Jeremy M. Sivak

The Aging Eye: Common Degenerative Mechanisms Between the Alzheimer's Brain and Retinal Disease

Alzheimers disease (AD) is a common, incurable, and progressive dementia, characterized by loss of learning and memory and the neuropathologic accumulation of amyloid plaques and neurofibrillary tangles in the brain. A number of similarities between AD pathology and several distinct retinal degenerations have been described, particularly with respect to either glaucoma or age-related macular degeneration (AMD), each a leading cause of vision loss and blindness worldwide. Although comparisons between these diseases may provide important new insights into their pathogenic mechanisms, glaucoma and AMD result in markedly different degenerations. Therefore, analyses of the differences and the similarities between these conditions may prove equally productive. Common mechanisms that appear to underlie all three diseases are explored here, as well as potential use of the retina as a biomarker for AD diagnosis and progression. Based on this comparison, past and current efforts to transfer therapeutic strategies between diseases are discussed.

Proteomics Analyses of Human Optic Nerve Head Astrocytes Following Biomechanical Strain

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: Towards non-invasive glaucoma gene therapy

UNLABELLED Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

ROS Detoxification and Proinflammatory Cytokines Are Linked by p38 MAPK Signaling in a Model of Mature Astrocyte Activation

Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL) and optic nerve head (ONH), and perform essential roles in maintaining retinal ganglion cell (RGC) detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS) we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs) were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1), with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.

Angiogenic Dysfunction in Bone Marrow-Derived Early Outgrowth Cells from Diabetic Animals Is Attenuated by SIRT1 Activation

Impaired endothelial repair is a key contributor to microvascular rarefaction and consequent end‐organ dysfunction in diabetes. Recent studies suggest an important role for bone marrow‐derived early outgrowth cells (EOCs) in mediating endothelial repair, but the function of these cells is impaired in diabetes, as in advanced age. We sought to determine whether diabetes‐associated EOC dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient‐dependent life span extension in mammals. Despite being cultured in normal (5.5 mM) glucose for 7 days, EOCs from diabetic rats expressed less SIRT1 mRNA, induced less endothelial tube formation in vitro and neovascularization in vivo, and secreted less of the proangiogenic ELR+ CXC chemokines CXCL1, CXCL3, and CXCL5. Ex vivo SIRT1 activation restored EOC chemokine secretion and increased the in vitro and in vivo angiogenic activity of EOC conditioned medium derived from diabetic animals to levels similar to that derived from control animals. These findings suggest a pivotal role for SIRT1 in diabetes‐induced EOC dysfunction and that its pharmacologic activation may provide a new strategy for the restoration of EOC‐mediated repair mechanisms.

Pharmacologic Uncoupling of Angiogenesis and Inflammation during Initiation of Pathological Corneal Neovascularization

Background: The mechanism initiating pathological corneal neovascularization (CoNV) remains unclear. Results: After injury, substantial CoNV occurs during an initial, VEGFR-2-dependent phase, prior to influence from inflammatory cells. Conclusion: Pathological CoNV can be pharmacologically uncoupled from inflammatory cell recruitment and may be coordinated by VEGF from repair epithelial cells. Significance: This work reveals a window in which angiogenesis and inflammation can be selectively targeted during injury repair. Pathological neovascularization occurs when a balance of pro- and anti-angiogenic factors is disrupted, accompanied by an amplifying inflammatory cascade. However, the interdependence of these responses and the mechanism triggering the initial angiogenic switch have remained unclear. We present data from an epithelial debridement model of corneal neovascularization describing an initial 3-day period when a substantial component of neovascular growth occurs. Administration of selective inhibitors shows that this initial growth requires signaling through VEGFR-2 (vascular endothelial growth factor receptor-2), independent of the accompanying inflammatory response. Instead, increased VEGF production is found prominently in repair epithelial cells and is increased prior to recruitment of neutrophil/granulocytes and macrophage/monocytes. Consequently, early granulocyte and monocyte depletion has little effect on corneal neovascularization outgrowth. These data indicate that it is possible to pharmacologically uncouple these mechanisms during early injury-driven neovascularization in the cornea and suggest that initial tissue responses are coordinated by repair epithelial cells.

Discovery of Oral VEGFR-2 Inhibitors with Prolonged Ocular Retention That Are Efficacious in Models of Wet Age-Related Macular Degeneration

The benefit of intravitreal anti-VEGF therapy in treating wet age-related macular degeneration (AMD) is well established. Identification of VEGFR-2 inhibitors with optimal ADME properties for an ocular indication provides opportunities for dosing routes beyond intravitreal injection. We employed a high-throughput in vivo screening strategy with rodent models of choroidal neovascularization and iterative compound design to identify VEGFR-2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate preferential ocular tissue distribution and efficacy after oral administration while minimizing systemic exposure.

An Effective Prodrug Strategy to Selectively Enhance Ocular Exposure of a Cannabinoid Receptor (CB1/2) Agonist

Glaucoma is a leading cause of vision loss and blindness, with increased intraocular pressure (IOP) a prominent risk factor. IOP can be efficaciously reduced by administration of topical agents. However, the repertoire of approved IOP-lowering drug classes is limited, and effective new alternatives are needed. Agonism of the cannabinoid receptors CB1/2 significantly reduces IOP clinically and experimentally. However, development of CB1/2 agonists has been complicated by the need to avoid cardiovascular and psychotropic side effects. 1 is a potent CB1/2 agonist that is highly excluded from the brain. In a phase I study, compound 1 eyedrops were well tolerated and generated an IOP-lowering trend but were limited in dose and exposure due to poor solubility and ocular absorption. Here we present an innovative strategy to rapidly identify compound 1 prodrugs that are efficiently metabolized to the parent compound for improved solubility and ocular permeability while maintaining low systemic exposures.

Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury

Astrocytes perform critical non–cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.

Biomechanical insult switches PEA-15 activity to uncouple its anti-apoptotic function and promote erk mediated tissue remodeling

Biomechanical insult contributes to many chronic pathological processes, yet the resulting influences on signal transduction mechanisms are poorly understood. The retina presents an excellent mechanotransduction model, as mechanical strain on sensitive astrocytes of the optic nerve head (ONH) is intimately linked to chronic tissue remodeling and excavation by matrix metalloproteinases (MMPs), and apoptotic cell death. However, the mechanism by which these effects are induced by biomechanical strain is unclear. We previously identified the small adapter protein, PEA-15 (phosphoprotein enriched in astrocytes), through proteomic analyses of human ONH astrocytes subjected to pathologically relevant biomechanical insult. Under resting conditions PEA-15 is regulated through phosphorylation of two key serine residues to inhibit extrinsic apoptosis and ERK1/2 signaling. However, we surprisingly observed that biomechanical insult dramatically switches PEA-15 phosphorylation and function to uncouple its anti-apoptotic activity, and promote ERK1/2-dependent MMP-2 and MMP-9 secretion. These results reveal a novel cell autonomous mechanism by which biomechanical strain rapidly modifies this signaling pathway to generate altered tissue injury responses.