Deletion of Thioredoxin Interacting Protein (TXNIP) Augments Hyperoxia-Induced Vaso-Obliteration in a Mouse Model of Oxygen Induced-Retinopathy

<div><p>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.</p></div

Deletion of TXNIP increases antioxidant defense level under normoxia and hyperoxia.

<p>In comparison to WT, retinas from TKO showed significantly higher level of reduced-GSH (<b>A</b>) and thioredoxin reductase activity (<b>B</b>) under normoxia. A 2×2 analysis showed a significant interaction between gene (TKO) and oxygen levels (hyperoxia) in both reduced-GSH and thioredoxin reductase activity measurements. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 6–8).</p

Deletion of TXNIP does not alter VEGF levels under normoxia or hyperoxia.

<p>(A) VEGF mRNA levels were detected from various groups using rt-PCR. (B) VEGF protein expression was examined using heparin-bound beads from p12 WT and TKO retinas. There was no change in levels of VEGF mRNA or VEGF expression between WT and TKO under normoxia. Hyperoxia caused significant decrease in VEGF mRNA compared to normoxia. Hyperoxia did not alter VEGF protein levels from normoxia in WT and TKO. (#P<0.05 Hyperoxia vs Normoxia, n = 4–6).</p

Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.

<p><b>A</b>) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. <b>B</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).</p

Deletion of TXNIP augments hyperoxia-induced vaso-obliteration compared to WT.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to hyperoxia (75% O2, p7–p12). Retinas were fixed and stained with iso-lectin B4 to quantify oxygen induced vaso-obliteration. <b>A</b>–<b>C</b>) Retinas from TKO mice exposed to hyperoxia showed significant increases in vaso-obliteration compared to WT. (*P<0.05 vs WT, n = 12). <b>D</b>) Hyperoxia stimulates TXNIP expression mRNA in WT but not in TKO mice. (*P<0.05 vs WT normoxia, n = 4)</p

Deletion of TXNIP augments hyperoxia-induced ASK-1 activation and apoptotic markers.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to 5 days hyperoxia (p7–12). Retinas were collected for protein ASK-1 (<b>A</b>) and apoptotic markers (Cleaved Caspase-3 and PARP) (<b>B</b>). A 2×2 analysis showed a significant interaction between gene (TKO) and oxygen levels (Hyperoxia) in activation of ASK-1. In parallen, Hyperoxia caused significant increase in cleaved caspase-3 and PARP expression compared to normoxia in WT and TKO. Reduced Trx levels were associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).</p

Deletion of TXNIP does not alter VEGFR2/Akt activation under hyperoxia.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to hyperoxia (75% O2, p7–p12). Activation of VEGFR2 (<b>A</b>) and Akt (<b>B</b>) were examined as downstream signal of VEGF in p12 WT and TKO retinas. TKO showed significant decrease in phosphorylation of VEGFR-2 and Akt compared to WT under normoxic condition. We did not detect significant change in the activation of VEGFR2 and its downstream Akt in retinas from TKO and WT in response to hyperoxia. (*P<0.05, TKO vs WT, n = 4–6).</p

Representative diagram shows the impact of TXNIP deletion on retina vasculature under both normoxia and hyperoxia.

<p>Under normoxia, retinas from TXNIP-deficient mice showed similar VEGF levels, less peroxynitrite (ONOO-) levels, less VEGF receptor-2 (pVEGFR2) activation and upregulated thioredoxin (Trx) that collectively lead to normal vascular development in comparison to WT mice. Under hyperoxia, retinas from WT mice showed higher peroxynitrite formation, less survival Akt activation (pAkt) and upregulated proapoptotic signal of ASK-1 resulting in vaso-obliteration. Retinas from TKO although showed less peroxynitrite levels and maintained Akt activation, retinas experienced significant decreases in thioredoxin (Trx) that shift the balance of the ASK-1-Trx inhibitory complex and increases the activation of the proapoptotic ASK-1 pathway leading to exacerbated vasoobliteration compared to WT.</p

Deletion of TXNIP decreases thioredoxin levels under hyperoxic conditions.

<p>WT and TKO mice were subjected to 5 days hyperoxia (p7–12). Retinas were collected for protein and mRNA levels of thioredoxin (cytoplasmic Trx-1) (<b>A</b>) as well as mitochondrial Trx-2 (<b>B</b>) and total thioredoxin protein (<b>C</b>). A 2×2 analysis showed a significant interaction between gene (TKO) and oxygen levels (Hyperoxia) in both Trx-1 and Trx-2 as well as Trx total expression. Exposure of TKO pups to hyperoxia significantly decreased the anti-apoptotic thioredoxin on both mRNA as well as protein level compared to WT. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).</p

ProNGF selectively activates RhoA kinase activation in vivo and in RGC cultures.

<p><b>A.</b> Pull-down assay of rat retinal lysate showed 2.3-fold increase in the expression of active Rho in diabetic rats as compared with the controls (n = 4–5). <b>B.</b> Pull-down assay of rat retinal lysate showed 1.7-fold increase in active RhoA expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 5). <b>C.</b> Pull-down assay of RGC-5 lysate showed 1.6-fold increase in RhoA expression in RGC-5 cells treated with proNGF as compared with the controls (n = 4). Treatment of rats or RGC-5 with Y27632 blocked RhoA activation proNGF-treated samples but not the control groups. <b>D.</b> Pull-down assay of RGC-5 showing that treatment of RGC-5 cells with TNF-α did not increase RhoA activation as compared with the control group (n = 4). <b>E.</b> Statistical analysis showing overexpression of proNGF in healthy retina induced 3-fold increase in TNF-α mRNA expression as compared with the control group. <b>F.</b> Statistical analysis of G-LISA showing 1.7-fold increase of RhoA in primary RGC cultures treated with proNGF as compared with the control. TNF-α caused modest increase in RhoA activation (20%) as compared with the controls. These effects were reduced by treatment with Y27632. * = significant difference as compared with the control group at p<0.05.</p