EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells.

Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.

Loss of cystatin C regulates permeability and inflammatory pathways in retina.

Cystatin C has been linked to inflammation in other diseases, such as epilepsy and Alzheimer's disease. These studies were designed to investigate whether Cystatin C regulates retinal inflammation and permeability. To address this question, we used Cystatin C knockout mice in a retinal ischemia/reperfusion model to determine whether Cystatin C regulated retinal damage, as well as inflammatory mediators and retinal permeability. To support the mouse work, we also used primary retinal endothelial cells cultured in normal and high glucose. Ischemia/reperfusion in Cystatin C knockout mice caused increased formation of degenerate capillaries. Loss of Cystatin C increased fluorescein leakage in the retina, which was accompanied by reduced levels of zonula occludin 1 (ZO-1) and occludin proteins. When REC were grown in high glucose, recombinant Cystatin C decreased retinal permeability, while Cystatin C siRNA increased dextran flux compared to high glucose alone. Recombinant Cystatin C decreased levels of interleukin-1-beta (IL-1ß) and high mobility group box 1 (HMGB1) levels. In conclusion, loss of Cystatin C increased vascular damage in response to ischemia/reperfusion. Cystatin C regulated permeability and inflammatory mediators in the retina in response to stressors. Cystatin C offers a new target for retinal disease therapeutic development.

TNFAIP3 is anti-inflammatory in the retinal vasculature.

Purpose: To determine whether tumor necrosis factor alpha-induced protein 3 (TNFAIP3) regulates inflammatory and permeability proteins in the retinal vasculature. Methods: We used retinal lysates from type 1 diabetic mice and endothelial cell-specific exchange protein for cAMP 1 (Epac1) knockout mice to determine the protein levels of TNFAIP3. We also treated retinal endothelial cells (RECs) in normal (5 mM) and high (25 mM) glucose with an Epac1 agonist or with TNFAIP3 siRNA. We performed western blotting for TNFAIP3 and inflammatory and permeability proteins after treatment. TNFAIP3 siRNA was used only in cells grown in high glucose. Immunostaining was performed for localization of ZO-1 and tight junction protein 1. Results: TNFAIP3 was reduced in the diabetic retinas and the retinas of the Epac1 conditional knockout mice. The Epac1 agonist increased TNFAIP3 levels in RECs grown in high glucose. Reduction of TNFAIP3 with siRNA led to increased levels of tumor necrosis factor alpha (TNFα) and phosphorylation of nuclear factor kappa beta (NF-kB), while decreasing occludin and zonula occludens 1 (ZO-1) protein levels and inhibitory kappa beta kinase (IkB) phosphorylation. Tumor receptor-associated factor 6 (TRAF6) levels were increased above high glucose levels. Conclusions: TNFAIP3 serves as an anti-inflammatory factor in the retinal vasculature. Epac1 regulates TNFAIP3. TNFAIP3 may offer a new mechanism for regulating inflammation and permeability in the retinal vasculature.

TNFAIP3 may be key to TLR4-activation of the inflammasome in the retinal vasculature.

The goal of these studies were to determine whether tumor necrosis factor, alpha-induced protein 3 (TNFAIP3) regulated toll-like receptor 4 (TLR4) actions on the NOD-like receptor protein 3 (NLRP3) inflammasome. Western blotting was done on retinal lysates from TLR4 floxed and endothelial cell specific TLR4 knockout mice for TNFAIP3, TLR4, and NLRP3 pathway proteins. Retinal endothelial cells (REC) were grown in normal (5 mM) and high glucose (25 mM) and treated with TNFAIP3 siRNA, followed by Western blotting for TLR4 and NLRP3 pathway proteins. Loss of TLR4 in endothelial cells increased TNFAIP3 levels, while decreasing NLRP3 pathway proteins. High glucose culturing conditions increased TLR4 and NLRP3 proteins, which were also increased by TNFAIP3 siRNA. Data demonstrate that TLR4 regulates NLRP3 pathway proteins. TNFAIP3 can regulate TLR4 and the NLRP3 pathway. TNFAIP3 may offer a new target for therapeutic development against retinal inflammation.

