Table 1.
Baseline characteristics of the AAA group and control group.
Figure 1.
Lymphatic microvessels in intima of abdominal aortic aneurysm.
EVG staining of normal aorta (A) and abdominal aortic aneurysm (AAA) wall (B). Immunohistochemistry for podoplanin corresponding to the areas outlined with a solid line in A and B (C, normal aorta; D, AAA). Podoplanin-positive microvessels were markedly increased in the intima in AAA wall. E, Higher magnification of the outlined area in D. Double immunofluorescence staining of lymphatic vessels in intima of AAA (F–I). F, Podoplanin (red); G, Prox-1 (green); H, DAPI (blue); I, merge of podoplanin, Prox-1, and DAPI. Double immunofluorescence staining of smooth muscle cells in intima of AAA (J–M). J, podoplanin (red); K, alpha smooth muscle isoform of actin (green), L, DAPI (blue); M, merge of podoplanin, alpha smooth muscle isoform of actin, and DAPI. Immunohistochemistry for podoplanin corresponding to the areas outlined with a dotted line in A and B (N; normal aorta, O; AAA). Lymphatic vasa vasorum was observed in adventitia in both normal aorta (N) and AAA (O) (black arrow: LVV). Scale bars indicate 100 µm (A, B), 50 µm (C, D, J–M, N, O), and 10 µm (E–I). Lymphatic microvessel density; the mean number of lymphatic microvessel (LMVs) in 10 microscopic fields at high magnification (×200) (P). Percentage of lymphatic microvessel area in 10 microscopic fields at high magnification (×200) (Q). Lymphatic microvessel density and percentage of lymphatic microvessel area were compared between normal aorta and AAA. (*P<0.001).
Figure 2.
Lymphangiogenesis and angiogenesis in abdominal aortic aneurysm walls.
A, Elastica van Gieson staining of the abdominal aortic aneurysm (AAA) wall. Microvessels in the intima/media are encircled by a red dotted-line. B, Higher magnification of the area outlined with a solid line in A. Immunohistochemistry for podoplanin (C), CD31 (D), VEGFR-1 (E), VEGFR-2 (F), VEGFR-3 (G), VEGF-A (H), VEGF-C (I), and Ki-67 (J) in the area outlined with a solid line in A. The microvessels encircled by the red dotted-line consisted of podoplanin-positive cells (C) and CD31-positive cells (D). These microvessels were positive for VEGFR-1, VEGFR-2, and VEGFR-3 (E–G). Expression of VEGF-A and C was increased within and around these microvessels (H, I). The nuclei of these microvessels were positive for Ki-67 (H). K, 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 (A), 50 µm (B–J).
Figure 3.
HIF-1 expression in normal aorta and AAA.
Elastica van Gieson staining of the intima/media in normal aorta (A) and abdominal aortic aneurysm (AAA) wall (C). Immunohistochemistry for HIF-1α in normal aorta (B) and AAA (D). Nuclear and cytoplasmic expression of HIF-1α was observed in intima/media in AAA (D). E, Double immunofluorescence staining for HIF-1α (green), CD11b (red), DAPI (blue), and the merged image of the outlined in D. F, Double immunofluorescence staining for HIF-1α (green), VEGF-C(red), DAPI(blue), and the merged image of the outlined in D. G, Double immunofluorescence staining for HIF-1α(green), MMP-9 (red), DAPI (blue), and the merged image of the outlined in D. Expression of HIF-1α was increased in CD11b positive macrophages. VEGF-C and MMP-9 were expressed in the HIF-1α–positive macrophages. Scale bars indicated 50 µm (A–D) and 10 µm (E–G).
Figure 4.
Macrophage infiltration of AAA microvessels.
A, Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin (B) and macrophages (C), CD19 (E), and myeloperoxidase (MPO) of the AAA wall. B, Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C, Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D, CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E, CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F, MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P, Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P, Macrophage: green, CD11b (G)/LYVE-1 (H)/VEGF-C (I)/MMP-9 (J)/TGF-β1 (K)/IL-4 (L)/IL-8 (M)/MIP-1α (N)/IFN-γ (O)/MCP-1 (P): 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. Q, 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. R, 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. S, 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 (A–F) and 10 µm (G–S).
Figure 5.
Fluorescence lymphography and microscopy of abdominal aortic aneurysm.
A, Three dimensional CT image of an infrarenal abdominal aortic aneurysm (AAA). B, Intraoperative photography of an infrarenal AAA. C, Intraoperative near-infrared fluorescence lymphography of indocyanine green (ICG) (red arrow: ICG fluorescence, yellow dotted-line: sample resection line). D, Macroscopic findings of the resected sample observed from the luminal side (yellow dotted line: cross-section line of the harvested sample). E, 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). F, Near-infrared fluorescence lymphography of ICG in the cross-sectioned AAA wall (red arrow: ICG fluorescence, up: luminal side, bottom: adventitial side). G, Fluorescence microscopy of ICG in the AAA wall (ICG: red, DAPI: blue). H, Serial frozen section with Elastica van Gieson staining. I, Higher magnification of the outlined area in G. J, Higher magnification of the outlined area in H. K, Immunohistochemistry for podoplanin in the outlined area in G and H. L, Immunohistochemistry to label macrophages in the outlined area in G and H. Scale bars indicate 50 µm (G–L).