Vitamin C Reduces Cholesterol-Induced Microcirculatory Changes in Rabbits
Abstract The microcirculation was studied for 10 weeks in untreated rabbits (n=12) and in rabbits treated with vitamin C in their drinking water (0.5 g/d; n=6), a 1% cholesterol diet (n=12), or a combination of the two treatments (n=11). The studies were performed by direct intravital microscopic imaging of the conjunctiva of both eyes to evaluate blood flow velocity, microvessel diameter, and microhemorheologic conditions. As we reported previously, changes occurred in all of the aforementioned variables as a consequence of cholesterol feeding. After 3 and 6 weeks of feeding, there was a marked and significant (P<.0001) decrease in blood flow velocity in third-order arterioles, which was accompanied by stasis and erythrocyte aggregation in the smaller conjunctival vessels. When cholesterol treatment was combined with vitamin C, blood flow was almost identical to that of controls and significantly (P<.0001) higher than that of rabbits treated with cholesterol alone. All other changes were also significantly reduced by the addition of vitamin C treatment to the cholesterol diet. Cholesterol-treated rabbits developed macroscopic arterial lesions that were not significantly reduced by vitamin C treatment. Neither circulating oxysterol levels nor atheromas were reduced by vitamin C treatment, which also had no significant effect on lipid or circulating vitamin E levels. We have previously shown that the lipid-soluble antioxidant BHT is able to prevent both cholesterol-induced microcirculatory changes and the development of arterial lesions in rabbits. This phenomenon is compatible with a critical oxidation step occurring in the lipid phase that is common to both processes. The finding that microcirculatory changes can be prevented by a water-soluble antioxidant is compatible with a role for water-soluble oxidants in this context. The possibility is discussed that vitamin C might also be important for the microcirculation in humans.
- Received March 22, 1996.
- Accepted July 31, 1996.
The hypothesis that LDL oxidation plays an important role in atherogenesis has been tested in a great number of clinical and animal studies (for a review, see Reference 11 ). The most convincing support for this hypothesis comes from studies with antioxidants. Studies with the antioxidant probucol have shown that it is a more efficient antiatherogenic drug than other comparable lipid-lowering drugs in rabbits.2 3 However, clinical studies with probucol have failed to show a preventive effect on the progression of manifest atherosclerosis.4 We have shown that the antioxidant BHT, despite its propensity to increase serum lipid levels, inhibited the development of atherosclerosis and intimal thickening in cholesterol-fed rabbits.5 6
Oxidative reactions seem to be important not only for atheroma development but also for the microcirculation. Recently, we demonstrated that dietary cholesterol caused a marked decrease in blood flow in third-order arterioles in the rabbit conjunctiva and that this effect could be prevented by treatment with BHT.7 Oxidized LDL has been shown to interfere with the function of NO,8 9 10 and thus, it is possible that the effect of BHT on cholesterol-induced changes in the microcirculation is secondary to the preventive effect of this antioxidant on LDL oxidation. In accordance with this finding, dietary vitamin E was recently shown to restore endothelium-dependent vasodilation in cholesterol-fed rabbits,11 and probucol preserved endothelial function in segments of thoracic aortas from such animals.12
Since oxidation of lipids in the LDL particle occurs in a lipid phase, lipid-soluble antioxidants have generally been used in attempts to interfere with the oxidative modification of LDL. Water-soluble antioxidants such as vitamin C may support the antioxidant effect of vitamin E to some extent, which is believed to be the major physiological antioxidant in LDL particles. If oxidative modification of LDL is a key event in both the development of arterial lesions and microcirculatory changes induced by dietary cholesterol, lipid-soluble antioxidants are likely to be more effective than water-soluble ones. Since the important oxidative event for the microcirculation is less well defined, it is not known whether water-soluble antioxidants can interfere with this process.
In the study presented here, we investigated whether vitamin C interfered with cholesterol-induced development of macroscopic arterial lesions and changes in the microcirculation in rabbits. In contrast to BHT, ascorbate had no effect on atheroma development under our experimental conditions. However, this water-soluble antioxidant did have a significant effect on cholesterol-induced changes in the microcirculation.