Prohibitin 1 Regulates Inflammatory Mediators and Reactive Oxygen Species in Retinal Endothelial Cells.

Diabetic retinopathy is associated with increased inflammatory mediator levels. In these studies, we focused on prohibitin 1. We performed western blotting for retinal lysates from diabetic mice and Epac1 floxed and cdh5Cre-Epac1 mice. We also grew primary retinal endothelial cells (REC) in normal (5 mM) and high (25 mM) glucose, and treated some cells with an Epac 1 agonist or prohibitin 1 siRNA. Western blotting was done to confirm knockdown of prohibitin 1 and Epac 1 agonism. We measured the tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1ß), phosphorylated prohibitin 1, phosphorylated nuclear factor kappa beta (NFkB), high mobility group box 1 (HMGB1) and reactive oxygen species (ROS) levels in REC after transfection with prohibitin 1 siRNA. Results showed that high glucose increased the inflammatory mediators, as well as HMGB1 and ROS. The levels of ROS, HMGB1, and inflammatory pathways were all reduced after cells were transfected with prohibitin 1 siRNA. Epac1 reduced prohibitin 1 phosphorylation. In conclusion, decreased prohibitin 1 significantly reduced the inflammatory mediator and ROS levels in REC. Epac1 regulates the prohibitin 1 levels in REC.

Epac1 and PKA regulate of P2X7 and NLRP3 inflammasome proteins in the retinal vasculature.

Others have shown that the purinergic 2X7 receptor (P2X7R) and the NOD-like receptor family protein 3 (NLRP3) inflammasome are involved in multiple inflammatory diseases. In this study, we tested whether Epac1 and PKA lie upstream of P2X7R actions on the NLRP3 inflammasome. We also evaluated whether eye drops of a P2X7R inhibitor protected the retina against ischemia/reperfusion (I/R) injury by measuring retinal thickness and degenerate capillary formation after exposure to I/R and treatment with A438079 eye drops. Mice were exposed to the I/R model followed by eye drops of A438079 for 2 or 10 days. Additionally, primary human retinal endothelial cells (REC) grown in normal and high glucose were treated with ATP (to stimulate P2X7R), an Epac1 agonist, or forskolin (to stimulate PKA), followed by measurements of P2X7R and NLRP3 inflammasome proteins. Eye drops containing A438079 protected the retina against neuronal and vascular damage after exposure to I/R. When REC were treated with ATP to stimulate P2X7R, NLRP3 inflammasome proteins were all increased compared to high glucose only. Epac1 and PKA agonists reduced P2X7R levels in REC grown in high glucose. In conclusion, these data suggest that P2X7 regulates retinal responses to the I/R stress, and that P2X7 increases NLRP3 inflammasome proteins in human REC. Epac1 and PKA can inhibit of P2X7, which will reduce NLRP3 inflammasome proteins in REC grown in high glucose.

PKA and Epac1 Reduce Nek7 to Block the NLRP3 Inflammasome Proteins in the Retinal Vasculature.

Purpose: To determine whether protein kinase a (PKA) and exchange protein for cAMP 1 (Epac1) inhibit NIMA-related kinase 7 (Nek7) to block the NOD-like receptor family pyrin domain-containing family member 3 (NLRP3) signaling pathway. Methods: Retinal endothelial cells (RECs) were grown in normal (5 mM) or high (25 mM) glucose. Some cells were treated with a Nek7 cDNA plasmid, Nek7 siRNA; an Epac1 agonist, forskolin; a PKA agonist; or an empty vector. Epac1 floxed and Cdh5-cre Epac1 mice and Nek7 floxed and Cdh5-cre Nek7 mice were also used. Western blot analyses were done on cell culture or whole retinal lysates for NLRP3, cleaved caspase 1, interleukin-1-beta (IL-1ß). A PKA activity assay was also done. Results: Nek7 cDNA increased NLRP3 signaling proteins, but Nek7 siRNA inhibited high-glucose induction of these proteins in retinal endothelial cells. Epac1 and forskolin both reduced Nek7 and NLRP3 pathway proteins, even when given in combination with Nek7 cDNA. Elimination of Nek7 in endothelial cells reduced NLRP3 signaling proteins in whole retinal lysates from mice. Conclusions: Nek7 regulated NLRP3 inflammasome protein levels both in vitro and in vivo. Both Epac1 and PKA lie upstream of Nek7 and NLRP3 and can overcome excessive Nek7 levels. These studies establish that cAMP proteins can inhibit Nek7 and block activation of the NLRP3 inflammasome proteins.

