). Moreover, the inhibitory effect of atorvastatin on PDB-stimulated O2- formation in the endothelium-intact segments was completely reversed (Figure 4cUp) in the presence of the HMG-CoA reductase product mevalonic acid at a concentration of 400 µmol/L. Compatible results were obtained with the cultured PAECs (Figure 5ADown) and RAW 264.7 cells (Figure 5BDown), in which atorvastatin also inhibited the phorbol ester–stimulated O2- formation in a mevalonate-sensitive manner.



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  		Figure 5. A, Effect of atorvastatin (10 µmol/L, 18 hours) alone (-atorva) and in combination with 400 µmol/L mevalonic acid (+atorva) on PDB (5 µmol/L)–stimulated O2- formation, estimated by lucigenin-enhanced chemiluminescence in PAECs (5x104 cells/well, n=4 to 9). *P<0.05 vs control; #P<0.05 vs atorvastatin alone. B, Effect of atorvastatin (10 µmol/L, 18 hours) alone (-atorva) and in combination with 400 µmol/L mevalonic acid (+atorva) on basal and tetradecanoylphorbol 13-acetate (TPA) (1 µmol/L)–stimulated O2- formation, estimated by lucigenin-enhanced chemiluminescence in murine RAW 264.7 macrophages (6x105 cells/assay, n=4 to 6). *P<0.05 vs control; #P<0.05 vs atorvastatin alone.

Effects of HCRIs on ecNOS Expression and Activity
To elucidate how the HCRIs affected PDB-stimulated O2- formation in the isolated rat aorta, expression of ecNOS mRNA in the segments after 6 or 12 hours exposure to atorvastatin (10 µmol/L), cerivastatin (1 µmol/L), or pravastatin (10 µmol/L) was monitored by RT-PCR analysis. All 3 HCRIs caused a small but significant increase in ecNOS mRNA after 6 hours’ exposure of the segments (Figure 6ADown), and this effect was maintained at 12 hours (1.7-fold to 2.1-fold increase in ecNOS mRNA). Exposure to atorvastatin (10 µmol/L) for 18 hours also upregulated calcium-dependent NOS activity in homogenates of these segments by {approx}2.5-fold (Figure 6BDown), indicative of an increase in ecNOS protein abundance.



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  		Figure 6. A, ecNOS mRNA expression in rat aortic rings with intact endothelium in the presence of atorvastatin (atorva, 10 µmol/L), cerivastatin (ceriva, 1 µmol/L), or pravastatin (prava, 10 µmol/L) for 6 hours. Statistical summary of 3 separate experiments after densitometric quantification of the ecNOS band (*P<0.05 vs control). B, Calcium-dependent (+Ca2+) and -independent (-Ca2+) NOS activity in homogenates of aortic segments incubated for 18 hours with or without 10 µmol/L atorvastatin (n=4). *P<0.05 vs control.

Effects of HCRIs on SOD and NADPH Oxidase Expression and Activity
None of the HCRIs significantly affected the expression of Cu2+/Zn2+-SOD or Mn2+-SOD mRNA after 6 (Figure 7ADown) or 12 hours exposure (97% to 115% of control) of the segments. Moreover, total SOD activity in homogenates of segments treated with atorvastatin for 18 hours did not differ significantly from that of control segments exposed to vehicle only (Figure 7ADown, inset).



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  		Figure 7. A, RT-PCR analysis of the effects of HCRIs on Cu2+/Zn2+-SOD and Mn2+-SOD mRNA expression in endothelium-intact rat aortic segments after 6 hours exposure to atorvastatin (atorva, 10 µmol/L), cerivastatin (ceriva, 1 µmol/L), or pravastatin (prava, 10 µmol/L) (n=4). The inset depicts the lack of effect of atorvastatin (10 µmol/L) on total SOD activity after 18 hours (n=4). B, Basal NADPH oxidase activity in homogenates of aortic segments incubated for 18 hours with atorvastatin (10 µmol/L) or pravastatin (10 µmol/L) (n=7). The inset shows the NADPH oxidase activity in homogenates of endothelium-intact (E+) and denuded (E-) aortic segments. The data are expressed as means of 2 separate experiments.

Expression of the NADPH oxidase subunit gp91-phox was also not significantly altered after exposure of the segments to the HCRIs for 3, 6, or 12 hours (atorvastatin: 86%, 130%, and 133% of control; cerivastatin: 124%, 135%, and 130% of control; pravastatin: 100%, 132%, and 143% of control; n=3, P>0.05). In addition, exposure to atorvastatin or pravastatin (10 µmol/L) for 18 hours showed no significant change of basal NADPH oxidase activity in homogenates of these segments (Figure 7BUp), which was largely (up to 80%) endothelium-dependent, as shown in the inset of Figure 7BUp.

