Inhibition of Mitochondrial Respiration Impairs Nutrient Consumption and Metabolite Transport in Human Retinal Pigment Epithelium.

Mitochondrial respiration in mammalian cells not only generates ATP to meet their own energy needs but also couples with biosynthetic pathways to produce metabolites that can be exported to support neighboring cells. However, how defects in mitochondrial respiration influence these biosynthetic and exporting pathways remains poorly understood. Mitochondrial dysfunction in retinal pigment epithelium (RPE) cells is an emerging contributor to the death of their neighboring photoreceptors in degenerative retinal diseases including age-related macular degeneration. In this study, we used targeted-metabolomics and 13C tracing to investigate how inhibition of mitochondrial respiration influences the intracellular and extracellular metabolome. We found inhibition of mitochondrial respiration strikingly influenced both the intracellular and extracellular metabolome in primary RPE cells. Intriguingly, the extracellular metabolic changes sensitively reflected the intracellular changes. These changes included substantially enhanced glucose consumption and lactate production; reduced release of pyruvate, citrate, and ketone bodies; and massive accumulation of multiple amino acids and nucleosides. In conclusion, these findings reveal a metabolic signature of nutrient consumption and release in mitochondrial dysfunction in RPE cells. Testing medium metabolites provides a sensitive and noninvasive method to assess mitochondrial function in nutrient utilization and transport.

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.

SURGICAL RETINAL EXPLANTS AS A SOURCE OF RETINAL PROGENITOR CELLS.

PURPOSE: To describe the novel observation of spontaneously migrating retinal cells from living donor surgical retinal explants that express progenitor cell markers in the absence of exogenous growth factors. METHODS: Surgical retinal explants were harvested from 5 consecutive patients undergoing 23 G pars plana vitrectomy for the management of rhegmatogenous detachment. During surgery, equatorial flap tears were trimmed with the vitreous cutter and aspirated. Excised tissue was then regurgitated into a syringe containing balanced salt solution and immediately transferred to tissue culture. Migrating cells subsequently underwent immunohistochemical staining and their characteristics were compared with those of a spontaneously immortalized Müller stem cell line. RESULTS: Spontaneously migrating cells were observed from samples taken from all 5 patients from Day 2 to 10 after transfer to culture. These cells were found to express embryonic cell markers, including paired box 6 (Pax6), sex-determining region Y-box 2 (Sox-2), nestin, cone-rod homeobox, and cyclin-dependent kinase inhibitor 1B (p27Kip1) as well as proteins consistent with early or retained differentiation down the Müller cell lineage, including glial fibrillary acidic protein and glutamine synthetase. CONCLUSION: After injury, the human equatorial retina is capable of spontaneously producing cells that demonstrate migration and that express progenitor cell markers. In addition, these cells express proteins consistent with Müller cell lineage. These initial observations support the assertion that the human retina may possess the potential for regeneration and that surgical retinal explants could also act as a ready source of retinal progenitor cells.

Preclinical and clinical studies of photobiomodulation therapy for macular oedema.

AIMS/HYPOTHESIS: Diabetic macular oedema (DME) is the leading cause of visual impairment in people with diabetes. Intravitreal injections of vascular endothelial growth factor inhibitors or corticosteroids prevent loss of vision by reducing DME, but the injections must be given frequently and usually for years. Here we report laboratory and clinical studies on the safety and efficacy of 670 nm photobiomodulation (PBM) for treatment of centre-involving DME. METHODS: The therapeutic effect of PBM delivered via a light-emitting diode (LED) device was tested in transgenic mice in which induced Müller cell disruption led to photoreceptor degeneration and retinal vascular leakage. We also developed a purpose-built 670 nm retinal laser for PBM to treat DME in humans. The effect of laser-delivered PBM on improving mitochondrial function and protecting against oxidative stress was studied in cultured rat Müller cells and its safety was studied in pigmented and non-pigmented rat eyes. We then used the retinal laser to perform PBM in an open-label, dose-escalation Phase IIa clinical trial involving 21 patients with centre-involving DME. Patients received 12 sessions of PBM over 5 weeks for 90 s per treatment at a setting of 25, 100 or 200 mW/cm2 for the three sequential cohorts of 6-8 patients each. Patients were recruited from the Sydney Eye Hospital, over the age of 18 and had centre-involving DME with central macular thickness (CMT) of >300 µm with visual acuity of 75-35 Log minimum angle of resolution (logMAR) letters (Snellen visual acuity equivalent of 20/30-20/200). The objective of this trial was to assess the safety and efficacy of laser-delivered PBM at 2 and 6 months. The primary efficacy outcome was change in CMT at 2 and 6 months. RESULTS: LED-delivered PBM enhanced photoreceptor mitochondrial membrane potential, protected Müller cells and photoreceptors from damage and reduced retinal vascular leakage resulting from induced Müller cell disruption in transgenic mice. PBM delivered via the retinal laser enhanced mitochondrial function and protected against oxidative stress in cultured Müller cells. Laser-delivered PBM did not damage the retina in pigmented rat eyes at 100 mW/cm2. The completed clinical trial found a significant reduction in CMT at 2 months by 59 ± 46 µm (p = 0.03 at 200 mW/cm2) and significant reduction at all three settings at 6 months (25 mW/cm2: 53 ± 24 µm, p = 0.04; 100 mW/cm2: 129 ± 51 µm, p < 0.01; 200 mW/cm2: 114 ± 60 µm, p < 0.01). Laser-delivered PBM was well tolerated in humans at settings up to 200 mW/cm2 with no significant side effects. CONCLUSIONS/INTERPRETATION: PBM results in anatomical improvement of DME over 6 months and may represent a safe and non-invasive treatment. Further testing is warranted in randomised clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02181400 Graphical abstract.

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.

Effects of VEGF inhibitors on human retinal pigment epithelium under high glucose and hypoxia.

BACKGROUND: Retinal pigment epithelium (RPE) is known to secrete factors important for retinal homeostasis. How this secretome changes in diabetic eyes treated with anti-vascular endothelial growth factor (VEGF) inhibitors is unclear. METHODS: Diabetic conditions were simulated in vitro using ARPE-19 cell-line culture, with high glucose (25 mM) culture media, and hypoxia was chemically induced using cobalt chloride. Stress was assessed using cell viability assays as well as Western blots and enzyme-linked immunosorbent assay (ELISA) for production of HIF-1a and VEGF-A. Production of neurotrophic factors was quantified once conditions were established using ELISA under stress with and without the addition of VEGF inhibitors. Changes were analysed with one-way ANOVA. RESULTS: Hypoxia downregulated pigment epithelium-derived factor (PEDF) expression. The addition of bevacizumab, ranibizumab and aflibercept in normoxic conditions all led to a significant downregulation of PEDF. Glucose concentration had no effect on secretion of PEDF. Brain-derived neurotrophic factor (BDNF) secretion was downregulated in high glucose and was upregulated in hypoxia. Placental growth factor (PlGF) secretion by ARPE-19 was undetectable by ELISA. CONCLUSIONS: We found that hypoxia, high glucose or VEGF inhibitors affected secretion of neurotrophic factors. This variation under different conditions may influence neuron and photoreceptor survival in the diabetic state and VEGF inhibitor treated eyes.

Characterization of canonical Wnt signalling changes after induced disruption of Müller cell in murine retina.

Müller cells are the primary glia in the retina, playing a critical role in retinal homeostasis and retinal pathology. This study evaluated the canonical Wnt signalling pathway and its downstream effects on retinal degeneration in a transgenic mouse model of inducible Müller cell disruption. Increased expression of the LacZ reporter gene in the retina suggested Wnt signalling had been activated after induced Müller cell disruption. Activation was validated by observing nuclear translocation of ß-Catenin. The mRNA expression of 80 Wnt related genes were assessed using real-time PCR. The Wnt signalling inhibitors Dkk1, Dkk3 and sFRP3 were significantly downregulated. Furthermore, the ubiquitin-mediated ß-Catenin proteolysis genes ß-TrCP and SHFM3, were also significantly downregulated. The downstream target genes of the Wnt signalling, including Fra1, CyclinD2 and C-Myc were upregulated. The changes of these genes at the protein level were validated by Western blot. Their distributions in the retina were evaluated by immunofluorescent staining. Our findings indicate that Müller cells are involved in retinal Wnt signalling. Activation of Wnt signalling and its downstream target genes may play important roles in photoreceptor degeneration and neovascularization occurring in the retina after induced disruption of Müller cells.