Epac1 and PKA agonists inhibit ROS to reduce NLRP3 inflammasome proteins in retinal endothelial cells.

Purpose: Reactive oxygen species (ROS) activate inflammatory pathways in several organs, including the retina. More recent work has shown that ROS activate the NOD-like receptor protein 3 (NLRP3) inflammasome pathway proteins. We recently showed that the exchange protein activated by cAMP 1 (Epac1) and protein kinase A (PKA) regulates NLRP3 proteins in the retina. Our goal was to determine whether Epac1 and PKA reduce ROS and NLRP3 inflammasome proteins. Methods: We used human primary retinal endothelial cells (RECs) grown in normal glucose (5 mM) and stimulated in normal glucose with hydrogen peroxide (H2O2) to induce ROS and measured NLRP3 pathway proteins. In some groups, we treated cells with an Epac1 or a PKA agonist in addition to H2O2 treatment to determine whether Epac1 and PKA reduced ROS and induced NLRP3 pathway proteins. Results: The data showed that 500 µM H2O2 was the optimal dose to increase ROS in RECs. In RECs treated with H2O2, NLRP3 pathway proteins were increased, which were significantly reduced by cotreatment with PKA or Epac1 agonists. H2O2 significantly increased NIMA-related kinase 7 (Nek7) and purinergic 2X7 receptor 7 (P2X7) levels, which were blocked by Epac1 and PKA agonists. Conclusions: Taken together, these data suggest that Epac1 and PKA reduce retinal inflammation through the reduced ROS-induced activation of NLRP3 pathway proteins.

Forskolin regulates retinal endothelial cell permeability through TLR4 actions in vitro.

To investigate whether forskolin, a protein kinase A agonist, regulates toll-like receptor 4 actions on retinal endothelial cell permeability in vitro. We also evaluated whether PKA could regulate TLR4 signaling independent of exchange protein activated by cAMP in REC in culture. REC were grown in normal (5 mM) or high (25 mM) glucose. Cells were treated with forskolin to increase PKA levels, siRNA against TLR4, siRNA against myeloid differentiation primary response 88, siRNA against translocating chain associated membrane protein 1, siRNA against epac1, or scrambled siRNA, or a combination of these treatments. Western blotting was done for zonula occludens 1 and occludin protein levels, as well as TLR4 signaling cascade proteins. Permeability measurements were done for REC in culture following inhibition of TLR4 or its signaling cascades. Forskolin restored high glucose-associated decreases in ZO-1 and occludin, which was associated with improved in vitro permeability levels. Both forskolin and TLR4 inhibition reduced high glucose-induced increases in REC permeability, but the actions were not cooperative. Forskolin regulated both MyD88-dependent and -independent signaling pathways, independent of Epac1. Finally, blockade of MyD88 or TRAM1 reduced permeability in REC grown in high glucose. A PKA agonist regulated TLR4 signaling independent of Epac1. PKA agonism or TLR4 inhibition is effective at reducing high glucose-induced permeability in REC in vitro. These studies offer new avenues for therapeutic development.

Forskolin eye drops improve retinal damage from ischemia/reperfusion.

Purpose: To determine whether forskolin, a protein kinase A (PKA) agonist, eye drops could reduce neuronal and vascular damage after exposure to ischemia/reperfusion (I/R). Methods: C57BL/6J mice were exposed to the I/R protocol. A group of mice were given forskolin eye drops (10 µM) daily. Two days after I/R, neuronal measurements were performed, while vascular measurements were performed at 10 days post-I/R. Western blotting was conducted to investigate whether forskolin could increase PKA levels and reduce the levels of inflammatory mediators. Results: Forskolin statistically significantly increased PKA levels, but not exchange protein activated by cAMP 1 (Epac1). The forskolin eye drops also reduced neuronal and vascular damage compared to I/R alone. Tumor necrosis factor alpha (TNF-α) and interleukin-1-ß (IL-1ß) levels were statistically significantly reduced after administration of forskolin eye drops compared to I/R alone. Conclusions: Forskolin eye drops were protective against I/R. The findings offer a new therapeutic for local delivery.