Animals and Treatments
Forty-one young, male, New Zealand White rabbits with an average initial weight of 3.0 kg were housed individually under conditions of 12-hour light/dark periods. Twelve rabbits were fed standard chow and normal drinking water for 10 weeks. Another 6 rabbits received standard chow and drinking water supplemented with 500 mg of vitamin C daily. Twelve rabbits were fed standard chow supplemented with l% (wt/wt) cholesterol. A fourth group of 11 rabbits received standard chow supplemented with 1% (wt/wt) cholesterol and drinking water supplemented with 500 mg of vitamin C daily. The rabbit chow pellets were produced by and purchased from AB AnalyCen. The vitamin C supplementation consisted of tablets (C-vitamin sockefri, ACO) dissolved in the first bottle of water each day, after which time the rabbits were given unsupplemented drinking water ad libitum. Two separate sets of experiments were performed. One set of experiments included all 4 rabbit groups (6 rabbits from each group) and measurements at weeks 0, 3, 6, and 10. The second set of experiments included only 3 groups (control, cholesterol-fed, and cholesterol-fed and vitamin C–supplement rabbits) with n=6, 6, and 5 animals, respectively. For the second set of experiments, measurements were made at weeks 0 and 10 only. The group of rabbits treated with cholesterol and vitamin C originally contained 6 rabbits, from which 1 was excluded from study because this animal refused to eat the diet. At the end of the study, the rabbits were killed by an intravenous overdose of fluanizone and fentanyl. The project was approved by the Animal Ethics Committee (Stockholm, Sweden).
Studies of Microvascular Changes
At the start of the study as well as at the end of the 10-week period, the microcirculatory vessels of the conjunctival plexus of both eyes of all rabbits were examined with a long-focus stereomicroscope. Six rabbits in each of the 4 groups were also examined after 3 and 6 weeks. The images were recorded with a video camera as described previously.7 Efforts were made to focus on the same area of conjunctiva on each occasion. A total observation time of at least 2 minutes was used for each rabbit eye. The observations were made without actually touching the conjunctiva while the rabbit was awake and without the use of anesthesia or other drugs. The animals were placed in a standard box for blood sampling during the observations. Recordings and evaluations were performed strictly without any knowledge of which treatment group each rabbit belonged to and with rabbits from the different groups mixed in random order. Measurements and studies were performed on third-order arterioles on the anterior conjunctival artery. The equipment and methods used for image processing have been described previously.7
Evaluation of Blood Flow Velocity
Blood flow velocity in third-order arterioles was measured by means of a computer-generated flying-spot technique.7 The accuracy of this method was found to be 0.07 mm/s at velocities ranging from 0 to 1 mm/s. On each occasion three different measurements were made in each eye, and the blood flow velocity thus obtained (in millimeters per second) was the mean of six measurements for each rabbit. The coefficient of variation was found to be <10% in our previous study.7
Measurement of Microvessel Diameter
An autotracking method was used for measuring microvessel diameter.13 Three independent measurements were made in each eye on each occasion. The arteriolar diameter (in microns) was calculated as the mean of six measurements for each rabbit on each occasion. The coefficient of variation was found to be <10% in our previous study.7
Evaluation of Stasis and Aggregation
The area of the plexus examined was 9 mm2, and efforts were made to focus on the same area of conjunctiva on each occasion. Observations were always made on both eyes in each rabbit. Stasis was defined as the condition in which cellular elements were retained as a mass of blood cells that interrupted blood flow. Stasis was further classified as (1) no stasis or the presence of regions of static blood in vessels with diameters <20 μm or with a flow velocity <300 μm/s in the capillaries; (2) the presence of continuously static blood in vessels with diameters <20 μm or extremely slow flow velocity in the capillaries; and (3) the presence of continuously static blood in the entire corneal plexus network (vessels beyond arterioles of the third degree).
