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Deletion of thioredoxin interacting protein (TXNIP) augments hyperoxia-induced vaso-obliteration in a mouse model of oxygen induced-retinopathy.

Abdelsaid MA, Matragoon S, Ergul A, El-Remessy AB - PLoS ONE (2014)

Bottom Line: Exposure of TKO to hyperoxia significantly decreased the anti-apoptotic thioredoxin protein (∼ 50%) level compared with WT.This effect was associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway.Our results showed that despite comparable VEGF level and signal in TKO, exposure to hyperoxia significantly decreased Trx expression compared to WT.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia, United States of America; Department of Physiology, Georgia Regents University, Augusta, Georgia, United States of America; Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America.

ABSTRACT
We have recently shown that thioredoxin interacting protein (TXNIP) is required for VEGF-mediated VEGFR2 receptor activation and angiogenic signal. Retinas from TXNIP knockout mice (TKO) exhibited higher cellular antioxidant defense compared to wild type (WT). This study aimed to examine the impact of TXNIP deletion on hyperoxia-induced vaso-obliteration in ischemic retinopathy. TKO and WT pups were subjected to oxygen-induced retinopathy model. Retinal central capillary dropout was measured at p12. Retinal redox and nitrative state were assessed by reduced-glutathione (GSH), thioredoxin reductase activity and nitrotyrosine formation. Western blot and QT-PCR were used to assess VEGF, VEGFR-2, Akt, iNOS and eNOS, thioredoxin expression, ASK-1 activation and downstream cleaved caspase-3 and PARP in retinal lysates. Retinas from TKO mice exposed to hyperoxia showed significant increases (1.5-fold) in vaso-obliteration as indicated by central capillary drop out area compared to WT. Retinas from TKO showed minimal nitrotyrosine levels (10% of WT) with no change in eNOS or iNOS mRNA expression. There was no change in levels of VEGF or activation of VEGFR2 and its downstream Akt in retinas from TKO and WT. In comparison to WT, retinas from TKO showed significantly higher level of GSH and thioredoxin reductase activity in normoxia but comparable levels under hyperoxia. Exposure of TKO to hyperoxia significantly decreased the anti-apoptotic thioredoxin protein (∼ 50%) level compared with WT. This effect was associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway. Our results showed that despite comparable VEGF level and signal in TKO, exposure to hyperoxia significantly decreased Trx expression compared to WT. This effect resulted in liberation and activation of the apoptotic ASK-1 signal. These findings suggest that TXNIP is required for endothelial cell survival and homeostasis especially under stress conditions including hyperoxia.

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Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.A) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. B) TKO showed higher eNOS mRNA level in normoxia. Hyperoxia significantly reduced eNOS mRNA levels. A 2×2 analysis showed a significant interaction between the gene (TKO) and the oxygen level (hyperoxia) in both nitrotyrosine and eNOS levels. C) We did not detect difference between TKO and WT in the expression of iNOS. Hyperoxia caused significant reduction of iNOS compared to normoxia. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).
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pone-0110388-g002: Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.A) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. B) TKO showed higher eNOS mRNA level in normoxia. Hyperoxia significantly reduced eNOS mRNA levels. A 2×2 analysis showed a significant interaction between the gene (TKO) and the oxygen level (hyperoxia) in both nitrotyrosine and eNOS levels. C) We did not detect difference between TKO and WT in the expression of iNOS. Hyperoxia caused significant reduction of iNOS compared to normoxia. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).

Mentions: We next examined the levels of nitrotyrosine (NY), the footprint of peroxynitrite which is believed to mediate the detrimental effects of hyperoxia. As shown in Fig. 2A, hyperoxia significantly increased retinal NY formation (3-fold) in WT compared to normoxia. TKO showed minimal level of NY (10%) at normoxia and 20% at hyperoxia compared to WT. The two-way ANOVA showed a significant interaction between TKO and WT in response to high levels of oxygen (Fig. 2A). We also examined the expression of eNOS and iNOS at the mRNA level. Our results showed a significant interaction between TKO and WT in eNOS mRNA levels. TKO retinas showed 1.6-fold increase under normoxic conditions compared to WT-normoxia. Hyperoxia induced significant reduction in retinas from WT and TKO 52% and 51%, respectively compared to retinas from WT-normoxia (Fig. 2B). There was no significant interaction between TKO and WT in iNOS mRNA levels. Hyperoxia caused significant decrease in iNOS mRNA levels in both WT and TKO 54% and 51%, respectively compared to normoxia (Fig. 2C). These results suggest that TKO retinas had less tyrosine nitration at both normoxia and hyperoxia compared to WT.


