Intravital Microscopy on Atherosclerosis in Apolipoprotein E–Deficient Mice Establishes Microvessels as Major Entry Pathways for Leukocytes to Advanced Lesions

Abstract

Background— There has been considerable speculation about the role of lesion microvessels in the accumulation of leukocytes in atherosclerosis. However, direct study of microvascular recruitment of leukocytes in lesions has not been performed, and the quantitative role for this route of entry is unclear. Methods and Results— Here, microvascular recruitment of leukocytes was studied in advanced lesions in 12- to 24-month-old apolipoprotein E–deficient (ApoE −/− ) mice. Histology and transmission electron microscopy demonstrated the presence of mainly adventitial, but also intimal, microvessels. Interactions between leukocytes and endothelium occurred in lesion venules. Leukocyte rolling was largely P-selectin dependent; however, residual rolling was mediated by L-selectin and endothelial P-selectin glycoprotein ligand 1. Leukocyte adhesion was significant and was attenuated in mice treated with antibodies against P-selectin, CD18, or both before preparation for intravital microscopy, suggesting acute activation of these 2 molecules by surgical trauma. Nonetheless, the density of firmly arrested leukocytes was 100-fold higher in lesion venules compared with the arterial lumen even in mice pretreated with antibodies against P-selectin and CD18, indicating strong recruitment of cells from venules that is unrelated to experimental manipulation. Fluorescent myelomonocytic cells in ApoE −/− mice carrying a knock-in mutation for enhanced green fluorescent protein (EGFP) in the lysozyme M locus (ApoE −/− /lysM EGFP/EGFP mice) were distributed specifically around lesion venules, but not around arterioles or capillaries, further indicating ongoing extravasation from venules into plaque tissue. Conclusions— These findings provide strong data for microvascular recruitment of leukocytes in atherosclerosis and indicate roles for L-selectin and P-selectin glycoprotein ligand 1 in this process.