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

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.

Pronerve growth factor induces angiogenesis via activation of TrkA: possible role in proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) is the leading cause of blindness in working age Americans. We demonstrated that diabetes disturbs the homeostasis of nerve growth factor (NGF) resulting in accumulation of its precursor proNGF. Increases in proNGF were positively correlated with progression of diabetic retinopathy, having the highest level in ocular fluids from PDR patients compared to nondiabetic patients. Here, we attempted to evaluate the contribution and the possible mechanism of proNGF to PDR. The angiogenic response of aqueous humor samples from PDR patients was examined in human retinal endothelial cells in the presence or absence of anti-proNGF antibody. Additional cultures were treated with mutant-proNGF in the presence of specific pharmacological inhibitors of TrkA and p75(NTR) receptors. PDR-aqueous humor samples exerted significant angiogenic response including cell proliferation, migration, and alignment into tube-like structures. These effects were significantly reduced by anti-proNGF antibody but not by IgG. Treatment of retinal endothelial cells with mutant-proNGF activated phosphorylation of TrkA and p38MAPK; however, it did not alter p75(NTR) expression. Inhibition of TrkA but not p75(NTR) significantly reduced mutant-proNGF-induced cell proliferation, cell migration, and tube formation. Taken together, these results provide evidence that proNGF can contribute to PDR at least in part via activation of TrkA.

Modulation of p75(NTR) prevents diabetes- and proNGF-induced retinal inflammation and blood-retina barrier breakdown in mice and rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by early blood-retina barrier (BRB) breakdown and neurodegeneration. Diabetes causes imbalance of nerve growth factor (NGF), leading to accumulation of the NGF precursor (proNGF), as well as the NGF receptor, p75 neurotrophin receptor (p75(NTR)), suggesting a possible pathological role of the proNGF-p75(NTR) axis in the diabetic retina. To date, the role of this axis in diabetes-induced retinal inflammation and BRB breakdown has not been explored. We hypothesised that modulating p75(NTR) would prevent diabetes- and proNGF-induced retinal inflammation and BRB breakdown. METHODS: Diabetes was induced by streptozotocin in wild-type and p75(NTR) knockout (p75KO) mice. After 5 weeks, the expression of inflammatory mediators, ganglion cell loss and BRB breakdown were determined. Cleavage-resistant proNGF was overexpressed in rodent retinas with and without p75(NTR) short hairpin RNA or with pharmacological inhibitors. In vitro, the effects of proNGF were investigated in retinal Müller glial cell line (rMC-1) and primary Müller cells. RESULTS: Deletion of p75(NTR) blunted the diabetes-induced decrease in retinal NGF expression and increases in proNGF, nuclear factor κB (NFκB), p-NFκB and TNF-α. Deletion of p75(NTR) also abrogated diabetes-induced glial fibrillary acidic protein expression, ganglion cell loss and vascular permeability. Inhibited expression or cleavage of p75(NTR) blunted proNGF-induced retinal inflammation and vascular permeability. In vitro, proNGF induced p75(NTR)-dependent production of inflammatory mediators in primary wild-type Müller and rMC-1 cultures, but not in p75KO Müller cells. CONCLUSIONS/INTERPRETATION: The proNGF-p75(NTR) axis contributes to retinal inflammation and vascular dysfunction in the rodent diabetic retina. These findings underscore the importance of p75(NTR) as a novel regulator of inflammation and potential therapeutic target in diabetic retinopathy.

Thioredoxin-interacting protein expression is required for VEGF-mediated angiogenic signal in endothelial cells.

AIMS: Thioredoxin-interacting protein (TXNIP) contributes to cellular redox-state homeostasis via binding and inhibiting thioredoxin (TRX). Increasing evidence suggests that cellular redox homeostasis regulates vascular endothelial growth factor (VEGF)-mediated signaling. This study aims to examine the redox-dependant role of TXNIP in regulating VEGF-mediated S-glutathionylation and angiogenic signaling. TXNIP-knockout mice (TKO) or wild-type (WT) treated with the reduced glutathione (GSH)-precursor, N-acetyl cysteine (WT-NAC, 500 mg/kg) were compared to WT using hypoxia-induced neovascularization model. RESULTS: In response to hypoxia, retinas from TKO and WT-NAC mice showed significant decreases in reparative revascularization and pathological neovascularization with similar VEGF expression compared with WT. VEGF failed to stimulate vascular sprouting from aortic rings of TKO compared to WT mice. TKO mice or WT+NAC experienced reductive stress as indicated by twofold increase in TRX reductase activity and fourfold increase in reduced-GSH levels compared with WT. In human microvascular endothelial (HME) cells, VEGF stimulated co-precipitation between vascular endothelial growth factor receptor 2 (VEGFR2) with low molecular weight protein tyrosine phosphatase (LMW-PTP). Silencing TXNIP expression blunted VEGF-induced oxidation of GSH and S-glutathionylation of the LMW-PTP in HME cells. These effects were associated with impaired VEGFR2 phosphorylation that culminated in inhibiting cell migration and tube formation. Overexpression of TXNIP restored VEGFR2 phosphorylation and cell migration in TKO-endothelial cells. INNOVATION: TXNIP expression is required for VEGF-mediated VEGFR2 activation and angiogenic response in vivo and in vitro. TXNIP expression regulates VEGFR-2 phosphorylation via S-glutathionylation of LMW-PTP in endothelial cells. CONCLUSION: Our results provide novel mechanistic insight into modulating TXNIP expression as a potential therapeutic target in diseases characterized by aberrant angiogenesis.