Effects of Ranibizumab and Aflibercept on Human Müller Cells and Photoreceptors under Stress Conditions.

Anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of retinal vascular diseases. However, constitutive VEGF also acts as a trophic factor on retinal nonvascular cells. We have studied the effects of aflibercept and ranibizumab on human Müller cells and photoreceptors exposed to starvation media containing various concentrations of glucose, with or without CoCl2-induced hypoxia. Cell survival was assessed by calcein-AM cell viability assays. Expression of heat shock proteins (Hsp) and redox proteins thioredoxin 1 and 2 (TRX1, TRX2) was studied by Western blots. The production of neurotrophic factors in Müller cells and interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors was measured by enzymelinked immunosorbent assays. Aflibercept and ranibizumab did not affect the viability of both types of cells. Neither aflibercept nor ranibizumab affected the production of neurotrophic factors or expression of Hsp60 and Hsp90 in Müller cells. However, aflibercept but not ranibizumab affected the expression of Hsp60, Hsp9, TRX1 and TRX2 in photoreceptors. Aflibercept and ranibizumab both inhibited the production of IRBP in photoreceptors, aflibercept more so than ranibizumab. Our data indicates that the potential influence of aflibercept and ranibizumab on photoreceptors should be specifically monitored in clinical studies.

Genome-wide association study for sight-threatening diabetic retinopathy reveals association with genetic variation near the GRB2 gene.

AIMS/HYPOTHESIS: Diabetic retinopathy is a serious complication of diabetes mellitus and can lead to blindness. A genetic component, in addition to traditional risk factors, has been well described although strong genetic factors have not yet been identified. Here, we aimed to identify novel genetic risk factors for sight-threatening diabetic retinopathy using a genome-wide association study. METHODS: Retinopathy was assessed in white Australians with type 2 diabetes mellitus. Genome-wide association analysis was conducted for comparison of cases of sight-threatening diabetic retinopathy (n = 336) with diabetic controls with no retinopathy (n = 508). Top ranking single nucleotide polymorphisms were typed in a type 2 diabetes replication cohort, a type 1 diabetes cohort and an Indian type 2 cohort. A mouse model of proliferative retinopathy was used to assess differential expression of the nearby candidate gene GRB2 by immunohistochemistry and quantitative western blot. RESULTS: The top ranked variant was rs3805931 with p = 2.66 × 10(-7), but no association was found in the replication cohort. Only rs9896052 (p = 6.55 × 10(-5)) was associated with sight-threatening diabetic retinopathy in both the type 2 (p = 0.035) and the type 1 (p = 0.041) replication cohorts, as well as in the Indian cohort (p = 0.016). The study-wide meta-analysis reached genome-wide significance (p = 4.15 × 10(-8)). The GRB2 gene is located downstream of this variant and a mouse model of retinopathy showed increased GRB2 expression in the retina. CONCLUSIONS/INTERPRETATION: Genetic variation near GRB2 on chromosome 17q25.1 is associated with sight-threatening diabetic retinopathy. Several genes in this region are promising candidates and in particular GRB2 is upregulated during retinal stress and neovascularisation.

Dysregulation of inter-photoreceptor retinoid-binding protein (IRBP) after induced Müller cell disruption.

Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter-photoreceptor retinoid-binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down-regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N-retinylidene-N-retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases. This study reports down-regulation of inter-photoreceptor retinoid-binding protein (IRBP) in photoreceptors and retinoid cycle derangement after Müller cell disruption in a transgenic mouse model. The findings indicate that Müller cells communicate with photoreceptors in response to stress by secreting soluble protein factor(s). We propose that down-regulation of IRBP may represent an early and novel pathogenic mechanism in degenerative retinal diseases.

Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model of selective Müller cell ablation.