Effects on PKC
To verify that the HCRIs have no direct effect on PKC activity, which may be inferred from the lack of effect of atorvastatin on PDB-stimulated O2- formation within 1 hour after its administration (see Figure 3BUp), we investigated the effect of atorvastatin on a semipurified PKC fraction from rat cerebellum as well as on the PKC activity present in homogenates prepared from the rat aorta and cultured PAECs. In concentrations of up to 100 µmol/L, atorvastatin revealed no inhibitory effect on PKC activity either immediately or after an 18-hour incubation period with the cells or segments (not shown). Moreover, as detected by Western blot analysis, exposure to atorvastatin for 18 hours also did not affect the abundance of PKC isoforms {alpha}, ß, {delta}, and {epsilon}, all of which have been implicated in phorbol ester–stimulated NADPH oxidase assembly, and hence activity,30 in the particulate and soluble fraction of the cultured PAECs (not shown).

Effects of Clostridium Toxins on PDB-Stimulated O2- Formation
Because there was no apparent effect of the HCRIs on the abundance of the major O2--metabolizing or -producing enzymes, the hypothesis was next investigated that HCRIs affect the PKC-dependent assembly of NADPH oxidase rather than its activity. To this end, the effect of Clostridium sordellii lethal toxin on PDB-stimulated O2- formation was investigated. This toxin glucosylates and thereby inactivates p21 Ras and Rac,31 32 the latter of which is essential for NADPH oxidase activity in intact phagocytes33 34 and therefore potentially mimics the effects of the HCRIs on endothelial NADPH oxidase activity. Indeed, exposure of the segments to the toxin for 4 hours resulted in a marked inhibition (86%) of PDB-stimulated O2- formation (Figure 8Down), whereas the heat-inactivated toxin had no such effect. In contrast, exposure of the segments for 18 hours to Clostridium botulinum exoenzyme (C3), which ADP ribosylates, hence inactivating p21 Rho, did not affect PDB-stimulated O2- formation (Figure 8Down).



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  		Figure 8. Effect of atorvastatin (atorva, 10 µmol/L, 18 hours, n=3), Clostridium sordellii lethal toxin (LT+) (0.5 µg/mL, 4 hours, n=9), the heat-inactivated toxin (LT-) (n=9), or Clostridium botulinum exoenzyme (C3) (10 µg/mL, 18 hours, n=3) on PDB-stimulated O2- formation in endothelium-intact rat aortic rings estimated by lucigenin-enhanced chemiluminescence. *P<0.05 vs control; {dagger}P<0.05 vs LT+.

Effects of Atorvastatin on NO-Mediated Endothelium-Dependent Relaxation
Finally, the effect of atorvastatin on endothelial function in the rat aorta was evaluated. Endothelium-intact segments were incubated with and without atorvastatin for 18 hours, after which the relaxant response to acetylcholine, which under the chosen experimental conditions is mediated almost exclusively by NO, was assessed. Pretreatment with atorvastatin indeed significantly augmented the acetylcholine-induced relaxation compared with control segments (Figure 9ADown), and this effect was no longer apparent when the control segments were assayed in the presence of SOD in the superfusate (Figure 9BDown). SOD, on the other hand, did not affect the relaxant response in segments pretreated with atorvastatin.



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  		Figure 9. Concentration-response curves showing relaxation to acetylcholine (ACh) of phenylephrine-contracted endothelium-intact rings of the rat aorta after 18 hours incubation in the presence or absence (control) of 10 µmol/L atorvastatin (atorva). A, Superfusion in the absence (n=11) and B, in the presence of SOD (100 U/mL) in the superfusate (n=8). *P<0.05 vs control.


