Sulindac inhibits neointimal formation after arterial injury in wild-type and apolipoprotein E-deficient mice

Abstract

Neointimal hyperplasia is a critical component of restenosis, a major complication of angioplasty and related therapeutic procedures. We studied the effects of hyperlipidemia and the nonsteroidal anti-inflammatory drugs, aspirin (acetyl-salicylic acid; ASA), and sulindac, on neointimal formation in a mouse femoral arterial injury model. At 2 months of age, normolipidemic, wild-type (WT), and hyperlipidemic, apolipoprotein E-deficient (apoE-/-) mice were divided into three treatment groups: Western-type diet (WD), WD + ASA (200 mg/kg food), and WD + sulindac (300 mg/kg food). After 1 week, mice underwent arterial injury and treatments were maintained for 4 weeks. Histomorphometry of the injured arteries showed striking effects of plasma cholesterol levels and drug treatment on neointimal hyperplasia. In the WD or WD + ASA groups, apoE-/- mice had twice the neointimal area than WT mice ( approximately 30,000 vs. 13,000 microm(2) per section; P < 0.0001). Compared with ASA or WD alone, sulindac treatment resulted in approximately 70% (P = 0.0001) and 50% (P = 0.01) reductions in the neointimal area in apoE-/- and WT mice, respectively. ASA, at a dose sufficient to inhibit platelet aggregation, did not affect neointimal formation in mice of either genotype. Evidence of macrophages was noted in the lesions of apoE-/- mice in the WD and WD + ASA groups, but remarkably, none was detectable with sulindac treatment, despite hyperlipidemia, suggesting early steps in the response to injury were abrogated. These results demonstrate sulindac reduces neointimal formation in both normolipidemic and hyperlipidemic settings and raise the possibility that similar benefits may be obtained in patients undergoing angioplasty and related procedures.

Figure 1

Plasma cholesterol levels 4 weeks after mouse femoral artery injury. ApoE−/− and WT mice were fed WD, WD + ASA, or WD + sulindac, 1 week before and 4 weeks after arterial injury. Venous blood samples were taken for cholesterol analysis. Bars represent means ± SEM based on results from 9–14 mice per group.

Figure 2

Histologic appearances of mouse femoral arteries 4 weeks after injury. Representative photomicrographs (×200; combined Masson-elastic stain) of lesions from mice in each treatment group are displayed. (A and B) WD alone. (C and D) WD + ASA. (E and F) WD + sulindac. In B and D, the looser packing of cells and the many lucent foci (“clefts”) are characteristic of increased deposition of extracellular matrix and cholesterol, respectively.

Figure 3

Effects of genotype and drug treatments on the I/M ratios of lesions from injured mouse femoral arteries. Four weeks after injury, femoral arterial sections were taken for morphometric analysis and the intimal and medial areas were determined. The ratio of these areas (I/M ratio) was calculated for each section. The means ± SEM (n = 9–14 for each group) are displayed.

Figure 4

Aggregation of platelets from apoE−/− mice. Before isolating platelet-rich plasma, mice were fed a WD with ASA or without (control) for 1 week. Arachidonic acid was added and the formation of aggregates was detected by an increase in light transmittance over time. Representative assays are shown.

Figure 5

Macrophage content of lesions from mouse femoral arteries 4 weeks after injury. Arterial sections were immunostained for the macrophage marker, MOMA-2, whose presence is indicated by a brown color. Note the substantial macrophage infiltration and cholesterol clefts (lucent foci) adjacent to the internal elastic lamina in the section from the apoE−/− mouse from the WD group (Middle). Original magnification: Left, ×200; and Right, ×630.