👤 Jena J Steinle

Our goal was to explore whether inhibition of sortilin could protect the retina against ischemia/reperfusion (I/R) damage, as well as explore whether this inhibition could reduce inflammatory mediators in retinal Müller cells. We used both primary human Müller cells and a rat Müller cell line (rMC-1) grown in normal (5 mM) or high (25 mM) glucose. Some cells were treated with AF38469, a small-molecule inhibitor of sortilin. We performed western blotting for the inflammatory mediators, tumor necrosis factor α, and NOD-like receptor protein 3. We also measured protein levels of lysosome-associated membrane glycoprotein 2 (LAMP2), a marker of autophagy, and cleaved caspase 3, a marker of apoptosis, in the cells. We then tested the actions of eye drops containing AF38469 on mice exposed to I/R. We assessed neuronal damage at 2 days post-I/R and vascular damage at 10 days post-I/R. High-glucose culturing conditions significantly increased inflammatory, autophagic, and apoptotic markers in both primary human Müller and rat Müller cells. All markers were reduced by treating the cells with AF38469. AF38469 eye drops also significantly reduced I/R-induced neuronal and vascular damage. These studies demonstrate that sortilin regulates the inflammatory, autophagic, and apoptotic pathways in Müller cells grown in high glucose. Inhibition of sortilin using AF38469 eye drops also reduced I/R-induced retinal damage. Show less

To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. 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. 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. 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. Show less

Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. Although it is clear that tumor necrosis factor-α is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase Toll-like receptor 4 (TLR4) signaling, which could be inhibited by a β-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate β-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (RECs) and rat retinal Müller cells (rMC-1) exposed to high glucose (25 mM), were treated with a novel β-adrenergic receptor agonist, Compound 49b (50 nM), or phosphate-buffered saline (control). TLR4 and its downstream signaling partners (MyD88, IL-1 receptor-associated kinase 1, TNF receptor-associated factor 6 and total and phosphorylated nuclear factor-κB) were examined. In addition, we assessed high-mobility group box 1 (HMGB1) protein levels. Our data showed that diabetes or high-glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in RECs and Müller cells grown in high-glucose culture conditions, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway, providing a potential mechanism for its protective actions. Show less

To establish the key insulin receptor substrate 1 (IRS-1) structural elements required in this insulin regulatory pathway, we investigated the effects of substituting alanine for serine 307 in IRS-1 on the ability of tumor necrosis factor-α (TNF-α) and a related mediator, suppressor of cytokine signaling 3 (SOCS3), to phosphorylate IRS-1 and regulate insulin signaling in the rat retinal Müller cell (rMC-1) cell line. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose medium and transfected with either normal IRS-1(Ser307)plasmid or a mutated IRS-1(Ser307Ala) plasmid. Cells were also treated with recombinant TNF-α or SOCS3 to induce increased levels of these proteins. In cells with IRS-1(Ser307Ala), TNF-α and SOCS3 failed to phosphorylate IRS-1. Likewise, resulting downstream effects, including changes in phosphorylation of insulin receptor(Tyr960), antiapoptotic Akt phosphorylation, and proapoptotic cleavage of caspase 3 were also blocked. We also report for the first time that SOCS3 and TNF-α are reciprocally stimulatory leading to a mutual enhancement of levels of both factors, thus forming a potential positive feedback loop that contributes to insulin receptor resistance. Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina. Show less

To determine whether beta-adrenergic receptors are involved in the modulation of inflammatory cytokines in Müller cells in a hyperglycemic environment. Rat Müller cells were grown in high (25 mM)- or low (5 mM)-glucose medium. Müller cells lysates were processed for real-time polymerase chain reaction to measure steady state mRNA expression for the following inflammatory markers: iNOS, TNF-alpha, IL-1B, and ICAM-1. Western blot analysis and ELISA assays were performed to determine the protein levels of these inflammatory markers and PGE2 content. Isoproterenol treatment significantly decreased protein levels of iNOS, TNF-alpha, and IL-1B, in rMC-1 cells cultured in high glucose as early as 1 hour, compared with cells receiving no treatment. PGE2 content was also reduced after isoproterenol treatment. There were no significant changes observed in protein levels of ICAM-1 production after isoproterenol treatment in high glucose. Steady state mRNA levels for iNOS were significantly decreased 1 hour after isoproterenol, whereas ICAM-1 gene expression was significantly increased after 1 hour. Isoproterenol significantly increased gene expression for IL-1B after 24 hours of treatment. These results suggest that stimulation of beta-adrenergic receptors with isoproterenol leads to decreased levels of PGE(2), TNF-alpha, and IL-1B protein content, and in both gene expression and protein levels of iNOS in Müller cells cultured in hyperglycemia. beta-Adrenergic receptor agonists had limited effects on ICAM-1 protein production. These results indicate that isoproterenol treatment reduces cytokine activation in cultured rat Müller cells. Show less