1. Excess Lymphangiogenesis Cooperatively Induced by Macrophages and CD4+ T Cells Drives the Pathogenesis of Lymphedema 
Fusa Ogata, Katsuhito Fujiu, Sahohime Matsumoto, Yukiteru Nakayama, Munehiko Shibata, Yuichi Oike, Isao Koshima, Tetsuro Watabe, Ryozo Nagai, Ichiro Manabe 
Journal of Investigative Dermatology 
Volume 136, Issue 3, Pages (March 2016)
DOI: /j.jid
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2. Figure 1
Lymphatic vessel obstruction induces edema and excessive lymphangiogenesis. (a) Picrosirius red staining of transverse sections 5 mm caudal to the inguinal lymph node after the axillary lymphatic obstruction (Supplementary Figure S1c). Sections from age-matched untreated (Normal) and sham-treated mice are also shown. Scale bars = 500 μm (lower magnification panels) or 100 μm (higher magnification panels). B: blood vessels, CL: major collector lymphatic vessels. (b) Skin thickness. n = 4 in each group. *P < 0.05 versus day 0. (c) Lymphatic vessels visualized using Evans blue. Shown are representative macroscopic views of subcutaneous tissue. Note the blue spots and light blue areas caused by leakage of Evans blue. Scale bars = 5 mm. (d) Lymphatic endothelial cells (LECs) and leukocytes in dermal tissues stained for podoplanin and CD45 (red), respectively, 7 days after lymphatic obstruction. Note that LECs in collector lymphatic vessels (CL) are not stained for podoplanin (Wick et al., 2008; Wollina et al., 2014). Scale bars = 100 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Lymphatic obstruction induces lymphangiogenesis via VEGF-C/VEGFR3. (a) Vegfc and Vegfd mRNA levels in edematous tissues. n = 3 for each group. mRNA expression levels were normalized first to those of 18s rRNA and then to the levels on day 0. *P < 0.05 versus day 0. (b, c) VEGF-C/VEGFR3 inhibition using a VEGFR3-Fc fusion protein. Control IgG-Fc or VEGFR3-Fc (6 μg) was injected into the tail vein on days −1 and 0. An additional 6 μg was administered subcutaneously into the region adjacent to the epigastric vein and collector lymphatic vessels intraoperatively. Seven days after the lymphatic obstruction, subcutaneous lymphatic vessels and edema were analyzed. n = 7 in each group. *P < Scale bars = 5 mm. (c) Picrosirius red-stained sections of edematous tissues 28 days after lymphatic obstruction. Scale bars = 100 μm. VEGF, vascular endothelial growth factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Macrophages promote lymphangiogenesis. (a) CD11b+F4/80+Ly6G− macrophage and CD3+ T-cell fractions among total live cells from edematous tissues. n = 3. *P < 0.05 versus day 0. Levels of mRNA expression were normalized first to those of 18s rRNA and then to the levels on day 0. (b) Vegfc expression in lesional macrophages. n = 3 in each. *P < (c, d) Effects of macrophage depletion induced by clodronate liposome treatment (c) and in Itgam-HBEGF transgenic mice (d). Lymphangiograms and skin thickness 7 days after lymphatic obstruction are shown. n = 4 in each group. Scale bars = 5 mm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
CD4+ T cells are essential for injury-induced lymphangiogenesis and edema. The lymphatic obstruction model was performed in (a) Rag2−/− and (b) Cd4−/− and Cd8a−/− mice. Seven days after the procedure, lymphatic vessels were visualized using Evans blue. Skin thickness is also shown. n = 3 in each group.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
CD4+ T-cell-macrophage interaction induces lymphangiogenesis. (a) Effects of coculturing lesional CD11b+ and/or CD4+ cells on human dermal lymphatic endothelial cell (HDLEC) tube formation. n = 3. *P < 0.05 versus HDLECs only. #P < (b) Lesional CD11b+ and CD4+ cells were cocultured in the presence of neutralizing antibodies, as indicated. Shown are levels of Vegfc mRNA in CD11b+ cells and VEGF-C in culture media. n = 3 in each group. *P < 0.05 versus CD11b+ cells only. #P < 0.05 versus CD11b+ and CD4+ cocultured with IgG. (c) Effects of IL-17A and IFN-γ on VEGF-C expression in lesional macrophages. Levels of Vegfc mRNA in the cells and VEGF-C in culture media are shown. *P < 0.05 versus vehicle. #P < n = 4–5. (d, e) Effects of IFN-γ and IL-17 inhibition on lymphangiogenesis and edema. (d) Lymphangiograms and skin thickness 7 days after lymphatic obstruction. n = 3 in each group. (e) Levels of Vegfc expression in subcutaneous tissues on day 7. n = 3. *P < VEGF, vascular endothelial growth factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Atorvastatin suppresses lymphedema development. (a–d) Atorvastatin (ATV) or vehicle was administered daily beginning 7 days before lymphatic obstruction. (a) Lymphangiograms and skin thickness on day 7. n = 4. *P < Scale bars = 5 mm. (b) Picrosirius red staining of skin sections 3 months after the operation and age-matched samples. Scale bars = 100 μm. (c) IFN-γ+ and IL-17+ CD3+ CD4+ cell fractions and CD11b+F4/80+ macrophage fractions in subcutaneous tissues 7 days after lymphatic obstructions. n = 4. (d) Vegfc mRNA levels in lesional CD11b+F4/80+ cells (n = 3) and dermal tissues (n = 9). (e) mRNA levels in the edematous tissues were analyzed. n = 8. *P < 0.05 versus day 0 of the same treatment. #P < (f, g) Lesional CD11b+ and CD4+ cells were treated with vehicle or ATV (10 μM). Shown are Vegfc mRNA levels in CD11b+ cells and VEGF-C levels in the culture media (f) as well as Ifng and Il17 mRNA levels in CD4+ cells (g). n = 9–10. *P < ATV, atorvastatin; VEGF, vascular endothelial growth factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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