Candesartan stimulates reparative angiogenesis in ischemic retinopathy model: role of hemeoxygenase-1 (HO-1).

Ischemic diseases such as stroke and proliferative retinopathy are characterized by hypoxia-driven release of angiogenic factors such as vascular endothelial growth factor (VEGF). However, revascularization of the ischemic areas is inadequate, resulting in impaired neuro-vascular function. We aim to examine the vascular protective effects of candesartan, an angiotensin receptor blocker, in an ischemic retinopathy mouse model. Vascular density, number of tip cells, and perfusions of capillaries were assessed. Activation of Muller glial cells and levels of peroxynitrite, VEGF, VEGFR2, inducible nitric oxide synthase, hemeoxygenase-1 (HO-1) were assessed. Proangiogenic effects of candesartan were examined in human endothelial cells (EC) that were cultured in normoxia or hypoxia and transduced with siRNA against HO-1. Candesartan (1 mg/kg) and (10 mg/kg) decreased hypoxia-induced neovascularization by 67 and 70%, respectively. Candesartan (10 mg/kg) significantly stimulated the number of tip cells and physiological revascularization of the central retina (45%) compared with untreated pups. The effects of candesartan coincided with reduction of hypoxia-induced Muller glial activation, iNOS expression and restoration of HO-1 expression with no significant change in VEGF levels. In vitro, silencing HO-1 expression blunted the ability of candesartan to induce VEGF expression under normoxia and VEGFR2 activation and angiogenic response under both normoxia and hypoxia. These findings suggest that candesartan improved reparative angiogenesis and hence prevented pathological angiogenesis by modulating HO-1 and iNOS levels in ischemic retinopathy. HO-1 is required for VEGFR2 activation and proangiogenic action of candesartan in EC. Candesartan, an FDA-approved drug, could be repurposed as a potential therapeutic agent for the treatment of ischemic diseases.

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.

Diabetes exacerbates retinal oxidative stress, inflammation, and microvascular degeneration in spontaneously hypertensive rats.

PURPOSE: Hypertension and diabetes are known risk factors for retinal microvascular damage. However, the combined effects of diabetes with early and established stages of hypertension on retinal microvascular degeneration remain incompletely understood. METHODS: Male spontaneously hypertensive rats (SHR) were compared to SHR with streptozotocin-induced diabetes (SHR+D) for 6 or 10 weeks and Wistar rats as controls. RESULTS: Hypertension alone (the SHR group) or in combination with diabetes (the SHR+D group) for 6 weeks induced additive increases in total retinal cell death, compared to the Wistar controls. This increase was associated with significant increases in phosphorylated-Jun N-terminal kinase (pJNK) activation, phosphorylated-Akt inhibition, plasma and retinal lipid peroxides, and soluble intracellular adhesion molecule-1 (sICAM-1) levels. After 10 weeks, a similar trend was still observed in retinal nitrotyrosine, nuclear factor kappaB p65, and tumor necrosis factor-α expression, associated with exacerbated pJNK activation and formation of acellular capillaries. CONCLUSIONS: In conclusion, combining diabetes and hypertension-potentiated retinal oxidative/inflammatory stress promoted imbalance between the JNK stress and survival Akt pathways resulting in accelerated retinal cell death and acellular capillary formation.

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.

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.

Silencing p75(NTR) 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 p75(NTR) 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/p75(NTR) 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 p75(NTR) 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 p75(NTR)shRNA. 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 p75(NTR) 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 p75(NTR) and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75(NTR) antagonist. Therefore, targeting p75(NTR) represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF.

MicroRNA-146b-3p regulates retinal inflammation by suppressing adenosine deaminase-2 in diabetes.

Hyperglycemia- (HG-) Amadori-glycated albumin- (AGA-) induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR). There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA) and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3'-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3'-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-α release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2.

Alternative splicing of the APC gene in the neural retina and retinal pigment epithelium.

