Silencing p75NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

Accumulation of the nerve growth factor precursor (proNGF) and its receptor p75NTR have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75NTR induce endothelial cell (EC) death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75NTR were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75NTRshRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75NTR using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75NTR and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75NTR antagonist. Therefore, targeting p75NTR represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF

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 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 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 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

Peroxynitrite mediates diabetes-induced endothelial dysfunction: Possible role of rho kinase activation. Exp. Diabetes Res

Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction

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