*    Discussion
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 *Discussion
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Starting from the finding that HCRIs improve endothelium-dependent vasodilatation in modestly hyperlipidemic patients within 1 month of therapy,2 35 we hypothesized that HCRIs influence the NO/O2- balance in the vessel wall, which seems to be shifted toward O2- in CHD.36 The present findings show that although they do not have a direct antioxidative effect, HCRIs effectively lower the NADPH oxidase–dependent O2--forming capacity of native endothelial cells. That endothelial NADPH oxidase was indeed the source of O2- in the rat aorta under the chosen experimental conditions was evidenced by the following findings: (1) O2- formation was almost exclusively endothelium-dependent; (2) according to CD62L RT-PCR, no appreciable leukocyte contamination was present; (3) O2- formation was stimulated by activation of PKC, which facilitates assembly of the active NADPH oxidase complex by phosphorylating p47-phox35 ; (4) PKC-mediated O2- formation, conversely, was sensitive to AEBSF and PAO, two inhibitors of NADPH oxidase assembly in phagocytes27 28 ; (5) it was substantially inhibited by SOD, hence arguing for a cell membrane–bound oxidase; and most importantly, (6) it was strongly attenuated by Clostridium sordellii lethal toxin but not by Clostridium botulinum C3 exoenzyme.

Rho GTPases are substrates for bacterial toxins. Whereas Clostridium botulinum C3 exoenzyme ADP-ribosylates Rho A, B, and C at Asn-41, Clostridium difficile toxins A and B monoglucosylate Rho, Rac, and Cdc42 at threonine-37/35.37 Clostridium sordellii lethal toxin is also a monoglucosyltransferase, which, however, glucosylates Rac but not Rho and Cdc42 and, in addition, GTPases from the Ras subfamily (Ras, Ral, Rap).31 32 Glucosylation renders the GTPases inactive by blocking the interaction with downstream effectors.20 37 Toxin A/B and Clostridium sordellii lethal toxin enter the cell via receptor-mediated endocytosis and exhibit their enzymatic activity in the cytosol of the target cells. The inhibition of PKC-mediated O2- formation by Clostridium sordellii lethal toxin but not Clostridium botulinum C3 exoenzyme, therefore, suggests that the rat aortic endothelium contains a phagocyte-like NADPH oxidase, the activation of which is also Rac-dependent. However, as previously demonstrated,38 this oxidase seems to prefer NADH over NADPH as the reducing equivalent for O2- formation. Recently, it was shown that a functional phagocyte-type NADPH oxidase is expressed in cultured rat coronary microvascular endothelial cells,39 whereas immunohistochemical staining localized several NADPH oxidase subunits exclusively to the adventitia and not to the media of the rat thoracic aorta.40 In our hands, NADPH oxidase–dependent O2- formation appeared to be localized predominantly to the luminal endothelium, because denuded segments displayed virtually no phorbol ester–inducible generation of O2-. The most likely reasons why we were able to detect luminal O2- formation in these segments are that (1) these were rather short (5 to 7 mm) and (2) we used a luminometer with a cavity lined with mirrors so that any flash of light is ultimately picked up by the photomultiplier.

Finally, the finding that the inhibitory effect of atorvastatin on PDB-stimulated O2- formation was prevented in the presence of an excess of the HMG-CoA reductase product mevalonic acid, not only in the endothelium-intact aortic segments but also in cultured endothelial cells and macrophages from different species, suggests that the HCRI class of compounds indeed targets the assembly of NADPH oxidase by preventing the isoprenylation of p21 Rac. This conclusion is also supported by the time-dependency of the inhibitory action of these drugs, which excludes a direct anti-oxidative or PKC-blocking effect. Moreover, there was indeed no effect of the HCRIs on PKC activity and expression of the PKC isoforms thought to be involved in phorbol ester–stimulated O2- formation,30 either immediately or after incubation of the rat aortic segments or cultured endothelial cells for 18 hours.

One may argue that if p21 Rac is the target of the HCRIs, there must be a concomitant decrease in phorbol ester–stimulated NADPH oxidase activity in homogenates of the rat aorta that we did not detect. The most likely reason for this result is that the phorbol ester–induced assembly of the enzyme complex is disrupted during homogenization and centrifugation of the tissue samples, so that only the residual NADPH oxidase activity (ie, without p21 Rac) can be detected, which may be considered an index for the abundance of gp91-phox and p22-phox rather than phorbol ester–stimulated NADPH oxidase activity.

One may further argue that in vivo, phorbol ester–stimulated O2- formation is a rare event that is rather different from basal NADPH oxidase activity, although it seems difficult to judge which type of O2- formation (basal or stimulated) is indeed relevant (eg, for patients with CHD). This is an important point, because physiological stimuli formerly thought to modulate only endothelial NO (and prostacyclin) formation, such as an increase in shear stress or pulsatile stretch, also enhance endothelial superoxide production,41 42 43 and PKC activation may play an important role therein.

Further evidence for the conclusion that the small G proteins, in particular p21 Rac, are the targets of the HCRIs comes from the recent finding of a prenylation inhibition–dependent proapoptotic effect of atorvastatin in rat aortic cultured smooth muscle cells that appears to involve p21 Rho.9 In addition, the prenylation inhibition–dependent increase in tissue plasminogen activator activity in rat aortic cultured endothelial cells elicited by lovastatin also seems to involve p21 Rho.10 Furthermore, an NO-mediated effect of the HCRIs on NADPH oxidase expression or activity is unlikely, because we could not detect an effect of L-NMMA and L-NNA (ie, inhibition of endogenous NO synthesis) on either basal or PDB-stimulated O2- formation even after prolonged incubation. Finally, the lack of effect of the HCRIs on the abundance of the major O2- metabolizing enzymes in the endothelium, Cu2+/Zn2+-SOD and Mn2+-SOD,44 as well as total SOD activity, excludes an inhibition of endothelial O2- formation by the HCRIs via this route.

Apart from reducing endothelial O2- formation, all three HCRIs also upregulated expression of endothelial ecNOS {approx}2-fold and increased calcium-dependent NOS activity in homogenates of the rat aorta to a similar extent, indicative of a parallel increase in NOS protein abundance. Similar findings have recently been described for lovastatin and simvastatin in cultured human saphenous vein endothelial cells,17 so that the increase in endothelial NOS activity can also be regarded as a class effect. Moreover, the fact that comparable findings were obtained with cultured and native endothelial cells from two different species suggests that the same mechanism is also likely to be operative in humans in vivo.

Because of the simultaneous effects of the HCRIs on endothelial NO and O2- formation, it seemed likely that this would result in an improvement of endothelial function. The evaluation of the relative contributions of either of these effects to the anticipated improvement of endothelial function seemed more difficult. Although the rat aorta is not an ideal preparation to answer these questions, because despite addition of polymyxin B, incubation of these segments for several hours frequently leads to an upregulation of inducible NOS expression in the smooth muscle that interferes with endothelium-dependent relaxation,45 the outcome of these experiments was surprisingly clear. Thus, the endothelium-dependent relaxant response to acetylcholine was indeed significantly enhanced after exposure to atorvastatin. Moreover, addition of SOD to the superfusate did not affect the acetylcholine-induced relaxation in atorvastatin-treated segments but significantly improved that in control segments, so that they were no longer distinguishable from each other. Because the relaxant response to acetylcholine under the chosen experimental conditions—there was no significant release of endothelium-derived hyperpolarizing factor in these segments,46 and the release of both prostacyclin and vasoconstrictor prostanoids was blocked by diclofenac—the latter findings suggest that it is the decrease in O2- formation that is responsible for the HCRI-mediated improvement of endothelium-dependent NO-mediated relaxation and not the increase in NO release.

It certainly remains to be determined whether the present findings in the isolated rat aorta can indeed be extrapolated to the situation in patients with CHD, even though all of the effects of the HCRIs have been observed at clinically relevant concentrations. Nonetheless, these data reveal that by affecting the same target, ie, HMG-CoA reductase activity, HCRIs can rapidly improve endothelial function (even in healthy blood vessels) independently of the concurrent decrease in serum LDL cholesterol. A note of caution may therefore be appropriate, that we should not interpret the proven beneficial effects of this class of compounds only in the latter direction. Conversely, the present findings also open up the possibility that, particularly when administered at high doses, HCRIs may produce adverse effects related to inhibition of function of the small G proteins, such as inhibition of cell replication in wound healing or the induction of apoptosis, eg, in vascular smooth muscle cells.9 10

In summary, the present findings demonstrate that HCRIs improve endothelial function, presumably by blocking the p21 Rac–mediated assembly of NADPH oxidase, thereby lowering the capacity of the endothelium to generate O2- and shifting the NO/O2- balance in the vessel wall toward NO. Apart from affecting vascular tone, this increased bioavailability of NO also has important implications for the synthesis of proatherosclerotic gene products by the endothelium (eg, chemokines and adhesion molecules), the expression of which is governed by NO-sensitive transcription factors, such as nuclear factor {kappa}B.47 It would thus appear that the therapeutic effects of HCRIs in patients with CHD are mediated both by the rapid restoration of endothelial function and the intermediate reduction of the lipid load of the vessel wall.


*    Acknowledgments
 
This work was supported by the Deutsche Forschungsgemeinschaft (He 1587/6-1) and a grant from Goedecke/Parke-Davis. The expert technical assistance of Renate Dohrmann is gratefully acknowledged.

Received December 10, 1998; accepted May 11, 1999.


*    References
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