PURPOSE: Hypertrophy and hyperplasia of the retinal pigment epithelium (RPE) is associated with an inherited predisposition to human familial adenomatous polyposis coli, suggesting that expression of the adenomatous polyposis coli (APC) tumor suppressor may regulate RPE proliferation/differentiation. Distinctive APC isoforms exist in different cell types due to alternative splicing of the APC transcripts. We hypothesize that differences in expression patterns of APC protein isoforms are critical to RPE proliferation/differentiation. METHODS: To investigate these relationships, APC gene expression was characterized in the retinas and RPE from fetal and adult human and mouse, and in the epiretinal membranes (ERM) from 5 patients with proliferative vitreoretinopathy (PVR). Expression patterns of alternative splice-forms of APC transcripts were evaluated by comparative quantitative RT-PCR. Exon 1 of APC encodes a heptad repeat that confers the ability of APC to homodimerize. APC protein isoforms containing or lacking this heptad were characterized by western blot analysis and immunohistochemistry. RESULTS: Comparative quantitative RT-PCR demonstrated a predominant exon 1 containing, conventional APC splice-form in the early developing fetal RPE and retina, and in all the tested ERM samples from patients with PVR. This method also demonstrated an increased level of exon 1 lacking APC splice-form in the mature RPE and retina. Western blot analysis and immunofluorescence microscopy demonstrated the conventional APC only in the RPE, and the APC isoform without the first heptad repeat in both the retina and RPE. Immunofluorescence microscopy also demonstrated only the conventional APC in the ERM samples tested. CONCLUSIONS: These results suggest that alternative splicing of APC leads to differential APC expression with potentially unique functions. APC isoform without the first heptad repeat may play a role in cell cycle cessation in the adult retina and RPE, and the down regulation of this APC isoform may contribute to the potential of RPE to migrate and proliferate.

HGF regulation of RPE proliferation in an IL-1beta/retinal hole-induced rabbit model of PVR.

PURPOSE: To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model. METHODS: Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography. RESULTS: Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery. CONCLUSIONS: These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

MAP kinase and beta-catenin signaling in HGF induced RPE migration.

PURPOSE: Hepatocyte growth factor (HGF) has been implicated in retinal pigment epithelial (RPE) cell proliferation and migration that occurs in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). The aim of this study is to investigate HGF induced signaling pathways that lead to RPE cell migration. METHODS: Localization of beta-catenin was determined by immunofluorescence. HGF induced migration of ARPE-19 cells was studied using a quantitative migration assay after wounding in the presence of a DNA polymerase inhibitor, and in the presence or absence of a mitogen activated protein kinase (MAP kinase) kinase inhibitor. C-jun expression was determined by semi-quantitative RT-PCR and by Northern blot analysis. P42/p44 MAP kinase activity was determined by western blot and by an immunoprecipitation kinase assay. Tyrosine phosphorylation of the HGF receptor (HGFR or c-met) and beta-catenin was determined by immunoprecipitation and western blot analysis. Transactivation activity of beta-catenin was determined by luciferase reporter gene analysis. RESULTS: Beta-catenin and E-cadherin were co-localized on the basal surface of the RPE in vivo. Diffusion of the cell surface-localized beta-catenin occurs in migratory cells in vitro in the presence of HGF. HGF induced a MAP kinase dependent ARPE-19 cell migration, which is accompanied with a transient increase of c-jun expression and concomitant increases of MAP kinase activity, tyrosine phosphorylation of HGFR and beta-catenin, increased cytosolic levels of beta-catenin, and transactivation activity of beta-catenin. Tyrosine phosphorylation of HGFR and beta-catenin occurs in the primary or passaged RPE cultures or proliferative ARPE-19 cells, but not freshly isolated RPE or differentiated ARPE-19 cells. CONCLUSIONS: This study defines the signal transduction pathways activated by HGF in RPE cells, leading to an increase in the MAP kinase activity and free pool of beta-catenin, and changes in gene expression. These findings are consistent with the hypothesis that both beta-catenin and MAP kinases are components of the HGF induced RPE migration that occurs in proliferative retinal diseases.