Aggregation was defined as the presence of clumps of cells that were sufficiently cohesive to circulate as a solid mass.7 Aggregation was further classified as (1) no aggregation or the presence of occasional aggregates in small microvessels with a diameter <30 μm; (2) aggregates in as much as 50% of microvessels with a diameter <30 μm; and (3) aggregation in >50% of microvessels with a diameter <30 μm.7
Measurement of Cholesterol and Lipid Levels
Blood samples were drawn from the central ear artery of each rabbit at 0 and 10 weeks. From 24 selected rabbits, blood samples were also drawn at 3 and 6 weeks. After centrifugation the plasma samples were analyzed for cholesterol and triglycerides by standard enzymatic procedures. Hyperlipidemic sera were diluted before assay, and under these conditions there was a linear relation between photometric response and the amount of serum. Commercially available enzymatic assays (Boehringer Mannheim GmbH) were used for quantification.
Measurement of Oxysterols
Oxysterol concentrations were measured in sera or arterial tissue by isotope dilution–mass spectrometry with the use of deuterium-labeled internal standards as described previously.14
Measurement of Vitamin C Levels
Vitamin C levels were determined as described by Vuilleumier and Keck.15 This method is specific for vitamin C itself and does not measure its semidehydro form.
Analysis of Lesion Extent in the Aorta
Immediately after each rabbit was killed, that portion of the aorta from the aortic valves to the renal arteries was removed and cut open longitudinally. The area between the first and seventh intercostal artery of each aorta was photographed and coded. The areas were subsequently evaluated by morphometric techniques without knowledge of the treatment group assignment for each rabbit. Lesion area was calculated as the percent of aortic area examined.
Systolic blood pressure (SBP) was measured in one of the forelimb extremities by the strain gauge technique. The strain gauge was placed around the distal part of the extremity. A BP cuff was placed around the extremity ≈3 cm proximal to the strain gauge. The size of the balloon in the cuff was 2×85 to 25×110 mm, depending on the size of the extremity. The cuff pressure was rapidly raised to a suprasystolic level and then slowly reduced. SBP was defined as the cuff pressure recorded when an increase in distal-extremity volume appeared.
All analyses were done with a parametric or nonparametric ANOVA as appropriate (ie, blood flow and diameter with a parametric ANOVA and stasis and aggregation with a nonparametric ANOVA). First, an overall factorial repeated-measures ANOVA was performed with the 4×6 rabbit groups. A detailed analysis was performed only if this overall ANOVA was significant, which was determined by parametric ANOVA or the Kruskal-Wallis test for the nonparametric situation.
During the experiment the weights of the 4 sets of rabbits increased from an average of 3.0 to 3.7 kg. This increase in weight was slightly lower for both rabbit groups that were given dietary cholesterol (average final weight, 3.5 kg) than those fed the control diet with or without vitamin C (average final weight, 4.0 kg). Plasma lipid levels increased in the 2 sets of rabbits given cholesterol and cholesterol plus vitamin C (P<.0001), whereas no effect was seen on either cholesterol or triglycerides in the plasma of rabbits treated with vitamin C only. During the 10-week study, the rabbits that were fed cholesterol-supplemented pellets without vitamin C increased their cholesterol levels from 1.0±0.1 to 68±6 mmol/L (P<.0001) and triglyceride levels from 1.3±0.1 to 4.5±1.0 mmol/L (mean±SEM, P=.01). The rabbits that were fed cholesterol-supplemented pellets and vitamin C (in their drinking water) increased their cholesterol levels to as much as 63±6 mmol/L (P<.0001) and triglycerides to 3.9±1.2 mmol/L (P>.05). Thus, vitamin C treatment had no significant effect on either serum cholesterol or serum triglyceride levels (P>.05). Vitamin C also had no significant effect on serum cholesterol and triglyceride levels in rabbits that were fed the control diet (P>.05).
Vitamin C Consumption and Circulating Antioxidant Levels
Rabbits that were treated with vitamin C consumed most of the drinking water that contained the vitamin. Vitamin C concentrations in the drinking water decreased gradually with time due to oxidation during the consumption period and in the last portions were about half of the original concentration. It was estimated that each rabbit consumed a minimum of 0.3 g daily of intact vitamin C. Treatment increased circulating levels of vitamin C from 16±2 to 38±8 μmol/L in the cholesterol-fed rabbits (P<.05). The vitamin E concentration was 7.6±1.8 μmol/L in the control group and 7.5±1.1 μmol/L in the rabbits treated with vitamin C only. It is known that hyperlipidemia increases circulating levels of fat-soluble vitamins,5 16 and in accordance with this, considerably higher serum levels of vitamin E were found in the cholesterol-treated than the control rabbits. Rabbits treated with cholesterol only had vitamin E levels of 31±3 μmol/L, and rabbits treated with both cholesterol and vitamin C had vitamin E levels of 26±4 μmol/L. Thus, vitamin C had no significant effect on vitamin E levels in any of the groups of animals (P>.05).
During the course of the study, several changes in the microcirculation were observed. It should be noted that 24 rabbits were investigated during weeks 3 and 6, whereas an additional 17 rabbits were investigated before treatment and after 10 weeks of treatment (see “Methods”). Among the microcirculatory variables, blood flow velocity can probably be regarded as the most reliable owing to the fact that accurate measurements can be made and relatively great effects obtained.7
As previously described,7 blood flow velocity decreased markedly during treatment with cholesterol compared with the other regimens (P=.0009; Fig 1⇓). This decrease was highly significant when compared with controls (P=.0002). The decrease was maintained through the sixth week of treatment but was attenuated after 10 weeks of treatment, possibly due to adaptive mechanisms. Blood flow, however, was still significantly lower in the cholesterol group than in all other groups. When cholesterol was combined with vitamin C treatment, blood flow was almost identical to that of controls and significantly (P<.0001) higher than that of rabbits treated with cholesterol alone. Vitamin C had no significant effect on blood flow in normolipidemic animals (results not shown).
In accordance with previous work, arteriolar diameter also decreased significantly during cholesterol treatment compared with the other regimens (P<.0001; Fig 2⇓). This decrease was also attenuated between weeks 6 and 10 and paralleled the changes in blood flow. After 10 weeks of treatment, there was still a significant difference in arteriolar diameter between the cholesterol-treated and the untreated rabbits (P=.01). Vitamin C treatment appeared to have a preventive effect on the cholesterol-induced reduction in arteriolar diameter after 3 and 6 weeks of treatment but not after 10 weeks of treatment (Fig 3⇓). The difference in arteriolar diameter between rabbits treated with cholesterol and those treated with cholesterol plus vitamin C was highly significant (P=.006) after 6 weeks of treatment. Vitamin C had no significant effect on arteriolar diameter in normolipidemic rabbits.
As expected,7 cholesterol-fed animals showed increased stasis in their blood vessels compared with control animals (Fig 3⇑). This stasis was maximal after 6 weeks of treatment and decreased slightly after 10 weeks of treatment. The changes in the cholesterol-treated rabbits were significantly different from those of control animals (P<.003). Vitamin C had a protective effect on cholesterol-induced changes, and the difference in stasis between rabbits treated with cholesterol and those treated with cholesterol and vitamin C was significantly different (P<.007 at 3 and 6 weeks and P=.04 at 10 weeks). Vitamin C had no effect on the occurrence of stasis in normolipidemic rabbits (Fig 3⇑).
Cholesterol treatment also induced erythrocyte aggregation in microvessels whose diameters were <30 μm (P=.0005; Fig 4⇓). In this case also, addition of vitamin C had a preventive effect (P=.02). Vitamin C had no effect on the occurrence of aggregation in normolipidemic animals (Fig 4⇓).
Effect of Cholesterol Treatment on Arterial Lesion Development
As expected, cholesterol treatment led to significant development of macroscopic arterial lesions. In the group of rabbits treated with cholesterol only, mean atherosclerotic involvement was 24±7%. Additional treatment with vitamin C did not have a preventive affect on atherosclerosis, and mean aortic involvement was 22±9% in this latter group of animals.
Effect of Cholesterol Treatment on BP and Sedimentation Rate
To evaluate whether cholesterol treatment had more general effects on the circulation, BP was measured in cholesterol-treated rabbits and matched controls. SBP was found to be 106±2 mm Hg in the cholesterol-treated rabbits and 119±6 mm Hg in controls (P=.04). To evaluate the possible effects of cholesterol treatment on erythrocyte aggregation in vitro, sedimentation rate was measured in blood from cholesterol-treated rabbits and matched controls. The sedimentation rate was found to be 2.1±0.1 mm in cholesterol-treated rabbits and 1.6±0.04 mm in controls (P>.05).
