Expression of Lectinlike Oxidized Low-Density Lipoprotein Receptor-1 in Human Atherosclerotic Lesions by Hiroharu Kataoka, Noriaki Kume, Susumu Miyamoto, Manabu Minami, Hideaki Moriwaki, Takatoshi Murase, Tatsuya Sawamura, Tomoh Masaki, Nobuo Hashimoto, and Toru Kita Circulation Volume 99(24):3110-3117 June 22, 1999 Copyright © American Heart Association, Inc. All rights reserved.

RT-PCR detection of LOX-1 mRNA in human atherosclerotic plaques. RT-PCR detection of LOX-1 mRNA in human atherosclerotic plaques. The total RNA (250 ng) from 8 atherosclerotic plaques and 2 aortas was first reverse-transcribed. cDNA fragments were amplified by 35-cycle PCR with set of primers for hLOX-1 (top) and β-actin (bottom). PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis. Lane 1, DNA size marker; lanes 2 and 3, normal aorta; lanes 4 through 11, atherosclerotic plaques. Hiroharu Kataoka et al. Circulation. 1999;99:3110-3117 Copyright © American Heart Association, Inc. All rights reserved.

Indirect immunofluorescence of hLOX-1–transfected CHO cells and untransfected CHO cells with anti-hLOX-1 monoclonal antibody and anti-bLOX-1 monoclonal antibody. Indirect immunofluorescence of hLOX-1–transfected CHO cells and untransfected CHO cells with anti-hLOX-1 monoclonal antibody and anti-bLOX-1 monoclonal antibody. Cells were fixed with cold acetone and incubated with antibodies as described in Methods. The anti-hLOX-1 monoclonal antibody recognized hLOX-1 transfected CHO cells (A) but not untransfected CHO cells (B). The anti-bLOX-1 monoclonal antibody also recognized hLOX-1–transfected CHO cells (D) but not untransfected CHO cells (E). Staining hLOX-1–transfected CHO cells with nonimmnune mouse IgG (C) and rat IgG (F) did not reveal any significant signals. Magnification ×200. Hiroharu Kataoka et al. Circulation. 1999;99:3110-3117 Copyright © American Heart Association, Inc. All rights reserved.

Immunohistochemically stained human aorta both without visible atherosclerotic changes (A and B) and with early atherosclerotic lesions (C and G). Immunohistochemically stained human aorta both without visible atherosclerotic changes (A and B) and with early atherosclerotic lesions (C and G). A and B, Frozen serial sections of a nonlesional aorta were stained with anti-hLOX-1 monoclonal antibody (A, ×200) and anti-von Willebrand factor antibody (B, ×200). LOX-1 was not expressed in luminal endothelial cells of nonlesional aorta (A). Serial sections showed uniform staining in luminal surface of the aorta with anti-von Willebrand factor antibody, indicating that intact endothelial linings were preserved (B). C through F, Frozen serial sections of early atherosclerotic lesions were stained with anti-hLOX-1 monoclonal antibody (C, ×200; D, ×1000), anti-von Willebrand factor antibody (E, ×200), and nonimmune mouse IgG (F, ×200). LOX-1 was expressed in luminal endothelial cells of early atherosclerotic lesions with subendothelial infiltrates. Some nonendothelial cells in intima were also positive for anti-hLOX-1 monoclonal antibody. G, Double immunohistochemical staining with anti-bLOX-1 antibody (dark blue) and anti-von Willebrand factor antibody (red) in early atherosclerotic lesions (×1000). LOX-1 expression in luminal endothelial cells was confirmed in early atherosclerotic lesions. Hiroharu Kataoka et al. Circulation. 1999;99:3110-3117 Copyright © American Heart Association, Inc. All rights reserved.

Immunohistochemical staining of advanced atherosclerotic lesions. Immunohistochemical staining of advanced atherosclerotic lesions. A through D, Frozen serial sections of advanced atherosclerotic lesions were stained with anti-hLOX-1 monoclonal antibody (A, ×100) and nonimmune mouse IgG (B, ×100). LOX-1 was expressed by nonendothelial cells in intima of advanced atherosclerotic lesions. Higher-power view of A showed that LOX-1–positive subendothelial cells included round cells consistent with morphology of macrophages (C, ×1000) and spindle-shaped cells consistent with morphology of smooth muscle cells (D, ×1000). E and F, Other serial sections of advanced atherosclerotic lesions were stained with anti-hLOX-1 monoclonal antibody (E, ×100) and anti-von Willebrand factor antibody (F, ×100). LOX-1 was not expressed by luminal endothelial cells stained with anti-von Willebrand factor antibody. Most intimal nonendothelial cells expressing LOX-1 were spindle-shaped, consistent with morphology of intimal smooth muscle cells. G and H, Double-labeled immunohistochemical staining with anti-hLOX-1 monoclonal antibody and antibodies for cell markers of advanced atherosclerotic lesions. Cell marker antibodies were visualized by alkaline phosphatase method and appeared red on slides, whereas the anti-bLOX-1 antibody was marked with avidin-biotin complex peroxidase technique and appeared dark blue. G, Double immunohistochemical staining with anti-hLOX-1 antibody (dark blue) and anti-CD68 antibody (red) (×1000). H, Double immunohistochemical staining with anti-hLOX-1 antibody (dark blue) and anti-smooth muscle α-actin antibody (red) (×1000). LOX-1 was expressed by some macrophages (G) and smooth muscle cells (H) in advanced atherosclerotic intima. Hiroharu Kataoka et al. Circulation. 1999;99:3110-3117 Copyright © American Heart Association, Inc. All rights reserved.

Single-labeled immunohistochemical staining of advanced lesion with intimal neovasculature. Single-labeled immunohistochemical staining of advanced lesion with intimal neovasculature. Sections were stained with anti-hLOX-1 monoclonal antibody. Intimal neovascular endothelium and intimal nonendothelial cells expressed hLOX-1 (A, ×100; B, ×400). Hiroharu Kataoka et al. Circulation. 1999;99:3110-3117 Copyright © American Heart Association, Inc. All rights reserved.