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. 1999 Feb;126(3):730–734. doi: 10.1038/sj.bjp.0702331

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

L Fontana 1, K L McNeill 1, J M Ritter 1, P J Chowienczyk 1,*
PMCID: PMC1565839  PMID: 10188985

Abstract

  1. Low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. The mechanism is uncertain, but increased production of superoxide anion O2− with inactivation of endothelium-derived NO and formation of toxic free radical species have been implicated. We investigated effects 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 O2− 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 μg protein ml−1 for 20 min) markedly inhibited relaxation of aortic rings (in Krebs' solution at 37°C and pre-constricted to 80% maximum tension with noradrenaline) to acetylcholine 82±10% (mean percentage difference between sum of relaxations after each concentration of acetylcholine in the presence and absence of LDL, ±s.e.mean, n=26, P<0.001) but not to the endothelium-independent agonist nitroprusside.

  3. MnTMPyP (10 μM) reduced inhibitory effects of LDL from 124±27 to 56±17% (n=6, P<0.05).

  4. Vitamin C (1  mM) reduced inhibitory effects of LDL from 59±8 to 22±5% (n=6, P<0.05).

  5. Inhibitory effects of LDL were similar in the absence or presence of arginine (84±12 vs 79±16%, n=14, P=0.55). Effects of L-arginine (10 mM) did not differ significantly 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 O2− generation.

Keywords: Antioxidants, arginine, endothelium, low density lipoprotein, nitric oxide, oxidative stress, superoxide anion, superoxide dismutase, vitamin C

Introduction

Low-density lipoprotein (LDL) acutely inhibits endothelium-dependent relaxation in rabbit aortic rings (Jacobs et al., 1990) and, in men with increased serum concentrations of LDL-cholesterol, 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 (O2−) (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 (García et al., 1995). This may be because Cu-Zn SOD does not gain access to intracellular O2−. To elucidate the role of O2− in lipoprotein induced endothelial dysfunction we examined effects of the cell permeable superoxide dismutase (SOD) mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) on the inhibitory effects of LDL on endothelium-dependent relaxation in an isolated rabbit aortic ring preparation. We also examined effects of vitamin C which may be effective in scavenging intra- and extra-cellular O2− (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 O2− production by constitutive NO synthase (cNOS) (Pritchard et al., 1995) or by an antioxidant effect (Nagasee et al., 1997). An antioxidant effect has been seen with both L- and D-arginine (Nagasee et al., 1997) whereas the effect on cNOS is assumed to be specific to L-arginine. We, therefore, examined the ability of L- and D-arginine to prevent inhibitory effects 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 EDTA. Plasma was separated by low-speed centrifugation at 4°C and LDL (density, 1.019–1.063 g ml−1) then isolated from the plasma by discontinuous density gradient ultracentrifugation (Chung et al., 1980). Isolated LDL was then dialysed with continuous stirring at 4°C for 24 h against two changes of 10 mM phosphate buffer solution (pH 7.4). The protein concentration was measured (Lowry et al., 1951) and the final concentration of LDL expressed as μg protein ml−1. Oxidative modification of LDL was prevented by the inclusion of EDTA (0.3 mM) in all buffers (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 Cu2+ (CuSO4, 1.7 μM) for 3 h.

Experimental protocols

New Zealand white male rabbits (2–2.5 Kg) were sacrificed 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 37°C. 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 μM) 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 (10−8–10−5 M) were then obtained. Following washout, preconstriction with noradrenaline and relaxation to acetylcholine were repeated during incubation with MnTMPyP (10 μM) alone, LDL (150 μg protein ml−1) alone and LDL with MnTMPyP. After a final 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 effects of LDL on relaxation to nitroprusside as a non endothelium-dependent control. Doses of MnTMPyP and vitamin C were chosen as the maximum dose which, in pilot studies, had no inhibitory effects on relaxation to acetylcholine. Effects of Cu-Zn SOD (20 units ml−1) were examined in these pilot studies; it was found to have no effect on relaxation to acetylcholine in the presence or absence of LDL.

Data analysis and statistical methods

Results are expressed as means±s.e.mean. The following summary measures were used to express inhibitory effects of drugs (D) alone, LDL (L) alone and LDL in the presence of drugs on percentage relaxation to acetylcholine (RACh):

graphic file with name 126-0702331e1.jpg

where Σ RACh is the sum of relaxations to each dose of acetylcholine. In addition we calculated values of EC50 and Emax for the acetylcholine relaxation curves. Differences in these summary statistics between the different drugs were sought using one way analysis of variance (ANOVA). Repeated measures ANOVA was used to assess whether inhibitory properties of LDL differed in the presence or absence of drugs and whether this effect differed between the different drugs. A non-parametric test (paired sign) was also used to test whether inhibitory effects of LDL differed in the presence and absence of each of the drugs. Differences were considered significant if P<0.05.

