Fig 2: BACE2-dependent changes in VEGFR3 signaling in LECs.(A) Schematic for VEGFR3 signaling. Upon ligand binding, VEGFR3 dimerizes, resulting in intracellular autophosphorylation and activation of the downstream genes FOXC2 and DLL4. V, verubecestat, inhibitor of BACE2. Gene expression levels of (B) DLL4 and FOXC2 and (C) VEGFR3 after the application of DMSO, 100 nM verubecestat (V), and VEGF-C. (D and E) Gene expression levels of DLL4, FOXC2, VEGFR3, BACE1, and BACE2 after BACE knockdown (siB1/siB2) without or with (+V) subsequent verubecestat application. All dot plots were normalized on the control mean and depict mean and SD alongside the P values calculated by unpaired t tests against the DMSO control (B and C) or by 1-way ANOVA (D and E), in both cases followed by Bonferroni’s multiple-comparison test. *P < 0.05; **P < 0.01; ****P < 0.0001. P values are only indicated where significance was observed. In B, 1 data point was excluded from the DLL4 expression/VEGF-C156S data set, since it was identified as an outlier via the ROUT method. Data are derived from n = 6 (B and C) or n = 4 (D and E) biological replicates.

Fig 3: BACE2 inhibition reduces murine plasma sVEGFR3.Volcano plots of proteomic analysis of murine plasma from (A) compound 89–treated (Cpd89) versus vehicle-treated (veh) mice and (B) LY2811376-treated versus vehicle-treated mice (n = 13, treated; n = 14, veh). VEGFR3 is highlighted in red. (C) Corresponding extracted LFQ intensities of sVEGFR3 and (D) MSD-assay quantifications of sVEGFR3. Plasma sVEGFR3 (E) and plasma sSEZ6L (F) levels in 8–10 B1KO, B2KO, and respective WT mice with (blue) or without (black) 3 days of 50 mg/kg per os twice a day verubecestat dosing. (G) Plasma levels of VEGFR3 and SEZ6L during 7 days of 0.1% dietary verubecestat (average drug intake, 97 mg/kg/d; n = 6 per group, all male, age: 7–10 weeks), respective to untreated control levels. Two-sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots (A and B). Proteins with P < 0.05 are shown as red circles. (C) Significance after FDR correction is indicated with plus signs. All dot plots were normalized on the control mean and depict the SD alongside the calculated P values, calculated by 1-way (D and G) or 2-way (E and F) ANOVA with Bonferroni’s multiple-comparison test. *P < 0.05; ***P < 0.001; ****P < 0.0001. P values are only indicated where significance could be observed. Number of biological replicates in E and F was 9, except for Bace1-WT + verubecestat (n = 8) and for Bace2WT without verubecestat (n = 10).

Fig 4: Identification of VEGFR3 as a BACE2 substrate candidate.(A) Volcano plot of proteomic analysis of murine plasma from WT and B2KO mice (n = 6). VEGFR3 (FLT4) is highlighted in red. (B) Normalized VEGFR3 LFQ intensities extracted from A. (C) MSD-assay quantifications of sVEGFR3 in the same plasma samples. (D) Immunoblot detection of sVEGFR3 ectodomain in mouse plasma from A, using nonreducing and reducing conditions. (E) Volcano plot of proteomic analysis of murine plasma from an independent B2KO line (n = 9) compared with WT (n =9) and (F) the extracted normalized LFQ values. Volcano plots of the proteomic analyses of Bace1/Bace2 double-knockout (BDKO) mice (n =9) compared with the WT line (n = 9) (G) (corresponding extracted LFQ intensities of sVEGFR3 in F) and B1KO (n = 9) compared with an individual control WT line (n = 9) (H). (I) Normalized LFQ values extracted from H. (J) Localization of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The signal peptide is shown in rose, the ectodomain is indicated in blue, the intracellular domain in green, and the transmembrane domain in yellow. Two sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots (A, E, G, and H). Proteins with P < 0.05 are shown as red circles. Extracted LFQ quantifications (B, F, and I) of VEGFR3 with significance after FDR correction are labeled with plus signs. All dot plots were normalized on the WT mean and depict mean and SD. MSD-assay data (C) additionally depicts the P value calculated by unpaired t test. ****P < 0.0001.

Fig 5: Inhibition of Bace2 in zebrafish embryos leads to enhanced facial lymphatic development.(A) Laterally imaged zebrafish embryos and representative images comparing the facial lymphatic development between vehicle control– (Veh) and verubecestat-treated (100 μM) (Verub) zebrafish embryos at 3 dpf in a flt4:mCit transgenic background. Asterisks represent the starting points of measurements, while arrowheads depict the end points of the respective measurements. (B) Quantification of LFL vessel length (in μm) in vehicle control– and verubecestat-treated (100 μM) zebrafish embryos at 3 dpf (n = 18 for vehicle, n = 20 for verubecestat-treated group). (C) Representative images comparing the facial lymphatic development between vehicle control– and verubecestat-treated (100 μM) zebrafish embryos at 5 dpf in a fli:nucGFP and lyve1:dsRed transgenic background. Enlarged image of the LFL depicts nuclei of all endothelial cells (green, fli:nucGFP), the cytoplasm of the facial lymphatic vessel (red, lyve1:dsRed) and other lymphatic vessels, and the nuclei present in the facial lymphatic vessel (yellow, colocalization). (D) Corresponding quantification of the number of fli:nucGFP+ nuclei present in the LFL of 5 dpf embryos (n = 23 for control; n = 29 for verubecestat-treated group). All images with anterior to the left. All dot plots depict mean and SD, alongside P values calculated by unpaired t test. ***P < 0.001; ****P < 0.0001.