Jena J. Steinle, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the role of Eph B4 receptors in various stages of angiogenesis. Angiogenesis, new blood vessel growth, is a complex process with a number of guided steps. Within the past decade, numerous growth factors have been shown to direct this process. Recently, Eph B4 receptor knockout mice were shown to die in utero due to cardiovascular defects. However, it is unclear what role this receptor plays in the cardiovascular system. Furthermore, this family of receptor tyrosine kinases has been reported to play significant roles in the retino-tectal systems of the nervous system through repulsion of neurite outgrowth. I would like to determine the role of Eph B4 receptor in the various stages of angiogenesis. Since we have pilot studies indicating phosphorylation of PKB/Akt, we would like to determine if MMP-2 and MMP-9 are increased to allow for endothelial cell movement. PKB/Akt is also implicated in endothelial cell migration. Because PKB/Akt also phosphorylates eNOS to activate nitric oxide, a known mitogen, we will determine if Eph B4 activation results in endothelial cell proliferation. Finally, the role of Eph B4 in tube formation will be investigated. These results from both mesenteric and retinal endothelial cells will greatly increase our understanding of Eph B4 receptor regulation of the various stages of angiogenesis.

protein quantitation /detection; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Diabetic retinopathy remains the fifth leading cause of preventable blindness worldwide. Interventions to prevent progression of diabetic retinopathy are limited to improved glycemic control (a challenging goal for all diabetic patients) and to lase photocoagulation (available only for advanced stages of retinopathy). We and others have reported that adrenergic signaling is lost in the diabetic retina, suggesting that development of novel agents to restore autonomic homeostasis is necessary. Unfortunately, currently available adrenergic agents are associated with adverse systemic or non-specific effects. These problems inspired our group to synthesize compound 49b, a novel and selective ß-adrenergic receptor agonist, as a potential paradigm shift in the prevention of diabetic retinopathy. Our preliminary data suggest that compound 49b prevents the formation of degenerate capillaries, which involves degenerate capillary formation, which are the hallmark pathology noted in the diabetic retinal vasculature. In addition to preventing degenerate capillaries in vivo, compound 49b prevents the cleavage of caspase 3, a well- established marker of apoptosis, in retinal endothelial cells (REC) in vitro, suggesting that Compound 49b can decrease apoptosis. In the oxygen-induced model of retinopathy, others have associated increased levels of insulin-like growth factor binding protein-3 (IGFBP-3) with protection from REC apoptosis. Furthermore, using the streptozotocin-induced diabetic rat model, we observed that chronic insulin deficiency reduced IGFBP-3 protein levels in whole retinal lysates, but topical application of compound 49b to the eye restored retinal IGFBP-3 to its control level in these insulin- deficient rats. Thus, we hypothesize that compound 49b prevents the critical vascular damage underlying diabetic retinopathy in part by restoring IGFBP-3 levels in retinal endothelial cells. This project focuses on a deeper understanding of the mechanisms underlying this protective action.

DESCRIPTION (provided by applicant): The proposed study will test the novel hypothesis that in the diabetic retina, hyperglycemia stimulates production of tumor necrosis factor a (TNFa), which in turn decreases insulin receptor binding leading to decreased signal transduction. The overall effect of this signaling cascade would be to create insulin resistance, exacerbate problems caused by limited insulin production in diabetes, and thus contribute to development of diabetic retinopathy seen in both type 1 and type 2 diabetes. While our preliminary data and previous reports by others support a major role for inflammatory mediators such as TNFa in diabetic retinopathy, the pathways involved are largely unknown. Our proposed studies will focus on one likely candidate, the suppressor of cytokine signaling 3 (SOCS3) pathway (Fig.1), which is poorly understood in retina and yet represents a promising therapeutic target in future treatments for diabetic retinopathy. Our overall goal is to 1) establish the role of the SOCS3 pathway in regulating insulin signaling (through insulin receptor substrate-1; IRS-1) and apoptosis in normal and diabetic rats and 2) evaluate effects of upstream drug targets on the SOCS3 pathway and their downstream effects on insulin signaling and retinal cell apoptosis.

Diabetic retinopathy remains the leading cause of vision loss in working age adults. Previous research has led to the development of anti-VEGF therapy, which is an effective treatment for proliferative diabetic retinopathy and macular edema in some patients, while other patients are unresponsive. For treatment of non-proliferative diabetic retinopathy, few options are available save good glycemic control, which is problematic for many patients. Recent discoveries offer new insights into the molecular mechanisms underlying diabetic retinopathy and suggest that in addition to oxidative stress, increased inflammation may be a major causative factor in diabetes-induced retinal damage. We recently reported that high glucose significantly increased high mobility group box 1 (HMGB1) protein levels, suggesting a potential role for the alarmin system in regulating retinal responses to high glucose. HMGB1 is extensively involved in inflammation; it can serve as a chaperone to regulate transcription in the nucleus, is secreted by immune cells, interacts with p53, and activates cytokine release. As such, it provides a promising target to blunt the inflammatory response in the retina. Due to its multiple mechanisms of activation and roles in various cell types, an improved understanding of the cellular regulation of HMGB1 actions in the retina becomes increasingly important. Our preliminary data demonstrate that insulin-like growth factor binding protein 3 (IGFBP-3) can inhibit high glucose-induced increases in HMGB1 levels in retinal endothelial cells (REC). We have previously reported that IGFBP-3 KO mice have retinal damage similar to rodent models of diabetic retinopathy, despite normal glucose levels. In addition to IGFBP-3, studies have shown that PKA can directly phosphorylate the Box A region of yeast HMGB1 leading to decreased HMGB1 actions. Studies also showed that increased SIRT1 promoted deacetylation of HMGB1, leading to reduced cytoplasmic translocation. Thus, there is scientific rationale to investigate the regulation of HMGB1 by PKA, Epac1, IGFBP-3, and SIRT1. Furthermore, inhibition of HMGB1 activity using an inhibitor (glycyrrhizin) restored retinal thickness and reduced retinal degenerate capillaries in an in vivo model of retinal ischemia/reperfusion injury in mice. These data have led to the hypothesis that inhibition of HMGB1 activity in the retina protects against diabetes-induced damage. Our overall goal is to determine the mechanisms by which the PKA and Epac1 pathways inhibit HMGB1/inflammation-induced retinal injury and serve as protective pathways that may block diabetic retinal damage.  

Project Summary (Tissue Culture/Molecular) Continuing funds are requested by 17 vision scientists (holding 13 eligible NEI R01 grants) and their personnel to support three resource/service cores and an administrative core in the newly merged Department of Ophthalmology, Visual and Anatomical Sciences (former Ophthalmology and Anatomy/Cell Biology Departments). The Tissue Culture/Molecular (TC/M) core will be directed by Dr. Jena Steinle, an R01-funded investigator with significant expertise in tissue culture and molecular biology techniques. Together with the PI, and a research assistant, the environment and capability to conduct vision research at Wayne State University and affiliated institutions will be enhanced through prioritization of the work of NEI R01 funded investigators, their staff and students (graduate and fellows) in numerous aspects of both tissue culture and molecular biology. The TC/M core will provide vision scientists (priority for NEI R01 funded) with facilities and technical assistance that will assure quality and cost effectiveness of the work performed. This core will maintain and propagate ocular and other cell types, as well as provide investigators with genotyping, reverse transcription-polymerase chain reaction (RT) and quantitative polymerase chain reaction (q)-PCR, gene silencing- siRNA, shRNA, CRISPR/Cas9, nested PCR and adherence assays. It will also provide for consultation/assistance on recombinant (r) AAV virus vector design and construction and assistance to the PI, students or staff for in vitro functional assessments. Overall, the TC/M core will assist NEI R01-funded investigators and their staff/students in studies that require the use of tissue culture and molecular biology techniques; foster collaboration between vision scientists using tissue culture and molecular biological techniques; and bring new investigators into vision research by providing assistance in tissue culture and molecular techniques that can expand their research focus.

Diabetic retinopathy is the leading cause of blindness in working age adults; however, much of this blindness occurs in the later phases of the disease due to proliferative disease or macular edema. Recently, the role of inflammation has become a focus of potential therapies targeted to treat earlier stages and/or prevent progression of the disease. While it is clear that a large number of cytokines/chemokines are increased in the diabetic retina, the role of innate immunity has only recently been investigated. Recent work has demonstrated that toll-like receptors (TLRs) are altered in diabetes. Work has also shown that TLR4 is increased in the streptozotocin-induced diabetic retina. Additionally, TLR4 may have actions in retinal endothelial cells (REC), as both TLR2/4 pathways are active in these retinal cells. Our preliminary data has expanded on those findings to demonstrate that ?-adrenergic receptors can decrease TLR4 signaling in the diabetic mouse retina, as well as in both REC and retinal Müller cells. Supporting our findings in retina, studies in macrophages also demonstrate that ?-adrenergic receptors can regulate TLR4. The response to Compound 49b was blocked when Epac1 or PKA were knocked down by siRNA, suggesting these proteins act as damage associated molecular pattern molecules (DAMPs) regulating TLR4 signaling in the diabetic retina. Our primary hypothesis for this proposal is that PKA and Epac1 can regulate TLR4 and may represent a key pathway that controls retina damage in diabetes. Our overall goal is to better understand the role of downstream mediators of ?-adrenergic receptors in the regulation of TLR4 signaling in the diabetic retina, with the intent of identifying key pathways in innate immunity that can be targeted for novel therapeutics. !