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EphrinB2 controls vessel pruning through STAT1-JNK3 signalling.

Salvucci O, Ohnuki H, Maric D, Hou X, Li X, Yoon SO, Segarra M, Eberhart CG, Acker-Palmer A, Tosato G - Nat Commun (2015)

Bottom Line: JNK3 activation causes endothelial cell death.This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels.Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda Maryland 20892, USA.

ABSTRACT
Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signalling repressing c-jun N-terminal kinase 3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

No MeSH data available.


Related in: MedlinePlus

Sustained EphrinB2 phosphorylation in endothelial cells within the retrolental mass of PHPV. (a) Retrolental mass and retinal degeneration in PHPV. Cross section of PHPV eyeball stained with H&E (representative of 6 samples). Center panel: tiled image of the entire eye section showing the characteristic retrolental mass and retinal detachment; scale bar: 5mm. Left panel: magnified image showing retinal degeneration; scale bar: 100μm. Righteft panel: magnified image showing fibrovascular tissue within the retrolental mass; scale bar: 100μm. (b) Endothelial cells (CD31+) within the PHPV retrolental mass are p-EphrinB+ and JNK3−. Staining with anti-EphrinB (green), anti-JNK3 (white), anti-CD31 (red) and DAPI (blue). Arrowheads point to CD31+p-EphrinB+JNK3− cells. Scale bars: 1mm (left panel), 100μm (right panels) (c) Proximity co-localization of p-EphrinB and SHP2 in endothelial cells in PHPV retrolental mass. Red: PLA signal from p-EphrinB/SHP2 in section of PHPV retrolental mass; green: CD31 immunostaining; blue: DAPI. V: vessel. Arrowheads point to p-EphrinB+SHP2+ cells. Scale bars: 10μm. (d) PLA showing co-localization of EphrinB2 and STAT1 in endothelial cells from PHPV retrolental mass. Red: PLA signal from EphrinB2/STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrowheads point to EphrinB2+STAT1+ cells. Scale bars: 50μm. (e) Co-localization of EphrinB2 and p-STAT1 is limited in endothelial cells in PHPV retrolental mass. Red: PLA signal from EphrinB2/p-STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrows point toEphrinB2+p-STAT1+ cells. Scale bars: 50μm.
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Figure 10: Sustained EphrinB2 phosphorylation in endothelial cells within the retrolental mass of PHPV. (a) Retrolental mass and retinal degeneration in PHPV. Cross section of PHPV eyeball stained with H&E (representative of 6 samples). Center panel: tiled image of the entire eye section showing the characteristic retrolental mass and retinal detachment; scale bar: 5mm. Left panel: magnified image showing retinal degeneration; scale bar: 100μm. Righteft panel: magnified image showing fibrovascular tissue within the retrolental mass; scale bar: 100μm. (b) Endothelial cells (CD31+) within the PHPV retrolental mass are p-EphrinB+ and JNK3−. Staining with anti-EphrinB (green), anti-JNK3 (white), anti-CD31 (red) and DAPI (blue). Arrowheads point to CD31+p-EphrinB+JNK3− cells. Scale bars: 1mm (left panel), 100μm (right panels) (c) Proximity co-localization of p-EphrinB and SHP2 in endothelial cells in PHPV retrolental mass. Red: PLA signal from p-EphrinB/SHP2 in section of PHPV retrolental mass; green: CD31 immunostaining; blue: DAPI. V: vessel. Arrowheads point to p-EphrinB+SHP2+ cells. Scale bars: 10μm. (d) PLA showing co-localization of EphrinB2 and STAT1 in endothelial cells from PHPV retrolental mass. Red: PLA signal from EphrinB2/STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrowheads point to EphrinB2+STAT1+ cells. Scale bars: 50μm. (e) Co-localization of EphrinB2 and p-STAT1 is limited in endothelial cells in PHPV retrolental mass. Red: PLA signal from EphrinB2/p-STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrows point toEphrinB2+p-STAT1+ cells. Scale bars: 50μm.

Mentions: The abnormal persistence of fetal hyaloid vasculature in humans is associated with a syndrome known as “persistent hyperplastic primary vitreous (PHPV)”, which includes microphthalmia and leukocoria 35,36. In humans, hyaloid vessels involute before birth. Consistent with previous descriptions, we find that PHPV eyes (6 examined) display an abnormal retrolental fibrovascular mass, which physically interacts with the neuro-retina causing tractional retinal detachment and degeneration (Fig. 10a). Human CD31 immunostaining revealed endothelial cells co-expressing p-EphrinB in a PHPV retrolental mass; JNK3 was not detected (Fig. 10b, Supplementary Fig. 8a–c). By PLA, p-EphrinB was specifically associated with SHP2 in retrolental CD31+ cells (Fig. 10c, Supplementary Fig. 8d,e). Also, EphrinB2 was specifically associated with STAT1 (Fig. 10d, Supplementary Fig. 8d,e), but minimally with p-STAT1 (Fig. 10e, Supplementary Fig. 8d,e) in retrolental CD31+ cells. Since we showed that p-EphrinB/SHP2 sustain endothelial cell viability preventing physiologic involution of hyaloid vessels, the detection of p-EphrinB/SHP2 in the retrolental mass of PHPV is consistent with a pathogenetic role of p-EphrinB in the abnormal persistence of hyaloid vessels in PHPV.


