VEGFR3/Flt-4 Fc Chimera Protein 743-R3-100 from R&D Systems, a Bio-Techne Brand

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Nature communications
June 08 2020

Fig 1: Migratory phenotype and VEGF-C-induced hyperactivation of Akt in PIK3CAH1047R -expressing LECs.a Experimental plan for short-term 4-OHT induction for the analysis of initiation of microcystic LM formation in PIK3CAH1047R;Vegfr3-CreERT2 mice. Asterisk indicates the date of birth. b Whole-mount immunofluorescence of ear skin of 4-OHT-treated (100 µg) P21 PIK3CAH1047R;Vegfr3-CreERT2 mice (on the left) and quantification of the morphology of vessel ends in control (n = 3) and PIK3CAH1047R (n = 6) mice (mean ± s.e.m., Two-tailed unpaired Student’s t-test) (on the right). Mutant shows increase in spiky vessel ends representing active sprouts (arrow) in comparison to blunt ends of normal capillaries (arrowhead). c Staining for phospho-S6 staining as a readout of PI3K activity, showing high signal in active lymphatic vessel sprouts (arrows) in 3-week-old mutant skin and embryonic back skin. No signal was detected in normal capillaries (arrowheads). d Staining for VE-cadherin showing straight zipper-like junctions, typical of lymphangiogenic vessels, in active lymphatic sprouts (arrows) in mutant skin as opposed to oakleaf shaped LECs with button junctions in normal capillaries (arrowhead). e Immunofluorescence of primary LECs isolated from PIK3CAH1047R;Vegfr3-CreERT2 mice, showing increase in stress fiber formation and disruption of cell-cell junctions in 4-OHT-treated (PIK3CAH1047R-expressing) compared with vehicle-treated (Ctrl) cells. Images are representative of three independent experiments. f IncuCyte scratch assay showing faster wound closure in 4-OHT-treated (PIK3CAH1047R-expressing) compared with vehicle-treated (Ctrl) primary LECs. Data is representative of two independent experiments showing mean relative wound density (n = 7 (Ctrl) or 5 (H1047R) wells ± s.d.). The difference between area under the curves: P < 0.0001 (Two-tailed unpaired Student’s t-test). Representative images of wells at t = 0 h and t = 48 h are shown on the right. Wound area is highlighted in light gray. g–i, Western blot analysis (g, h) and quantification of phospho-Akt (Ser473) (i) in primary dermal LECs from PIK3CAH1047R;Vegfr3-CreERT2 mice treated in vitro with 4-OHT and/or VEGF-C for indicated times. Data are representative of one experiment (g) or three independent experiments (h). Data in (i) represent mean (n = 3 independent experiments) ± s.e.m. Two-tailed one-sample t-test (untreated Ctrl vs. PIK3CA in (i)) or Two-tailed unpaired Student’s t-test (all others). Scale bars: 50 µm (b–e), 300 µm (f). Source data are provided as a Source Data file.
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Nature communications
June 08 2020

Fig 2: PIK3CA mutations underlie both micro- and macrocystic LM.a Hematoxylin and eosin stained sections of three microcystic (on the left) and two macrocystic (on the right) LMs. Note RBCs inside and outside of the malformations (asterisks), and lymphoid cell infiltration (yellow arrows). b On the left: hematoxylin and eosin stained sections of macrocystic and microcystic LM. On the right: immunofluorescence staining showing VEGFR3 expression in the endothelium lining both macrocystic and microcystic LM (arrows), except for areas associated with smooth muscle cells (arrowhead). Boxed area is shown in both hematoxylin and eosin staining and immunofluorescence staining. Note high expression of VEGFR3 in small-caliber lymphatic vessels in areas containing infiltrated CD45+ cells (asterisks). lum = vessel/cyst lumen. Images in (a, b) are representative of n = 3 microcystic LM and n = 2 macrocystic LM lesions. Scale bars: 200 µm (a, b).
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Nature communications
June 08 2020

