Supplementary Material for:

Leukotriene B₄ antagonism ameliorates experimental lymphedema

Wen Tian, Stanley G. Rockson,* Xinguo Jiang, Jeanna Kim, Adrian Begaye, Eric M. Shuffle, Allen B. Tu, Matthew Cribb, Zhanna Nepiyushchikh, Abdullah H. Feroze, Roham T. Zamanian, Gundeep S. Dhillon, Norbert F. Voelkel, Marc Peters-Golden, Jan Kitajewski, J. Brandon Dixon, Mark R. Nicolls*

*Corresponding author. Email: rockson{at}stanford.edu (S.G.R.); mnicolls{at}stanford.edu (M.R.N.)

Published 10 May 2017, Sci. Transl. Med. 9, eaal3920 (2017)
DOI: 10.1126/scitranslmed.aal3920

This PDF file includes:

• Materials and Methods
• Fig. S1. Mouse tail model of acquired lymphedema.
• Fig. S2. Analysis of mouse tail edema and LTB₄ and BLT1 expression after LTB₄ antagonism started on postoperative day 3.
• Fig. S3. Bestatin treatment reduces inflammation.
• Fig. S4. Macrophage depletion does not resolve mouse tail lymphedema.
• Fig. S5. Bestatin treatment reduces microvascular permeability in mouse tail lymphedema.
• Fig. S6. LTB₄ inhibits in vivo and in vitro HLEC lymphangiogenesis.
• Fig. S7. LTB₄ (400 nM) damages HLEC junctions and reduces connexin mRNA transcript.
• Fig. S8. 5-LO expression in neutrophils and macrophages is increased in human lymphedema.
• Fig. S9. Increased LTB₄ and decreased PGE2 signaling in mouse tail lymphedema.
• Fig. S10. LTB₄ antagonism before initial lymphangiogenesis period is not therapeutic.
• Fig. S11. LTB₄ exerts concentration-dependent effects on HLEC VEGFR2 signaling.
• Fig. S12. Overexpressing 5-LO interferences lymphatic drainage and promotes microvascular leakage in mouse tail.
• Fig. S13. LTB₄ (200 nM) inhibits Notch signaling in HLECs.
• Fig. S14. Confirmation of the generation of Prox1-specific, Notch1-deficient (Notch1^LECKO) mice.
• Fig. S15. Bestatin does not rescue Notch signaling or limit lymphatic dilation in Notch1^LECKO mice.
• Fig. S16. Effects of bestatin on inflammation and microvascular permeability in Notch1^LECKO mice.
• Fig. S17. Bestatin does not rescue tail lymphedema in mice treated with DAPT.
• Table S1. Demographics of healthy controls and lymphedema patients—LTB₄ and PGE₂ analysis.
• Table S2. Summary of lymphangiogenesis and angiogenesis microarray results.
• Legends for movies S1 to S3

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Other Supplementary Material for this manuscript includes the following:

• Table S3 (Microsoft Excel format). Primary data.
• Movie S1 (.mp4 format). NIR imaging of collecting lymphatics in the healthy control mouse.
• Movie S2 (.mp4 format). NIR imaging of collecting lymphatics in the bestatin-treated mouse after lymphatic ablation surgery.
• Movie S3 (.mp4 format). NIR imaging of collecting lymphatics in the saline-treated mouse after lymphatic ablation surgery.