EphrinB2 controls vessel pruning through STAT1-JNK3 signalling.

Salvucci O, Ohnuki H, Maric D, Hou X, Li X, Yoon SO, Segarra M, Eberhart CG, Acker-Palmer A, Tosato G - Nat Commun (2015)

Sustained EphrinB2 phosphorylation in endothelial cells within the retrolental mass of PHPV. (a) Retrolental mass and retinal degeneration in PHPV. Cross section of PHPV eyeball stained with H&E (representative of 6 samples). Center panel: tiled image of the entire eye section showing the characteristic retrolental mass and retinal detachment; scale bar: 5mm. Left panel: magnified image showing retinal degeneration; scale bar: 100μm. Righteft panel: magnified image showing fibrovascular tissue within the retrolental mass; scale bar: 100μm. (b) Endothelial cells (CD31+) within the PHPV retrolental mass are p-EphrinB+ and JNK3−. Staining with anti-EphrinB (green), anti-JNK3 (white), anti-CD31 (red) and DAPI (blue). Arrowheads point to CD31+p-EphrinB+JNK3− cells. Scale bars: 1mm (left panel), 100μm (right panels) (c) Proximity co-localization of p-EphrinB and SHP2 in endothelial cells in PHPV retrolental mass. Red: PLA signal from p-EphrinB/SHP2 in section of PHPV retrolental mass; green: CD31 immunostaining; blue: DAPI. V: vessel. Arrowheads point to p-EphrinB+SHP2+ cells. Scale bars: 10μm. (d) PLA showing co-localization of EphrinB2 and STAT1 in endothelial cells from PHPV retrolental mass. Red: PLA signal from EphrinB2/STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrowheads point to EphrinB2+STAT1+ cells. Scale bars: 50μm. (e) Co-localization of EphrinB2 and p-STAT1 is limited in endothelial cells in PHPV retrolental mass. Red: PLA signal from EphrinB2/p-STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrows point toEphrinB2+p-STAT1+ cells. Scale bars: 50μm.
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Figure 10: Sustained EphrinB2 phosphorylation in endothelial cells within the retrolental mass of PHPV. (a) Retrolental mass and retinal degeneration in PHPV. Cross section of PHPV eyeball stained with H&E (representative of 6 samples). Center panel: tiled image of the entire eye section showing the characteristic retrolental mass and retinal detachment; scale bar: 5mm. Left panel: magnified image showing retinal degeneration; scale bar: 100μm. Righteft panel: magnified image showing fibrovascular tissue within the retrolental mass; scale bar: 100μm. (b) Endothelial cells (CD31+) within the PHPV retrolental mass are p-EphrinB+ and JNK3−. Staining with anti-EphrinB (green), anti-JNK3 (white), anti-CD31 (red) and DAPI (blue). Arrowheads point to CD31+p-EphrinB+JNK3− cells. Scale bars: 1mm (left panel), 100μm (right panels) (c) Proximity co-localization of p-EphrinB and SHP2 in endothelial cells in PHPV retrolental mass. Red: PLA signal from p-EphrinB/SHP2 in section of PHPV retrolental mass; green: CD31 immunostaining; blue: DAPI. V: vessel. Arrowheads point to p-EphrinB+SHP2+ cells. Scale bars: 10μm. (d) PLA showing co-localization of EphrinB2 and STAT1 in endothelial cells from PHPV retrolental mass. Red: PLA signal from EphrinB2/STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrowheads point to EphrinB2+STAT1+ cells. Scale bars: 50μm. (e) Co-localization of EphrinB2 and p-STAT1 is limited in endothelial cells in PHPV retrolental mass. Red: PLA signal from EphrinB2/p-STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrows point toEphrinB2+p-STAT1+ cells. Scale bars: 50μm.
Mentions: The abnormal persistence of fetal hyaloid vasculature in humans is associated with a syndrome known as “persistent hyperplastic primary vitreous (PHPV)”, which includes microphthalmia and leukocoria 35,36. In humans, hyaloid vessels involute before birth. Consistent with previous descriptions, we find that PHPV eyes (6 examined) display an abnormal retrolental fibrovascular mass, which physically interacts with the neuro-retina causing tractional retinal detachment and degeneration (Fig. 10a). Human CD31 immunostaining revealed endothelial cells co-expressing p-EphrinB in a PHPV retrolental mass; JNK3 was not detected (Fig. 10b, Supplementary Fig. 8a–c). By PLA, p-EphrinB was specifically associated with SHP2 in retrolental CD31+ cells (Fig. 10c, Supplementary Fig. 8d,e). Also, EphrinB2 was specifically associated with STAT1 (Fig. 10d, Supplementary Fig. 8d,e), but minimally with p-STAT1 (Fig. 10e, Supplementary Fig. 8d,e) in retrolental CD31+ cells. Since we showed that p-EphrinB/SHP2 sustain endothelial cell viability preventing physiologic involution of hyaloid vessels, the detection of p-EphrinB/SHP2 in the retrolental mass of PHPV is consistent with a pathogenetic role of p-EphrinB in the abnormal persistence of hyaloid vessels in PHPV.

Bottom Line: JNK3 activation causes endothelial cell death.This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels.Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda Maryland 20892, USA.

ABSTRACT
Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signalling repressing c-jun N-terminal kinase 3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

No MeSH data available.


Related in: MedlinePlus