Platelet-Derived Growth Factor Preserves Retinal Synapses in a Rat Model of Ocular Hypertension.

PURPOSE: Platelet-derived growth factor (PDGF) promotes neuronal survival in experimental glaucoma and recruits glial cells that regulate synapses. We investigated the effects of intravitreal PDGF on the inflammatory milieu and retinal synapses in the presence of raised IOP. METHODS: Animals with laser-induced IOP elevation received intravitreal injections of either saline or 1.5 μg PDGF. At 7 days, a further intravitreal injection was administered so groups received "PDGF-saline" (n = 15), "PDGF-PDGF" (n = 13), or "saline-saline" (n = 20). Platelet-derived growth factor receptor activation was assessed after 2 weeks using Western blot for PI3 kinase. Immunohistochemistry was performed for markers of synapses in the inner plexiform layer (IPL): PSD-95, GluR1, SY38; RGCs: βIII-tubulin, and glial cells: Iba-1, CD45. Real-time quantitative polymerase chain reaction (qPCR) was performed for Arc, selp, MCP-1, IL-6, IL-10, and CX3CR1 (n = 13). RESULTS: A single injection of PDGF increased IPL synaptic density in high IOP eyes (PSD-95 = 8.65 ± 0.43, SY38 = 8.68 ± 0.51, GluR1 = 9.03 ± 0.60 puncta/μm3, P < 0.001) and expression of synaptic modulator Arc (6.92 ± 3.71-fold change/control, P < 0.05) in comparison with vehicle (PSD-95 = 4.59 ± 0.41, SY38 = 4.46 ± 0.38, GluR1 = 5.94 ± 0.50 puncta/μm3, Arc = 1.46 ± 0.31-fold/control). This was associated with more resident microglia (8.16 ± 1.34-fold change/control, P < 0.001) and infiltrating monocyte-derived macrophages in the retina as well as increased Selp expression (26.8 ± 14.12-fold change/control, P < 0.05). Optic nerve head (ONH) showed an increased microglia (saline = 1.44 ± 0.13 versus PDGF = 2.23 ± 0.18-fold change/control, P < 0.01) but not infiltrating macrophages. IL-10 expression was significantly increased in PDGF-treated eyes (5.43 ± 0.47-fold change/control, P < 0.05) relative to vehicle (2.51 ± 0.67-fold change/control). CONCLUSIONS: Platelet-derived growth factor increased microglial and monocyte-derived macrophage populations in the eye and protected intraretinal synapses from degeneration in our experimental glaucoma model.Supported by the Agency for Science Technology and Research Singapore (RC), the Cambridge Eye Trust, the HB Allen Charitable Trust and the Jukes Glaucoma Research Fund, and by Grant 1868 from Fight for Sight (KM).This is the final version of the article. It first appeared from the Association for Research in Vision and Opthalmology via http://dx.doi.org/10.1167/iovs.15-1786

Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism.

There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2(Akita) diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina.This work was funded by the Medical Research Council (G0901303) of the UK. We also wish to thank the Cambridge Eye Trust for their support.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/cddis.2016.11

The seawater neodymium and lead isotope record of the final stages of Central American Seaway closure

Key Points: Seawater Nd and Pb isotope records for the Pliocene Caribbean and EEP Caribbean Nd isotope composition became more UNADW-like during the Pliocene Short term changes support link between CAS closure and strength of AMOC The shoaling and final closure of the Central American Seaway (CAS) resulted in a major change of the global ocean circulation and has been suggested as an essential driver for strengthening of Atlantic Meridional Overturning Circulation (AMOC). The exact timing of CAS closure is key to interpreting its importance. Here we present a reconstruction of deep and intermediate water Nd and Pb isotope compositions obtained from fossil fish teeth and the authigenic coatings of planktonic foraminifera in the eastern equatorial Pacific (ODP Site 1241) and the Caribbean (ODP Sites 998, 999 and 1000) covering the final stages of CAS closure between 5.6 and 2.2 Ma. The data for the Pacific site indicate no significant Atlantic/Caribbean influence over this entire period. The Caribbean sites show a continuous trend to less radiogenic Nd isotope compositions during the Pliocene, consistent with an enhancement of Upper North Atlantic Deep Water (UNADW) inflow and a strengthening of the AMOC. Superimposed onto this long-term trend, shorter-term changes of intermediate Caribbean Nd isotope signatures approached more UNADW-like values during intervals when published reconstructions of seawater salinity suggested complete closure of the CAS. The data imply that significant deep water exchange with the Pacific essentially stopped by 7 Ma and that shallow exchange, which still occurred at least periodically until approximately 2.5 Ma, may have been linked to the strength of the AMOC but did not have any direct effect on the intermediate and deep Caribbean Nd isotope signatures through mixing with Pacific waters

