Effects of vitamin C treatment on collar-induced intimal thickening

Abstract

Vitamin C has efficient antioxidant properties and is involved in important physiological processes such as collagen synthesis. As such, vitamin C deficiency leads to serious complications, including vascular diseases. The aim of this study was to investigate the effects of vitamin C treatment on collar-induced intimal thickening. Rabbits were fed a normocholesterolemic diet and a non-occlusive silicon collar was placed around the left carotid artery for 3, 7, and 14 days. The rabbits were treated with or without vitamin C (150 mg/kg/day). Collar-induced intimal thickening became apparent at day 7. The effect of the collar on intimal thickening was more prominent at day 14. Vitamin C treatment significantly inhibited collar-induced intimal thickening at day 14. The placement of the collar around the carotid artery decreased maximum contractile responses against contractile agents (KCl, phenylephrine, 5-hydroxytryptamine). The effect of the collar on contractile responses was enhanced as days elapsed. Decreased contractile responses of collared carotid arteries normalized at day 14 in the vitamin C treatment group. Vitamin C treatment also restored sensitivity to phenylephrine. The collar also significantly decreased acetylcholine-induced relaxations at day 3 and day 7. Acetylcholine-induced relaxations normalized in collared-arteries in the placebo group at day 14. Vitamin C treatment significantly increased acetylcholine-induced relaxations of both normal and collared carotid arteries at day 14. MMP-9 expression increased in collared arteries at day 3 and day 7 but did not change at day 14. MMP-2 expression increased in collared arteries at day 14. However, vitamin C treatment reduced collar-stimulated expression of MMP-2 at day 14. These findings indicate that vitamin C may have potentially beneficial effects on the early stages of atherosclerosis. Furthermore these results, for the first time, may indicate that vitamin C can also normalize decreased contractile response through perivascular collar placement.

Keywords: MMP; collar; intimal thickening; rabbit carotid artery; vascular reactivity; vitamin C.

Figure 1

Effects of collar and vitamin C on vascular morphometry. Notes: Cross-sectional areas of lumen (A), media (B), and EEL (C). (D) Images of hematoxylin-eosin stained rabbit carotid arteries on day 14. Original magnification ×4. Data are expressed as mean ± SEM. *P≤0.05, **P≤0.01 sham-operated vs collared; Wilcoxon signed rank test. Abbreviations: EEL, external elastic lamina; SEM, standard error of the mean.

Figure 2

Effects of collar and vitamin C on intimal thickening. Notes: (A–C) Images of hematoxylin-eosin stained rabbit carotid arteries. (A) Collared arteries from placebo group. (B) Collared arteries from vitamin C group. (C) Sham-operated collared artery from placebo group (14th day). Original magnification ×10. (D, E) Effects of collar and vitamin C on area intima (D) and index value (E). *P≤0.01, **P≤0.01, ***P≤0.001 sham-operated vs collared; Wilcoxon matched signed-rank test. ⁺⁺P≤0.01 placebo vs vitamin C; Mann–Whitney U-test. Data are expressed as mean ± SEM. Abbreviation: SEM, standard error of the mean.

Figure 3

The effects of collar and vitamin C on the vascular contractions. Notes: The effects of collar and vitamin C on the vascular contractions to KCl (A), phenylephrine (B), and serotonin (C). Cumulative dose-response curves (B, C) were made both in placebo-treated sham (○) and collar (□) rings and vitamin C-treated sham (•) and collar (■) rings. Data are expressed as mean ± SEM. **P≤0.01, ***P≤0.001 sham-operated vs collared; Wilcoxon matched signed-rank test. ⁺⁺P≤0.01, ⁺⁺⁺P≤0.001 placebo vs vitamin C; Mann–Whitney U-test. Abbreviations: SEM, standard error of the mean; 5-HT, 5-hydroxytryptamine.

Figure 4

The effects of collar and vitamin C on the vascular relaxations to acetylcholine and nitroglycerin. Notes: Effects of collar and vitamin C on the vascular relaxations to acetylcholine (A) and nitroglycerin (B). Cumulative dose-response curves were made both in placebo-treated sham (○) and collar (□) rings and vitamin C-treated sham (•) and collar (■) rings. Data are expressed as mean ± SEM. ***P≤0.001 sham-operated vs collared; Wilcoxon matched signed-rank test. ⁺P≤0.05 placebo vs vitamin C; Mann–Whitney U-test. Abbreviation: SEM, standard error of the mean.

Figure 5

Effects of collar and vitamin C on MMP-2 expression. Notes: Images of MMP-2 immunohistochemically stained rabbit carotid arteries for MMP-2. (A) Collared arteries from placebo group. (B) Collared arteries from vitamin C group. (C) Sham-operated collared artery from placebo group (14th day). Original magnification ×10. Data are expressed as mean ± SEM. **P≤0.01 sham-operated vs collared; chi-square test. (D) Immunopositivity scores of MMP-2 at day 3, 7 and 14. Abbreviation: SEM, standard error of the mean.

Figure 6

Effects of collar and vitamin C on MMP-9 expression. Notes: Images of MMP-9 immunohistochemically stained rabbit carotid arteries for MMP-9. (A) Collared arteries from placebo group. (B) Collared arteries from vitamin C group. (C) Sham-operated collared artery from placebo group (14th day). Original magnification ×10. Data are expressed as mean ± SEM. *P≤0.05 sham-operated vs collared; chi-square test. (D) Immunopositivity scores of MMP-9 at day 3, 7 and 14. Abbreviation: SEM, standard error of the mean.