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Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model 

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Presentation on theme: "Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model "— Presentation transcript:

1 Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model  Dai Yamanouchi, MD, PhD, Stephanie Morgan, BS, Colin Stair, BS, Stephen Seedial, BS, Justin Lengfeld, BS, K. Craig Kent, MD, Bo Liu, PhD  Journal of Vascular Surgery  Volume 56, Issue 2, Pages (August 2012) DOI: /j.jvs Copyright © 2012 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Apoptosis induced by calcium phosphate (CaPO4) treatment in vitro. Cultured mouse aortic smooth muscle cells (SMCs) were treated with calcium chloride (CaCl2) or CaPO4 as described in the Methods section. A, In vitro apoptosis determined by DNA fragmentation enzyme-linked immunosorbent assay. Fold-change was determined with comparison to untreated cell group (control; n = 4). *P < B, Representative results of flow cytometry analyses. Apoptotic and necrotic cells were identified by annexin V and 7-aminoactinomycin D (7AAD), respectively (n = 4). The error bars show the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Mice were subjected to abdominal aortic aneurysm (AAA) induction with calcium chloride (CaCl2) or calcium phosphate (CaPO4) and were euthanized at indicated times as described in the Methods section. A, Representative pictures of arterial dilation 7 days after surgery. B, Quantification of arterial expansion measured 3, 7, 28, and 42 days after surgery (n = 6). Fold-change calculated as maximum diameter/presurgery diameter. *P < .05. C, Van Gieson stain shows elastin layer degradation in representative treated arteries 7 days after surgery. Scale bar = 100 μm. D, Semiquantification of elastin degradation in arteries harvested 7 days after surgery. The error bars show the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Calcium phosphate (CaPO4)-induced aneurysm displays apoptosis. Mice underwent abdominal aortic aneurysm induction with calcium chloride (CaCl2) or CaPO4 and were euthanized at indicated times as described in the Methods section. A, Representative images of immunohistochemistry for apoptosis, as measured by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL; red), nuclei shown by 4′,6-diamidino-2-phenylindole (DAPI) stain (blue) in treated arteries 3 days after injury. Scale bar = 100 μm. B, TUNEL index as determined by TUNEL-positive cells/nuclei. Measurements taken from CaCl2-treated (black bar) and CaPO4-treated (white bars) arteries harvested 24, 48, or 72 hours after surgery (n = 3). *P < .05. The error bars show the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Inflammation accompanies calcium phosphate (CaPO4)-induced aneurysm. Mice underwent abdominal aortic aneurysm induction with calcium chloride (CaCl2) or CaPO4 and were euthanized at indicated intervals as described in the Methods section. A, Representative images are shown of immunohistochemistry for macrophage marker CD68 (green) in CaCl2-treated and CaPO4-treated aortas 3 days after injury. Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 100 μm. B, Macrophage infiltration as determined by CD68-positive cells/nuclei in treated arteries 24, 48, and 72 hours after treatment (n = 3). *P < .05. The error bars show the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Calcium phosphate (CaPO4)-induced aneurysm samples contain medial calcification. A, Arterial sections were stained with Alizarin Red for calcium deposit detection. Calcium appears red on a pink and yellow background. The control sections were harvested from animals treated with saline only. Sections were harvested at 48 hours, 72 hours, and at 7 days after CaPO4 treatment. Scale bar = 200 μm for all images. B, Quantification of calcium content in arterial sections harvested from arteries with the conventional calcium chloride (CaCl2) or the CaPO4 model. Data are expressed as a ratio of the total calcified media divided by the total medial area of each arterial section (n = 4). The error bars show the standard error. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

7 Supplemental Fig 1 Representative images show immunofluorescent-stained arteries treated with calcium phosphate harvested 3 days after injury. A, Apoptosis as identified by cleaved caspase 3 (red), overlay with nuclei (4',6-diamidino-2-phenylindole [DAPI];, blue). Scale bar = 100 μm. B, Colocalization of the smooth muscle cell marker smooth muscle actin (green) with terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL; red). Overlay with DAPI (blue). Scale bar = 200 μm. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

8 Supplemental Fig 2 Representative images of calcium phosphate-treated arteries harvested 7 days after injury stained for macrophages (Mac3), T lymphocytes (CD3), and the inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1). Scale bar = 100 μm. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

9 Supplemental Fig 3 Mice underwent calcium phosphate treatment and were euthanized 7 days later. A, Inflammatory cells are localized to adventitia, with monocytes and macrophages marked by anti-monocyte/macrophage-specific antibody (MOMA) and the medial layer delineated by smooth muscle actin (SMA; red), overlay with 4',6-diamidino-2-phenylindole [DAPI]; blue). Left panel scale bar = 200 μm; right panel scale bar = 100 μm. B, A representative costaining image shows apoptosis (cleaved caspase-3–positive cells, red) and inflammation (MOMA, green). Overlay with DAPI. Scale bar =100 μm. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

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