Endothelial dysfunction in small arteries and early signs of atherosclerosis in ApoE knockout rats

Abstract

Endothelial dysfunction is recognized as a major contributor to atherosclerosis and has been suggested to be evident far before plaque formation. Endothelial dysfunction in small resistance arteries has been suggested to initiate long before changes in conduit arteries. In this study, we address early changes in endothelial function of atherosclerosis prone rats. Male ApoE knockout (KO) rats (11- to 13-weeks-old) were subjected to either a Western or standard diet. The diet intervention continued for a period of 20-24 weeks. Endothelial function of pulmonary and mesenteric arteries was examined in vitro using an isometric myograph. We found that Western diet decreased the contribution of cyclooxygenase (COX) to control the vascular tone of both pulmonary and mesenteric arteries. These changes were associated with early stage atherosclerosis and elevated level of plasma total cholesterol, LDL and triglyceride in ApoE KO rats. Chondroid-transformed smooth muscle cells, calcifications, macrophages accumulation and foam cells were also observed in the aortic arch from ApoE KO rats fed Western diet. The ApoE KO rats are a new model to study endothelial dysfunction during the earlier stages of atherosclerosis and could help us improve preclinical drug development.

Figure 1

Monthly plasma cholesterol, LDL and HDL level and fasted triglyceride level vs diets in ApoE KO and Sprague Dawley rats. Cholesterol and LDL levels were significantly higher before the start of diet in ApoE KO rats compared to the Sprague Dawleys rats, (**) P < 0.0001; After 5 months of diet, cholesterol, LDL and triglyceride levels were significantly higher in ApoE KO rats fed on western diet compared to the ApoE KO and Sprague Dawley rats on standard diet, (**) P < 0.0001; HDL level was significantly higher in Sprague Dawley rats before and after control diet compared to the ApoE KO rats, (**) P < 0.0001; ANOVA, n = 8, n = 7.

Figure 2

Microscopic images of the aortic arch from ApoE KO rat fed Western diet; (A) H & E staining at 60 × magnification showed chondroid-transformed smooth muscle cells; (B) H & E staining in Sprague Dawley rat; (C) CD-68 staining revealed accumulation of macrophages and small foam cell lesions.

Figure 3

U46619 concentration–response curves of pulmonary arteries from different experimental groups under different experimental conditions; control conditions, incubation for 20 min with non-selective inhibitor of NO synthase, L-NAME (100 µM), incubation for 20 min with both L-NAME (100 µM) and indomethacin (3 µM); 20 min incubation with Cocktail: L-NAME, indomethacin, TRAM-34 and apamin, i.e. pharmacological inhibition of all major pathways for endothelium-dependent relaxation; (**), P < 0.001, effect of L-NAME; (+ +), P = 0.007, effect of indomethacin, F test, n = 8, 7.

Figure 4

Percentage of relaxation in pulmonary arteries in response to 10–5 M concentration of ACh under different experimental conditions; (A) control conditions, (**), p < 0.01, ApoE KO-western diet vs ApoE KO-control diet; (B) incubation for 20 min with non-selective inhibitor of NO synthase, 100 µML-NAME, (**), p < 0.001, ApoE KO-Western diet vs ApoE KO-standard diet; (C) incubation for 20 min with both L-NAME and 3 µM indomethacin, (**), p < 0.001, ApoE KO-standard diet vs Sprague Dawley ; (D) 20 min incubation with Cocktail: L-NAME, indomethacin, TRAM-34 and apamin, i.e. pharmacological inhibition of all major pathways for endothelium-dependent relaxation. ANOVA, n = 8, 7.

Figure 5

SNP concentration–response curves of (A) pulmonary arteries, (**), p = 0.03, ApoE KO Western diet vs Sprague Dawley, (+ +), P = 0.0002, ApoE KO Western diet vs ApoE KO-standard diet; (B) mesenteric arteries, (**), P = 0.02, ApoE KO Western diet vs Sprague Dawley, (xx), P = 0.0003, ApoE KO-standard diet vs Sprague Dawley; n = 8, 7.

Figure 6

Concentration–response curves to noradrenaline of mesenteric arteries. (***), P < 0.05 ApoE KO Western diet vs Sprague Dawley rats. n = 8, n = 7.

Figure 7

ACh concentration–response curves of mesenteric arteries from different experimental groups under; control conditions; after 20 min incubation with non-selective inhibitor of NO synthase, L-NAME (100 µM); after the incubation with both L-NAME (100 µM) and indomethacin (3 µM); after 20 min incubation with Cocktail: L-NAME, indomethacin, TRAM-34 and apamin, i.e. pharmacological inhibition of all major pathways for endothelium-dependent relaxation; (**), P < 0.001, effect of L-NAME; (+ +), P < 0.001, effect of indomethacin, (xx), P < 0.001, effect of Cocktail, n = 8, n = 7.