Exposure methods used as experimental plans: The transgenic Tg (kdrl;EGFP) zebrafish embryos were treated with the optimal dose (10 µg/mL) of snail extracts (LH, LM, LH3, and LM2) from 4 to 48 hpf (Method I) or from 4 to 72 hpf (Method II). The embryos treated via Method I were used to establish the dose curve and the mortality rate. Embryos treated with optimal doses via Method I were subjected to the analysis.

(Top) HPLC chromatograms of lyophilized extracts of H. aspersa (LH) and (Bottom) mucus of H. aspersa (LM) solubilized in 0.1% TFA (trifluoroacetic acid) and obtained at 200 nm.

Purified extracts from H. aspersa caused pro-angiogenetic effects on ISVs and intercapillary spaces in the CVP: (A–E) Representative images of zebrafish trunk ISVs of controls (fish water and 0.1% DMSO) (A) and treatments with 10 µg/mL of LH (B), LM (C), LH3 (D), and LM2 (E). All figures are lateral views with dorsal to the top and anterior to the left, and fluorescent vessels of embryos were observed at 48 hpf. White arrowheads indicate the CVP, showing an increase in the angiogenesis process. Enlarged white boxes indicate the magnification of ISVs and CVPs (F–J). Asterisks (*) indicate the intercapillary spaces. The experiments were repeated twice. Images were taken with a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Jena, Germany), using an objective Achroplan 10× and 20×/0.25 and a 488 nm laser, and represent 1 embryo out of 30 with the same phenotype (for more details, see Section 2).

The graphs represent the mean values obtained for ISV length (A) and the average numbers of intercapillary spaces in control and treated embryos (B). Data are representative of two replicates, and shown as the mean ± standard deviation; *** p < 0.001 vs. control group; ** p < 0.005 vs. control group; * p < 0.05 vs. control group; n = 15 from two independent experiments. Statistical analysis was performed with GraphPad version 8.3.3 (GraphPad Software, Inc., La Jolla, CA, USA).

Purified extracts from Helix aspersa caused an increased number of interconnecting vessels in the SIVP: Images from (A) to (J) show representative staining of an AP assay performed in control and treated embryos, with enlargement of the SIVP region at 72 hpf. (A–E) lateral view; (F–J) dorsal view. The scheme in (K) depicts the counting of ectopic sprouts. The graph in (L) shows the average number of SIVP branches. Data are representative of two replicates (n = 20 for each group) and are shown as the mean ± standard deviation; *** p < 0.001 vs. control group. Statistical analysis was performed with GraphPad version 8.3.3 (GraphPad Software, Inc., La Jolla, CA, USA).

Expression level analysis of vegfa as an angiogenesis marker: Embryos were treated with purified extracts from H. aspersa (LH, LM, LH3, and LM2) and analyzed at 48 hpf. Gene expression was normalized using rpl13a as a reference gene and expressed as the mRNA fold increase. Data are representative of three replicates, and are shown as the mean ± standard deviation; *** p < 0.001 vs. control group; * p < 0.05 vs. control group. Statistical analysis was performed with GraphPad version 8.3.3 (GraphPad Software, Inc., La Jolla, CA, USA).

Pretreatment with a VEGF pathway inhibitor prevented the formation of intersomitic vessels: The panels show representative images of a WISH assay performed with fli1 as an endothelial marker, at 36 hpf, in untreated (A,C,E,G,I) and treated embryos (B,D,F,H,J) with SUGEN 5416. Previously, some embryos were treated with snail derivatives via Method I and controls with fish water and 0.1% DMSO (see Materials and Methods, Section 2.4 and Section 2.5). Magnification of the trunk region (32×). Two replicates were performed (n = 15). Ratios at the bottom-left part of each picture specify the number of embryos showing the same staining pattern, compared to the total number of embryos used for each experiment. The images were taken in lateral position at 32× magnification with a Zeiss Axiozoom V13 (Zeiss, Jena, Germany) microscope, equipped with a PlanNeoFluar Z 1×/0.25 FWD 56 mm lens and Zen Pro software.