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.

Systemic effects of ophthalmic cyclopentolate on body weight in neonatal mice.

BACKGROUND: Cyclopentolate is standardly used in ophthalmologic examinations of neonates to facilitate screening for retinopathy of prematurity. Reports of systemic effects have raised concerns of an increased risk of feeding intolerance after the examinations. OBJECTIVES: The goal of this study was to evaluate systemic concentrations of cyclopentolate after ophthalmic administration, as well as assess changes in weight as an indirect measure of alteration in feeding. METHODS: Neonatal mice were randomized into three groups to simulate a neonatal model for ophthalmic medication administration. The cyclopentolate group received a one-time administration of tetracaine, cyclopentolate, and phenylephrine ophthalmologic solutions in accordance with the protocol used at the children's hospital. The placebo group received the same ophthalmic drop administration, except for normal saline in place of cyclopentolate, and the control group received no ophthalmic drops and minimal handling. Daily weights and serum samples to measure systemic concentrations of cyclopentolate post-ophthalmic administration were assessed at baseline and for 7 days following drop administration. RESULTS: Analysis of serum levels demonstrated detectability of systemic cyclopentolate after ophthalmic administration as early as 30 min (86 ng/ml), 1 h (60 ng/ml), and 24 h (6.2 ng/ml). There were also differences in weight gained on following ophthalmic administration observed between the cyclopentolate group and placebo group, with the cyclopentolate group weighing significantly less on days 3 and 7 (p = 0.02). CONCLUSIONS: RESULTS indicate cyclopentolate is absorbed systemically and instillation of cyclopentolate decreases weight gain in neonatal mice compared to placebo. These preclinical findings provide rationale for further studies in neonatal patients.

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.

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.

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.

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.

Retinal microglial activation and inflammation induced by amadori-glycated albumin in a rat model of diabetes.

OBJECTIVE: During diabetes, retinal microglial cells are activated to release inflammatory cytokines that initiate neuronal loss and blood-retinal barrier breakdown seen in diabetic retinopathy (DR). The mechanism by which diabetes activates microglia to release those inflammatory mediators is unclear and was therefore elucidated. RESEARCH DESIGN AND METHODS: Microglia activation was characterized in streptozocin-injected rats and in isolated microglial cells using immunofluorescence, enzyme-linked immunosorbent assay, RT-PCR, and Western blot analyses. RESULTS: In 8-week diabetic retina, phospho-extracellular signal-related kinase (ERK) and P38 mitogen-activated protein kinases were localized in microglia, but not in Mueller cells or astrocytes. At the same time, Amadori-glycated albumin (AGA)-like epitopes were featured in the regions of microglia distribution, implicating a pathogenic effect on microglial activation. To test this, diabetic rats were treated intravitreally with A717, a specific AGA-neutralizing antibody, or murine IgG. Relative to nondiabetic rats, diabetic rats (IgG-treated) manifested 3.9- and 7.9-fold increases in Iba-1 and tumor necrosis factor (TNF)-α mRNAs, respectively. Treatment of diabetic rats with A717 significantly attenuated overexpression of these mRNAs. Intravitreal injection of AGA per se in normal rats resulted in increases of Iba-1 expression and TNF-α release. Guided by these results, a cultured retinal microglia model was developed to study microglial response after AGA treatment and the mechanistic basis behind this response. The results showed that formation of reactive oxygen species and subsequent activation of ERK and P38, but not Jun NH2-terminal kinase, are molecular events underpinning retinal microglial TNF-α release during AGA treatment. CONCLUSIONS: These results provide new insights in understanding the pathogenesis of early DR, showing that the accumulated AGA within the diabetic retina elicits the microglial activation and secretion of TNF-α. Thus, intervention trials with agents that neutralize AGA effects may emerge as a new therapeutic approach to modulate early pathologic pathways long before the occurrence of vision loss among patients with diabetes.

Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

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.