Results

In all experiments incubation with LDL markedly inhibited relaxation to acetylcholine (Figures 1234). The mean inhibition for all experiments was 82±9.8% (n=26, P<0.001) and did not differ significantly between experiments in which different drugs (MnTMPyP, vitamin C, arginine) were used. Incubation with LDL did not significantly inhibit relaxation to nitroprusside (per cent inhibition 14±6%, n=6, Figure 5). Relaxation to acetylcholine was significantly reduced after incubation with drugs (MnTMPyP, vitamin C, arginine) alone (mean inhibition for all drugs: 18±2%, n=26, P<0.001). However, the magnitude of the effect was small in comparison with the inhibition produced by LDL (82 vs 18%, P<0.001) and did not differ significantly for different drugs. Inhibitory effects of LDL were, overall, less marked in the presence of the drugs (MnTMPyP, vitamin C, arginine: P<0.001, for all drugs). There was a significant difference between drugs in their effects on preventing inhibition by LDL (P<0.01). MnTMPyP produced the most marked effects reducing inhibitory effects of LDL from 124±27 to 56±17% (n=6, P<0.05, Figure 1). Vitamin C also reduced inhibitory effects of LDL from 59±8 to 22±5% (n=6, P<0.05, Figure 2). Inhibitory effects 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 EC50 following incubation with LDL (3.4±0.76 μM in the absence and 0.67±0.24 μM in the presence of MnTMPyP, n=6, P<0.01) and the change in Emax (51.3±10.9% in the absence and 19.9±8.2% in the presence of MnTMPyP, n=6, P<0.05). Vitamin C also reduced the change in EC50 following incubation with LDL (0.54±0.17 μM in the absence and 0.14±0.03 μM in the presence of vitamin C, n=6, P<0.05) and the change in Emax (26.2±5.8% in the absence and 6.1±2.2% in the presence of vitamin C, n=6, P<0.01). L- and D-arginine had no significant effect on the change in EC50 or Emax induced by LDL. Non-parametric testing confirmed significant effects of MnTMPyP and vitamin C but not L- or D-arginine on each measure of LDL induced inhibition of relaxation. Following the final washout and pre-contraction with noradrenaline, relaxation responses to each dose of acetylcholine were not significantly different from those obtained at baseline.

Figure 1.

Figure 1

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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 μM), LDL alone (150 μg protein ml−1) and LDL plus MnTMPyP.

Figure 2.

Figure 2

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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 μg protein ml−1) and LDL plus vitamin C.

Figure 3.

Figure 3

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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 μg protein ml−1) and LDL plus L-arginine.

Figure 4.

Figure 4

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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 μg protein ml−1) and LDL plus D-arginine.

Figure 5.

Figure 5

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Relaxation of pre-contracted rabbit aortic rings (n=6) to sodium nitroprusside before and after 20 min incubation with LDL (150 μg protein ml−1).

Discussion

We have examined effects of lipoproteins on endothelium-dependent relaxation in vessels not previously exposed to raised concentrations of LDL. This allows an important distinction between acute effects of LDL and those related to established hypercholesterolaemia. In the latter condition responsiveness to endothelium-dependent and -independent agonists may be influenced by development of atherosclerosis (Verbeuren et al., 1986) and the oxidation of LDL within the vessel wall which may influence signal transduction mechanisms (Liao & Clark, 1995) and the expression of NO synthase (Liao et al., 1995; Hirata et al., 1995). Our findings 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 effects of LDL were seen at a lower concentration (150 μg protein ml−1, within the physiological range for the rabbit) of LDL than those used in most previous studies (⩾500 μg protein ml−1) and although not as marked were highly significant. 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 different donors) may have accounted for the variability in inhibitory effects of LDL which tended to be greater in the experiments on MnTMPyP. These factors might also explain why not all investigators have observed inhibitory effects of LDL on endothelium-dependent relaxation (Galle et al., 1995). In experiments where LDL produced greater inhibitory effects some contraction was observed in response to acetylcholine. This may have resulted from unopposed actions of endothelium derived constricting factors such as prostaglandin H2 (Kato et al., 1990).

The major novel finding 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 effect of LDL on endothelium-dependent relaxation. MnTMPyP is capable of catalyzing the dismutation of O2− in vitro with a rate constant ∼107 M−1 s−1 and in vivo the reduced form of MnTMPyP combines with O2− with a rate constant ∼109 M−1 s−1 (Faulkner et al., 1994). The effect of MnTMPyP supports the possibility that intracellular generation of O2− is responsible, at least in part, for LDL induced endothelial dysfunction. Our findings 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 O2− (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 effect was less striking than that obtained with MnTMPyP, perhaps because at this dose vitamin C produced some impairment of endothelium-dependent 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 effect 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 α-tocopherol (Keaney et al., 1994). Our findings 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 O2− 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 O2− generation increases. This is thought to be due to uncoupling of electron transport within cNOS with cNOS catalyzing formation of O2− rather than NO (Pritchard et al., 1995). The effect 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 effective 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-Böger 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 O2−. Superoxide-generating 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 O2− is an NADPH oxidase located in medial smooth muscle and adventitia (Pagano et al., 1995). However in hypercholesterolaemic rabbits increased O2− derives mainly from the endothelium (Ohara et al., 1993). It is likely, therefore, that lipoproteins stimulate the activity of one or more of these systems or, alternatively, that endogenous SOD activity is compromised.

In conclusion we have demonstrated that the cell permeable SOD mimetic MnTMPyP attenuates the inhibitory effects of LDL on endothelium-dependent relaxation. This suggests a possible therapeutic role for effective intracellular scavenging of O2−.

Acknowledgments

This work was supported by a Lilly Diabetes Grant.

Abbreviations

cNOS

constitutive NO synthase

LDL

low density lipoprotein

MnTMPyP

manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin

NO

nitric oxide

O2−

superoxide anion

SOD

superoxide dismutase

TBARS

thiobarbituric acid reactive substances

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