Research ArticleVascular Biology

Leukotriene B4 antagonism ameliorates experimental lymphedema

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Science Translational Medicine  10 May 2017:
Vol. 9, Issue 389, eaal3920
DOI: 10.1126/scitranslmed.aal3920
  • Fig. 1. Ketoprofen efficacy in a preclinical model of lymphedema can be attributed to its inhibition of LTB4.

    (A) Mouse tail model of acquired lymphedema was surgically induced in the tails of female C57BL/6J mice through thermal ablation of lymphatic trunks (lymphatic surgery). Skin incision alone was performed in the sham surgery group (sham). (B) Natural progression of mouse tail model of lymphedema. Tail volume at each measurement time point was calculated as Δ volume from day 0. Cartoon representations of the cross-sectional view of lymphedematous tails were created to illustrate lymphedema progression. Mice without surgery (control) or sham groups were compared to the categories subjected to lymphatic surgery; n = 8. (C) Overview of the eicosanoid pathway. Therapies, targeting different eicosanoid pathways, tested in the study were marked in blue. (D to J) Serial tail volume measurements at each time point over 24 days. Treatments targeting both 5-LO and COX1/2 (ketoprofen, n = 15) (D), 5-LO (zileuton, n = 10) (F), LTA4H (bestatin, n = 14) (H), BLT1 (Ly293111, n = 10) (I), or Ltb4r1 (local administering of lentiviral shLtb4r1, n = 6) (J) were compared with ibuprofen (inhibits COX1/2, n = 13) (E) and montelukast (antagonizes CysLT, n = 10) (G) therapies. All therapies started on postsurgical day 3. Cartoon representations in red demonstrate the cross-sectional view of the lymphedematous tails in the saline-treated groups; cartoon in blue illustrates the eicosanoid inhibitor–treated animals after lymphatic ablation. Quantification of dermal (K) and epidermal (L) skin thickness and lymphatic area (M) in the day 24 mouse tail skin for (D) to (J); n = 5. In (B) and (D) to (M), data are presented as means and SEM; ns, not significant, Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses.

  • Fig. 2. Bestatin treatment improves tail anatomy and restores lymphatic function.

    (A) Representative histology of mouse tail harvested on day 24 comparing samples from sham surgery control (sham) and animals treated with saline or bestatin after lymphatic ablation surgery (lymphatic surgery). Yellow arrowheads point at lymphatic dilation. Cutaneous dimension is indicated by yellow arrows. Scale bar, 200 μm; n = 6. (B) Fluorescence dextran microlymphangiography in the Prox1-Cre-ERT2-tdTomato mouse tail. Lymphatics are genetically marked by tdTomato (red) and outlined with a white dashed line. Fluorescein isothiocyanate (FITC)–dextran is shown in green. FITC-dextran not taken up by lymphatics is indicated by a white asterisk. Scale bar, 100 μm; n = 5. (C) Representative still photographs from movies S2 and S3 captured by a near-infrared (NIR) imaging system with a controlled pressure cuff. The collecting lymphatic function was tracked by imaging the transportation of a NIR dye in the vessels. Collecting lymphatics and the surgical wound are marked. Direction of lymph flow from the distal to the proximal part of the mouse tail is indicated. Scale bar, 500 μm; n = 3. (D) Trafficking ability of collecting lymphatics as quantified by the rate of NIR packet movement; n = 3; data are presented as means and SEM, Mann-Whitney test. (E) Representative images showing extravasation of Evans Blue dye from the lymphatics distal to the wound in the saline-treated mouse tail after lymphatic surgery; n = 3.

  • Fig. 3. LTB4 exhibits concentration-dependent effects on HLEC lymphangiogenesis and survival.

    (A) Representative images of HLEC network formation, fibrin gel sprouting, and 3D spheroid sprouting assays. HLECs were treated with various concentrations of LTB4: 5.0 to 10 nM LTB4 has prolymphangiogenic activity, and 200 to 400 nM is the antilymphangiogenic concentration. Scale bars, 100 or 25 μm, as indicated; n = 5. (B to E) Quantitative analysis of (A). (F to I) Quantitative analysis of HLECs subjected to VEGF-C (50 ng/ml) and 400 nM LTB4 with or without 10 μM U75302 (a BLT1 inhibitor) or lentiviral shLtb4r1 in network formation, migration, wound healing, and fibrin gel sprouting assays in fig. S6B. Lentiviral short hairpin RNA (shRNA) transduction particles targeting turbo green fluorescent protein (shGFP) were used as controls; n = 5. (J) Quantification of Matrigel plug assay in fig. S6F. Growth factor–reduced Matrigel containing HLECs pretreated with VEGF-C (50 ng/ml) and 400 nM LTB4 with or without 10 μM U75302 was injected subcutaneously into SCID mice. Lymphangiogenesis in vivo was determined as percentage of lymphatic vascular area; n = 5. (K) Analysis of HLEC viability, apoptosis, and cytotoxicity 24 hours after LTB4 culture; n = 6. (L) Western blotting of cleaved caspase 3 in HLECs; n = 3. In (B) to (J) and (L), data are presented as means and SEM; comparisons with the control groups were made using Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses. In (K), mean fluorescence readings are shown.

  • Fig. 4. LTB4 production is elevated in preclinical and clinical lymphedema.

    (A) LTB4 concentrations in the lymph fluid, measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) over time as mouse tail lymphedema developed; n = 6. (B) Mouse serum LTB4 concentrations, measured by LC-MS/MS. Blood serum was collected on postsurgical day 24; n = 6. (C) Transcripts of Ltb4r1 and Ltb4r2 in the tail skin, measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR); n = 5. (D) Quantification of 5-LO–positive macrophages in the mouse tail skin in fig. S3B; n = 5. (E) LTB4 concentrations in the blood serum of healthy controls (n = 21) and lymphedema (n = 18) patients, measured by LC-MS/MS. Demographic data are shown in table S1. (F) Quantification of 5-LO–expressing macrophages in patient skin samples in fig. S8; n = 5. For (A), (B), and (E), data are presented in box-and-whiskers plots showing minimal to maximal values and all data points; for (C), (D), and (F), data are presented as means and SEM; comparisons with day 0 samples in (A) and (C) were made using Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses; comparisons as indicated in (B), (D), (E), and (F) used the Mann-Whitney test.

  • Fig. 5. Blocking LTB4 during initial lymphangiogenesis period abrogates the therapeutic benefit of LTB4 antagonism.

    (A to E) Serial tail volume measurements of conditions with LTB4 antagonism before initial lymphangiogenesis period: Alox5−/− mice (n = 25) (A), Ltb4r1−/− mice (n = 10) (B), WT mice treated with shLtb4r1 lentivirus on day(−7) (shLtb4r1 pretx, n = 8) (C), and WT animals treated with bestatin started on day 0 (bestatin pretx, n = 6) (D) or with Ly293111 started on day 0 (Ly293111 pretx, n = 6) (E). (F and G) Quantification of dermal thickness (F) and lymphatic area (G) in the day 24 tail skin for (A) to (E); n = 5. (H) Quantitations of tail volume on postsurgical day 24 for various groups. (I) Relative mRNA expression of key lymphangiogenic factors in the mouse tail skin harvested on day 24 were measured by qRT-PCR. Results were normalized to the saline-treated group. Green indicates an increased average fold change; red indicates a decreased fold change; n = 5. For (A) to (G), data are presented as means and SEM; for (H), data are presented in box-and-whiskers plots showing minimal to maximal values and all data points; comparisons with the saline-treated groups were made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses.

  • Fig. 6. LTB4 exerts concentration-dependent effects on VEGFR3 and Notch signaling.

    (A) qRT-PCR analysis measured the relative transcripts of Vegfr3 in HLECs after the treatment of LTB4; n = 5. (B) Western blots detecting phospho-VEGFR3 and total VEGFR3 in HLECs; n = 3. (C) qRT-PCR analysis measured the relative transcription of Vegfr2 in HLECs after the treatment of LTB4; n = 5. (D) Luciferase activity of HLECs cotransfected with Notch reporter plasmid pG13-11-CSL and pRL-SV40; n = 5. Luciferase activity (E) or relative transcript expression of EFNB2, Hes1, and Hey1 in HLECs (F to H) was subjected to the treatment of recombinant Dll4 (50 μg/ml; Notch ligand), 25 μM DAPT (Notch inhibitor), 200 nM LTB4, 200 nM LTB4 + shLtb4r1, or 200 nM LTB4 + Dll4 (50 μg/ml); n = 5. All data are presented as means and SEM; comparisons with the control groups were made using Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses.

  • Fig. 7. Bimodal effects of LTB4 on lymphangiogenesis are regulated by VEGFR3 and Notch signaling.

    (A to D) Representative fibrin gel and 3D spheroid sprouting assay images (A and B) and quantification of bimodal effects of LTB4 on HLEC lymphangiogenesis through regulation of VEGFR3 and Notch signaling (C and D). HLECs were treated with VEGF-C (50 ng/ml), recombinant Dll4 (50 μg/ml), 25 μM DAPT, 5 nM LTB4, 5 nM LTB4 + shLtb4r1, 5 nM LTB4 + 5 μM MAZ 51(VEGFR3 inhibitor), 200 nM LTB4, 200 nM LTB4 + shLtb4r1, 200 nM LTB4 + Dll4 (50 μg/ml), 200 nM LTB4 + VEGF-C (50 ng/ml), or 200 nM LTB4 + Dll4 (50 μg/ml) + VEGF-C (50 ng/ml. Scale bar, 25 μm. Data are presented as means and SEM; n = 5; comparisons with the control group were made using Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses. (E) HLEC viability, apoptosis, and cytotoxicity. Mean fluorescence readings are presented; n = 3.

  • Fig. 8. Loss of Notch signaling in LECs abrogates effectiveness of LTB4 antagonism in experimental lymphedema.

    (A) Representative histology and fluorescence microlymphangiography of mouse tails of the lymphatic endothelial cell-specific, Notch1-deficient (Notch1LECKO) mice after sham or lymphatic ablation surgery. Lymphatic dilation is indicated by yellow arrow. Lymphatics are marked by tdTomato and outlined with a white dashed line. FITC-dextran is shown in green. FITC-dextran not taken up by lymphatics is indicated by a white asterisk. White arrowheads point at vessel hypersprouting. Scale bars, 300 or 100 μm, as indicated; n = 5. (B) Day 24 tail volume measurements of Notch1LECKO mice subjected to lymphatic ablation surgery, treated with saline (n = 7) or bestatin (n = 7), compared with sham controls (n = 7). Quantification of dermal skin thickness (C) and lymphatic dilation (D) of WT or Notch1LECKO mice in (B); n = 5. (E to G) Relative whole-tail gene transcripts of Vegfr3 (E), NRP2 (F), and EFNB2 (G) of WT or Notch1LECKO mice in (B); n = 5. For (B), data are presented in box-and-whiskers plots showing minimal to maximal values and all data points; for (C) to (G), data are presented as means and SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test for post hoc analyses.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/389/eaal3920/DC1

    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 (Notch1LECKO) mice.

    Fig. S15. Bestatin does not rescue Notch signaling or limit lymphatic dilation in Notch1LECKO mice.

    Fig. S16. Effects of bestatin on inflammation and microvascular permeability in Notch1LECKO 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.

    Table S3. Primary data.

    Movie S1. NIR imaging of collecting lymphatics in the healthy control mouse.

    Movie S2. NIR imaging of collecting lymphatics in the bestatin-treated mouse after lymphatic ablation surgery.

    Movie S3. NIR imaging of collecting lymphatics in the saline-treated mouse after lymphatic ablation surgery.

  • 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 (Notch1LECKO) mice.
    • Fig. S15. Bestatin does not rescue Notch signaling or limit lymphatic dilation in Notch1LECKO mice.
    • Fig. S16. Effects of bestatin on inflammation and microvascular permeability in Notch1LECKO 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

    [Download PDF]

    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.