Midkine is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits Hiroshi Narita, MD, Sen Chen, PhD, Kimihiro Komori,

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Midkine is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits Hiroshi Narita, MD, Sen Chen, PhD, Kimihiro Komori, MD, PhD, Kenji Kadomatsu, MD, PhD Journal of Vascular Surgery Volume 47, Issue 6, Pages 1322-1329 (June 2008) DOI: 10.1016/j.jvs.2007.12.037 Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 1 Induction of neointimal hyperplasia by bare metal stent implantation. A, The arrows in the (a) plain radiographs and (b) angiography after stent implantation into the right iliac artery indicate the stented lesion. B, Neointimal hyperplasia was induced moderately in a (a) rabbit fed a normal diet, but was markedly induced in a (b) rabbit fed a hypercholesterol diet. Bar, 500 μm. Journal of Vascular Surgery 2008 47, 1322-1329DOI: (10.1016/j.jvs.2007.12.037) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 2 Midkine expression during neointima formation. Right iliac arteries of hypercholesterolemic rabbits were obtained at the indicated number of days after stenting. The graphs show relative densitometric density ratios presented as the average ± SE (n = 4). *P < .01; **P < .0001 (Student t test). Western blot and reverse transcription–polymerase chain reaction (RT-PCR) data were obtained from two individual animals, respectively, at each time point (except for control and day 7: n = 4). Because the results of each set of experiments were similar, representative data are shown. A, Midkine protein expression revealed by Western blot analysis. B, Midkine messenger RNA expression estimated by RT-PCR. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Vascular Surgery 2008 47, 1322-1329DOI: (10.1016/j.jvs.2007.12.037) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 3 Protein localization in neointima hyperplasia. Immunohistochemistry data were obtained from three individual animals at each time point. Because the results of each set of experiments were similar, representative data are shown. A, Chicken antihuman midkine antibody (upper) or control chicken immunoglobulin (Ig) Y was used as the first antibody to localize midkine protein expression in an artery obtained 14 days after operation. Note that IgY did not show any signals. Bar, 100 μm. B, The indicated antibodies were used to localize their target proteins in the specimen. The midkine protein localization overlapped that of macrophages (RAM11). Bars, 100 μm. α-SMA, α-Smooth muscle actin. C, The double immunofluorescent staining for midkine and macrophage was performed on specimens 7 days after stenting. S indicates stent strut. Bar, 20 μm. D, Midkine protein was localized to the endothelium. Magnified photos of B, c and B, l are shown. Bar, 20 μm. Journal of Vascular Surgery 2008 47, 1322-1329DOI: (10.1016/j.jvs.2007.12.037) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 4 Midkine expression during differentiation of monocytic cells. A, Phase-contrast photos are shown. THP-1 cells were treated with either (left) dimethyl sulfoxide (DMSO) or (right) phorbol myristate acetate (PMA) for 24 hours. B, Western blot analysis for midkine protein expression. The graph at right shows the average ± SE values of the relative densitometric density ratio (n = 4). Journal of Vascular Surgery 2008 47, 1322-1329DOI: (10.1016/j.jvs.2007.12.037) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 5 Effect of oxided low-density lipoprotein cholesterol (oxLDL) on midkine expression in macrophages. A, Peritoneal macrophages were exposed to 100 μg/mL of LDL or oxLDL for 24 hours. Cells were then stained with oil red O to detect lipid droplets in the cell. Note that oxLDL was significantly more endocytosed by the cell. B, Midkine expression was estimated by Western blot analysis. The right graph shows the average ± SE values of the relative densitometric density ratio (n = 4). There were no significant differences among these three groups. Journal of Vascular Surgery 2008 47, 1322-1329DOI: (10.1016/j.jvs.2007.12.037) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions