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c-abl is required for the development of hyperoxia-induced retinopathy.

Nunes I, Higgins RD, Zanetta L, Shamamian P, Goff SP - J. Exp. Med. (2001)

Bottom Line: When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16.However, retinal VEGF expression in hyperoxia-treated homozygous mice did not decrease and remained at control levels.These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

ABSTRACT
The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl-deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

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RNase protection assays of retinal VEGF and ET-1 expression in wt hybrid mice. Total retinal RNA (3–5 μg) was prepared from P6 and P17 wt mice exposed to room air only (control animals) and from mice throughout the hyperoxia regimen (experimental animals). The hyperoxia treatment consisted of a 5-d exposure to 75% oxygen between P7 to P12 followed by the return to normoxia from P12 to P16. RNAs were hybridized with VEGF, ET-1, and actin riboprobes (arrows). Riboprobes alone were treated with (+) or without (−) RNase to verify that digestions were complete. The protected fragments were resolved by urea/SDS-PAGE and visualized by PhosphorImager. Band density was quantified using ImageQuant software. The normoxia and hyperoxia hybridization experiments were performed using riboprobes prepared on separate occasions. Therefore, the specific activities differed and expression levels between these two groups should not be directly compared.
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Figure 4: RNase protection assays of retinal VEGF and ET-1 expression in wt hybrid mice. Total retinal RNA (3–5 μg) was prepared from P6 and P17 wt mice exposed to room air only (control animals) and from mice throughout the hyperoxia regimen (experimental animals). The hyperoxia treatment consisted of a 5-d exposure to 75% oxygen between P7 to P12 followed by the return to normoxia from P12 to P16. RNAs were hybridized with VEGF, ET-1, and actin riboprobes (arrows). Riboprobes alone were treated with (+) or without (−) RNase to verify that digestions were complete. The protected fragments were resolved by urea/SDS-PAGE and visualized by PhosphorImager. Band density was quantified using ImageQuant software. The normoxia and hyperoxia hybridization experiments were performed using riboprobes prepared on separate occasions. Therefore, the specific activities differed and expression levels between these two groups should not be directly compared.

Mentions: Constriction of retinal vessels is characteristic of the retinopathy and has been proposed to be ET-1 mediated 13. However, there are no reports that describe retinal ET-1 mRNA expression in response to oxygen or in the murine ROP model we employed. To determine if ET-1 mRNA expression is differentially regulated in wt mice by oxygen, we assessed ET-1 mRNA levels in retinas of normoxia control and hyperoxia-treated experimental animals using RNase protection assays. Retinas from P6 and P17 control (normoxia) mice and from P7 to P16 experimental mice, i.e., mice exposed to the hyperoxia regimen, were used (Fig. 4). ET-1 was found to be expressed constitutively and levels were unchanged in the normoxia and hyperoxia-exposed retinas harvested throughout the hyperoxia regimen. These data suggest that retinal ET-1 mRNA levels are refractory to changes in inspired oxygen tension.


c-abl is required for the development of hyperoxia-induced retinopathy.

Nunes I, Higgins RD, Zanetta L, Shamamian P, Goff SP - J. Exp. Med. (2001)

RNase protection assays of retinal VEGF and ET-1 expression in wt hybrid mice. Total retinal RNA (3–5 μg) was prepared from P6 and P17 wt mice exposed to room air only (control animals) and from mice throughout the hyperoxia regimen (experimental animals). The hyperoxia treatment consisted of a 5-d exposure to 75% oxygen between P7 to P12 followed by the return to normoxia from P12 to P16. RNAs were hybridized with VEGF, ET-1, and actin riboprobes (arrows). Riboprobes alone were treated with (+) or without (−) RNase to verify that digestions were complete. The protected fragments were resolved by urea/SDS-PAGE and visualized by PhosphorImager. Band density was quantified using ImageQuant software. The normoxia and hyperoxia hybridization experiments were performed using riboprobes prepared on separate occasions. Therefore, the specific activities differed and expression levels between these two groups should not be directly compared.
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Related In: Results  -  Collection

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Figure 4: RNase protection assays of retinal VEGF and ET-1 expression in wt hybrid mice. Total retinal RNA (3–5 μg) was prepared from P6 and P17 wt mice exposed to room air only (control animals) and from mice throughout the hyperoxia regimen (experimental animals). The hyperoxia treatment consisted of a 5-d exposure to 75% oxygen between P7 to P12 followed by the return to normoxia from P12 to P16. RNAs were hybridized with VEGF, ET-1, and actin riboprobes (arrows). Riboprobes alone were treated with (+) or without (−) RNase to verify that digestions were complete. The protected fragments were resolved by urea/SDS-PAGE and visualized by PhosphorImager. Band density was quantified using ImageQuant software. The normoxia and hyperoxia hybridization experiments were performed using riboprobes prepared on separate occasions. Therefore, the specific activities differed and expression levels between these two groups should not be directly compared.
Mentions: Constriction of retinal vessels is characteristic of the retinopathy and has been proposed to be ET-1 mediated 13. However, there are no reports that describe retinal ET-1 mRNA expression in response to oxygen or in the murine ROP model we employed. To determine if ET-1 mRNA expression is differentially regulated in wt mice by oxygen, we assessed ET-1 mRNA levels in retinas of normoxia control and hyperoxia-treated experimental animals using RNase protection assays. Retinas from P6 and P17 control (normoxia) mice and from P7 to P16 experimental mice, i.e., mice exposed to the hyperoxia regimen, were used (Fig. 4). ET-1 was found to be expressed constitutively and levels were unchanged in the normoxia and hyperoxia-exposed retinas harvested throughout the hyperoxia regimen. These data suggest that retinal ET-1 mRNA levels are refractory to changes in inspired oxygen tension.

Bottom Line: When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16.However, retinal VEGF expression in hyperoxia-treated homozygous mice did not decrease and remained at control levels.These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

ABSTRACT
The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl-deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

Show MeSH
Related in: MedlinePlus