Diabetes and overexpression of proNGF cause retinal neurodegeneration via activation of RhoA pathway.

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotocin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75(NTR), cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75(NTR) and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75(NTR) expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.

S-glutathionylation of LMW-PTP regulates VEGF-mediated FAK activation and endothelial cell migration.

Although promising, the ability to regulate angiogenesis through delivery of VEGF remains an unrealized goal. We have shown previously that physiological levels of peroxynitrite (1 µM) are required for a VEGF-mediated angiogenic response, yet the redox-regulated mechanisms that govern the VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, the level of reduced-glutathione (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), which are key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) cells with VEGF (20 ng/ml) or peroxynitrite (1 µM) caused an immediate and reversible negative-shift in the cellular redox-state and thiol oxidation of LMW-PTP, which culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP, which inhibits its phosphorylation and activity, and causes the transient activation of FAK. Modulating the redox-state using decomposing peroxynitrite (FeTPPS, 2.5 µM) or the GSH-precursor [N-acetylcysteine (NAC), 1 mM] caused a positive-shift of the redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented the activation of FAK, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. Although mild oxidative stress achieved by combining VEGF with 0.1-0.2 mM peroxynitrite augmented cell migration, an acute shift to oxidative stress achieved by combining VEGF with 0.5 mM peroxynitrite induced and sustained FAK activation, and LMW-PTP S-glutathionylation, resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that a balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting the redox-state to reductive stress or oxidative stress inhibited the VEGF-mediated angiogenic response.

Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration.

PURPOSE: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. METHODS: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then electroporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. RESULTS: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75(NTR) and tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. CONCLUSIONS: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity.

BACKGROUND AND PURPOSE: Up-regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti-apoptotic defences and stimulates pro-inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression. EXPERIMENTAL APPROACH: Retinal neurotoxicity was induced by intravitreal injection of NMDA in Sprague-Dawley rats, which received verapamil (10 mg·kg(-1), p.o.) or vehicle. Neurotoxicity was examined by terminal dUTP nick-end labelling assay and ganglion cell count. Expression of TXNIP, apoptosis signal-regulating kinase 1 (ASK-1), NF-κB, p38 MAPK, JNK, cleaved poly-ADP-ribose polymerase (PARP), caspase-3, nitrotyrosine and 4-hydroxy-nonenal were examined by Western and slot-blot analysis. Release of TNF-α and IL-1ß was examined by elisa. KEY RESULTS: NMDA injection enhanced TXNIP expression, decreased Trx activity, causing increased oxidative stress, glial activation and release of TNF-α and IL-1ß. Enhanced TXNIP expression disrupted Trx/ASK-1 inhibitory complex leading to release of ASK-1 and activation of the pro-apoptotic p38 MAPK/JNK pathway, as indicated by cleaved PARP and caspase-3 expression. Treatment with verapamil blocked these effects. CONCLUSION AND IMPLICATIONS: Elevated TXNIP expression contributed to retinal neurotoxicity by three different mechanisms, inducing release of inflammatory mediators such as TNF-α and IL-1ß, altering antioxidant status and disrupting the Trx-ASK-1 inhibitory complex leading to activation of the p38 MAPK/JNK apoptotic pathway. Targeting TXNIP expression is a potential therapeutic target for retinal neurodegenerative disease.

Neurovascular protective effect of FeTPPs in N-methyl-D-aspartate model: similarities to diabetes.

We have previously shown a causal role of peroxynitrite in mediating retinal ganglion cell (RGC) death in diabetic and neurotoxicity models. In the present study, the role of peroxynitrite in altering the antioxidant and antiapoptotic thioredoxin (Trx) system will be investigated as well as the subsequent effects on glial activation and capillary degeneration. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-d-aspartate (NMDA) in rats, which also received the peroxynitrite decomposition catalyst FeTPPs. RGC loss was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and GC count. Glial activation and nitrotyrosine were assessed by immunohistochemistry. Acellular capillaries and pericytes were counted in retinal trypsin digest. NMDA-induced peroxynitrite formation caused RGC loss, which was associated with enhanced expression of Trx and its endogenous inhibitor thioredoxin interacting protein. The results also showed enhanced thioredoxin interacting protein/Trx binding and disruption of the Trx/apoptosis signal-regulating kinase 1 "inhibitory complex," leading to release of apoptosis signal-regulating kinase 1 and activation of the apoptotic pathway, as evidenced by p38 MAPK and poly-ADP-ribose polymerase activation. Furthermore, NMDA caused glial activation and compromised retinal vasculature, as indicated by acellular-capillary formation and pericyte loss. Treatment with FeTPPs blocked these effects. In conclusion, NMDA-induced retinal neuro/vascular injury is mediated by peroxynitrite-altered Trx antioxidant defense, which in turn activates the apoptosis signal-regulating kinase-1 apoptotic pathway. In addition to acute RGC death, an NMDA model can be a useful tool to study glial activation and capillary degeneration in retinal neurodegenerative disorders, including diabetic retinopathy.

Early intervention of tyrosine nitration prevents vaso-obliteration and neovascularization in ischemic retinopathy.

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso-obliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.