Fig 3: Developmental timing of activation of PIK3CA determines LM subtype.a Genetic constructs and experimental plan for tamoxifen-inducible activation of PIK3CAH1047R expression in lymphatic endothelia at early embryonic (blue, 4-OHT 2 mg) or postnatal (gray, 4-OHT dose as indicated) development. Asterisk indicates the date of birth. b–d Characterization of PIK3CAH1047R-driven macrocystic LM. Whole mount immunofluorescence of back skin from an E17 PIK3CAH1047R;Vegfr3-CreERT2 embryo (b). Antibodies and regions of skin are indicated, and boxed areas (i, ii) are magnified below. Arrows point to cyst-like malformations in the neck (cervical) region of the skin that were quantified in (c). Data represent mean ± s.e.m. Immunofluorescence analysis of paraffin-embedded sections of lymphatic lesions showing overgrowth of lymphatic (VEGFR3+) but not blood vessels (EMCN+) (d). e, f Characterization of PIK3CAH1047R -driven microcystic LM. Immunofluorescence analysis of skin whole-mounts (e; Vegfr3-CreERT2 model) or paraffin sections (f; Prox1-CreERT2 model), and boxed areas (e) are magnified. 4-OHT dosage, timing of administration and analysis are indicated. Inset in (e) shows bleeding in the lesion area. Dotted lines in (d, f) indicate epidermis. Scale bars: 50 µm (d, f), 250 µm (b (i, ii), e), 2 mm (overview images in b, e). Source data are provided as a Source Data file.
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Nature communications
June 08 2020

Fig 4: PIK3CAH1047R-driven LM is dependent on VEGF-C signaling.a Whole-mount immunofluorescence of ear skin of 5 weeks old PIK3CAH1047R;Vegfr3-CreERT2 (left panels) mice showing upregulation of VEGFR3 and NRP2 expression but unchanged VEGFR2 levels in abnormal sprouts (arrows) in comparison to capillaries with normal appearance (arrowheads), and those in wild-type mice (right panels). 4-OHT (100 µg) was administered at P21. b Quantification of VEGFR3 staining intensity in the dermal lymphatic vasculature in the ear skin of 4-OHT-treated PIK3CAH1047R;Vegfr3-CreERT2 mice (n = 5), compared with littermate controls (n = 4) ± s.d. c Immunofluorescence staining of paraffin sections of ear skin showing increase in CD45+ immune cells around LYVE1+ lymphatic lesions in a PIK3CAH1047R;Prox1-CreERT2 mouse compared with a control (Ctrl). d Percentage of CD45+CD11b+F4/80+ macrophages relative to all CD45+ cells in the ear skin of 5 weeks old PIK3CAH1047R;Vegfr3-CreERT2 mice treated with the vehicle (n = 6) or 4-OHT (100 µg, PIK3CAH1047R; n = 7) at P21 (mean ± s.d.). e Experimental plan for the induction of progressive microcystic LM and inhibition using the soluble VEGF-C trap (AAV-VEGFR3-Ig; AAV-sR3) or vehicle (PBS). f Whole-mount staining of ears from untreated PIK3CAH1047R;Vegfr3-CreERT2 mice (Ctrl), and mice treated with 4-OHT (100 µg) and AAV-sR3 or vehicle, and analyzed at different stages after induction. Images in red frame show immunofluorescence of ears from AAV-sR3 treated mice. g Quantification of lymphatic vessel branching in the progressive LM model. Red squares represent data from AAV-sR3 treated mice. Data represent mean (n = number of ears as indicated) ± s.d. Two-tailed unpaired Student’s t-test. Scale bars: 50 µm (a), 200 µm (c, f). Source data are provided as a Source Data file.
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Nature communications
June 08 2020

Fig 5: Co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream mTOR signaling pathways promotes LM regression.a Experimental plan for the induction and treatment of progressive microcystic LM using a combination therapy with VEGF-C trap and Rapamycin. b Whole-mount staining of ears from PIK3CAH1047R;Vegfr3-CreERT2 control (Ctrl) and 4-OHT-treated (100 µg) mice at 5 weeks of age, or following a 1.5-week treatment with Rapamycin, AAV-sR3 and/or vehicles at 6.5 weeks of age. c Quantification of lymphatic vessel branching in the PIK3CAH1047R;Vegfr3-CreERT2 mice. Stages of analysis and treatments are indicated. Data represent mean (n = number of ears as indicated) ± s.e.m. d Flow cytometry analysis of dermal LEC proliferation in the PIK3CAH1047R;Vegfr3-CreERT2 mice treated as indicated. Data represent mean (n = number of mice as indicated) ± s.d. e Lymphatic vessel morphology in areas of hypersprouting in 6.5-week-old 4-OHT-treated PIK3CAH1047R;Vegfr3-CreERT2 ears. Note blunt morphology of lymphatic sprouts in mice treated with Rapamycin and AAV-sR3 compared with vehicle-treated mice (yellow arrows). f Quantification of all vessel ends and their morphology (upper graph), with data shown separately for spiky ends representing active sprouts (lower graph), in the indicated groups. Data represent mean (n = number of mice as indicated ± s.e.m.) Two-tailed unpaired Student’s t-test. Scale bars: 200 µm (b), 100 µm (e). Source data are provided as a Source Data file.