HPLC and 1HNMR data of GAH. (a) Peak graph of standard and Agaricus bisporus-derived (sample) GAH. (b) Related parameter of (a). (c) Proton NMR spectra. (d) Chemical shifts of the 1H of standard and sample GAH.

GAH promotes zebrafish vascular development. (a,b) Vascular development status of transgenic fish with labeled vascular endothelial cells (red) and their nuclei (green) after treatment with different concentrations of GAH as indicated at 48 hpf using a laser scanning confocal microscope. Yellow arrows indicated: CtA, central arteries; PHBC, primordial hindbrain channel; MsV, mesencephalic vein; MtA, metencephalic artery; PHS, primary head sinus; VA, ventral aorta; AA, branchial arch; IOC, inner optic circle; OV, optic vein; PrA, prosencephalic artery; Se, intersegmental vessel; DLAV, dorsal longitudinal anastomotic vessel; CCV, common cardinal vein; CA, caudal artery; CV, caudal vein; PCeV, posterior (caudal) cerebral vein. Column 1 (vertical row) was a diagram of the blood vessels in the head region; Column 2 was a diagram of intersegmental vessels. Column 3 was a diagram of the nuclei of the vascular endothelial cells in the head; Column 4 was a diagram of the intersegmental vessels’ vascular endothelial cell nuclei. The experiment was repeated two times. (c–e) Statistical data of (a,b). (f) Expression of kdrl at 24 hpf and 48 hpf by ISH with increasing concentrations of GAH as indicated. Red arrows indicated MCeV, middle cerebral vein; PMBC, primordial midbrain channel; DA, dorsal aorta; CA, caudal artery; PCV, posterior (caudal) cardinal vein; CV, caudal vein; Se, intersegmental vessel. (g) Statistical data of (f) based on three (24 hpf) and four (48 hpf) repetitions. (h) BrdU incorporation assay to investigate cell proliferation activity. Green fluorescence indicated a vascular endothelial nucleus; red fluorescence indicated BrdU-positive cells. The experiment was repeated twice. Scale bars: 100 μm. (i) Statistical data of the number of BrdU-positive cells co-located with GFP in (h). p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***); NS, nonsignificant.

GAH repairs vascular impairment by regulating the VEGF signal. (a1–d3) The vascular development of Tg(kdrl: mCherry) zebrafish at 72 hpf under a laser scanning confocal microscope. Zebrafish embryos were exposed to 0.15 mmol/L VRI to induce vascular impairment and then treated with GAH for recovery. Type 1~Type 3 indicated different vascular impairment phenotypes, from slight to severe. Arrows indicated: Se, intersegmental vessel; CCV, common cardinal vein; CV, caudal vein; MsV, mesencephalic vein; PCeV, posterior cerebral vein; CtA, central artery; PrA, prosencephalic artery; IOC, inner optic circle; AA3–AA6, first to fourth branchial arch; PHBC, primordial hindbrain channel; DLAV, dorsal longitudinal anastomotic vessel; CA, caudal artery. (a1–d1): diagram of whole-body blood vessels. (a2–d2): enlarged diagram of the blood vessels in the head. (a3–d3): enlarged diagram of intersegmental vessels. (e1,e2): classification criteria and statistics of different types for head and trunk. p < 0.001 (***). (f) Statistical data of different types of vascular phenotypes across different treatment groups. The experiment was repeated three times.

GAH restores BMP signaling and facilitates the repair of vascular impairment induced by DMH1 and DM. Tg (BRE: GFP; kdrl: mCherry) double transgenic embryos were was treated with 10 μM DMH1 (a1–e3) or 10 μM DM (f1–j3) and then treated with GAH as indicated. BMP (green) activity and vascular development (red) and their colocalization were observed by confocal at 48 hpf. Arrows indicated: Se, intersegmental vessel; MsV, mesencephalic vein; PCeV, posterior cerebral vein; CtA, central artery; PCV, posterior cardinal vein; PHS, primary head sinus; VA, ventral aorta; CCV, common cardinal vein. The experiment was repeated twice for each inhibitor. (k,l) Statistical data of BRE and mCherry fluorescent intensity after DMH1 inhibition (a1–e1) and (a2–e2). (m,n) Statistical data of (f1–j1,f2–j2). Compared with the ctr group, p < 0.05 (#), p < 0.01 (##); compared with the 24 h + 10 μM DMH1/DM group, p < 0.05 (*), p < 0.01 (**), and nonsignificant (NS).

GAH repairs vascular impairment by upregulating the mRNA and protein levels of BMP and VEGF signaling pathway members. (a) Spatiotemporal expression of kdrl by ISH in different groups. Red arrows indicated the specific expression of kdrl in PMBC, PCV, CV, DA, and MCeV. Scale bars: 100 μm. The proportion of embryonic expression patterns in each group was shown as indicated and based on four (for DMH1) and three (for DM) repetitions. (b,d) Relative mRNA expression levels of bmp2b, bmp4, vegfaa, kdrl, and fli1 by quantitative PCR in different treatment groups as indicated. gapdh: reference gene. Each gene was tested at least five times. (c,e) Protein levels of Bmp2b, Bmp4, Vegfa, and Vegfr2 in different groups by ELISA assay. Each protein was repeated 2–3 times. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and NS, nonsignificant.

Working model of GAH-dependent vascular repair. In zebrafish, GAH activates BMP signaling transduction through non-Smad pathways to stimulate VEGF signaling or to activate the transcriptional levels of VEGF members via Smad1/5/9, which in turn promotes vascular development and impairment repair.