Insulin Signal Transduction is Impaired in the Type 2 Diabetic Retina.

Rates of type 2 diabetes are reaching epidemic levels. Yet, the tissue specific alterations due to insulin resistance are only recently being investigated. The goal of the present study was to evaluate retinal insulin signal transduction in a common mouse model of type 2 diabetes, the db/db mouse. Retinal lysates from five month old male db/db and db/+ (control) mice were collected and processed for Western blotting or ELISA analyses for insulin receptor, insulin receptor substrate-1 (IRS-1), Akt, tumor necrosis factor alpha (TNFα) and caspase 3 levels. Data demonstrate increased TNFα and IRS-1 phosphorylation on serine 307. This led to decreased Akt phosphorylation on serine 473 and increased cleavage of caspase 3. Taken together, the data suggest dysfunctional insulin signaling in the retina of the db/db mouse. insulin.

Glycyrrhizin protects IGFBP-3 knockout mice from retinal damage.

We previously reported that insulin-like growth factor binding protein 3 (IGFBP-3) knockout (KO) mice have neuronal and vascular damage to the retina. We also reported that glycyrrhizin, a high mobility growth factor binding protein 1 (HMGB1) inhibitor, is protective to the diabetic retina. In this study, we investigated whether glycyrrhizin could reduce neuronal and vascular damage in the IGFBP-3 KO mouse retina. We used measurements of retinal thickness, cell number in the ganglion cell layer, degenerate capillaries, reactive oxygen species (ROS) and protein levels of HMGB1, tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1ß) and sirtuin 1 (SIRT1) to determine whether glycyrrhizin could protect the retina. Data show that glycyrrhizin in the drinking water was effective in reducing neuronal damage at 2 months and vascular damage at 6 months. Glycyrrhizin reduced ROS levels at 6 months, and reduced levels of HMGB1, TNFα, and IL-1ß at both 2 and 6 months. Taken together, the data suggest that glycyrrhizin is protective to the retina of IGFBP-3 KO mice through anti-inflammatory mechanisms.

Glycyrrhizin Protects the Diabetic Retina against Permeability, Neuronal, and Vascular Damage through Anti-Inflammatory Mechanisms.

Damage associated molecular pattern (DAMPs), such as high mobility group box 1 (HMGB1), may be involved in retinal inflammation in response to high glucose. To test whether HMGB1 inhibition could protect the diabetic retina, C57BL/6J mice were made diabetic and treated with glycyrrhizin, a HMGB1 inhibitor, for up to six months. Measurements of permeability, neuronal, and vascular changes were done, as well as assessments of HMGB1, tumor necrosis factor alpha (TNFα), and interleukin-1-beta (IL1ß) levels. Retinal endothelial cells (REC) treated with glycyrrhizin had reduced IL1ß and cleaved caspase 3 levels. Data also demonstrate that glycyrrhizin effectively reduced HMGB1 levels throughout the retina, as well as maintained normal retinal permeability and retinal capillary coverage. Glycyrrhizin maintained normal cell numbers in the ganglion cell layer and prevented thinning of the retina at two months. These histological changes were associated with reduced reactive oxygen species, as well as reduced HMGB1, TNFα, and IL1ß levels. The data strongly imply that HMGB1 inhibition prevented diabetic retinal changes through anti-inflammatory pathways.

Epac1 and Glycyrrhizin Both Inhibit HMGB1 Levels to Reduce Diabetes-Induced Neuronal and Vascular Damage in the Mouse Retina.

