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Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Bramall AN, Szego MJ, Pacione LR, Chang I, Diez E, D'Orleans-Juste P, Stewart DJ, Hauswirth WW, Yanagisawa M, McInnes RR - PLoS ONE (2013)

Bottom Line: Together, these findings suggest that increased Edn2 expression is protective to mutant PRs.Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas.Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b(rd1/rd1) ; Edn2(-/-) mice. This finding, together with systemic abnormalities in Edn2(-/-) mice, suggested that the increased survival of mutant PRs in the Edn2(-/-) background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b(rd1/rd1) ; Edn2(-/-) PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b(rd1/rd1) retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

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The effect of EDN2 loss on mutant PR survival in vivo and in retinal explants.(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained Edn2+/+ and Edn2−/− retinas. (B,C) The loss of EDN2 in Tg(RHO P347S) retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in Pde6brd1/rd1 retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, Pde6brd1/rd1 and Tg(RHO P347S) retinas in mice expressing or lacking EDN2 (**p<0.005). (E) qRT−PCR assays of the Edn2 mRNA, normalized to Gapdh mRNA, in in vivo WT, WT explants and Pde6brd1/rd1 explants (n = 3;*p<0.05). Values were compared to the mean Edn2 mRNA levels in WT in vivo samples (arbitrarily given a value of 1). Edn2 transcripts were significantly increased in WT as well as Pde6brd1/rd1 explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured ex vivo from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in Pde6brd1/rd1; Edn2−/− retinal explants did not increase PR survival. Both Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.
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pone-0058023-g002: The effect of EDN2 loss on mutant PR survival in vivo and in retinal explants.(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained Edn2+/+ and Edn2−/− retinas. (B,C) The loss of EDN2 in Tg(RHO P347S) retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in Pde6brd1/rd1 retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, Pde6brd1/rd1 and Tg(RHO P347S) retinas in mice expressing or lacking EDN2 (**p<0.005). (E) qRT−PCR assays of the Edn2 mRNA, normalized to Gapdh mRNA, in in vivo WT, WT explants and Pde6brd1/rd1 explants (n = 3;*p<0.05). Values were compared to the mean Edn2 mRNA levels in WT in vivo samples (arbitrarily given a value of 1). Edn2 transcripts were significantly increased in WT as well as Pde6brd1/rd1 explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured ex vivo from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in Pde6brd1/rd1; Edn2−/− retinal explants did not increase PR survival. Both Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.

Mentions: To determine whether EDN2 promotes, resists, or has no influence on PR survival in IPDs, we used Edn2−/− mice to generate Tg(RHO P347S); Edn2−/− and Pde6brd1/rd1; Edn2−/− animals. We first determined that EDN2 is not required for normal retinal or PR formation or survival by examining the morphology of adult Edn2−/− retinas. No significant difference in gross morphology, or in ONL thickness (n = 5; p>0.05), was observed between WT and Edn2−/− retinas at PN40 (Fig. 2A). Edn2−/− mice were born at normal Mendelian ratios but exhibited runting by PN7 and a survival rate of only 25% at PN20 (unpublished observations). Edn2+/− mice displayed no overt phenotype. To allow observation of the effects of EDN2 loss in the slower degenerating Tg(RHO P347S) retinas, we were able to extend the lifespan of Edn2−/− mice to a maximum of PN50 by using a liquid diet and daily subcutaneous injections of normal saline to maintain fluid and electrolyte balance.


Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Bramall AN, Szego MJ, Pacione LR, Chang I, Diez E, D'Orleans-Juste P, Stewart DJ, Hauswirth WW, Yanagisawa M, McInnes RR - PLoS ONE (2013)

The effect of EDN2 loss on mutant PR survival in vivo and in retinal explants.(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained Edn2+/+ and Edn2−/− retinas. (B,C) The loss of EDN2 in Tg(RHO P347S) retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in Pde6brd1/rd1 retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, Pde6brd1/rd1 and Tg(RHO P347S) retinas in mice expressing or lacking EDN2 (**p<0.005). (E) qRT−PCR assays of the Edn2 mRNA, normalized to Gapdh mRNA, in in vivo WT, WT explants and Pde6brd1/rd1 explants (n = 3;*p<0.05). Values were compared to the mean Edn2 mRNA levels in WT in vivo samples (arbitrarily given a value of 1). Edn2 transcripts were significantly increased in WT as well as Pde6brd1/rd1 explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured ex vivo from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in Pde6brd1/rd1; Edn2−/− retinal explants did not increase PR survival. Both Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585171&req=5

pone-0058023-g002: The effect of EDN2 loss on mutant PR survival in vivo and in retinal explants.(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained Edn2+/+ and Edn2−/− retinas. (B,C) The loss of EDN2 in Tg(RHO P347S) retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in Pde6brd1/rd1 retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, Pde6brd1/rd1 and Tg(RHO P347S) retinas in mice expressing or lacking EDN2 (**p<0.005). (E) qRT−PCR assays of the Edn2 mRNA, normalized to Gapdh mRNA, in in vivo WT, WT explants and Pde6brd1/rd1 explants (n = 3;*p<0.05). Values were compared to the mean Edn2 mRNA levels in WT in vivo samples (arbitrarily given a value of 1). Edn2 transcripts were significantly increased in WT as well as Pde6brd1/rd1 explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured ex vivo from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in Pde6brd1/rd1; Edn2−/− retinal explants did not increase PR survival. Both Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.
Mentions: To determine whether EDN2 promotes, resists, or has no influence on PR survival in IPDs, we used Edn2−/− mice to generate Tg(RHO P347S); Edn2−/− and Pde6brd1/rd1; Edn2−/− animals. We first determined that EDN2 is not required for normal retinal or PR formation or survival by examining the morphology of adult Edn2−/− retinas. No significant difference in gross morphology, or in ONL thickness (n = 5; p>0.05), was observed between WT and Edn2−/− retinas at PN40 (Fig. 2A). Edn2−/− mice were born at normal Mendelian ratios but exhibited runting by PN7 and a survival rate of only 25% at PN20 (unpublished observations). Edn2+/− mice displayed no overt phenotype. To allow observation of the effects of EDN2 loss in the slower degenerating Tg(RHO P347S) retinas, we were able to extend the lifespan of Edn2−/− mice to a maximum of PN50 by using a liquid diet and daily subcutaneous injections of normal saline to maintain fluid and electrolyte balance.

Bottom Line: Together, these findings suggest that increased Edn2 expression is protective to mutant PRs.Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas.Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b(rd1/rd1) ; Edn2(-/-) mice. This finding, together with systemic abnormalities in Edn2(-/-) mice, suggested that the increased survival of mutant PRs in the Edn2(-/-) background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b(rd1/rd1) ; Edn2(-/-) PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b(rd1/rd1) retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

Show MeSH
Related in: MedlinePlus