Deletion of thioredoxin interacting protein (TXNIP) augments hyperoxia-induced vaso-obliteration in a mouse model of oxygen induced-retinopathy.

Abdelsaid MA, Matragoon S, Ergul A, El-Remessy AB - PLoS ONE (2014)

Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.A) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. B) TKO showed higher eNOS mRNA level in normoxia. Hyperoxia significantly reduced eNOS mRNA levels. A 2×2 analysis showed a significant interaction between the gene (TKO) and the oxygen level (hyperoxia) in both nitrotyrosine and eNOS levels. C) We did not detect difference between TKO and WT in the expression of iNOS. Hyperoxia caused significant reduction of iNOS compared to normoxia. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199686&req=5

pone-0110388-g002: Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.A) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. B) TKO showed higher eNOS mRNA level in normoxia. Hyperoxia significantly reduced eNOS mRNA levels. A 2×2 analysis showed a significant interaction between the gene (TKO) and the oxygen level (hyperoxia) in both nitrotyrosine and eNOS levels. C) We did not detect difference between TKO and WT in the expression of iNOS. Hyperoxia caused significant reduction of iNOS compared to normoxia. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).
Mentions: We next examined the levels of nitrotyrosine (NY), the footprint of peroxynitrite which is believed to mediate the detrimental effects of hyperoxia. As shown in Fig. 2A, hyperoxia significantly increased retinal NY formation (3-fold) in WT compared to normoxia. TKO showed minimal level of NY (10%) at normoxia and 20% at hyperoxia compared to WT. The two-way ANOVA showed a significant interaction between TKO and WT in response to high levels of oxygen (Fig. 2A). We also examined the expression of eNOS and iNOS at the mRNA level. Our results showed a significant interaction between TKO and WT in eNOS mRNA levels. TKO retinas showed 1.6-fold increase under normoxic conditions compared to WT-normoxia. Hyperoxia induced significant reduction in retinas from WT and TKO 52% and 51%, respectively compared to retinas from WT-normoxia (Fig. 2B). There was no significant interaction between TKO and WT in iNOS mRNA levels. Hyperoxia caused significant decrease in iNOS mRNA levels in both WT and TKO 54% and 51%, respectively compared to normoxia (Fig. 2C). These results suggest that TKO retinas had less tyrosine nitration at both normoxia and hyperoxia compared to WT.

Bottom Line: Exposure of TKO to hyperoxia significantly decreased the anti-apoptotic thioredoxin protein (∼ 50%) level compared with WT.This effect was associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway.Our results showed that despite comparable VEGF level and signal in TKO, exposure to hyperoxia significantly decreased Trx expression compared to WT.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia, United States of America; Department of Physiology, Georgia Regents University, Augusta, Georgia, United States of America; Charlie Norwood VA Medical Center, Augusta, Georgia, United States of America.

ABSTRACT
We have recently shown that thioredoxin interacting protein (TXNIP) is required for VEGF-mediated VEGFR2 receptor activation and angiogenic signal. Retinas from TXNIP knockout mice (TKO) exhibited higher cellular antioxidant defense compared to wild type (WT). This study aimed to examine the impact of TXNIP deletion on hyperoxia-induced vaso-obliteration in ischemic retinopathy. TKO and WT pups were subjected to oxygen-induced retinopathy model. Retinal central capillary dropout was measured at p12. Retinal redox and nitrative state were assessed by reduced-glutathione (GSH), thioredoxin reductase activity and nitrotyrosine formation. Western blot and QT-PCR were used to assess VEGF, VEGFR-2, Akt, iNOS and eNOS, thioredoxin expression, ASK-1 activation and downstream cleaved caspase-3 and PARP in retinal lysates. Retinas from TKO mice exposed to hyperoxia showed significant increases (1.5-fold) in vaso-obliteration as indicated by central capillary drop out area compared to WT. Retinas from TKO showed minimal nitrotyrosine levels (10% of WT) with no change in eNOS or iNOS mRNA expression. There was no change in levels of VEGF or activation of VEGFR2 and its downstream Akt in retinas from TKO and WT. In comparison to WT, retinas from TKO showed significantly higher level of GSH and thioredoxin reductase activity in normoxia but comparable levels under hyperoxia. Exposure of TKO to hyperoxia significantly decreased the anti-apoptotic thioredoxin protein (∼ 50%) level compared with WT. This effect was associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway. Our results showed that despite comparable VEGF level and signal in TKO, exposure to hyperoxia significantly decreased Trx expression compared to WT. This effect resulted in liberation and activation of the apoptotic ASK-1 signal. These findings suggest that TXNIP is required for endothelial cell survival and homeostasis especially under stress conditions including hyperoxia.

Show MeSH
Related in: MedlinePlus