Olivier Goureau

Nitric oxide in the eye : Multifaceted roles and diverse outcomes

Recent works have highlighted the role of nitric oxide in a wide array of disease entities, including septic shock, hypertension, cerebral ischemia, and chronic degenerative diseases of the nervous system. The functions of nitric oxide appear very diverse, having actions on vascular tone, neurotransmission, immune cytotoxicity, and many others. Nitric oxide is an important mediator of homeostatic processes in the eye, such as regulation of aqueous humor dynamics, retinal neurotransmission and phototransduction. Changes in its generation or actions could contribute to pathological states such as inflammatory diseases (uveitis, retinitis) or degenerative diseases (glaucoma, retinal degeneration). Localization in the eye and biochemical characteristics of nitric oxide will be reviewed. A better understanding of the nitric oxide pathway will be the key to the development of new approaches to the management and treatment of various ocular diseases.

Induction and Regulation of Nitric Oxide Synthase in Retinal Müller Glial Cells

Abstract: Müller glial cells from the rat retina were examined for their capacity to produce nitric oxide (NO). Treatment of retinal Müller glial (RMG) cells with lipopolysaccharide (LPS), interferon‐γ, and tumor necrosis factor‐α induced NO synthesis as determined by nitrite release in media. Simultaneous addition of LPS, interferon‐γ, and tumor necrosis factor‐α caused the largest increase in NO synthesis. NO biosynthesis was detected after 12 h and was dependent on the dose of LPS, interferon‐γ, and tumor necrosis factor‐α. Stereoselective inhibitors of NO synthase (NOS), cycloheximide and transforming growth factor‐β, blocked cytokine‐induced NO production. Cytosol from LPS/cytokine‐treated RMG cultures, but not from unstimulated cultures, produced a calcium/calmodulin‐independent conversion of l‐arginine to l‐citrulline that was completely blocked by NOS inhibitor. The expression of NOS in RMG cells was confirmed by northern blot analysis, in which stimulation of these cells led to an increase in NOS mRNA levels. We conclude that RMG cells can express an inducible form of NOS similar to the macrophage isoform. High NO release from activated RMG cells might represent a protection from infection but may also contribute to the development of retinal pathologies.

Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy

Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Significance Human induced pluripotent stem cells (hiPSCs) could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. The production of retinal cells from hiPSCs for personalized therapeutic approaches must comply with certain criteria, such as safety, efficiency, reproducibility, and low production cost. Here, we report a simple and scalable retinal differentiation process for the generation of retinal pigmented epithelial cells and neural retinal tissues containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This method also provides an accessible in vitro model to investigate mechanisms involved in human retinogenesis and retinal diseases. Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

Cytokine-Induced Activation of Signal Transducer and Activator of Transcription in Photoreceptor Precursors Regulates Rod Differentiation in the Developing Mouse Retina

Ciliary neurotrophic factor (CNTF) exhibits multiple biological effects during vertebrate retinogenesis, including regulation of photoreceptor cell differentiation. In the early postnatal mouse retina, CNTF induces rapid and transient phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3 and the extracellular signal-regulated kinase (ERK). Although both proliferating progenitor cells and postmitotic neurons respond directly to cytokine signals, CNTF elicits distinct phosphorylation patterns of STAT3 and ERK. CNTF stimulation induces low levels of STAT3 phosphorylation in progenitors and differentiated neurons but a robust STAT3 activation among postmitotic photoreceptor precursors expressing the cone-rod homeobox gene Crx and newly differentiated rod photoreceptors. In contrast, CNTF causes preferential phosphorylation of ERK in progenitor cells and photoreceptor precursors. Inhibition of the cytokine receptor gp130 using neutralizing antibodies reveals that gp130 is required for both CNTF-induced STAT3 and ERK phosphorylation. Perturbation of STAT signaling by a STAT inhibitor peptide or a dominant-negative STAT3 mutant causes enhanced production of rod photoreceptors in the absence of exogenous cytokines, whereas inhibiting ERK activation by a MEK (mitogen-activated protein kinase kinase)-specific inhibitor has no effect on rod photoreceptor differentiation in vitro. Furthermore, disrupting the function of epidermal growth factor (EGF) receptors, which modulate rod development in vivo, indicates that the EGF family of ligands does not mediate the inhibitory effect of cytokine on rod differentiation. These results demonstrate that cytokine signal transduction is dynamic and heterogeneous in the developing retina, and that endogenous ligand-induced STAT activation in retinal progenitor and/or photoreceptor precursor cells plays an important role in regulating photoreceptor development.

Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Müller Glial Cells

Abstract: Retinal Müller glial cells express the inducible isoform (‐2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS‐2 [NOS‐2 (‐/‐)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN‐γ‐stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS‐2 (‐/‐) mice and because inhibition of NOS‐2 induction by transforming growth factor‐β or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP‐ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells in responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.

Role of interferon regulatory factor-1 and mitogen-activated protein kinase pathways in the induction of nitric oxide synthase-2 in retinal pigmented epithelial cells.

Bovine retinal pigmented epithelial cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Experiments were performed to investigate the involvement of interferon regulatory factor-1 (IRF-1) on NOS-2 induction and its regulation by NOS-2 inhibitors such as pyrrolidine dithiocarbamate (PDTC), an antioxidant, or protein kinase inhibitors. Analysis by transitory transfections showed that LPS, alone or with IFN-γ, stimulated activity of the murine NOS-2 promoter fragment linked upstream of luciferase and its suppression by PDTC and by the different protein kinase inhibitors, genistein (tyrosine kinase inhibitor), PD98059 (mitogen-actived protein (MAP) kinase kinase inhibitor), and SB 203580 (p38 MAP inhibitor). Using specific antibodies, we have confirmed that extracellular signal-regulated kinases and p38 MAP kinase were activated by LPS and IFN-γ in retinal pigmented epithelial cells. Analysis by reverse transcriptase-polymerase chain reaction, Western blot, and electrophoretic mobility shift assay demonstrated that IFN-γ alone or combined with LPS induced an accumulation of IRF-1 mRNA and protein and IRF-1 DNA binding. Transfections assays with the IRF-1 promoter showed an induction of this promoter with IFN-γ, potentiated by LPS. The decrease of LPS/IFN-γ-induced IRF-1 promoter activity, IRF-1 synthesis, and IRF-1 activation, by PDTC, genistein, PD98059, and SB 203580, could explained in part the inhibition of the NOS-2 induction by these compounds. Our results demonstrate that IRF-1 is necessary for NOS-2 induction by LPS and IFN-γ and that its synthesis requires the involvement of a redox-sensitive step, the activation of tyrosine kinases, and extracellular signal-regulated kinases 1/2 and p38 MAP kinases.

Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients

The inducible isoform of nitric oxide synthase has been detected in cytomegalovirus (CMV)-infected retinas from acquired immunodeficiency syndrome (AIDS) patients by immunohistochemistry and NADPH-diaphorase staining. Subsequent immunohistochemistry using antibodies against CMV antigens and glial fibrillary acidic protein (GFAP) demonstrated that inducible NOS was localized in CMV-infected glial cells, particularly Müller cells. These findings indicate that inducible NOS is expressed in vivo in the human retina as a result of viral infection, and suggest that high levels of NO production might be involved in CMV-induced retinitis.

Protection against light-induced retinal degeneration by an inhibitor of NO synthase

The existence of nitric oxide synthase (NOS) in retinal rod outer segments and pigmented epithelial cells suggests that NO in excess could impair the interaction between these cells, resulting in photoreceptor degeneration. To test this hypothesis, NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, was intraperitoneally injected daily into rats subjected to constant illumination for 7 days in order to destroy their photoreceptors. By measuring photoreceptor nuclear layer thicknesses, we found that L-NAME partially protects (by up to 35%) against the degeneration of photoreceptors and acts to maintain their organization. Thus NO may be involved in the process by which photoreceptor degeneration results from constant illumination of the retina.

Lipopolysaccharide and cytokines induce a macrophage-type of nitric oxide synthase in bovine retinal pigmented epithelial cells

The present study demonstrates that bovine retinal pigmented epithelial cells, which are neuroectodermal in origin, produce nitric oxide (NO) upon treatment with interferon-gamma in the presence of lipopolysaccharide or tumor necrosis factor-alpha. NO production was measured by the accumulation of the stable endproduct NO2-. The biosynthesis of NO requires an induction period of approximately 12 hours and continues for at least 96 hours. The synthesis was abolished by the stereoselective inhibitors of NO synthase, NG-monomethyl-L-arginine and NG-nitro-L-arginine-benzylester. Cycloheximide and dexamethasone blocked cytokine-induced NO production. The results indicate that endotoxin and cytokines are capable of inducing NO synthase of the macrophage type, in retinal pigmented epithelial cells.