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TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP - Exp Diabetes Res (2012)

Bottom Line: We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
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.

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HG-induced TXNIP expression correlates with proapoptotic caspase-3 expression in rMC1. (a) IHC of TXNIP. Txnip staining is enhanced in rMC1 under HG exposure for 5 days, which correlates with increased (b) proapoptotic caspase 3 staining in similar duration of HG using a caspase-3 FLICA staining kit. A representative of n = 3 is shown here. (c–f) HG induces TXNIP and pro-inflammatory gene expression in rMC1. Total RNA was isolated with TRIZOL and mRNA levels for (c) TXNIP, (d) iNOS, (e) Cox-2, and (f) VEGF-A were measured by qRT-PCR at various time periods of HG exposure (n = 3-4). P values were compared against respective controls at time 0.
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fig7: HG-induced TXNIP expression correlates with proapoptotic caspase-3 expression in rMC1. (a) IHC of TXNIP. Txnip staining is enhanced in rMC1 under HG exposure for 5 days, which correlates with increased (b) proapoptotic caspase 3 staining in similar duration of HG using a caspase-3 FLICA staining kit. A representative of n = 3 is shown here. (c–f) HG induces TXNIP and pro-inflammatory gene expression in rMC1. Total RNA was isolated with TRIZOL and mRNA levels for (c) TXNIP, (d) iNOS, (e) Cox-2, and (f) VEGF-A were measured by qRT-PCR at various time periods of HG exposure (n = 3-4). P values were compared against respective controls at time 0.

Mentions: Autophagy/mitophagy is initially a mechanism for cell survival; however excessive and continuous autophagy may remove vital proteins and organelles and induce cell apoptosis [36, 37]. Therefore, we examined whether proapoptotic caspase-3 is activated under chronic HG in rMC1. As shown in Figures 7(a) and 7(b), HG increases TXNIP and caspase-3 staining in rMC1 when compared with LG, suggesting cells undergoing a path of apoptotic cell death. Furthermore, injured and dying cells induce the expression of pro-inflammatory genes significantly (P < 0.05) for TXNIP (~36.55 ± 12.45 at day 3), iNOS (14.47 ± 4.8 at day 3), Cox-2 (4.49 ± 0.88 at day 5) and VEGF-A mRNA (2.15 ± 0.27 day 5) (Figures 7(c)–7(f)). These results suggest that under HG and oxidative stress, rMC1 cells promote an inflammatory and autophagy/mitophagy response to remove defective organelles.


TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP - Exp Diabetes Res (2012)

HG-induced TXNIP expression correlates with proapoptotic caspase-3 expression in rMC1. (a) IHC of TXNIP. Txnip staining is enhanced in rMC1 under HG exposure for 5 days, which correlates with increased (b) proapoptotic caspase 3 staining in similar duration of HG using a caspase-3 FLICA staining kit. A representative of n = 3 is shown here. (c–f) HG induces TXNIP and pro-inflammatory gene expression in rMC1. Total RNA was isolated with TRIZOL and mRNA levels for (c) TXNIP, (d) iNOS, (e) Cox-2, and (f) VEGF-A were measured by qRT-PCR at various time periods of HG exposure (n = 3-4). P values were compared against respective controls at time 0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7: HG-induced TXNIP expression correlates with proapoptotic caspase-3 expression in rMC1. (a) IHC of TXNIP. Txnip staining is enhanced in rMC1 under HG exposure for 5 days, which correlates with increased (b) proapoptotic caspase 3 staining in similar duration of HG using a caspase-3 FLICA staining kit. A representative of n = 3 is shown here. (c–f) HG induces TXNIP and pro-inflammatory gene expression in rMC1. Total RNA was isolated with TRIZOL and mRNA levels for (c) TXNIP, (d) iNOS, (e) Cox-2, and (f) VEGF-A were measured by qRT-PCR at various time periods of HG exposure (n = 3-4). P values were compared against respective controls at time 0.
Mentions: Autophagy/mitophagy is initially a mechanism for cell survival; however excessive and continuous autophagy may remove vital proteins and organelles and induce cell apoptosis [36, 37]. Therefore, we examined whether proapoptotic caspase-3 is activated under chronic HG in rMC1. As shown in Figures 7(a) and 7(b), HG increases TXNIP and caspase-3 staining in rMC1 when compared with LG, suggesting cells undergoing a path of apoptotic cell death. Furthermore, injured and dying cells induce the expression of pro-inflammatory genes significantly (P < 0.05) for TXNIP (~36.55 ± 12.45 at day 3), iNOS (14.47 ± 4.8 at day 3), Cox-2 (4.49 ± 0.88 at day 5) and VEGF-A mRNA (2.15 ± 0.27 day 5) (Figures 7(c)–7(f)). These results suggest that under HG and oxidative stress, rMC1 cells promote an inflammatory and autophagy/mitophagy response to remove defective organelles.

Bottom Line: We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus