Effect of decorin administration on AAA development in mice.

<p>(<b>A-B</b>) Immediately after CaCl₂ treatment, Gelfoam patches loaded with PBS (CaCl₂+PBS, n = 9) or 20 (g decorin (CaCl₂+decorin, n = 10) were placed into the periaortic spaces between the left renal vein (lt RV) and bifurcation. AA: abdominal aorta, IVC: inferior vena cava, lt RA: left renal artery. Untreated mice were used as controls (Control, n = 6). The mice were sacrificed 42 days after CaCl₂ treatment. (<b>C</b>) Representative photographs show aortas 42 days after CaCl₂ treatment. (<b>D</b>) Quantitative analysis of the maximum external diameters of abdominal aortas. Data are mean ± SD. **<i>p</i><0.01 compared to Control; ##<i>p</i><0.01 compared to CaCl₂+PBS. (<b>E</b>) Representative histological and immunohistochemical stains of abdominal aorta specimens from mice treated as indicated. HE and EVG stains depict cell nuclei (blue-black) and elastin network (black), respectively. The localization of MMP-9 is indicated with red staining. M: media, A: adventitia. (<b>F</b>) Infiltrating mononuclear cells were counted in five high-power fields. Data are mean ± SD. **<i>p</i><0.01 compared to Control; #<i>p</i><0.05 compared to CaCl₂+PBS. (<b>G-H</b>) The degree of medial layer elastin disruption was graded as mild (grade I), moderate (grade II), high (grade III), or severe (grade IV) based on EVG-stained sections. Yellow arrows indicate disruption of elastic lamellae. White asterisks indicate preserved elastic lamellae.</p

Role of decorin in macrophage secretion of MMP-9.

<p>(<b>A</b>) Cultured mouse macrophages were pre-treated with or without 0.4, 4, or 40 μg/ml decorin, then stimulated with or without 100 ng/ml lipopolysaccharide (LPS) for 48 h. (<b>B</b>) MMP-9 protein levels in the conditioned media were determined by gelatin zymography. Representative results are shown. (<b>C-D</b>) Cultured mouse macrophages were pre-treated with or without 40 μg/ml decorin, then stimulated with or without 100 ng/ml LPS for 48 h. Quantitative analyses of MMP-9 (<b>C</b>) and TGF-β (<b>D</b>) are shown. TGF-β protein levels in the conditioned media were determined by enzyme-linked immunosorbent assay (ELISA). Data are mean ± SD. **<i>p</i><0.01 compared to Control; ##<i>p</i><0.01 compared to LPS.</p

Expression of decorin in human AAA.

<p>(<b>A-B</b>) Protein samples were obtained from the aortic walls of human AAA specimens (n = 47) and non-aneurysmal specimens (Control, n = 4). Representative results from western blot detection of decorin are shown (<b>A</b>) with the corresponding quantitative analysis (<b>B</b>). GAPDH served as an internal control. Data are mean ± SD. *<i>p</i><0.05 compared to Control. (<b>C</b>) The correlation between the protein levels of decorin and MMP-9 was examined and the quantitative analysis is shown (Pearson r = 0.68, n = 51, <i>p</i>< 0.001). (<b>D</b>) The correlation between the protein levels of decorin and TGF-β was examined and the quantitative analysis is shown (Pearson r = 0.39, n = 51, <i>p</i>< 0.01). (<b>E</b>) Representative histological and immunohistochemical stains are shown for human AAA wall specimens. The luminal surface is oriented toward the top of each panel. HE and EVG stains depict cell nuclei (blue-black) and elastin network (black), respectively. The localization of decorin and MMP-9 is indicated by red staining. M: media, A: adventitia. (<b>F-G</b>) Representative images of immunofluorescence staining for decorin (red) and CD68 (macrophage marker, green, <b>F</b>) or α-smooth muscle actin (α-SMA) (smooth muscle cell marker, green, <b>G</b>). Yellow in the merged images indicates overlapping localization of the red and green signals.</p

Role of decorin in VSMC secretion of MMP-9.

<p>(<b>A</b>) Cultured rat vascular smooth muscle cells (VSMCs) were pre-treated with or without 0.4, 4, or 40 μg/ml decorin, then stimulated with or without 100 ng/ml lipopolysaccharide (LPS) for 48 h. Protein levels of MMP-9 and MMP-2 in the conditioned media were determined by gelatin zymography. (<b>B</b>) Representative results are shown. (<b>C-D</b>) Cultured rat VSMCs were pre-treated with or without 40 μg/ml decorin, then stimulated with or without 100 ng/ml LPS for 48 h. Quantitative analyses for MMP-9 (<b>C</b>) and MMP-2 (<b>D</b>) are shown. Data are mean ± SD. **<i>p</i><0.01 compared to Control; ##<i>p</i><0.01 compared to LPS.</p

Role of periostin/FAK axis in mechanotransduction signaling pathway in VSMCs.

