Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: 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 ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining