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.

Ephrin B1 Regulates Inflammatory Pathways in Retinal Müller Cells.

The role of inflammation has been accepted as a factor in the complications of diabetic retinopathy. Discovery of the upstream regulation of these inflammatory factors has remained a challenge. In this study, we explored the actions of ephrin B1 in retinal Müller cells and their actions on inflammatory proteins. We used diabetic human and mouse samples, as well as Müller cells in culture to measure ephrin B1 in Müller cells. We then generated Müller cell specific ephrin B1 knockout mice. We measure levels of key inflammatory proteins, including high mobility group box 1 (HMGB1) and NOD-like receptor protein 3 (NLRP3) pathway proteins in retinal lysates from the ephrin B1 floxed and ephrin B1 Müller cell specific knockout mice. Data show that ephrin B1 is significantly increased in the retina of diabetic humans and mice, as well as in Müller cells grown in high glucose. Elimination of ephrin B1 in mouse Müller cells led to a significant decline in all inflammatory proteins studied. In conclusion, a reduction in ephrin B1 in the diabetic retina may offer a new therapeutic modality for diabetic retinopathy.

Semaphorin 7a regulates inflammatory mediators and permeability in retinal endothelial cells.

Research supports a key role for inflammation in damaging the retinal vasculature. Current work is designed to investigate regulation of key inflammatory pathways. In this study, we hypothesized that semaphorin 7a (Sema7a) was involved in the increased inflammatory mediators and permeability changes in retinal endothelial cells (REC) grown in high glucose. For these studies, we used diabetic mouse samples and REC to investigate our hypothesis. Primary retinal endothelial cells were grown in normal (5 mM) or high glucose (25 mM glucose) for measurements. In a subset of cells grown in high glucose, cells were transfected with Sema7a siRNA or scrambled siRNA. We measured levels of key inflammatory mediators and zonula occludens-1 (ZO-1) and occludin levels by Western blot. Data suggest that high glucose increased inflammatory mediators and reduced the tight junction proteins, which follows what is often observed in cells grown in high glucose. Sema7a siRNA significantly decreased inflammatory proteins and increased levels of ZO-1 and occludin. These data suggest that Sema7a mediates the actions of high glucose in REC. Use of Sema7a siRNA may offer a new avenue for treatment.

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.

Loss of TLR4 in endothelial cells but not Müller cells protects the diabetic retina.

Others have previously reported that global loss of toll-like receptor 4 (TLR4) reduced retinal inflammation. To determine cell specific actions of TLR4 in the retina, we generated diabetic endothelial cell specific and Müller cell specific TLR4 knockout mice. Diabetic Cdh5-Cre TLR4 mice, PDGFRα-Cre TLR4 mice, and TLR4 floxed mice were evaluated for retinal permeability, neuronal damage, and numbers of degenerate capillaries, all changes commonly observed in the diabetic retina. We also measured protein levels of key inflammatory mediators. We found that diabetes increased permeability, neuronal, and vascular damage in all mice. Loss of TLR4 in the retinal endothelial cells protected against these changes when compared to diabetic TLR4 floxed mice. In contrast, loss of TLR4 in Müller cells did not reduce diabetes-induced increases in permeability or neuronal and vascular damage. Elimination of TLR4 in either mouse model reduced inflammatory mediators, as well as VEGF levels. Taken together, our findings suggest that loss of TLR4 in endothelial cells is protective against diabetic-induced damage, while Müller cell TLR4 is not involved in the damage.

Epac1 Requires AMPK Phosphorylation to Regulate HMGB1 in the Retinal Vasculature.

Purpose: To investigate whether AMP-activated protein kinase (AMPK) is required for the reduction of high mobility group box 1 (HMGB1) by exchange proteins activated by cAMP 1 (Epac1) in the retinal vasculature. Methods: We measured AMPK phosphorylation in normal and diabetic Epac1 floxed and cdh5/Epac1 Cre mice. We also treated primary human retinal endothelial cells (RECs) in normal (5-mM) or high (25-mM) glucose with an Epac1 agonist and AMPK or insulin-like growth factor receptor binding protein 3 siRNA. We measured protein levels of AMPK, sirtuin 1 (SIRT1), and HMGB1. Results: AMPK phosphorylation was reduced in cdh5/Epac1 Cre mice, suggesting that Epac1 regulated AMPK actions. High-glucose culturing conditions reduced AMPK levels in RECs, but the levels were increased by the Epac1 agonist, supporting the idea that Epac1 regulates AMPK. The Epac1 agonist was not able to reduce HMGB1 levels or increase SIRT1 when AMPK was blocked by AMPK siRNA, thus demonstrating that Epac1 requires AMPK to regulate SIRT1 and HMGB1. Conclusions: Epac1 requires AMPK to increase SIRT1 and reduce HMGB1 in the diabetic retinal vasculature. This finding provides another pathway by which Epac1 may protect the retina during diabetes.

Systemic alterations in leukocyte subsets and the protective role of NKT cells in the mouse model of diabetic retinopathy.

The involvement of leukocytes in the pathophysiology of DR has mostly examined the role of monocytes and neutrophils with little emphasis on other immune cell types. In this study, we determined the systemic alterations in T cell subsets, myeloid cell types, NK cells, and NKT cells in the streptozotocin (STZ) mouse model of diabetic retinopathy (DR), and the role of NKT cells on retinal leukostasis and permeability changes. C57BL/6 J mice were made diabetic with 60 mg/kg dose of STZ given for 5-days. Flow cytometry assay measured the frequency of leukocyte subsets in the peripheral blood, spleen, and bone marrow of STZ- and vehicle-treated C57BL/6 J mice. Our results showed an increased proportion of memory CD8 T cells and interferon-gamma (IFN-γ) secreting CD8 T cells in the bone marrow of STZ-treated compared to control mice. Subsequently, increased production of inflammatory monocytes in the bone marrow and an enhanced frequency of CD11b + cells in the diabetic retina were seen in STZ-treated compared to control mice. The diabetic mice also exhibited a decrease in total NKT and CD4+NKT cells. A monoclonal antibody-based approach depleted NKT cells from STZ-treated mice, followed by measurements of retinal vascular permeability and leukostasis. The depletion of NKT cells in STZ-treated mice resulted in a significant increase in vascular permeability in the retinal tissue. Together, our results strongly imply the involvement of NKT cells in regulating the pathophysiology of the diabetic retina.

Role of HMGB1 signaling in the inflammatory process in diabetic retinopathy.

High mobility group box 1 (HMGB1) is a key player in retinal inflammation. HMGB1 is a danger associated protein pattern receptor which can sense high glucose as a stressor. Increased HMGB1 levels have been found in patients with late stage diabetic retinopathy. HMGB1 can bind toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE), leading to increased inflammation commonly through nuclear factor kappa beta (NFkB). Because diabetic patients have been found to have increased HMGB1 and RAGE levels, as well as polymorphisms of TLR4, a number of investigations have focused on inhibition of these pathways in the diabetic retina. Work in diabetic animal models and cell culture have demonstrated a number of factors that can inhibit HMGB1/TLR4/RAGE signaling. This regulation offers potential new avenues for therapeutic development. This review is focused on HMGB1 signaling and downstream pathways leading to inflammation in the diabetic retina.

Review: Role of cAMP signaling in diabetic retinopathy.

Despite decades of research, diabetic retinopathy remains the leading cause of blindness in working age adults. Treatments for early phases for the disease remain elusive. One pathway that appears to regulate neuronal, vascular, and inflammatory components of diabetic retinopathy is the cyclic adenosine 3', 5'-monophosphate (cAMP) pathway. In this review, we discuss the current literature on cAMP actions on the retina, with a focus on neurovascular changes commonly associated with preproliferative diabetic retinopathy models.

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.

Role of Insulin-Like Growth Factor Binding Protein-3 in the Pathogenesis of Herpes Stromal Keratitis.

Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). Methods: In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro- and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3-/-) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. Results: Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3-/- than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3-/- than B6 mice. Conclusions: Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.

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.