Raw Data

Raw dat

IL-4 and IL-13 blockade does not increase corneal angiogenesis.

<p><b>A.</b> Representative immunofluorescent cornea whole mount images stained for CD31 and LYVE-1. Scale bar = 100μm. <b>B.</b> Quantification of blood vessel area per 0.25mm². Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). <b>C.</b> Quantification of blood vessel volume per 0.25mm². Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS).</p

IL-4 or IL-13 blockade increases inflammatory lymphangiogenesis.

<p><b>A.</b> Representative gross images of corneas 14 days after suture placement. <b>B.</b> Representative immunofluorescent cornea whole mount images for LYVE-1. Scale bar = 100μm. <b>C.</b> Quantification of lymphatic vessel area per 0.25mm². Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). <b>D.</b> Quantification of lymphatic vessel volume per 0.25mm². Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; *p<0.05).</p

LNT decreases the pathological changes of lymphedema.

<p>A) <i>Left panel</i>: Representative H&E stain of the hindlimbs of mice with or without LNT. Cross-sections were obtained 2 mm proximal to the tarsal joint. The dotted black line indicates the area of fibroadipose deposition. The area highlighted by the blue dotted box is shown in high-power view in part B. <i>Right panel</i>: Quantification of the percentage of fibroadipose deposition area of hindlimbs of mice with and without LNT. B) <i>Left panel</i>: Representative high-power view of the areas indicated in the blue dotted boxes in part A. Note the decreased hyperkeratosis and dermal thickness in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of epidermal and dermal thickness in hindlimbs of mice with and without LNT. C) <i>Left panel</i>: Representative immunofluorescent images of hindlimbs stained for type I collagen (red) and nuclear DAPI (blue). Note decreased type I collagen deposition in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of type I collagen deposition (measured as a percentage of the total slide stained area) after surgery with and without LNT.</p

rhIL-4 and rhIL-13 decrease LEC proliferation and downregulate expression of Prox-1.

<p><b>A.</b> Flow cytometry analysis for incorporated EdU by cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative histograms. Quantification of EdU positive hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.01); control vs. rhIL-13 (n = 5 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). <b>B.</b> Quantification of hLEC proliferation using a WST-1 cell assay kit. Control vs. rhIL-4 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-4 vs. 100 ng/ml rhIL-4 (n = 10 in each; *p<0.05); control vs. rhIL-13 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-13 vs. 100 ng/ml rhIL-13 (n = 10 in each; p = NS). <b>C.</b> Representative immunofluorescent staining for Ki67 and VEGF-R3 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Ki67⁺ cells per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). <b>D.</b> Representative immunofluorescent staining for Prox-1 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Prox-1 signal intensity using MetaMorph analysis per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).</p

LNT promotes regeneration of collecting lymphatic vessels.

<p>A) <i>Upper panel</i>: Representative images of ICG lymphangiograms of normal mice (i.e., Cre-Lox mice without DT ablation), mice that had undergone local DT ablation followed by PLND but no LNT (-LNT), and mice that had undergone local DT ablation followed by PLND then LNT (+LNT). Main lymphatic collecting vessels can be seen in the normal hindlimb as white parallel linear structures, whereas no visible collecting vessels are noted in mice with ablated lymphatic circulation and no LNT (-LNT). Also note the dermal reflux as represented by accumulation of white dye diffusely in the mice without LNT (-LNT). In contrast, mice treated with LNT after lymphatic ablation (+LNT) have abnormal hyperplastic lymphatic vessels that converging toward the transplanted node, as indicated by the bright white spot in the blue dotted box. <i>Lower panel</i>: Immunofluorescent image of the transplanted lymph node indicated by the blue dotted box in the upper panel. Lymphatic collecting vessels indicated by podoplanin (green) and α-SMA (red). The inset in the lower left corner represents the magnified view of the lymphatic vessel adjacent to the transplant lymph node as indicated by the white dotted line. B) Representative immunofluorescent images of collecting lymphatics in mice with and without LNT. Note the collapsed lumen and proliferation of α-SMA⁺ cells in mice without LNT (-LNT). <i>C)</i> Quantification of collecting vessels in a 0.25 mm² area, collecting vessel diameter, and α-SMA thickness in mice with and without LNT. Note the significant increase in number of collecting vessels and luminal diameter with a corresponding decrease in α-SMA thickness in mice treated with LNT (+LNT).</p

IL-4 and IL-13 blockade increases LEC proliferation <i>in vivo</i>.

<p><b>A.</b> Representative immunofluorescent cornea whole mount images stained for Ki67 and LYVE-1. Scale bar = 100μm. <b>B.</b> Quantification of Ki67⁺/LYVE-1⁺ cells (n/0.25mm²). Control vs. IL-4mAb (n = 5 in each; *p<0.01); control vs. IL-13mAb (n = 5 in each; *p<0.01); IL-4mAb vs. IL-13mAb (n = 5 in each; *p<0.01).</p

LNT improves T cell-mediated immune responses.

<p>A) Schematic diagram of experimental protocol. Ten weeks after surgery with or without LNT, mice were sensitized with topical 0.5% DNFB to the ipsilateral hindlimb once daily for three days. T cell response was elicited by challenging the contralateral ear with topical 0.3% DNFB 5 days later. The ears were harvested for analysis 3 days later. B) <i>Left panel</i>: Representative H&E stain of the ear skin from mice with and without LNT. Note the increased inflammatory reaction in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of epidermal and dermal thickness of ear skin. Note significant increase in both epidermal and dermal thickness in mice treated with LNT (+LNT). C) <i>Left panel</i>: Representative images demonstrating immunofluorescent localization of CD45⁺ cells (red) in ear skin of mice with and without LNT. <i>Right panel</i>: Quantification of CD45⁺ cells per HPF (80x) in mice treated with and without LNT. Note the more robust inflammatory response as indicated by the greater amount of CD45⁺ cells in mice treated with LNT (+LNT).</p

rhIL-4 and rhIL-13 inhibit hLEC tubule formation.

<p><b>A.</b> Representative images of cultured hLECs tubule formation on matrigel after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 12 hours. <b>B.</b> Quantification of tubule formation per high-power field. Control vs. rhIL-4 (n = 5–8 in each; *p<0.01); control vs. rhIL-13 (n = 5–8 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5–8 in each; p = NS).</p

rhIL-4 and rhIL-13 inhibit hLEC migration.

<p><b>A.</b> Representative images of cultured hLECs after scratch wound and treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) at 0, 12 and 24 hours. White dotted lines represent LEC migration borders. Black solid lines represent initial gap created by scratch. <b>B.</b> Quantification of hLEC migration at 12 and 24 hours. Control vs. rhIL-4 (n = 5 in each; *p<0.01 for both time points); control vs. rhIL-13 (n = 5 in each; *p<0.01 for both time points); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS for both time points).</p