This Article Abstract Full Text Full Text (PDF) View Accompanying Videos Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (32) Citing Articles via Google Scholar Google Scholar Articles by Tanaka, K. Articles by Nagai, R. Search for Related Content PubMed PubMed Citation Articles by Tanaka, K. Articles by Nagai, R.

Age-Associated Aortic Stenosis in Apolipoprotein E-Deficient Mice

Kimie Tanaka, MD^_*, Masataka Sata, MD^_*,,,_*, Daiju Fukuda, MD^_*, Yoshihiro Suematsu, MD, Noboru Motomura, MD, Shinichi Takamoto, MD, Yasunobu Hirata, MD^_* and Ryozo Nagai, MD^_*

^_* Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Department of Cardiothoracic Surgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Department of Advanced Clinical Science and Therapeutics, University of Tokyo Graduate School of Medicine, Tokyo, Japan
PRESTO, JST, Kawaguchi, Japan

View larger version (68K): [in a new window]   Figure 1 Age-associated increase in transaortic flow velocity in wild-type mice and in apolipoprotein E (ApoE)–/– mice. (A and B) Mice were anesthetized by intraperitoneal injection of nembutal. Transaortic flow signals were evaluated by continuous waves recorded through a near apical approach with a 12-MHz sector probe and an echocardiography imaging apparatus (EnVisor M2540A, PHILLIPS, Tokyo, Japan) in wild-type (A, 8- to 120-week-old, male n = 18, female n = 2) and ApoE–/– mice (B, 9- to 115-week-old, male n = 23, female n = 22). As the mice grew older, the velocity increased in both groups. There was a significant correlation between age and transaortic valve flow velocity ([AV] flow velocity) in both groups. (C and D) Transaortic flow patterns of a 98-week-old male C57BL/6 mouse (C) and a 103-week-old female ApoE–/– mouse (D). The maximum aortic flow velocity was 427 cm/s in the ApoE–/– mouse. (E) B-mode (upper panels) and color Doppler (lower panels) images were obtained through a parasternal approach with a 12-MHz linear probe and an ultrasound imaging system (LOGIQ 7, GE Medical Systems, Tokyo, Japan). Functional aortic regurgitation could be detected in senile ApoE–/– mice (arrowheads). Ao = aorta; AR = aortic regurgitation signal; LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.

View larger version (87K): [in a new window]   Figure 2 Sclerotic changes of aortic valves of senile wild-type and apolipoprotein E (ApoE)–/– mice. (A) Gross appearance of aortic valves of 96-week-old wild-type mice and 95-week-old ApoE–/– mice. Scale bar, 500 µm. (B) Hearts were taken from 94- to 98-week-old wild-type (male n = 2, femalen = 1) and 74- to 97-week-old ApoE–/– (male n = 5, female n = 5) mice, and embedded in OCT compound. Frozen sections containing aortic valves were stained by von Kossa method (VK) to detect ectopic calcification. Antiosteocalcin (OCL) immunostaining was performed on the frozen sections obtained from wild-type and ApoE–/– mice. Arrows indicate the positive area. Scale bars, 100 µm (upper panel) or 20 µm (middle and lower panels). (C) Immunohistochemical studies were performed on the frozen sections. Endothelial cells were detected by anti-CD31 immunostaining (CD31). Arrowheads indicate the sites of endothelial denudation. Macrophages and T cells were detected by immunostaining with anti-MOMA-2 and anti-CD3 antibodies, respectively. Smooth muscle-like cells were identified using an anti--smooth muscle actin (SMA) antibody. Arrows indicate the positive area. Scale bar, 20µm.

View larger version (171K): [in a new window]   Figure 3 Electron micrographic observation of the aortic valves of senile ApoE–/– mice. (A and B) Electron micrographs of the aortic valve of a 50-week-old ApoE–/– mouse. There are a macrophage (A, left), an osteoblast-like cell (A, right), and a smooth muscle-like cell (B). Scale bar = 5µm. (C) A macrophage observed in the valve leaflet (A). Scale bar = 1µm. (D) Higher magnification image of a smooth muscle-like cell (B) that has muscle fibers, basement membrane, and mitochondria. Scale bar = 1µm. (E) An osteoblast-like cell (A) that has a well-developed Golgi apparatus and rough endoplasmic reticulum. Scale bar = 1µm. (F) Calcium deposition in the sclerotic valve leaflet. Scale bar = 1µm.

View larger version (80K): [in a new window]   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 BMTGFPApoE 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 BMTLacZApoE 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 (BMTGFPwild-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 BMTGFPwild-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.

View larger version (79K): [in a new window]   Figure 5 Cell death and cytokine expression in sclerotic aortic valves of apolipoprotein E (ApoE)–/– mice. (A) Hearts were harvested from 8-week-old wild-type mice (n = 3) and 97- to 108-week-old ApoE–/– mice (n = 3) and snap-frozen in OCT compound. Frozen sections (4 µm) were stained for MCP-1, PDGF, VEGF, and SDF-1 using the avidin-biotin complex technique and vector red substrate (red). Nuclei were counter-stained with hematoxylin. Arrows indicate positive cells. Scale bar = 20 µm. (B) Apoptotic cell death was detected by TUNEL staining. Frozen sections (4 µm) were fixed with 4% paraformaldehyde and permeabilized. TUNEL staining (green) was performed according to the manufacturer’s specification. Arrows indicate apoptotic cells. Scale bar = 20 µm. (C) Co-localization of TUNEL-positive cells and cytokine expression in the sclerotic aortic valve of 104-week-old ApoE–/– mice. Immunofluorescence staining for chemokine or cytokines (red) was performed on the frozen sections after TUNEL staining. Scale bar, 20µm.