Figure 4

Sub-lethal HHcy causes vascular abnormality in vivo without suppressing VEGF/VEGFR transcripts and ROS level change. (A) Upper panel: schematic illustration of transgenic zebrafish line Tg(fli1a:EGFP;gata1a:DsRed) showing anatomical position (rectangle) of CVP and ISV forming area in 36 hpf and 48 hpf embryos respectively. Left panel: representative images revealing impairment in sprouting angiogenesis during CVP formation of CBS morphants. Arrows are depicting CVP sprouts. Scale bar, 0.05 mm. Right panel: representative confocal images showing abnormal ISV formation in CBS morphants compared with SC MO injected embryos. Arrows indicating incomplete, irregular, and abnormally branched ISVs. Scale bar, 50 μm. (B,C) Bar plots respectively showing CVP sprout number and percentage of normal ISV are significantly reduced in CBS morphants. (D) Bar diagram depicting percentage of embryos with angiogenic abnormality is significantly higher in CBS MO injected embryos compared with SC MO injected ones. (E) Representative fluorescent images showing no observable difference in the vascular anatomy of SC MO and CGL MO injected embryos at 2 dpf. Scale bar, 0.2 mm. (F) Bar plot depicting no significant change in percentage of embryos with observable angiogenic abnormality in CGL morphants compared with SC MO injected ones. (G) Bars showing mRNA expression levels of canonical VEGF signaling markers in CBS MO and SC MO injected embryos at 2 dpf. 18S was used as internal control. (H) Representative images depicting location of oxidative stress in 2 dpf zebrafish embryos, detected using general ROS indicator CM-H2DCFDA. White arrows showing drastic increase in ROS levels in the pericardial area of CBS morphants and H₂O₂ treated embryos. White boxes of the tail and regions representing sites of previously observed CVP and ISV angiogenesis, respectively. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, *** p ≤ 0.001 and ns is non-significant (p > 0.05).