Zebrafish stocks and chemical library screening
The wild-type AB strain zebrafish and transgenic lines Tg(kdrl:GRCFP)zn1
, and Tg(gata1:dsRed)sd2
were used in this study. Embryos were raised under standard condition and staged according to description by Kimmel et al
. Live embryos were placed into 96-well plates, 6 embryos per well with 200 μl fresh fish water containing 1× Antibiotic-Antimycotic Solution (Mediatech, Manassas, VA). Chemical libraries were added to the embryos at the concentration of approximately 10 μM at 30 hpf, and circulation and morphology of the embryos were observed at 48 hpf.
Chemical libraries and compounds
BIOMOL chemical library consisting of ~500 compounds of bioactive lipids, endocannabinoid, ion-channel ligands, enzyme inhibitors, phosphatase and kinase inhibitors, and orphan ligands (BIOMOL); and Prestwick library of 1120 compounds consisting of 85% FDA-approved drugs (Prestwick Chemical, Inc) were screened for compounds that regulate blood vessel function after circulation was established. PP1 was purchased from BIOMOL; Dasatinib, PTK787 and Sunitinib were purchased from Selleck; U0126 and BDM were purchased from Sigma-Aldrich; ZM323881 was purchased from Tocris; SU6656 and Bcr-abl Inhibitor were purchased from Calbiochem. High-concentration stocks of these organic compounds were made in DMSO. Working solutions were diluted from DMSO stocks into fish water.
Apoptosis in the endothelial cells of Tg(fli1a:nEGFP)y7 transgenic embryos was examined using terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay as per manufacturer's protocol with some modifications (In situ Cell Death Detection Kit, TMR Red, Roche Applied Science). After PP1 treatment, 4 dpf embryos were fixed overnight in 2% paraformaldehyde in PBS at 4 °C. Embryos were then washed with PBST buffer twice and stored in methanol at −20 °C overnight. Embryos were rehydrated, permeabilized by proteinase K (Sigma) (50 μg/ml) for 40 min, and refixed in 2% paraformaldehyde in PBS for 10 min at room temperature. Embryos were then washed 3 × 5 min in PBST and incubated in the TUNEL reaction mix for 3 h at 37 °C in darkness. After reaction, embryos were washed 3 × 30 min in PBST at room temperature and stored in PBST at 4 °C.
Tubular network degeneration assay with HUVEC
Matrigel (growth factor reduced) was thawed at 4 °C overnight, and each well of prechilled 96-well plates was coated with 50 μl PBS-diluted Matrigel (1:1) and incubated at 37 °C for 30 min. HUVECs ( 15 000 cells per well) were added into each Matrigel-treated well and cultured in 0.1 ml ECM (supplemented with 0.5% FBS and 40 ng/ml VEGF). After incubation at 37 °C and 5% CO2 for 4 h, tube-like structures were formed, and then treated by PP1 at different concentrations for 18 h. The density of tubular structure was quantified by manual counting of the length of endothelial network in high-power fields (200×).
A panel of 22 representative mammalian kinases was tested for inhibition by PP1 using SelectScreen Kinase Profiling Service (Invitrogen, Carlsband, CA). The concentration of PP1 tested was 5 μM in 1% DMSO.
Fluorescein isothiocyanate dextran (MW=2 000 000 Da, Sigma) or tetramethylrhodamine dextran (MW=2 000 000 Da, Invitrogen) dissolved in double-distilled water was microinjected into the sinus venous of zebrafish embryos at 48 hpf or 4 dpf, respectively.
Pictures of zebrafish embryos were taken with either a confocal microscope (Zeiss LSM510 Meta and Axiovert 200M), or AxioImager A1 microscope and AxioCam digital camera (Zeiss, Oberkochen, Germany), and edited with Photoshop 7.0 (Adobe Systems, San Jose, CA).
Embryos for the transmission electron microscopy were fixed in 2% (v/v) glutaraldehyde (Sigma-Aldrich) and 2% (w/v) paraformaldehyde (Sigma-Aldrich) dissolved in 0.1 M sodium cacodylate buffer (pH 7.4) at 4 °C overnight, and post-fixed in 2% (w/v) osmium tetroxide at room temperature for 4 h. After dehydrating through serial ethanol (15%, 30%, 50%, 75%, 85%, 95%, and 100%), the embryos were embedded in Spurr's resin (SPI-Chem). Sections of 75 nm were obtained with a microtome (Leica), and stained with 1% (w/v) uranyl acetate and 0.5% (w/v) lead citrate. Images were obtained with an electron microscope (JEOL).