Retinal diseases such as macular telangiectasis type 2 (MacTel), age-related macular degeneration (AMD) and diabetic retinopathy (DR) affect both neurons and blood vessels. Treatments addressing both at the same time might have advantages over more specific approaches, such as vascular endothelial growth factor (VEGF) inhibitors, which are used to treat vascular leak but are suspected to have a neurotoxic effect. Here, we studied the effects of an intravitreal injection of triamcinolone acetonide (TA) in a transgenic model in which patchy Müller cell ablation leads to photoreceptor degeneration, vascular leak, and intraretinal neovascularization. TA was injected 4 days before Müller cell ablation. Changes in photoreceptors, microglia and Müller cells, retinal vasculature, differential expression of p75 neurotrophin receptor (p75(NTR) ), tumor necrosis factor-α (TNFα), the precursor and mature forms of neurotrophin 3 (pro-NT3 and mature NT3) and activation of the p53 and p38 stress-activated protein kinase (p38/SAPK) signaling pathways were examined. We found that TA prevented photoreceptor degeneration and inhibited activation of microglial and Müller cells. TA attenuated Müller cell loss and inhibited overexpression of p75(NTR) , TNFα, pro-NT, and the activation of p53 and p38/SAPK signaling pathways. TA not only prevented the development of retinal vascular lesions but also inhibited fluorescein leakage from established vascular lesions. TA inhibited overexpression of VEGF in transgenic mice but without affecting its basal level expression in the normal retina. Our data suggest that glucocorticoid treatment may be beneficial for treatment of retinal diseases such as MacTel, AMD, and DR that affect both neurons and the vasculature.

Differential gene expression in Lin-/VEGF-R2+ bone marrow-derived endothelial progenitor cells isolated from diabetic mice.

BACKGROUND: Diabetes is known to impair the number and function of endothelial progenitor cells in the circulation, causing structural and functional alterations in the micro- and macro-vasculature. The aim of this study was to identify early diabetes-related changes in the expression of genes that have been reported to be closely involved in endothelial progenitor cell migration and function. METHODS: Based on review of current literature, this study examined the expression level of 35 genes that are known to be involved in endothelial progenitor cell migration and function in magnetically sorted Lin-/VEGF-R2+ endothelial progenitor cells obtained from the bone marrow of Akita mice in the early stages of diabetes (18 weeks) using RT-PCR and Western blotting. We used the Shapiro-Wilk and D'Agostino & Pearson Omnibus tests to assess normality. Differences between groups were evaluated by Student's t-test for normally distributed data (including Welch correction in cases of unequal variances) or Mann-Whitney test for not normally distributed data. RESULTS: We observed a significant increase in the number of Lin-/VEGF-R2+ endothelial progenitor cells within the bone marrow in diabetic mice compared with non-diabetic mice. Two genes, SDF-1 and SELE, were significantly differentially expressed in diabetic Lin-/VEGF-R2+ endothelial progenitor cells and six other genes, CAV1, eNOS, CLDN5, NANOG, OCLN and BDNF, showed very low levels of expression in diabetic Lin-/VEGF-R2+ progenitor cells. CONCLUSION: Low SDF-1 expression may contribute to the dysfunctional mobilization of bone marrow Lin-/VEGF-R2+ endothelial progenitor cells, which may contribute to microvascular injury in early diabetes.

Conditional Müllercell ablation causes independent neuronal and vascular pathologies in a novel transgenic model.

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller cell ablation and the consequences of primary Müller cell dysfunction have been studied in adult mice. We found that selective ablation of Müller cells led to photoreceptor apoptosis, vascular telangiectasis, blood-retinal barrier breakdown and, later, intraretinal neovascularization. These changes were accompanied by impaired retinal function and an imbalance between vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor. Intravitreal injection of ciliary neurotrophic factor inhibited photoreceptor injury but had no effect on the vasculopathy. Conversely, inhibition of VEGF-A activity attenuated vascular leak but did not protect photoreceptors. Our findings show that Müller glial deficiency may be an important upstream cause of retinal neuronal and vascular pathologies in retinal diseases. Combined neuroprotective and anti-angiogenic therapies may be required to treat Müller cell deficiency in retinal diseases and in other parts of the CNS associated with glial dysfunction.

Involvement of NT3 and P75(NTR) in photoreceptor degeneration following selective Müller cell ablation.

