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Figure 4 Bone marrow-derived cells detected in sclerotic aortic valve. (A) Bone marrow transplantation (BMT) was performed from GFP mice to 59-week-old apolipoprotein E (ApoE)–/– mice. Hearts of 93-week-old BMT^GFPApoE mice (n = 3) were fixed in 4% paraformaldehyde and embedded in plastic resin. Plastic-embedded sections containing aortic valve were observed under a confocal microscope (FLUOVIEW FV300, Olympus, Tokyo, Japan). Scale bar, 100 µm. Adjacent sections were used for double-immunofluorescence study (B to D). Nuclei were counterstained with Hoechst 33258 (blue). (B) GFP-positive cells (green) that expressed -smooth muscle actin (SMA) (red). Arrows indicate double-positive cells. Scale bar = 10 µm. (C) GFP-positive endothelial-like cells (green) that expressed MECA32 or CD31 (red). Arrowheads indicate the surface of the aortic valves. Arrows indicate double-positive cells (yellow). Scale bar = 10 µm. (D) GFP-positive cells (green) that expressed osteopontin (OPN) or osteocalcin (OCL) (red). Arrows indicate double-positive cells (yellow). Scale bar = 10 µm. (E) Aortic valves of 80-week-old BMT^LacZApoE mice. Bone marrow transplantation was performed from LacZ mice to ApoE–/– mice (n = 3). Frozen sections were stained with polyclonal anti-LacZ antibody, ABC technique, and Vector red substrate (red). Adjacent sections were used for double immunofluorescence study (F). Scale bar = 50 µm. (F) LacZ-positive cells (green) that expressed CD31 (red). Nuclei were counterstained with Hoechst 33258 (blue). Arrows indicate double-positive cells (yellow). Scale bar = 10 µm. (G) Bone marrow transplantation was performed from GFP-mice to C57BL/6 mice (BMT^GFPwild-type mice, n = 3). A total of 44 weeks after BMT, the hearts were harvested and embedded in plastic resin. Plastic-embedded sections containing aortic valve were stained for alpha-SMA (red) under a confocal microscope. Nuclei were counterstained with Hoechst 33258 (blue). Left: Lower magnification image. Scale bar = 50 µm. The arrow indicates the -SMA-positive cells that were negative for GFP. Right: Higher magnification image. Scale bar = 10 µm. (H and I) Bone marrow-derived endothelial-like cell (H) or macrophages (I) observed in the aortic valve of BMT^GFPwild-type mice. Plastic-embedded sections were stained for endothelial cells (MECA32, red) or macrophages (MOMA2, red). Nuclei were counterstained with Hoechst 33258 (blue). Arrows indicate the double-positive cells (yellow). Arrowheads indicate the surface of the valve. Scale bar = 10 µm. DIC = differential interference contrast.

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