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UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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See next page for figure legend.UXT potentiates angiogenesisthroughNotch signaling. (A-C) UXT potentiates angiogenesis in vitro. (A) Knocking down UXT by shRNAs or ectopically expressing wild-type or mutant UXT (phage plasmids) was performed in HUVECs, followed by the in vitro angiogenesis assay. Fluorescence images were taken (A) and quantified (B,C). Scale bar: 500 µm. Relative mean mesh area (B) and relative branching length (C) were calculated by using Image J. (D-F) 3D in vitro angiogenesis with collagen gel-embedded cytodex beads coated with HUVECs, treated with shNT, shUXT, phage-vector or phage-UXT. Cumulative length and the numbers of all sprouts originating from an individual cytodex bead were quantified using ImageJ after 24 h, with ten cytodex beads analyzed per experimental group. Scale bar: 200 µm. (G-I) Fluorescence images (G) and quantification (H,I) of the in vitro angiogenesis assay on HUVECs treated with DMSO or DAPT. Wild-type (shNT) or UXT-deficient (shUXT) HUVECs were seeded on Matrigel and then treated with either DMSO or DAPT (1.5 µM) for 6 h. Scale bar: 500 µm. Relative mean mesh area (H) and relative branching length (I) were calculated using ImageJ. The data were normalized on the basis of the corresponding input control. (J-Q) Tg(kdrl:EGFP)s843 embryos were treated with either 60 µM DAPT or DMSO. (J,K) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino (Ctrl MO) were treated with DMSO. (N,O) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino were treated with DAPT. (L,M) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino (UXT MO) were treated with DMSO. (P,Q) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino were treated with DAPT. All images were taken at 30 hpf. The confocal images in K,O,M,Q show higher magnification of areas of the bright-field images to the left. The red arrowheads indicate the robust angiogenesis in ISVs, whereas the yellow arrowheads highlight the DLAV growth rescued by DAPT treatment. Scale bar: 100 µm. (R-U) 2 ng of RBP-Jκ morpholino were injected into Tg(kdrl:EGFP)s843 embryos without (R,S) or with (T,U) 4 ng of UXT morpholino. All the images were taken at 30 hpf. The confocal images in S,U show higher magnification of areas of the bright-field images to the left. Red arrowheads indicate excessive angiogenesis in ISVs, and yellow arrowheads denote DLAV growth rescued by co-injection of the RBP-Jκ morpholino. Scale bar: 100 µm. (V) Quantification of J-U. The ISV branching points per segment were analyzed. Five different embryos were analyzed per experimental group. The data were normalized on the basis of the corresponding input control. All quantitative data are presented as the mean±s.e.m. (at least three independent experiments); *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.
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DEV112532F7: See next page for figure legend.UXT potentiates angiogenesisthroughNotch signaling. (A-C) UXT potentiates angiogenesis in vitro. (A) Knocking down UXT by shRNAs or ectopically expressing wild-type or mutant UXT (phage plasmids) was performed in HUVECs, followed by the in vitro angiogenesis assay. Fluorescence images were taken (A) and quantified (B,C). Scale bar: 500 µm. Relative mean mesh area (B) and relative branching length (C) were calculated by using Image J. (D-F) 3D in vitro angiogenesis with collagen gel-embedded cytodex beads coated with HUVECs, treated with shNT, shUXT, phage-vector or phage-UXT. Cumulative length and the numbers of all sprouts originating from an individual cytodex bead were quantified using ImageJ after 24 h, with ten cytodex beads analyzed per experimental group. Scale bar: 200 µm. (G-I) Fluorescence images (G) and quantification (H,I) of the in vitro angiogenesis assay on HUVECs treated with DMSO or DAPT. Wild-type (shNT) or UXT-deficient (shUXT) HUVECs were seeded on Matrigel and then treated with either DMSO or DAPT (1.5 µM) for 6 h. Scale bar: 500 µm. Relative mean mesh area (H) and relative branching length (I) were calculated using ImageJ. The data were normalized on the basis of the corresponding input control. (J-Q) Tg(kdrl:EGFP)s843 embryos were treated with either 60 µM DAPT or DMSO. (J,K) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino (Ctrl MO) were treated with DMSO. (N,O) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino were treated with DAPT. (L,M) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino (UXT MO) were treated with DMSO. (P,Q) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino were treated with DAPT. All images were taken at 30 hpf. The confocal images in K,O,M,Q show higher magnification of areas of the bright-field images to the left. The red arrowheads indicate the robust angiogenesis in ISVs, whereas the yellow arrowheads highlight the DLAV growth rescued by DAPT treatment. Scale bar: 100 µm. (R-U) 2 ng of RBP-Jκ morpholino were injected into Tg(kdrl:EGFP)s843 embryos without (R,S) or with (T,U) 4 ng of UXT morpholino. All the images were taken at 30 hpf. The confocal images in S,U show higher magnification of areas of the bright-field images to the left. Red arrowheads indicate excessive angiogenesis in ISVs, and yellow arrowheads denote DLAV growth rescued by co-injection of the RBP-Jκ morpholino. Scale bar: 100 µm. (V) Quantification of J-U. The ISV branching points per segment were analyzed. Five different embryos were analyzed per experimental group. The data were normalized on the basis of the corresponding input control. All quantitative data are presented as the mean±s.e.m. (at least three independent experiments); *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.

Mentions: To substantiate whether UXT modulates angiogenesis by targeting Notch signaling, an in vitro angiogenesis assay was employed. HUVECs were seeded on Matrigel and they migrated to establish capillary-like structures with a lumen. As expected, control HUVECs formed normal capillary-like structures (Fig. 7A, shNT and phage-vec). By contrast, UXT-deficient HUVECs became rounded as isolated cells and failed to establish contacts with adjacent cells (Fig. 7A, sh-UXT). When HUVECs ectopically expressed UXT (Fig. 7A, phage-UXT), these cells formed more-robust capillaries and cord-like structures (Fig. 7A, phage-UXT). Quantitatively, ectopic expression of UXT induced a 1.5-fold increase in the mean mesh area (Fig. 7B) and branching length (Fig. 7C), as compared with the control HUVECs. Consistently, ectopic expression of UXT-2M caused a marginal dominant-negative effect on the angiogenesis (Fig. 7A, phage-UXT-2M; Fig. 7B,C).Fig. 7.


UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

See next page for figure legend.UXT potentiates angiogenesisthroughNotch signaling. (A-C) UXT potentiates angiogenesis in vitro. (A) Knocking down UXT by shRNAs or ectopically expressing wild-type or mutant UXT (phage plasmids) was performed in HUVECs, followed by the in vitro angiogenesis assay. Fluorescence images were taken (A) and quantified (B,C). Scale bar: 500 µm. Relative mean mesh area (B) and relative branching length (C) were calculated by using Image J. (D-F) 3D in vitro angiogenesis with collagen gel-embedded cytodex beads coated with HUVECs, treated with shNT, shUXT, phage-vector or phage-UXT. Cumulative length and the numbers of all sprouts originating from an individual cytodex bead were quantified using ImageJ after 24 h, with ten cytodex beads analyzed per experimental group. Scale bar: 200 µm. (G-I) Fluorescence images (G) and quantification (H,I) of the in vitro angiogenesis assay on HUVECs treated with DMSO or DAPT. Wild-type (shNT) or UXT-deficient (shUXT) HUVECs were seeded on Matrigel and then treated with either DMSO or DAPT (1.5 µM) for 6 h. Scale bar: 500 µm. Relative mean mesh area (H) and relative branching length (I) were calculated using ImageJ. The data were normalized on the basis of the corresponding input control. (J-Q) Tg(kdrl:EGFP)s843 embryos were treated with either 60 µM DAPT or DMSO. (J,K) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino (Ctrl MO) were treated with DMSO. (N,O) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino were treated with DAPT. (L,M) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino (UXT MO) were treated with DMSO. (P,Q) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino were treated with DAPT. All images were taken at 30 hpf. The confocal images in K,O,M,Q show higher magnification of areas of the bright-field images to the left. The red arrowheads indicate the robust angiogenesis in ISVs, whereas the yellow arrowheads highlight the DLAV growth rescued by DAPT treatment. Scale bar: 100 µm. (R-U) 2 ng of RBP-Jκ morpholino were injected into Tg(kdrl:EGFP)s843 embryos without (R,S) or with (T,U) 4 ng of UXT morpholino. All the images were taken at 30 hpf. The confocal images in S,U show higher magnification of areas of the bright-field images to the left. Red arrowheads indicate excessive angiogenesis in ISVs, and yellow arrowheads denote DLAV growth rescued by co-injection of the RBP-Jκ morpholino. Scale bar: 100 µm. (V) Quantification of J-U. The ISV branching points per segment were analyzed. Five different embryos were analyzed per experimental group. The data were normalized on the basis of the corresponding input control. All quantitative data are presented as the mean±s.e.m. (at least three independent experiments); *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.
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DEV112532F7: See next page for figure legend.UXT potentiates angiogenesisthroughNotch signaling. (A-C) UXT potentiates angiogenesis in vitro. (A) Knocking down UXT by shRNAs or ectopically expressing wild-type or mutant UXT (phage plasmids) was performed in HUVECs, followed by the in vitro angiogenesis assay. Fluorescence images were taken (A) and quantified (B,C). Scale bar: 500 µm. Relative mean mesh area (B) and relative branching length (C) were calculated by using Image J. (D-F) 3D in vitro angiogenesis with collagen gel-embedded cytodex beads coated with HUVECs, treated with shNT, shUXT, phage-vector or phage-UXT. Cumulative length and the numbers of all sprouts originating from an individual cytodex bead were quantified using ImageJ after 24 h, with ten cytodex beads analyzed per experimental group. Scale bar: 200 µm. (G-I) Fluorescence images (G) and quantification (H,I) of the in vitro angiogenesis assay on HUVECs treated with DMSO or DAPT. Wild-type (shNT) or UXT-deficient (shUXT) HUVECs were seeded on Matrigel and then treated with either DMSO or DAPT (1.5 µM) for 6 h. Scale bar: 500 µm. Relative mean mesh area (H) and relative branching length (I) were calculated using ImageJ. The data were normalized on the basis of the corresponding input control. (J-Q) Tg(kdrl:EGFP)s843 embryos were treated with either 60 µM DAPT or DMSO. (J,K) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino (Ctrl MO) were treated with DMSO. (N,O) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of control morpholino were treated with DAPT. (L,M) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino (UXT MO) were treated with DMSO. (P,Q) Tg(kdrl:EGFP)s843 embryos injected with 4 ng of UXT morpholino were treated with DAPT. All images were taken at 30 hpf. The confocal images in K,O,M,Q show higher magnification of areas of the bright-field images to the left. The red arrowheads indicate the robust angiogenesis in ISVs, whereas the yellow arrowheads highlight the DLAV growth rescued by DAPT treatment. Scale bar: 100 µm. (R-U) 2 ng of RBP-Jκ morpholino were injected into Tg(kdrl:EGFP)s843 embryos without (R,S) or with (T,U) 4 ng of UXT morpholino. All the images were taken at 30 hpf. The confocal images in S,U show higher magnification of areas of the bright-field images to the left. Red arrowheads indicate excessive angiogenesis in ISVs, and yellow arrowheads denote DLAV growth rescued by co-injection of the RBP-Jκ morpholino. Scale bar: 100 µm. (V) Quantification of J-U. The ISV branching points per segment were analyzed. Five different embryos were analyzed per experimental group. The data were normalized on the basis of the corresponding input control. All quantitative data are presented as the mean±s.e.m. (at least three independent experiments); *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.
Mentions: To substantiate whether UXT modulates angiogenesis by targeting Notch signaling, an in vitro angiogenesis assay was employed. HUVECs were seeded on Matrigel and they migrated to establish capillary-like structures with a lumen. As expected, control HUVECs formed normal capillary-like structures (Fig. 7A, shNT and phage-vec). By contrast, UXT-deficient HUVECs became rounded as isolated cells and failed to establish contacts with adjacent cells (Fig. 7A, sh-UXT). When HUVECs ectopically expressed UXT (Fig. 7A, phage-UXT), these cells formed more-robust capillaries and cord-like structures (Fig. 7A, phage-UXT). Quantitatively, ectopic expression of UXT induced a 1.5-fold increase in the mean mesh area (Fig. 7B) and branching length (Fig. 7C), as compared with the control HUVECs. Consistently, ectopic expression of UXT-2M caused a marginal dominant-negative effect on the angiogenesis (Fig. 7A, phage-UXT-2M; Fig. 7B,C).Fig. 7.

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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