Molecular Mechanisms Mediating Retinal Reactive Gliosis Following Bone Marrow Mesenchymal Stem Cell Transplantation.

A variety of diseases lead to degeneration of retinal ganglion cells (RGCs) and their axons within the optic nerve resulting in loss of visual function. Although current therapies may delay RGC loss, they do not restore visual function or completely halt disease progression. Regenerative medicine has recently focused on stem cell therapy for both neuroprotective and regenerative purposes. However, significant problems remain to be addressed, such as the long-term impact of reactive gliosis occurring in the host retina in response to transplanted stem cells. The aim of this work was to investigate retinal glial responses to intravitreally transplanted bone marrow mesenchymal stem cells (BM-MSCs) to help identify factors able to modulate graft-induced reactive gliosis. We found in vivo that intravitreal BM-MSC transplantation is associated with gliosis-mediated retinal folding, upregulation of intermediate filaments, and recruitment of macrophages. These responses were accompanied by significant JAK/STAT3 and MAPK (ERK1/2 and JNK) cascade activation in retinal Muller glia. Lipocalin-2 (Lcn-2) was identified as a potential new indicator of graft-induced reactive gliosis. Pharmacological inhibition of STAT3 in BM-MSC cocultured retinal explants successfully reduced glial fibrillary acidic protein expression in retinal Muller glia and increased BM-MSC retinal engraftment. Inhibition of stem cell-induced reactive gliosis is critical for successful transplantation-based strategies for neuroprotection, replacement, and regeneration of the optic nerve.This work was support by funding from the Biotechnology and Biological Sciences Research Council (BBSRC), the HB Allen Charitable Trust, the Cambridge Eye Trust, the Jukes Glaucoma Research Fund and by Pfizer, Neusentis. We thank Dr. Andras Lakatos from the University of Cambridge (UK) for donating the GFAP-STAT3 CKO mice, Prof. Verdon Taylor from the University of Basel (CH) for the Hes5 GFP+ve mice, Dr. Stefano Pluchino from the University of Cambridge (UK) for donating the mouse neural precursor cell (NPC) line and Prof. Astrid Limb from UCL, London (UK) for the MIO-M1 cell line.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/stem.209

Diabetic retinopathy: a complex pathophysiology requiring novel therapeutic strategies.

INTRODUCTION: Diabetic retinopathy (DR) is the leading cause of vision loss in the working age population of the developed world. DR encompasses a complex pathology, and one that is reflected in the variety of currently available treatments, which include laser photocoagulation, glucocorticoids, vitrectomy and agents which neutralize vascular endothelial growth factor (VEGF). Whilst these options demonstrate modest clinical benefits, none is yet to fully attenuate clinical progression or reverse damage to the retina. This has led to an interest in developing novel therapies for the condition, such as mediators of angiopoietin signaling axes, immunosuppressants, nonsteroidal anti-inflammatory drugs (NSAIDs), oxidative stress inhibitors and vitriol viscosity inhibitors. Further, preclinical research suggests that gene therapy treatment for DR could provide significant benefits over existing treatments options. AREAS COVERED: Here we review the pathophysiology of DR and provide an overview of currently available treatments. We then outline recent advances made towards improved patient outcomes and highlight the potential of the gene therapy paradigm to revolutionize DR management. EXPERT OPINION: Whilst significant progress has been made towards our understanding of DR, further research is required to enable the development of a detailed spatiotemporal model of the disease. In addition, we hope that improvements in our knowledge of the condition facilitate therapeutic innovations that continue to address unmet medical need and improve patient outcomes, with a focus on the development of targeted medicines.Cambridge Eye Trust Research Councils UK - Medical Research Council the Wellcome Trus

Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons.

Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide-induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future

Multiplayer Cost Games with Simple Nash Equilibria

Multiplayer games with selfish agents naturally occur in the design of distributed and embedded systems. As the goals of selfish agents are usually neither equivalent nor antagonistic to each other, such games are non zero-sum games. We study such games and show that a large class of these games, including games where the individual objectives are mean- or discounted-payoff, or quantitative reachability, and show that they do not only have a solution, but a simple solution. We establish the existence of Nash equilibria that are composed of k memoryless strategies for each agent in a setting with k agents, one main and k-1 minor strategies. The main strategy describes what happens when all agents comply, whereas the minor strategies ensure that all other agents immediately start to co-operate against the agent who first deviates from the plan. This simplicity is important, as rational agents are an idealisation. Realistically, agents have to decide on their moves with very limited resources, and complicated strategies that require exponential--or even non-elementary--implementations cannot realistically be implemented. The existence of simple strategies that we prove in this paper therefore holds a promise of implementability.Comment: 23 page

Angiopoietins in Diabetic Retinopathy: Current Understanding and Therapeutic Potential.

Diabetic retinopathy (DR) is the commonest cause of blindness in the working-age population of the developed world. The molecular pathophysiology of DR is complex, and a complete spatiotemporal model of the disease is still being elucidated. Recently, a role for angiopoietin (Ang) proteins in the pathophysiology of DR has been proposed by several research groups, and several aspects of Ang signalling are being explored as novel therapeutic strategies. Here, we review the role of the Ang proteins in two important forms of DR, diabetic macular oedema and proliferative diabetic retinopathy. The function of the Ang proteins in regulating blood vessel permeability and neovascularisation is discussed, and we also evaluate recent preclinical and clinical studies highlighting the potential benefits of modulating Ang signalling as a treatment for DR.MW and PYWM are funded by Medical Research Council (UK) grants. MW, PYWM and AO 386 receive support from The Novo Nordisk UK Research Foundation, the Cambridge Eye Trust and 387 the Jukes Glaucoma Research Fund. AO, PYWM and KRM also hold Fight for Sight (UK) awards. 388 PYWM is also supported by a Clinician Scientist Fellowship Award (G1002570), the Isaac Newton 389 Trust, the UK National Institute of Health Research (NIHR) as part of the Rare Diseases 390 Translational Research Collaboration, and the NIHR Biomedical Research Centre based at 391 Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. Research in 392 the laboratory is supported by core funding from Wellcome and MRC to the Wellcome-MRC 393 Cambridge Stem Cell Institute.Peer Reviewe

Neuroprotective Effects of Human Mesenchymal Stem Cells and Platelet-Derived Growth Factor on Human Retinal Ganglion Cells.

Optic neuropathies such as glaucoma occur when retinal ganglion cells (RGCs) in the eye are injured. Strong evidence suggests mesenchymal stem cells (MSCs) could be a potential therapy to protect RGCs; however, little is known regarding their effect on the human retina. We, therefore, investigated if human MSCs (hMSCs), or platelet-derived growth factor (PDGF) as produced by hMSC, could delay RGC death in a human retinal explant model of optic nerve injury. Our results showed hMSCs and the secreted growth factor PDGF-AB could substantially reduce human RGC loss and apoptosis following axotomy. The neuroprotective pathways AKT, ERK, and STAT3 were activated in the retina shortly after treatments with labeling seen in the RGC layer. A dose dependent protective effect of PDGF-AB was observed in human retinal explants but protection was not as substantial as that achieved by culturing hMSCs on the retina surface which resulted in RGC cell counts similar to those immediately post dissection. These results demonstrate that hMSCs and PDGF have strong neuroprotective action on human RGCs and may offer a translatable, therapeutic strategy to reduce degenerative visual loss. Stem Cells 2018;36:65-78

Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings