Vitamin C Deficiency Exacerbates Dysfunction of Atherosclerotic Coronary Arteries in Guinea Pigs Fed a High-Fat Diet

Abstract

Vitamin C (vitC) deficiency has been associated with an increased risk of cardiovascular disease; while several putative mechanistic links have been proposed, functional evidence supporting a causal relationship is scarce. In this study, we investigated how vitC deficiency affects coronary artery vasomotor function and the development of coronary atherosclerotic plaques in guinea pigs subjected to chronic dyslipidemia by a high-fat diet regime. Female Hartley guinea pigs were fed either a control (low-fat diet and sufficient vitC) (N = 8) or a high-fat diet with either sufficient (N = 8) or deficient (N = 10) vitC for 32 weeks. Guinea pigs subjected to the high-fat diet developed significant atherosclerotic plaques in their coronary arteries, with no quantitative effect of vitC deficiency. In isolated coronary arteries, vasomotor responses to potassium, carbachol, nitric oxide, or bradykinin were studied in a wire myograph. Carbachol, bradykinin, and nitric oxide mediated relaxation in the coronary arteries of the control group. While vasorelaxation to carbachol and nitric oxide was preserved in the two high-fat diet groups, bradykinin-induced vasorelaxation was abolished. Interestingly, bradykinin induced a significant contraction in coronary arteries from vitC-deficient guinea pigs (p < 0.05). The bradykinin-induced contraction was unaffected by L-NAME but significantly inhibited by both indomethacin and vitC, suggesting that, during vitC deficiency, increased release of arachidonic acid metabolites and vascular oxidative stress are involved in the constrictor effects mediated by bradykinin. In conclusion, the present study shows supporting evidence that poor vitC status negatively affects coronary artery function.

Keywords: atherosclerosis; coronary arteries; guinea pig model; vitamin C; wire myography.

Figure 1

Experimental design of in vivo study. Created using Biorender.

Figure 2

Wire myography studies in coronary arteries precontracted with 40 mM potassium and stimulated with (A) the muscarinic receptor agonist (carbachol) or (B) the nitric oxide donor (sodium nitroprusside) to induce endothelium-independent relaxation. (A) Carbachol induced similar relaxation in coronary arteries of the control (CTRL), HFD, and HFDLoC groups, whereas arteries from the HFD group contracted significantly higher concentrations of carbachol compared with the HFDLoC group. (B) Sodium-nitroprusside-induced relaxation was not different between the coronary arteries of the three diet groups. Mean ± SEM, * p < 0.05, ** p < 0.01 HFDLoC vs. HFD determined by 2-way ANOVA and Tukey’s multiple-comparisons test. N, number of animals; n, number of investigated segments.

Figure 3

Concentration–response curves to bradykinin in guinea pig coronary arteries contracted by 40 mM K⁺. Results are expressed as percent change from the plateau contraction to 40 mM K⁺. * Significantly different from HFD. Mean ± SEM, * p < 0.05, ** p < 0.01, **** p < 0.0001 HFDLoC or CTRL vs. HFD determined by 2-way ANOVA and Tukey’s multiple-comparisons test. N, number of animals; n, number of investigated segments.

Figure 4

Concentration–response curves to bradykinin obtained in the absence or (A–C) presence of L-NAME (10 µM), (D–F) indomethacin (10 µM), and (G–I) vitC (75 µM L-ascorbic acid and 100 µM 2-phosphoascorbic acid). Results are expressed as percent change from the plateau contraction to 40 mM K⁺. Mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 without vs. with L-NAME, Indo, or vitC, determined by mixed-effects analysis and Šídák’s comparisons test. N, number of animals; n, number of investigated segments.

Figure 5

Representative examples of coronary arteries from guinea pigs fed with (A) control diet sufficient with vitC (CTRL); atherosclerotic lesions from coronary artery sections (B,E,F) following a fed high-fat diet sufficient in vitC (HFD) or (C,D,G,H) from animals fed a high-fat diet with low vitC (HFDLoC); (A–C,E,G) H&E and (D,F,H) PAS staining. Scale bar =100 µm.

Figure 6

Quantification of intima, lumen, and media as percent of vessel diameter calculated from lamina elastic externa. (A) Intima % versus diameter shows increased intima (above approximately 25%) in coronary arteries from HFD and HFDLoC animals. (B) Average of all measured artery segments from each animal and (C) average of the most stenotic arteries from each animal shows that the average relative intima thickness and the average of the most stenotic arteries from each animal is significantly increased in HFD and HFDLoC compared with CTRL animals. (D) Lumen % versus Intima % shows a negative linear relationship between relative intima thickness and lumen. Increasing intima > 25% further decreases the lumen in HFD and HFDLoC. (G) Media % versus intima % shows a positive correlation between intima and media in CTRL, in contrast to HFD and HFDLoC animals, where increasing intima >25% results in decreasing media, suggesting an atrophic remodeling. The average of (E) the arteries with smallest lumen and (F) the smallest media from each animal shows no significant difference between the groups. (H) Intima (top) % versus vitC (total) concentration shows a significant negative correlation between intima% and vitC concentration (Pearson’s r = −0.36, p < 0.05); (I) Intima (top) % versus cholesterol concentration shows a significant positive correlation (Pearson’s r = 0.54, p < 0.01). Columns represent average ± SEM of each animal. * p < 0.05, *** p < 0.001 HFD and HFDLoC vs. CTRL determined by one-way ANOVA and Tukey’s post hoc test.