Takhellambam S. Devi

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009.

TXNIP Links Innate Host Defense Mechanisms to Oxidative Stress and Inflammation in Retinal Muller Glia under Chronic Hyperglycemia: Implications for Diabetic Retinopathy

Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1β) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

Inhibition of TXNIP expression in vivo blocks early pathologies of diabetic retinopathy

Evidence is mounting that proinflammatory and proapoptotic thioredoxin-interacting protein (TXNIP) has a causative role in the development of diabetes. However, there are no studies investigating the role of TXNIP in diabetic retinopathy (DR). Here, we show that, in diabetic rats, TXNIP expression and hexosamine biosynthesis pathway (HBP) flux, which regulates TXNIP, are elevated in the retina and correlates well with the induction of inflammatory cyclooxygenase 2 (Cox-2) and sclerotic fibronectin (FN). We blocked the expression of TXNIP in diabetic rat retinas by: (i) inhibiting HBP flux; (ii) inducing post-transcriptional gene silencing (PTGS) for TXNIP mRNA; and (iii) performing an in vivo transcriptional gene silencing (TGS) approach for TXNIP knockdown by promoter-targeted small interfering RNAs and cell-penetrating peptides as RNA interference (RNAi) transducers. Each of these methods is efficient in downregulating TXNIP expression, resulting in blockade of its target genes, Cox-2 and FN, demonstrating that TXNIP has a causative role in aberrant gene induction in early DR. RNAi TGS of TXNIP abolishes diabetes-induced retinal gliosis and ganglion injury. Thus, TXNIP has a critical role in inflammation and retinal injury in early stages of DR. The successful employment of TXNIP TGS and amelioration of its pathological effects open the way for novel therapeutic strategies aimed to block disease onset and progression of DR.

RAGE-TXNIP axis is required for S100B-promoted Schwann cell migration, fibronectin expression and cytokine secretion.

During peripheral nerve injury, Schwann cells (SCs) adopt a migratory phenotype and remodel the extracellular matrix and provide a supportive activity for neuron regeneration. SCs synthesize neurotrophic factors and cytokines that are crucial for the repair of the injured nerve. The receptor for advanced glycation end products (RAGE) and its ligand S100B, which are secreted by SCs, are required for the repair of the injured peripheral nerve in vivo. However, the precise intracellular pathways involved have not been completely elucidated. Here, we show that RAGE-induced S100B secretion involves the recruitment of S100B in lipid rafts and caveolae. Moreover, we demonstrate for the first time that RAGE induces the expression of thioredoxin interacting protein (TXNIP) in SCs and the injured sciatic nerve in vivo. TXNIP is involved in the activation of p38 MAPK, CREB and NFκB in SCs. TXNIP silencing partially inhibits RAGE-induced SC migration and completely abolishes RAGE-induced fibronectin and IL-1β expression. Our results support a model in which TXNIP mediates in part RAGE-induced SC migration and is required for the expression of provisional ECM and pro-inflammatory IL-1β. We provide new insight on the role of the SC RAGE–TXNIP axis in the repair of injured peripheral nerves.

Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: Implications for diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by early loss of retinal capillary pericytes and microvascular dysfunction. We recently showed that pro-oxidative stress and pro-apoptotic thioredoxin interacting protein (TXNIP) is significantly up-regulated in rat retinas in experimental diabetes and mediates inflammation and apoptosis. Therefore, we hypothesize here that TXNIP up-regulation in pericyte plays a causative role in oxidative stress and apoptosis under sustained high glucose exposure in culture. We maintained a rat retinal capillary pericyte cell line (TR-rPCT1) for 5 days under low glucose (LG, 5.5mM) or high glucose (HG, 25 mM) with or without anti-oxidant N-acetylcysteine (5mM, NAC), Azaseine (2 μM, AzaS), an inhibitor of TXNIP, and TXNIP siRNA (siTXNIP3, 20 nM). The results show that HG increases TXNIP expression in TR-rPCT1, which correlates positively with ROS generation, protein S-nitrosylation, and pro-apoptotic caspase-3 activation. Furthermore, pericyte apoptosis is demonstrated by DNA fragmentation (alkaline comet assay) and a reduction in MTT survival assay. Treatment of TR-rPCT1 with NAC or an inhibition of TXNIP by AzaS or siTXNIP3 each reduces HG-induced ROS, caspase-3 activation and DNA damage demonstrating that TXNIP up-regulation under chronic hyperglycemia is critically involved in cellular oxidative stress, DNA damage and retinal pericyte apoptosis. Thus, TXNIP represents a novel gene and drug target to prevent pericyte loss and progression of DR.

GSK-3β/CREB axis mediates IGF-1-induced ECM/adhesion molecule expression, cell cycle progression and monolayer permeability in retinal capillary endothelial cells: Implications for diabetic retinopathy

Various growth factors and cytokines are implicated in endothelial dysfunction and blood-retinal barrier (BRB) breakdown in early diabetic retinopathy (DR). However, cellular and molecular mechanisms that may underlie the pathology of DR are not fully understood yet. We therefore examined the effect of insulin-like growth factor (IGF)-1 on ECM/adhesion molecule expression, cell cycle regulation and monolayer permeability in an endothelial cell line (TR-iBRB2). We investigate whether the action of IGF-1 (1) involves glycogen synthase kinase 3beta (GSK-3β) and cAMP responsive transcription factor (CREB) and (2) alters ECM/adhesion molecule gene expression. Treatment of TR-iBRB2 cell with IGF-1 (100ng/ml for 0-24h) increases phosphorylation of (i) Akt Thr308, and its substrates including GSK-3β at Ser9, which inactivates its kinase function, and (ii) CREB at Ser133 (activation). These phosphorylations correlate positively with enhanced expression of CREB targets such as ECM protein fibronectin and cell cycle progression factor cyclin D1. However, stable transfection of a mutant GSK3β(S9A) or a dominant negative K-CREB in TR-iBRB2 prevents IGF-1-induced fibronectin and cyclin D1 expression. Furthermore, IGF-1 reduces the level of intercellular adherence molecule VE-cadherin and increases monolayer permeability in TR-iBRB2 cells when measured by FITC-dextran leakage. The effect of IGF-1 on VE-cadherin and membrane permeability is absent in TR-iBRB2 cells expressing the GSK-3β(S9A). Similarly, K-CREB reverses IGF-1 down-regulation of VE-cadherin and up-regulation of fibronectin. These results indicate that GSK-3β/CREB axis alters ECM/adhesion molecule expression and cell cycle progression in retinal endothelial cells, and may potentially contribute to endothelial dysfunction and BRB leakage in DR.

Theophylline regulates inflammatory and neurotrophic factor signals in functional recovery after C2-hemisection in adult rats

Recovery of respiratory activity in an upper cervical hemisection model (C2H) of spinal cord injury (SCI) can be induced by systemic theophylline administration 24-48 h after injury. The objectives in the present study are (1) to identify pro-inflammatory and neurotrophic factors expressed after C2H and (2) molecular signals involved in functional recovery. Four groups of adult female rats classified as (i) sham (SH) controls, (ii) subjected to a left C2 hemisection (C2H) only, (iii) C2H rats administered theophylline for 3 consecutive days 2 days after C2H (C2H-T day 5) and (iv) C2H rats treated with theophylline for 3 consecutive days 2 days after C2H and then weaned for 12 days (C2H-T day 17) prior to assessment of respiratory function and molecular analysis were employed. Corresponding sham controls, C2H untreated (vehicle only controls) and C2H treated (theophylline) rats were sacrificed, C3-C6 spinal cord segments quickly dissected and left (ipsilateral) hemi spinal cord and right (contralateral) hemi spinal cord were separately harvested 2 days post surgery. Sham operated and C2H untreated-controls corresponding to C2H-T day 5 and C2H-T day 17 rats, respectively, were prepared similarly. Messenger RNA levels for pro-inflammatory genes (TXNIP, IL-1β, TNF-α and iNOS) and neurotrophic and survival factors (BDNF, GDNF, and Bcl2) were analyzed by real time quantitative PCR. Gene expression pattern was unaltered in SH rats. TXNIP, iNOS, BDNF, GDNF and Bcl2 mRNA levels were significantly increased in the ipsilateral hemi spinal cord in C2H rats. BDNF, GDNF and Bcl2 levels remained elevated in the ipsilateral hemi spinal cord in C2H-T day 5 rats. In this same group, there was further enhancement in TXNIP and IL-1β while iNOS returned to basal levels. Theophylline increased DNA binding activity of transcription factors - cyclic AMP responsive element (CRE) binding protein (CREB) and pro-inflammatory NF-κB. Messenger RNA levels for all genes returned to basal levels in C2H-T day 17 rats. However, BDNF mRNA levels remained significantly elevated after weaning from the drug. Our results suggest that enhanced resolution of early inflammatory processes and expression of pro-survival factors may underlie theophylline-induced respiratory recovery. The results identify potential targets for gene and drug therapies.