The role of high mobility group box 1 (HMGB1) in acute diabetic retinal damage has been demonstrated. We recently reported that glycyrrhizin, a HMGB1 inhibitor, protected the diabetic retina against neuronal, vascular, and permeability changes. In this study, we wanted to investigate the role of exchange protein for cAMP 1 (Epac1) on HMGB1 and the actions of glycyrrhizin. Using endothelial cell specific knockout mice for Epac1, we made some mice diabetic using streptozotocin, and treated some with glycyrrhizin for up to 6 months. We measured permeability, neuronal, and vascular changes in the Epac1 floxed and knockout mice. We also investigated whether Epac1 and glycyrrhizin work synergistically to reduce the retinal inflammatory mediators, tumor necrosis factor alpha (TNFα) and interleukin-1-beta (IL1ß), as well as sirtuin 1 (SIRT1) levels. Epac1 and glycyrrhizin reduced inflammatory mediators with synergistic actions. Glycyrrhizin also increased SIRT1 levels in the Epac1 mice. Overall, these studies demonstrate that glycyrrhizin and Epac1 can work together to protect the retina. Finally, glycyrrhizin may regulate HMGB1 through increased SIRT1 actions.

Epac1 deacetylates HMGB1 through increased IGFBP-3 and SIRT1 levels in the retinal vasculature.

Purpose: Inflammation is a key component of retinal disease. We previously reported that exchange protein for cAMP 1 (Epac1) reduced inflammatory mediators, including total levels of high mobility group box 1 (HMGB1) in retinal endothelial cells (RECs) and the mouse retina. The goal of this study was to determine intermediate pathways that allow Epac1 to reduce HMGB1, which could lead to novel targets for therapeutics. Methods: We used endothelial cell-specific conditional knockout mice for Epac1 and RECs to investigate whether Epac1 requires activation of insulin like growth factor binding protein 3 (IGFBP-3) and sirtuin 1 (SIRT1) to reduce acetylated HMGB1 levels with immunoprecipitation, western blot, and enzyme-linked immunosorbent assay (ELISA). Results: Data showed that high glucose reduced IGFBP-3 and SIRT1 levels, and increased acetylation of HMGB1 in RECs. An Epac1 agonist reduced acetylated HMGB1 levels in high glucose. The Epac1 agonist could not reduce HMGB1 or SIRT1 levels when IGFBP-3 siRNA was used. The agonist also could not reduce HMGB1 when SIRT1 siRNA was used. The mouse retina showed that loss of Epac1 increases acetylated HMGB1 levels and reduces IGFBP-3 and SIRT1 levels. Conclusions: Taken together, the data suggest that Epac1 activates IGFBP-3 to increase SIRT1, leading to a significant reduction in acetylated HMGB1. These findings provide novel therapeutic targets for reducing key inflammatory cascades in the retina.

Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines.

We have previously reported that Epac1 reduced inflammatory cytokines, which is protective to the diabetic retina. We have also published that impaired insulin signaling occurs in the diabetic retina. A reduction in interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα) by Epac1 could potentially restore normal insulin signal transduction. Confocal microscopy was performed to localize the insulin receptor in the retina of Epac1 floxed and endothelial cell-specific Epac1 knockout mice. Whole retinal lysates from Epac1 floxed and endothelial cell-specific Epac1 knockout mice were used to investigate proteins involved in the insulin signaling cascade. Primary human REC were cultured in normal and high glucose followed by Epac1 agonist treatment or transfection with IL-1ß or TNFα siRNA for protein analyses of insulin signaling proteins. Decreased expression of the insulin receptor was observed in the Epac1 knockout mouse retinal vasculature compared to floxed littermates. Work in mice showed that loss of Epac1 decreased insulin signaling proteins. Treatment with an Epac1 agonist decreased p38 and JNK signaling and increased insulin signaling, as did inhibition of IL-1ß or TNFα using siRNA when added to REC grown in high glucose. Taken together, Epac1 can restore normal insulin signaling in the retinal vasculature through reductions in inflammatory cytokines.

miRNA15a regulates insulin signal transduction in the retinal vasculature.

We previously reported that tumor necrosis factor alpha (TNFα) could inhibit insulin signal transduction in retinal cells. We recently found that miR15a/16 also reduced TNFα in retinal endothelial cells (REC) and in vascular specific miR15a/16 knockout mice. Since in silico programs suggested that miR15a could directly bind the insulin receptor, we wanted to determine whether miR15a altered insulin signal transduction. We used a luciferase-based binding assay to determine whether miR15a directly bound the insulin receptor. We then used Western blotting, ELISA, and qPCR to investigate whether miR15a altered insulin signaling proteins in REC and in both miR15a/16 endothelial cell knockout and overexpressing mice. We also treated some REC with resveratrol to determine if resveratrol could increase miR15a expression, since resveratrol is protective to the diabetic retina. We found that miR15a directly bound the 3'UTR of the insulin receptor. Treatment with resveratrol increased miR15a expression in REC grown in high glucose. While total insulin receptor levels were not altered, insulin signal transduction was reduced in REC grown in high glucose and was restored with treatment with resveratrol. miR15a knockout mice had reduced insulin receptor phosphorylation and Akt2 levels, with increased insulin receptor substrate 1 (IRS-1) phosphorylation on serine 307, a site known to inhibit insulin signaling. In contrast, overexpression of miR15a increased insulin signal transduction. Taken together, these data suggest that miR15a binds the insulin receptor and indirectly regulates insulin receptor actions. It also offers an additional mechanism by which resveratrol is protective to the diabetic retina.

Toll-Like Receptor 4 Reduces Occludin and Zonula Occludens 1 to Increase Retinal Permeability Both in vitro and in vivo.

We reported that ß-adrenergic receptors regulate toll-like receptor 4 (TLR4) signaling in the retina of diabetic mice and in retinal endothelial cells (REC) and Müller cells. We hypothesized that TLR4 regulates retinal permeability both in vitro and in vivo in the retinal vasculature. We used REC cultured in normal and high-glucose conditions and TLR4 siRNA treatments for cell culture studies of permeability and protein analyses of tumor necrosis factor α, occludin, and zonula occludens 1 (ZO-1). We used endothelial cell (EC)-specific Cre-Lox TLR4 knockout mice to study retinal permeability and neuronal and vascular changes following exposure to ocular ischemia/reperfusion (I/R) used as a retinal stressor. We found that the loss of TLR4 in the EC led to the reduced permeability following I/R and fewer degenerate capillaries. Retinal permeability was increased in REC grown in high-glucose conditions but was inhibited by TLR4 siRNA treatment. High-glucose culture conditions significantly reduced occludin and ZO-1 levels in REC, and TLR4 siRNA treatment restored levels to baseline. In conclusion, these studies demonstrate that TLR4 in EC strongly regulates retinal permeability and neuronal and vascular changes following exposure to stressors such as I/R.

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina.

OBJECTIVE AND DESIGN: Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that ß-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling. MATERIALS AND METHODS: We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies. METHODS: Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina. RESULTS: We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1Ser307 levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3. CONCLUSIONS: Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.

Inhibition of HMGB1 protects the retina from ischemia-reperfusion, as well as reduces insulin resistance proteins.

The role of inflammation in diabetic retinal amage is well accepted. While a number of cytokines and inflammatory mediators are responsible for these changes, upstream regulators are less well studied. Additionally, the role for these upstream mediators in retinal health is unclear. In this study, we hypothesized that inhibition of high mobility group box 1 (HMGB1) could restore normal insulin signaling in retinal endothelial cells (REC) grown in high glucose, as well as protect the retina against ischemia/reperfusion (I/R)-induced retinal damage. REC were grown in normal (5mM) or high glucose (25mM) and treated with Box A or glycyrrhizin, two different HMGB1 inhibitors. Western blotting was done for HMGB1, toll-like receptor 4 (TLR4), insulin receptor, insulin receptor substrate-1 (IRS-1), and Akt. ELISA analyses were done for tumor necrosis factor alpha (TNFα) and cleaved caspase 3. In addition, C57/B6 mice were treated with glycyrrhizin, both before and after ocular I/R. Two days following I/R, retinal sections were processed for neuronal changes, while vascular damage was measured at 10 days post-I/R. Results demonstrate that both Box A and glycyrrhizin reduced HMGB1, TLR4, and TNFα levels in REC grown in high glucose. This led to reduced cleavage of caspase 3 and IRS-1Ser307 phosphorylation, and increased insulin receptor and Akt phosphorylation. Glycyrrhizin treatment significantly reduced loss of retinal thickness and degenerate capillary numbers in mice exposed to I/R. Taken together, these results suggest that inhibition of HMGB1 can reduce retinal insulin resistance, as well as protect the retina against I/R-induced damage.