E ects of vitamin C and of a cell permeable superoxide dismutase mimetic on acute lipoprotein induced endothelial dysfunction in rabbit aortic rings

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1 British Journal of Pharmacology (1999) 126, 730 ± 734 ã 1999 Stockton Press All rights reserved 0007 ± 1188/99 $ E ects of vitamin C and of a cell permeable superoxide dismutase mimetic on acute lipoprotein induced endothelial dysfunction in rabbit aortic rings 1 L. Fontana, 1 K.L. McNeill, 1 J.M. Ritter & *,1 P.J. Chowienczyk 1 Department of Clinical Pharmacology, Guy's, King's and St Thomas' School of Biomedical Sciences, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, England 1 Low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. The mechanism is uncertain, but increased production of superoxide anion O 27 with inactivation of endothelium-derived NO and formation of toxic free radical species have been implicated. We investigated e ects of the cell permeable superoxide dismutase mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), the free radical scavenger vitamin C and arginine (which may reduce O 27 formation) on acute LDL-induced endothelial dysfunction in rabbit aortic rings, using LDL prepared by ultracentrifugation of plasma from healthy men and aortic rings from New Zealand white rabbits. 2 LDL (150 mg protein ml 71 for 20 min) markedly inhibited relaxation of aortic rings (in Krebs' solution at 378C and pre-constricted to 80% maximum tension with noradrenaline) to acetylcholine 82+10% (mean percentage di erence between sum of relaxations after each concentration of acetylcholine in the presence and absence of LDL, +s.e.mean, n=26, P50.001) but not to the endothelium-independent agonist nitroprusside. 3 MnTMPyP (10 mm) reduced inhibitory e ects of LDL from to 56+17% (n=6, P50.05). 4 Vitamin C (1 mm) reduced inhibitory e ects of LDL from 59+8 to22+5% (n=6, P50.05). 5 Inhibitory e ects of LDL were similar in the absence or presence of arginine (84+12 vs 79+16%, n=14, P=0.55). E ects of L-arginine (10 mm) did not di er signi cantly from those of D-arginine (10 mm). 6 Acute (20 min) exposure of aortic rings to LDL impairs endothelium-dependent relaxation which can be partially restored by MnTMPyP and vitamin C. This is consistent with LDL causing increased O 27 generation. Keywords: Antioxidants; arginine; endothelium; low density lipoprotein; nitric oxide; oxidative stress; superoxide anion; superoxide dismutase; vitamin C Abbreviations: cnos, constitutive NO synthase; LDL, low density lipoprotein; MnTMPyP, manganese (III) tetrakis (1-methyl- 4-pyridyl) porphyrin; NO, nitric oxide; O 7 2, superoxide anion; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances Introduction Low-density lipoprotein (LDL) acutely inhibits endotheliumdependent relaxation in rabbit aortic rings (Jacobs et al., 1990) and, in men with increased serum concentrations of LDLcholesterol, impaired endothelium-dependent relaxation occurs before development of structurally apparent atherosclerotic lesions (Chowienczyk et al., 1992; Zeiher et al., 1991). Since endothelium-derived relaxing factors, nitric oxide (NO) in particular, have antiatherogenic actions (Cooke & Tsao, 1994), LDL induced endothelial dysfunction may play a causal role in atherogenesis. The mechanism whereby LDL inhibits endothelium-dependent relaxation remains uncertain, but may involve increased formation of superoxide anion (O 27 ) (Ohara et al., 1993) with consequent inactivation of NO (Gryglewski et al., 1986). However Cu-Zn superoxide dismutase (SOD) does not restore endothelial function either in vitro following acute exposure to LDL (Plane et al., 1993) or in vivo (GarcõÂ a et al., 1995). This may be because Cu-Zn SOD does not gain access to intracellular O 27. To elucidate the role of O 27 in lipoprotein induced endothelial dysfunction we examined e ects of the cell permeable superoxide dismutase (SOD) mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) * Author for correspondence; p.chowienczyk@umds.ac.uk porphyrin (MnTMPyP) on the inhibitory e ects of LDL on endothelium-dependent relaxation in an isolated rabbit aortic ring preparation. We also examined e ects of vitamin C which 7 may be e ective in scavenging intra- and extra-cellular O 2 (Som et al., 1983; Halliwell & Gutteridge, 1989). Acute administration of L-arginine restores endothelium-dependent relaxation in both hypercholesterolaemic animals (Cooke et al., 1991) and humans (Drexler et al., 1991), possibly as a result of decreasing O 27 production by constitutive NO synthase (cnos) (Pritchard et al., 1995) or by an antioxidant e ect (Nagasee et al., 1997). An antioxidant e ect has been seen with both L- and D-arginine (Nagasee et al., 1997) whereas the e ect on cnos is assumed to be speci c to L-arginine. We, therefore, examined the ability of L- and D-arginine to prevent inhibitory e ects of LDL on endothelium-dependent relaxation in this preparation. Methods Preparation of lipoproteins Native LDL was isolated from healthy men (aged 24 ± 55 years). Venous blood was collected into vacutainers containing

2 EDTA. Plasma was separated by low-speed centrifugation at 48C and LDL (density, ± g ml 71 ) then isolated from the plasma by discontinuous density gradient ultracentrifugation (Chung et al., 1980). Isolated LDL was then dialysed with continuous stirring at 48C for 24 h against two changes of 10 mm phosphate bu er solution (ph 7.4). The protein concentration was measured (Lowry et al., 1951) and the nal concentration of LDL expressed as mg protein ml 71. Oxidative modi cation of LDL was prevented by the inclusion of EDTA (0.3 mm) in all bu ers (Jacobs et al., 1990). Oxidation of LDL was sought by measurement of thiobarbituric acid reactive substances (TBARS) (Yagi, 1976). TBARS were below the limit of detection being less than 1% of values obtained when samples were oxidized by exposure to Cu 2+ (CuSO 4, 1.7 mm) for 3 h. Experimental protocols L. Fontana et al New Zealand white male rabbits (2 ± 2.5 Kg) were sacri ced to obtain the descending thoracic aorta, which were trimmed of adhering tissue and fat. Transverse 2-mm-wide rings were cut and mounted in 3-ml organ baths containing oxygenated Krebs' solution (+EDTA, 0.3 mm), at 378C. Tissues were placed under 2 g resting tension for 60 min and tension adjusted to 2 g for a further 30 min. Isometric measurements were recorded via force transducers (Grass FT03, Austria). Tissues were contracted with increasing doses of noradrenaline (0.06 ± 0.12 mm) to determine a concentration which gave 80% maximum contraction. Repeated (2 ± 4) exposures to this concentration were performed to establish that reproducible contractions were obtained. Relaxation dose-response curves to acetylcholine (10 78 ±10 75 M) were then obtained. Following washout, preconstriction with noradrenaline and relaxation to acetylcholine were repeated during incubation with MnTMPyP (10 mm) alone, LDL (150 mg protein ml 71 ) alone and LDL with MnTMPyP. After a nal washout preconstriction and relaxation to acetylcholine was repeated to ensure that baseline responses were maintained over the time course of the experiment. Similar experiments were performed using vitamin C (1 mm), L-arginine (10 mm) and D-arginine (10 mm). A similar protocol was also used to examine e ects of LDL on relaxation to nitroprusside as a non endotheliumdependent control. Doses of MnTMPyP and vitamin C were chosen as the maximum dose which, in pilot studies, had no inhibitory e ects on relaxation to acetylcholine. E ects of Cu- Zn SOD (20 units ml 71 ) were examined in these pilot studies; it was found to have no e ect on relaxation to acetylcholine in the presence or absence of LDL. Antioxidants lipoproteins and endothelial function 731 these summary statistics between the di erent drugs were sought using one way analysis of variance (ANOVA). Repeated measures ANOVA was used to assess whether inhibitory properties of LDL di ered in the presence or absence of drugs and whether this e ect di ered between the di erent drugs. A non-parametric test (paired sign) was also used to test whether inhibitory e ects of LDL di ered in the presence and absence of each of the drugs. Di erences were considered signi cant if P Results In all experiments incubation with LDL markedly inhibited relaxation to acetylcholine (Figures 1 ± 4). The mean inhibition for all experiments was % (n=26, P50.001) and did not di er signi cantly between experiments in which di erent drugs (MnTMPyP, vitamin C, arginine) were used. Incubation with LDL did not signi cantly inhibit relaxation to nitroprusside (per cent inhibition 14+6%, n=6, Figure 5). Relaxation to acetylcholine was signi cantly reduced after incubation with drugs (MnTMPyP, vitamin C, arginine) alone (mean inhibition for all drugs: 18+2%, n=26, P50.001). However, the magnitude of the e ect was small in comparison with the inhibition produced by LDL (82 vs 18%, P50.001) and did Figure 1 Relaxation of pre-contracted rabbit aortic rings (n=6) to acetylcholine at baseline and after 20 min incubation with the cell permeable SOD mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) alone (10 mm), LDL alone (150 mg protein ml 71 ) and LDL plus MnTMPyP. Data analysis and statistical methods Results are expressed as means+s.e.mean. The following summary measures were used to express inhibitory e ects of drugs (D) alone, LDL (L) alone and LDL in the presence of drugs on percentage relaxation to acetylcholine (R ACh ): %inhibition by drug alone = [(S R ACh 7S R ACh+D )/S R ACh ]6100% %inhibition by LDL alone= [(S R ACh 7S R ACh+L )/S R ACh ]6100% %inhibition by LDL in presence of drug= [(S R ACh+D 7S R ACh+D+L )/S R ACh+D ]6100% where S R ACh is the sum of relaxations to each dose of acetylcholine. In addition we calculated values of EC 50 and E max for the acetylcholine relaxation curves. Di erences in Figure 2 Relaxation of pre-contracted rabbit aortic rings (n=6) to acetylcholine at baseline and after 20 min incubation with vitamin C alone (1 mm), LDL alone (150 mg protein ml 71 ) and LDL plus vitamin C.

3 732 L. Fontana et al not di er signi cantly for di erent drugs. Inhibitory e ects of LDL were, overall, less marked in the presence of the drugs (MnTMPyP, vitamin C, arginine: P50.001, for all drugs). There was a signi cant di erence between drugs in their e ects on preventing inhibition by LDL (P50.01). MnTMPyP Figure 3 Relaxation of pre-contracted rabbit aortic rings (n=8) to acetylcholine at baseline and after 20 min incubation with L-arginine alone (10 mm), LDL alone (150 mg protein ml 71 ) and LDL plus L- arginine. Antioxidants lipoproteins and endothelial function produced the most marked e ects reducing inhibitory e ects of LDL from to 56+17% (n=6, P50.05, Figure 1). Vitamin C also reduced inhibitory e ects of LDL from 59+8 to 22+5% (n=6, P50.05, Figure 2). Inhibitory e ects of LDL were similar in the absence or presence of L-arginine (78+12 vs 77+22%, n=8, Figure 3) and in the absence or presence of D-arginine (92+24 vs 80+26%, n=6, Figure 4). MnTMPyP reduced the change in EC 50 following incubation with LDL ( mm in the absence and mm in the presence of MnTMPyP, n=6, P50.01) and the change in E max ( % in the absence and % in the presence of MnTMPyP, n=6, P50.05). Vitamin C also reduced the change in EC 50 following incubation with LDL ( mm in the absence and mm in the presence of vitamin C, n=6, P50.05) and the change in E max ( % in the absence and % in the presence of vitamin C, n=6, P50.01). L- and D-arginine had no signi cant e ect on the change in EC 50 or E max induced by LDL. Nonparametric testing con rmed signi cant e ects of MnTMPyP and vitamin C but not L- or D-arginine on each measure of LDL induced inhibition of relaxation. Following the nal washout and pre-contraction with noradrenaline, relaxation responses to each dose of acetylcholine were not signi cantly di erent from those obtained at baseline. Discussion Figure 4 Relaxation of pre-contracted rabbit aortic rings (n=6) to acetylcholine at baseline and after 20 min incubation with D-arginine alone (10 mm), LDL alone (150 mg protein ml 71 ) and LDL plus D- arginine. Figure 5 Relaxation of pre-contracted rabbit aortic rings (n=6) to sodium nitroprusside before and after 20 min incubation with LDL (150 mg protein ml 71 ). We have examined e ects of lipoproteins on endotheliumdependent relaxation in vessels not previously exposed to raised concentrations of LDL. This allows an important distinction between acute e ects of LDL and those related to established hypercholesterolaemia. In the latter condition responsiveness to endothelium-dependent and -independent agonists may be in uenced by development of atherosclerosis (Verbeuren et al., 1986) and the oxidation of LDL within the vessel wall which may in uence signal transduction mechanisms (Liao & Clark, 1995) and the expression of NO synthase (Liao et al., 1995; Hirata et al., 1995). Our ndings that incubation of rabbit aortic rings with native LDL for 20 min inhibits relaxation to acetylcholine but not relaxation to nitroprusside are in agreement with those of other investigators (Jacobs et al., 1990; Plane et al., 1993). Inhibitory e ects of LDL were seen at a lower concentration (150 mg protein ml 71, within the physiological range for the rabbit) of LDL than those used in most previous studies (5500 mg protein ml 71 ) and although not as marked were highly signi cant. The LDL used in this study was likely to be representative of native LDL because it was protected from oxidation and no oxidation products (TBARS) were detectable. Furthermore the characteristics of the inhibition were similar to those reported by other investigators (Jacobs et al., 1990; Plane et al., 1993) for native LDL. In contrast to the inhibitory properties of native LDL observed in this study, oxidized LDL produces an inhibition of relaxation to acetylcholine which is only partially reversible and it also inhibits relaxation to nitrovasodilators (Jacobs et al., 1990). We cannot, however, exclude the presence of minor degrees of oxidation. Variations in the initial state of spontaneous oxidation and in the antioxidant content of the LDL (obtained from di erent donors) may have accounted for the variability in inhibitory e ects of LDL which tended to be greater in the experiments on MnTMPyP. These factors might also explain why not all investigators have observed inhibitory e ects of LDL on endothelium-dependent relaxation (Galle et al., 1995). In experiments where LDL produced greater inhibitory e ects some contraction was observed in response

4 L. Fontana et al to acetylcholine. This may have resulted from unopposed actions of endothelium derived constricting factors such as prostaglandin H 2 (Kato et al., 1990). The major novel nding of the present study is that, in contrast to Cu-Zn SOD (Plane et al., 1993), the cell permeable SOD mimetic MnTMPyP attenuates the inhibitory e ect of LDL on endothelium-dependent relaxation. MnTMPyP is capable of catalyzing the dismutation of O 27 in vitro with a rate constant *10 7 M 71 s 71 and in vivo the reduced form of MnTMPyP combines with O 27 with a rate constant *10 9 M 71 s 71 (Faulkner et al., 1994). The e ect of MnTMPyP supports the possibility that intracellular generation of O 27 is responsible, at least in part, for LDL induced endothelial dysfunction. Our ndings are consistent with the recent report that, in contrast to authentic SOD, membrane permeable SOD mimetics are capable of restoring endothelium-dependent relaxation following inhibition by an intracellular oxidant stress (MacKenzie & Martin, 1998). The non-enzymic antioxidant vitamin C is also capable of scavenging O 27 (Som et al., 1983). In the present study we found that a high dose of vitamin C partially reversed LDL induced impairment of endothelium-dependent relaxation. The e ect was less striking than that obtained with MnTMPyP, perhaps because at this dose vitamin C produced some impairment of endotheliumdependent relaxation in the absence of LDL. In pilot studies we found that higher doses of both vitamin C and MnTMPyP markedly inhibited relaxation to acetylcholine. We do not have a ready explanation for this e ect but speculate that, at high concentrations, these antioxidants react directly with NO thus impairing endothelium-dependent relaxation as has been demonstrated in the case of a-tocopherol (Keaney et al., 1994). Our ndings are consistent with the observation that brachial artery infusion of vitamin C to achieve concentrations within the forearm that approximate 1 mm improves endothelial function within forearm vasculature of hypercholesterolaemic subjects (Ting et al., 1996) whereas oral administration of vitamin C (producing only a modest increase in plasma concentrations) does not improve endothelial function in such subjects (Gilligan et al., 1994). One possible source of O 27 in hypercholesterolaemia is cnos: when cultured endothelial cells are incubated with LDL for 4 days the activity of cnos remains constant but NO production decreases and O 27 generation increases. This is Antioxidants lipoproteins and endothelial function 733 thought to be due to uncoupling of electron transport within cnos with cnos catalyzing formation of O 27 rather than NO (Pritchard et al., 1995). The e ect is reversed by increasing concentrations of L-arginine. Administration of L-arginine in vivo to the cholesterol-fed rabbit (Cooke et al., 1991) and to hypercholesterolaemic patients (Drexler et al., 1991; Chowienczyk et al., 1994) is capable of restoring endothelial function. D-arginine also has potential antioxidant activity (Nagasee et al., 1997) and in one clinical study tended to improve endothelial function in hypercholesterolaemic patients (Casino et al., 1994). In the present study we found that L- and D- arginine at a dose similar to that used in in vivo studies was not e ective in reversing endothelial dysfunction resulting from a short period of incubation with LDL. One possible reason for this discrepancy is that, over longer periods, LDL results in increased generation of an endogenous inhibitor of cnos which competes with L-arginine as a substrate for cnos (Vallance et al., 1992). Increased concentrations of such inhibitors have been demonstrated in cholesterol-fed rabbits (Yu et al., 1994; Bode-BoÈ ger et al., 1996). The restoration of endothelial function by MnTMPyP but not by L-arginine in the present preparation suggests that cnos may not be the source of the O 27. Superoxidegenerating systems involving xanthine oxidase and NADH oxidoreductase have been described in rabbit aortic and bovine coronary endothelium (Ohara et al., 1993; Mohazzab-H et al., 1994). In the aorta of normocholesterolaemic rabbits the major source of O 27 is an NADPH oxidase located in medial smooth muscle and adventitia (Pagano et al., 1995). However in hypercholesterolaemic rabbits increased O 27 derives mainly from the endothelium (Ohara et al., 1993). 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