Content of Oxysterols in the Circulation and Atheromas
As expected (see Reference 77 ), cholesterol treatment led to increased levels of oxysterols (Table⇓). Vitamin C, however, had no effect on these oxysterol levels. Atheromas from rabbits treated with cholesterol had significant amounts of oxysterols. In accordance with our previous work,7 aortas from the cholesterol-treated rabbits were found to contain both 7-oxygenated oxysterols (7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-oxo-cholesterol) and 5,6-oxygenated oxysterols (cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, and cholestan-3β,5α,6β-triol). The amount of 7-oxygenated oxysterols was found to be 0.90±0.05 ng/μg cholesterol, whereas the amount of 5,6-oxygenated oxysterols was 0.64±0.15 ng/μg cholesterol. Corresponding levels in the aortas of rabbits treated with both cholesterol and vitamin C were 1.16±0.16 and 0.62±0.07 ng/μg cholesterol. The differences between the two groups of animals were not statistically significant (P>.05).
The cholesterol-induced effects on the conjunctival microcirculation in the rabbit were the same as those demonstrated in our previous work, ie, reduced blood flow, decreased diameter of the arterioles, increased aggregation of erythrocytes, and increased stasis.7 Blood flow velocity is the most reliable and sensitive quantitative microcirculatory parameter. It is noteworthy that the cholesterol-induced microcirculatory changes were not associated with marked changes in SBP. The preventive effect of vitamin C treatment on the cholesterol-induced changes in blood flow velocity was highly significant. As expected, relative changes in the diameter of the microvessels induced by cholesterol were smaller but still statistically significant. However, the results of these measurements also support the contention that vitamin C has a preventive effect on cholesterol-induced changes.
It is well established that vitamin C is important for cholesterol metabolism in mammals (for a general review see Reference 1717 ). The role of vitamin C is most easily evaluated in species like guinea pigs, which are unable to synthesize vitamin C. Under normal conditions, species like rats and rabbits seem to synthesize vitamin C in sufficient amounts. Cholesterol feeding, however, may induce oxidative stress, and it has been shown that intake of exogenous cholesterol causes increased utilization of the vitamin.18 These same studies have also shown that metabolic stress caused by dietary cholesterol can be counteracted by consuming larger amounts of vitamin C.18 Very recently, it has been reported that utilization of vitamin C in guinea pigs is markedly enhanced by the combined effects of cholesterol and other constituents (eg, fat or sugar) of the Western diet.19 The clear effect of vitamin C supplementation on the microcirculation of hyperlipidemic rabbits in the present study is consistent with an increased requirement for the vitamin under certain conditions. It has been reported in some17 20 but not all21 studies that elevated serum cholesterol levels in rabbits after a load of dietary cholesterol is significantly reduced by vitamin C supplements. In the present study, administration of vitamin C had no significant effect on circulating cholesterol levels.
The effect of vitamin C may be due to interference with either LDL oxidation or a process that is induced by oxidized LDL. A key factor controlling the rate of LDL oxidation seems to be its content of peroxy radical scavengers. It has been shown that 80% of the antioxidant capacity of the LDL particle can be accounted for by α-tocopherol.22 There is, however, a synergistic interaction between vitamin C and α-tocopherol, and α-tocopherol can be regenerated from the corresponding radical by vitamin C.23 24 The resulting oxidation product of vitamin C, the semidehydroascorbate radical, can be converted back to the parent compound by the enzyme semidehydroascorbate reductase. In our study, vitamin C treatment had no significant effect on the levels of circulating vitamin E.
Both water- and lipid-soluble peroxy radicals have been detected during LDL peroxidation, but the relative importance of these radicals for propagation of the chain reaction in the lipid phase of the particle is unclear. It has been suggested that vitamin C has an inhibitory effect on LDL oxidation by either scavenging peroxy radicals directly or acting synergistically with the α-tocopherol endogenous to the particle. In accordance with this scenario, vitamin C treatment has been found to increase LDL resistance to oxidation in vitro in rabbits.25 Vitamin C deficiency is known to increase CCl4-induced lipid peroxidation in vivo in guinea pigs,26 and vitamin C supplementation decreases lipid peroxidation in vivo in rats that have an iron overload.27 It has also been shown that vitamin C, in both its reduced and oxidized forms, may act as a preventive antioxidant against metal ion–mediated LDL oxidation.28 Human subjects with low vitamin C levels have been reported to have higher amounts of lipid peroxides in plasma than do subjects with high vitamin levels.24
Supplementation with vitamin C in the drinking water did not prevent development of cholesterol-induced atheromas in rabbits in the present work. These results are in accordance with those obtained in a recent similar study in rabbits, in which a combination of α-tocopherol and vitamin C was used.25 Furthermore, circulating α-tocopherol levels were not affected by ascorbate treatment, and the amounts of oxidized lipids in the circulation and atheromas were about the same as in animals fed cholesterol alone. In a previous study,5 we showed that the lipid-soluble antioxidant BHT was able to reduce both the development of arterial lesions and the levels of oxysterols in atheromas in rabbits treated with dietary cholesterol. It is thus plausible that under the specific conditions employed here, the scavenging of radicals in the lipid phase is less effective by vitamin C than by BHT. Oxidants occurring in a lipid phase may thus be more important than those confined to an aqueous phase for development of atheromas in the experimental model used.
The preventive effect of vitamin C observed on cholesterol-induced microcirculatory changes might be ascribed to a scavenging of radicals in an aqueous phase. We can only speculate about the nature of these radicals. Since it is well documented that endothelium-dependent vasodilation mediated by NO is inhibited by oxidized LDL8 9 and that a lipid-soluble antioxidant is able to prevent microcirculatory changes,7 it seems likely that the critical oxidant in the aqueous phase is primarily derived or induced by oxidized lipid products. Among the different oxidants that may be derived from lipid oxidation and are possibly reducible by ascorbate in an aqueous phase, superoxide anion is a likely candidate. It has been shown that hypercholesterolemia increases superoxide anion production, probably in part by its effects on xanthine oxidase.30 31 The superoxide anions produced may then reduce the endothelium-dependent vasodilation mediated by NO, most probably by oxidative inactivation. In accordance with this hypothesis, treatment of cholesterol-fed rabbits with polyethyleneglycolated superoxide dismutase has been shown to improve the microvascular response to acetylcholine.32 Furthermore, the preserving effect of probucol on endothelial function in cholesterol-feed rabbits seems to be associated with reduced generation of superoxide anions.12 In view of all this, it seems likely that the positive effect of ascorbate on cholesterol-induced microcirculatory changes may be due at least in part to reduction of superoxide anion.
It should be pointed out that NO itself has lipophilic properties and can penetrate plasma membranes. However, the NO generated from endothelial cells can be released into the aqueous phase, diffuse outward, and interact with platelets and vascular smooth muscle cells.31 Thus, it is possible that superoxide anions or some other oxidants may interact with NO in the aqueous phase and that this interaction can be modified by water-soluble antioxidants.
To summarize, it seems possible to interfere with cholesterol-induced microcirculatory changes by two different strategies: reduction of lipid oxidation by lipid-soluble antioxidants and scavenging of oxidizing radicals in a water phase.
Vitamin C had little or no effect on the microcirculation in rabbits that were fed the control diet, indicating that the levels of water-soluble antioxidants are not critical to the microcirculation under normal conditions in this species. During periods of oxidative stress, however, levels of such antioxidants may be critical. In species that do not synthesize vitamin C, like humans and guinea pigs, the importance of vitamin C status may be even greater. Experiments designed to study this possibility are in progress.
This work was supported by the Marianne and Marcus Wallenberg Foundation, King Gustaf V and Queen Victoria Foundation, the Swedish Margarine Industry's Association for Nutritional Physiological Research, the Swedish Lung Foundation, Forenade Liv Mutual Group Life Insurance Co, and the Swedish Medical Research Council.
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