BACKGROUND: Neurotrophins can regulate opposing functions that result in cell survival or apoptosis, depending on which form of the protein is secreted and which receptor and signaling pathway is activated. We have recently developed a transgenic model in which inducible and patchy Müller cell ablation leads to photoreceptor degeneration. This study aimed to examine the roles of mature neurotrophin-3 (NT3), pro-NT3 and p75 neurotrophin receptor (P75(NTR)) in photoreceptor degeneration in this model. METHODS: Transgenic mice received tamoxifen to induce Müller cell ablation. Changes in the status of Müller and microglia cells as well as expression of mature NT3, pro-NT3 and P75(NTR) were examined by immunohistochemistry and Western blot analysis. Recombinant mature NT3 and an antibody neutralizing 75(NTR) were injected intravitreally 3 and 6 days after Müller cell ablation to examine their effects on photoreceptor degeneration and microglial activation. RESULTS: We found that patchy loss of Müller cells was associated with activation of surviving Müller cells and microglial cells, concurrently with reduced expression of mature NT3 and upregulation of pro-NT3 and P75(NTR). Intravitreal injection of mature NT3 and a neutralizing antibody to P75NTR, either alone or in combination, attenuated photoreceptor degeneration and the beneficial effect was associated with inhibition of microglial activation. CONCLUSIONS: Our data suggest that Müller cell ablation alters the balance between the protective and deleterious effects of mature NT3 and pro-NT3. Modulation of the neuroprotective action of mature NT3 and pro-apoptotic pro-NT3/P75(NTR) signaling may represent a novel pharmacological strategy for photoreceptor protection in retinal disease.

Isolation and characterization of mouse bone marrow-derived Lin⁻/VEGF-R2⁺ progenitor cells.

Circulating endothelial progenitor cells (EPCs) in the peripheral blood (PB) have physiological roles in the maintenance of the existing vascular beds and rescue of vascular injury. In this study, we have evaluated the properties of Lin⁻/VEGF-R2⁺ progenitor cells isolated from the mouse bone marrow (BM) and further studied their distribution and integration in an animal model of laser-induced retinal vascular injury. Lin⁻/VEGF-R2⁺ cells were enriched from C57BL/6 mice BM using magnetic cell sorting with hematopoietic lineage (Lin) depletion followed by VEGF-R2 positive selection. Lin⁻/VEGF-R2⁺ BM cells were characterized using flow cytometry and immunocytochemistry and further tested for colony formation during culture and tube formation on Matrigel®. Lin⁻/VEGF-R2⁺ BM cells possessed typical EPC properties such as forming cobble-stone shaped colonies after 3 to 4 weeks of culture, CD34⁺ expression, take up of Dil-acLDL and binding to Ulex europaeus agglutinin. However, they did not form tube-like structures on Matrigel®. The progenitor cells retained their phenotype over extended period of culture. After intravitreal transplantation in eyes subjected to the laser-induced retinal vascular injury, some Lin⁻/VEGF-R2⁺ cells were able to integrate into the damaged retinal vasculature but the level of cell integration seemed less efficient when compared with previous reports in which EPCs from the human PB were employed. Our results indicate that Lin⁻/VEGF-R2⁺ cells isolated from the mouse BM share some similarities to EPCs from the human PB but most of them are at a very early stage of maturation and remain quiescent during culture and after intravitreal transplantation.

Optogenetic restoration of high sensitivity vision with bReaChES, a red-shifted channelrhodopsin.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.

Retinal vascular changes after glial disruption in rats.

Glial dysfunction is found in a number of retinal vascular diseases but its link with blood-retinal barrier (BRB) breakdown remains poorly understood. The present study tested the hypothesis that glial dysfunction is a major contributor to the BRB breakdown that is a hallmark of retinal vascular diseases. We investigated the specificity of the purportedly selective glial toxin, DL-alpha-aminoadipic acid (DL-alpha-AAA) on different types of ocular cells in vitro and then tested the effect of glial disruption on retinal vasculature after intraocular injection of DL-alpha-AAA or siRNA targeting glutamine synthetase (GS) in rats. DL-alpha-AAA was toxic to astrocytes and Müller cells but not to other types of BRB-related cells in vitro. Subretinal injection of DL-alpha-AAA disrupted retinal glial cells, induced vascular telangiectasis and increased vascular permeability from 4 days to over 2 months post-injection. Vascular changes induced by DL-alpha-AAA were observed predominantly in regions of glial disruption, as reflected by reduced expression of GS and increased expression of glial fibrillary acidic protein and vimentin. Confocal microscopy showed changes in all three layers of the retinal vasculature, which co-localised with areas of Müller cell disruption. Double labeling immunohistochemistry revealed that retinal glial disruption after DL-alpha-AAA injection was accompanied by increased expression of vascular endothelial growth factor and reduced expression of the tight junction protein claudin-5. Intravitreal injection of GS siRNA induced similar changes in Müller cells and BRB breakdown. Our data are consistent with the hypothesis that glial dysfunction is a primary contributor to the BRB breakdown in retinal vascular diseases.

