Fluorescence lymphography and microscopy of abdominal aortic aneurysm.

<p><b>A</b>, Three dimensional CT image of an infrarenal abdominal aortic aneurysm (AAA). <b>B</b>, Intraoperative photography of an infrarenal AAA. <b>C</b>, Intraoperative near-infrared fluorescence lymphography of indocyanine green (ICG) (red arrow: ICG fluorescence, yellow dotted-line: sample resection line). <b>D</b>, Macroscopic findings of the resected sample observed from the luminal side (yellow dotted line: cross-section line of the harvested sample). <b>E</b>, Near-infrared fluorescence lymphography of ICG in the resected aneurysmal wall observed from the luminal side (red arrow: ICG fluorescence, yellow dotted line: cross-section line of the harvested sample). <b>F</b>, Near-infrared fluorescence lymphography of ICG in the cross-sectioned AAA wall (red arrow: ICG fluorescence, up: luminal side, bottom: adventitial side). <b>G</b>, Fluorescence microscopy of ICG in the AAA wall (ICG: red, DAPI: blue). <b>H</b>, Serial frozen section with Elastica van Gieson staining. <b>I</b>, Higher magnification of the outlined area in <b>G</b>. <b>J</b>, Higher magnification of the outlined area in <b>H</b>. <b>K</b>, Immunohistochemistry for podoplanin in the outlined area in <b>G</b> and <b>H</b>. <b>L</b>, Immunohistochemistry to label macrophages in the outlined area in <b>G</b> and <b>H</b>. Scale bars indicate 50 µm (<b>G–L</b>).</p

Baseline characteristics of the AAA group and control group.

<p>The definition of each item: hypertension (medication for hypertension or a systolic blood pressure ≧140 mmHg and/or a diastolic blood pressure ≧90 mmHg), hypercholesterolemia (medication for hypercholesterolemia or total cholesterol concentration in the serum of ≧220 mg/dL), hypertriglyceridemia (medication for hypertriglyceridemia or total triglyceride in the serum of ≧150 mg/dL), diabetes (present or past medication for diabetes), smoking (present or past smoking history).</p

Lymphatic microvessels in intima of abdominal aortic aneurysm.

<p>EVG staining of normal aorta (<b>A</b>) and abdominal aortic aneurysm (AAA) wall (<b>B</b>). Immunohistochemistry for podoplanin corresponding to the areas outlined with a solid line in <b>A</b> and <b>B</b> (<b>C</b>, normal aorta; <b>D</b>, AAA). Podoplanin-positive microvessels were markedly increased in the intima in AAA wall. <b>E</b>, Higher magnification of the outlined area in <b>D</b>. Double immunofluorescence staining of lymphatic vessels in intima of AAA (<b>F</b>–<b>I</b>). <b>F</b>, Podoplanin (red); <b>G</b>, Prox-1 (green); <b>H</b>, DAPI (blue); <b>I</b>, merge of podoplanin, Prox-1, and DAPI. Double immunofluorescence staining of smooth muscle cells in intima of AAA (<b>J–M</b>). <b>J</b>, podoplanin (red); <b>K</b>, alpha smooth muscle isoform of actin (green), <b>L</b>, DAPI (blue); <b>M</b>, merge of podoplanin, alpha smooth muscle isoform of actin, and DAPI. Immunohistochemistry for podoplanin corresponding to the areas outlined with a dotted line in <b>A</b> and <b>B</b> (<b>N</b>; normal aorta, <b>O</b>; AAA). Lymphatic vasa vasorum was observed in adventitia in both normal aorta (<b>N</b>) and AAA (<b>O</b>) (black arrow: LVV). Scale bars indicate 100 µm (<b>A, B</b>), 50 µm (<b>C, D, J–M, N, O</b>), and 10 µm (<b>E–I</b>). Lymphatic microvessel density; the mean number of lymphatic microvessel (LMVs) in 10 microscopic fields at high magnification (×200) (<b>P</b>). Percentage of lymphatic microvessel area in 10 microscopic fields at high magnification (×200) (<b>Q</b>). Lymphatic microvessel density and percentage of lymphatic microvessel area were compared between normal aorta and AAA. (*<i>P</i><0.001).</p

Macrophage infiltration of AAA microvessels.

<p><b>A</b>, Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin (<b>B</b>) and macrophages (<b>C</b>), CD19 (<b>E</b>), and myeloperoxidase (MPO) of the AAA wall. <b>B</b>, Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). <b>C</b>, Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). <b>D</b>, CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. <b>E</b>, CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. <b>F</b>, MPO-positive neutrophils infiltration in the intima/media or in adventitia. <b>G–P</b>, Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). <b>G–P</b>, Macrophage: green, CD11b (<b>G</b>)/LYVE-1 (<b>H</b>)/VEGF-C (<b>I</b>)/MMP-9 (<b>J</b>)/TGF-β1 (<b>K</b>)/IL-4 (<b>L</b>)/IL-8 (<b>M</b>)/MIP-1α (<b>N</b>)/IFN-γ (<b>O</b>)/MCP-1 (<b>P</b>): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. <b>Q</b>, Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. <b>R</b>, Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. <b>S</b>, Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm (<b>A–F</b>) and 10 µm (<b>G–S</b>).</p

Lymphangiogenesis and angiogenesis in abdominal aortic aneurysm walls.

<p><b>A</b>, Elastica van Gieson staining of the abdominal aortic aneurysm (AAA) wall. Microvessels in the intima/media are encircled by a red dotted-line. <b>B</b>, Higher magnification of the area outlined with a solid line in <b>A</b>. Immunohistochemistry for podoplanin (<b>C</b>), CD31 (<b>D</b>), VEGFR-1 (<b>E</b>), VEGFR-2 (<b>F</b>), VEGFR-3 (<b>G</b>), VEGF-A (<b>H</b>), VEGF-C (<b>I</b>), and Ki-67 (<b>J</b>) in the area outlined with a solid line in <b>A</b>. The microvessels encircled by the red dotted-line consisted of podoplanin-positive cells (<b>C</b>) and CD31-positive cells (<b>D</b>). These microvessels were positive for VEGFR-1, VEGFR-2, and VEGFR-3 (<b>E–G</b>). Expression of VEGF-A and C was increased within and around these microvessels (<b>H, I</b>). The nuclei of these microvessels were positive for Ki-67 (<b>H</b>). <b>K</b>, Comparison of mRNA expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2, and VEGFR-3 between normal aorta and AAA. Data were obtained from 10 AAA patients and 9 autopsied cases (controls). Data were analyzed by comparative Ct method. All of the mRNAs except VEGF-D were upregulated in AAA tissues. Standard deviation is indicated by bar errors. * indicates p<0.05 vs control. Scale bars indicated 200 µm (<b>A</b>), 50 µm (<b>B–J</b>).</p