<p>Rat VSMCs were treated with or without of FAK inhibitor (PF573228, 10 µM, FAK-I) and stimulated with cyclic uniaxial strain (20% elongation in length at a frequency of 30 cycles/min for 48 h, n = 5). Levels of phosphorylated FAK (<b>A, B</b>), ERK (<b>A, C</b>) and JNK (<b>A, D</b>) in cell lysates were determined by western blotting. Protein levels of MCP-1 (<b>A, E</b>) and periostin (<b>A, G</b>) in cell lysates, and active MMP-2 (<b>A, F</b>) in conditioned media were determined by western blotting and gelatin zymography, respectively. GAPDH served as an internal control. Data are mean ± SE. *<i>p</i><0.05 and **<i>p</i><0.01 compared to Control; #<i>p</i><0.05 and ##<i>p</i><0.01 compared to VSMCs under mechanical strain.</p

Role of periostin in linking mechanical strain to inflammatory responses in VSMCs.

<p>Cultured rat VSMCs treated with or without periostin-neutralizing antibody (PN-nAb, 1 µg/ml) were stimulated with cyclic uniaxial strain (20% elongation in length at a frequency of 30 cycles/min for 48 h, n = 6). Protein levels of periostin (<b>A</b>) and MCP-1 (<b>B</b>) in cell lysates were determined by western blotting. Levels of active MMP-2 in the conditioned media were determined by gelatin zymography (<b>C</b>). Levels of phosphorylated FAK (<b>D</b>), ERK (<b>E</b>) and JNK (<b>F</b>) in cell lysates were determined by western blotting. Levels of total FAK (<b>G</b>), ERK (<b>H</b>) and JNK (<b>I</b>) in cell lysates were determined by western blotting. GAPDH served as an internal control. Rat VSMCs were stimulated with recombinant periostin (rPeriostin; 1 µg/ml for 48 h; n = 4). Protein levels of MCP-1 in conditioned media were determined by ELISA (<b>J</b>). Data are mean ± SE. *<i>p</i><0.05 and **<i>p</i><0.01 compared to Control; #<i>p</i><0.05 compared to VSMCs under mechanical strain.</p

Temporal pattern of periostin expression during the development of abdominal aortic aneurysm (AAA) in a mouse model.

<p>A: Representative photographs show mouse abdominal aortas 42 days after periaortic application of saline (control) or CaCl₂ (for AAA induction). B: Changes in the diameters of abdominal aortas are shown for the indicated time points after application of saline or CaCl₂ (3 days, n = 3; 7 days, n = 4; 14 days, n = 4; 28 days, n = 3–4; 42 days, n = 7–8). Data are mean ± SE. *<i>p</i><0.05 compared to saline controls. C: Representative images show aortic walls stained with hematoxylin/eosin (HE), elastica van-Gieson (EVG), or antibody against periostin at the indicated time points after application of saline or CaCl₂.</p

Role of periostin in inducing inflammatory responses <i>in vivo</i>.

<p><b>A-B</b>: Gelfoam patches containing PBS (Control, n = 6) or recombinant periostin (rPeriostin, n = 7) were placed into periaortic spaces of mice for 7 days. Representative images show mouse aortic walls, stained with hematoxylin/eosin (HE) or with antibodies against periostin, phosphorylated FAK (pFAK), or MCP-1 (<b>A</b>). Infiltrating cells were counted in 5-10 high-power fields (<b>B</b>). Data are mean ± SE. *<i>p</i><0.05 compared to Control. <b>C</b>: Schematic diagram represents the proposed vicious cycle of periostin upregulation driven by mechanical strain through activation of FAK, resulting in the maintenance of inflammation in AAA.</p

sj-docx-1-jet-10.1177_15266028241248311 – Supplemental material for Early Clinical Results From the Japanese Prospective Multicenter Study to Evaluate Zenith Alpha Abdominal Stent Graft for Abdominal Aortic Aneurysm (JUSTICE) Registry Demonstrate Acceptable Limb Patency at 12 Months

Supplemental material, sj-docx-1-jet-10.1177_15266028241248311 for Early Clinical Results From the Japanese Prospective Multicenter Study to Evaluate Zenith Alpha Abdominal Stent Graft for Abdominal Aortic Aneurysm (JUSTICE) Registry Demonstrate Acceptable Limb Patency at 12 Months by Naoki Fujimura, Shigeo Ichihashi, Tsuyoshi Shibata, Hitoshi Matsumura, Michiko Watanabe, Noriyasu Morikage, Yoshihiko Kurimoto, Hiroshi Banno, Hidetoshi Uchiyama and Hideaki Obara in Journal of Endovascular Therapy</p

sj-tiff-3-jet-10.1177_15266028241248311 – Supplemental material for Early Clinical Results From the Japanese Prospective Multicenter Study to Evaluate Zenith Alpha Abdominal Stent Graft for Abdominal Aortic Aneurysm (JUSTICE) Registry Demonstrate Acceptable Limb Patency at 12 Months

Supplemental material, sj-tiff-3-jet-10.1177_15266028241248311 for Early Clinical Results From the Japanese Prospective Multicenter Study to Evaluate Zenith Alpha Abdominal Stent Graft for Abdominal Aortic Aneurysm (JUSTICE) Registry Demonstrate Acceptable Limb Patency at 12 Months by Naoki Fujimura, Shigeo Ichihashi, Tsuyoshi Shibata, Hitoshi Matsumura, Michiko Watanabe, Noriyasu Morikage, Yoshihiko Kurimoto, Hiroshi Banno, Hidetoshi Uchiyama and Hideaki Obara in Journal of Endovascular Therapy</p