Selective knockdown of hexokinase 2 in rods leads to age-related photoreceptor degeneration and retinal metabolic remodeling.

Photoreceptors, the primary site of phototransduction in the retina, require energy and metabolites to constantly renew their outer segments. They preferentially consume most glucose through aerobic glycolysis despite possessing abundant mitochondria and enzymes for oxidative phosphorylation (OXPHOS). Exactly how photoreceptors balance aerobic glycolysis and mitochondrial OXPHOS to regulate their survival is still unclear. We crossed rhodopsin-Cre mice with hexokinase 2 (HK2)-floxed mice to study the effect of knocking down HK2, the first rate-limiting enzyme in glycolysis, on retinal health and metabolic remodeling. Immunohistochemistry and Western blots were performed to study changes in photoreceptor-specific proteins and key enzymes in glycolysis and the tricarboxylic acid (TCA) cycle. Changes in retinal structure and function were studied by optical coherence tomography and electroretinography. Mass spectrometry was performed to profile changes in 13C-glucose-derived metabolites in glycolysis and the TCA cycle. We found that knocking down HK2 in rods led to age-related photoreceptor degeneration, evidenced by reduced expression of photoreceptor-specific proteins, age-related reductions of the outer nuclear layer, photoreceptor inner and outer segments and impaired electroretinographic responses. Loss of HK2 in rods led to upregulation of HK1, phosphorylation of pyruvate kinase muscle isozyme 2, mitochondrial stress proteins and enzymes in the TCA cycle. Mass spectrometry found that the deletion of HK2 in rods resulted in accumulation of 13C-glucose along with decreased pyruvate and increased metabolites in the TCA cycle. Our data suggest that HK2-mediated aerobic glycolysis is indispensable for the maintenance of photoreceptor structure and function and that long-term inhibition of glycolysis leads to photoreceptor degeneration.

Targeting the Notch and TGF-ß signaling pathways to prevent retinal fibrosis in vitro and in vivo.

Rationale: The Notch and transforming growth factor-ß (TGFß) signaling pathways are two intracellular mechanisms that control fibrosis in general but whether they play a major role in retinal fibrosis is less clear. Here we study how these two signaling pathways regulate Müller cell-dominated retinal fibrosis in vitro and in vivo. Methods: Human MIO-M1 Müller cells were treated with Notch ligands and TGFß1, either alone or in combination. Western blots were performed to study changes in γ-secretase proteases, Notch downstream effectors, endogenous TGFß1, phosphorylated Smad3 (p-Smad3) and extracellular matrix (ECM) proteins. We also studied the effects of RO4929097, a selective γ-secretase inhibitor, on expression of ECM proteins after ligand stimulation. Müller cell viability was studied by AlamarBlue and cytotoxicity by lactate cytotoxicity assays. Finally, we studied changes in Notch and TGFß signaling and tested the effect of intravitreal injections of the Notch pathway inhibitor RO4929097 on retinal fibrosis resulted from Sodium iodate (NaIO3)-induced retinal injury in mice. We also studied the safety of intravitreal injections of RO4929097 in normal mice. Results: Treatment of Müller cells with Notch ligands upregulated γ-secretase proteases and Notch downstream effectors, with increased expression of endogenous TGFß1, TGFß receptors and p-Smad3. TGFß1 upregulated the expression of proteins associated with both signaling pathways in a similar manner. Notch ligands and TGFß1 had additive effects on overexpression of ECM proteins in Müller cells which were inhibited by RO4929097. Notch and TGFß ligands stimulated Müller cell proliferation which was inhibited by RO4929097 without damaging the cells. NaIO3-induced retinal injury activated both Notch and TGFß signaling pathways in vivo. Intravitreal injection of RO4929097 prevented Müller cell gliosis and inhibited overexpression of ECM proteins in this murine model. We found no safety concerns for up to 17 days after an intravitreal injection of RO4929097. Conclusions: Inhibiting Notch signaling might be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing a treatment for preventing fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.

Differentiation of Retinal Glial Cells From Human Embryonic Stem Cells by Promoting the Notch Signaling Pathway.

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.