Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease

Abstract

Monocytes participate importantly in the pathogenesis of atherosclerosis, but their spatial and temporal recruitment from circulation remains uncertain. This study tests the hypothesis that monocyte accumulation in atheroma correlates with the extent of disease by using a sensitive and simple quantitative assay that allows tracking of highly enriched populations of blood monocytes. A two-step isolation method yielded viable and functionally intact highly enriched peripheral blood monocyte populations (>90%). Recipient mice received syngeneic monocytes labeled in two ways: by transgenically expressing EGFP or with a radioactive tracer [(111)In]oxine. After 5 days, more labeled cells accumulated in the aorta, principally the aortic root and ascending aorta, of 10-wk-old ApoE(-/-) compared with 10-wk-old C57BL/6 mice (223 +/- 3 vs. 87 +/- 22 cells per aorta). Considerably more monocytes accumulated in 20-wk-old ApoE(-/-) mice on either chow (314 +/- 41 cells) or high-cholesterol diet (395 +/- 53 cells). Fifty-week-old ApoE(-/-) mice accumulated even more monocytes in the aortic root, ascending aorta, and thoracic aorta after both chow (503 +/- 67 cells) or high-cholesterol diet (648 +/- 81 cells). Labeled monocyte content in the aorta consistently correlated with lesion surface area. These data indicate that monocytes accumulate continuously during atheroma formation, accumulation increases in proportion to lesion size, and recruitment is augmented with hypercholesterolemia. These results provide insights into mechanisms of atherogenesis and have implications for the duration of therapies directed at leukocyte recruitment.

Fig. 1.

Monocyte isolation. (A) The monocyte fraction (Left) and the depleted fraction (Right) were stained with anti-CD11b, CD90, B220, DX5, NK1.1, and Ly-6G mAbs. (B) Representative cytospin preparations of the cell fractions. (C) Proportion of monocytes and other cells based on morphology and calculated by counting 10 high-power fields. Three independent experiments yielded similar results.

Fig. 2.

Detection of monocytes in aortic lesions. (A) Assessment of purity of isolated monocytes from EGFP-expressing mice stained with anti CD11b. (B) Monocytes were injected i.v. into 20-wk-old ApoE^−/− mice on a high-cholesterol diet. Five days after injection, the aortas were excised, and immunohistochemistry was conducted with Abs against Mac-3 and EGFP. High-power fields of the lesion area at the root from mice that did not receive EGFP⁺ cells (Left) and from mice that received EGFP⁺ cells (Center and Right representing different sections) stained with anti-Mac-3 (Upper) and anti-EGFP (Lower). Data are representative of three independent experiments.

Fig. 3.

Labeling, excretion, and organ distribution of ¹¹¹In species. (A) MTS assay of monocytes labeled with increasing doses of decayed [¹¹¹In]oxine (squares), [¹¹¹In]oxine (triangles), or [¹¹¹In]chloride (inverted triangles). (B) Biodistribution of [¹¹¹In]oxine-labeled monocytes compared with biodistribution of ¹¹¹In alone 5 d after i.v. injection. (C) Retention of monocytes in the circulation. [¹¹¹In]oxine-labeled monocytes were injected i.v., and radioactivity was measured in blood withdrawals. Biodistribution measured immediately after injection was normalized to 100%. The range of monocyte blood half-life is calculated from a 95% confidence interval, as determined from a polynomial second-order fit. (n = 2–8; mean ± SEM).

Fig. 4.

Accumulation of monocytes in the aorta. [¹¹¹In]oxine-labeled monocytes were injected into WT or ApoE^−/− mice (ApoE: + or −, respectively) of different age [Age (wk): 10, 20, or 50] and on either chow or high-cholesterol diets (diet C or H, respectively). Five days after injection, aortas were excised. (A) Percent injected dose per aorta. The data above the graph represent mean monocyte number ± SEM for each group. (B) Statistics (P value) for each group were calculated by using one-way ANOVA with Tukey’s multiple-comparison test.

Fig. 5.

Localization of monocytes in aorta. [¹¹¹In]oxine-labeled monocytes were injected into 10-wk-old WT (A Left) or ApoE^−/− (A Right) mice and 20-wk-old (B Upper) and 50-wk-old (B Lower) ApoE^−/− mice on chow (B Left) or high-cholesterol (B Right) diet. Five days after injection, aortas were excised, exposed to imaging plates, and subsequently read out by a PhosphorImager. Representative aortas are shown. (C) ORO staining of excised aorta from a 50-wk-old mouse on a high-cholesterol diet. Highest ORO staining typically mapped to highest radioactivity (circles).

Fig. 6.

Correlation of monocyte accumulation with serum cholesterol and lesion surface area. (A) Correlation of serum cholesterol levels with percent lesion area (n = 8). (B) Serum cholesterol levels in WT or ApoE^−/− mice at different ages and on different diets, as identified in Fig. 4 (n = 4–14 per group). (C and D) Correlation between serum cholesterol and aortic accumulation in 20-wk-old (C) (n = 12) and 50-wk-old (D) (n = 14) mice, respectively. (E) Correlation of percent lesion area with aortic monocyte accumulation (n = 9). Linear regression was conducted to determine confidence of linear fit. P values are shown.