High-cholesterol diet-induced vascular defects and decreased ETFA expression in zebrafish larvae. Tg Flk1: EGFP zebrafish larvae were fed with different concentrations of high-cholesterol-containing diet (HCD) from 5 days postfertilization (dpf) to 15 dpf, followed by various evaluations. (a, b) HCD induced shorten body length of zebrafish embryos. (a) Representative phage-contrast images of 15dpf larvae fed with different concentrations of HCD (0%, 4%, 6%, 8%, and 10%), scale ; (b) statistical analysis of the total body length (mm) of the zebrafish larvae (one-way ANNOVA, , ); (c–e) HCD-induced developmental vascular defects in zebrafish larvae. (c) Representative image of vessel morphologies in caudal fins of 15dpf Tg Flk1: EGFP zebrafish larvae. Green EGFP signals represent VEGR2⁺ vessels, scale . (d) Statistical analysis of vessel length (μm) in caudal fins of HCD-fed zebrafish larvae (one-way ANNOVA, , ); (e) statistical analysis of the vessel number in caudal fins of HCD-fed zebrafish larvae (one-way ANNOVA, , , ); (f) RT-qPCR analysis of ETFA gene expression in HCD-fed zebrafish larvae. (one-way ANNOVA, ); (g) RT-qPCR analysis of HIF1 gene expression in HCD-fed zebrafish larvae. (one-way ANNOVA, ).

ETFα knockdown caused developmental delay and deformities. Tg Flk1: EGFP zebrafish embryos were injected with 5 ng blank control, control morpholino (Ctl-MO), or ETFA target morpholino (ETFA-MO) and were inspected at 8, 24, 32, or 48 hpf, respectively. (a) The mortality was calculated and compared among blank, control MO-treated, and ETFA MO-treated groups (one-way ANNOVA, , ); (b) developmental status of embryos injected with blank, control MO, and ETFA MO was observed at 8, 24, and 32 hpf, respectively, under a bright field. The photographs in the black box exhibit different deformities of ETFA MO-treated embryos; scale ; (c) developmental defects were observed at 32 hpf under a confocal microscope. Data presented in (c) were representative fluorescent images of malformation in ETFA-MO-injected embryos at 32 hpf, which include defect sprouting (DS) alone, DS with a short body, DS with spinal curvature, and DS with a curly tail; scale ; (d) quantitative data of the percentage of malformation in different groups (one-way ANNOVA, , ).

ETFα knockdown caused defective vascular sprouting at 32 hpf. (a) Representative fluorescent images showing different types of vascular defects in ETFA-MO-injected Tg Flk1: EGFP embryos at 32 hpf, including slight (incomplete DLAVs), moderate (short ISVs), and severe (inadequate sprouting); scale ; (b) quantitative data of the different vascular defects at 32 hpf (one-way ANNOVA, ); (c) the total numbers of intact intersegmental vessels (ISVs) (one-way ANNOVA, , ).

ETFα knockdown induced deformities at 48 hpf. (a) Representative bright field images showing different types of deformities in ETFA-MO-injected Tg Flk1: EGFP embryos at 48 hpf. The photographs in the black box exhibit different deformities of ETFA MO-treated larvae; scale ; (b) representative fluorescent images showing different types of vascular defects in ETFA-MO-injected Tg Flk1: EGFP embryos at 32 hpf. These defects were classified as slight (intact ISVs less than 27 but more than 25), moderate (intact ISVs less than 25 but more than 20), and severe defect (intact ISVs less than 20). The white arrows indicate where the vessel is defective. DLAV: dorsal longitudinal anastomotic vessel; ISVs: intersegmental vessels; DA: dorsal aorta, scale ; (c) ETFα inhibition impaired segmental vessel sprouting and their fusion with DLAV in Tg Flk1: EGFP zebrafish larvae at 48 hpf. Data presented here were representative fluorescent images of vascular sprouting defects of segmental vessels; (d) quantitative data of the different vascular defects at 48 hpf (one-way ANNOVA, ); (e) quantitative data of intact ISVs in zebrafish larvae at 48 hpf (one-way ANNOVA, , ).

ETFα overexpression rescued vascular defects in ETFA-MO-injected Tg flK1: EGFP embryos. Tg Flk1: EGFP zebrafish embryos were coinjected 5 ng MO with different concentrations of ETFA mRNA as indicated. Embryos were inspected and imaged at 48 hpf. Data presented here were the representative fluorescent images of vascular sprouting (a) and quantitative data of the intact ISVs in different groups (b) (one-way ANNOVA, , , ); (c) representative immunoblot for ETFα in zebrafish embryos with the indicated treatments. β-Actin was included as the loading control; (d) RT-qPCR analysis of HIF1 gene expression in zebrafish embryos with the indicated treatments. , , and (one-way ANNOVA, ).

The role of ETFα in vessel sprouting in vitro. (a) Representative immunoblot of ETFα in HUVECs stimulated by 7-keto cholesterol (8, 10, 15, and 20 μM). GAPDH was used as loading control; (b) representative immunoblot of ETFα in HUVECs stimulated by 20 μM 7-keto cholesterol or 7-keto with different doses of MG-132 (5, 15, and 20 nM). β-Actin was used as loading control; (c) quantitative analysis of the total tube length and (d) number of junctions in the tube formation assay (one-way ANNOVA, , , ); (e) quantitative analysis of the wound closure area in HUVECs with the indicated treatments at different time points (one-way ANNOVA, , ); (f) quantitative analysis of transwell migration assay in HUVECs with the indicated treatments (one-way ANNOVA, , , ); (g) quantitative analysis of Edu proliferation assay in HUVECs with the indicated treatments (one-way ANNOVA, , ).

Knockdown of ETFα in HUVECs affects mitochondrial bioenergetics and oxygen consumption. (a) Bioenergetic profile of control HUVECs measured by the Seahorse XF analyzer, in which the oxygen consumption rate (OCR) over time is determined. BSA was used to assess utilization of endogenous fatty acid; Palm-BSA was used to assess utilization of exogenous fatty acid. Etomoxir is an inhibitor of fatty acid oxidation (FAO). Oligomycin, an ATP synthase (complex V) inhibitor, was added to detect cellular ATP production; FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, was added to assess maximal respiration of HUVECS; a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor, was included to measure nonmitochondrial respiration in cells. (b) OCR profiles in ETFα knockdown HUVCEs; (c) basal respiration of control and ETFα knockdown HUVECs. OCRs in different groups were analyzed with one-way ANNOVA, , ; (d) maximal respiration of control and ETFA knockdown HUVECs. OCRs in different groups were analyzed with one-way ANNOVA, , , ; (e) representative immunoblot for HIF1α and ETFα in control-, scramble shRNA-, and ETFA shRNA lentivirus treated HUVECs. Cell lysates were collected at 24 hours or 48 hours after lentivirus transfection. Acetate was added to ETFα knockdown HUVECs. β-Actin was the loading control; (f) representative immunoblot for HIF1α and ETFα in control-, scramble shRNA-, and ETFA shRNA lentivirus-treated HUVECs. HUVECs were exposed to hypoxic condition for 12 hours before collection. Cell lysates were collected at 24 hours or 48 hours after lentivirus transfection.