Pluripotent human stem cells for the treatment of retinal disease

Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.     

Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration

Age-related macular degeneration (AMD) is a complex, degenerative and progressive eye disease that usually does not lead to complete blindness, but can result in severe loss of central vision. Risk factors for AMD include age, genetics, diet, smoking, oxidative stress and many cardiovascular-associated risk factors. Autophagy is a cellular housekeeping process that removes damaged organelles and protein aggregates, whereas heterophagy, in the case of the retinal pigment epithelium (RPE), is the phagocytosis of exogenous photoreceptor outer segments. Numerous studies have demonstrated that both autophagy and heterophagy are highly active in the RPE. To date, there is increasing evidence that constant oxidative stress impairs autophagy and heterophagy, as well as increases protein aggregation and causes inflammasome activation leading to the pathological phenotype of AMD. This review ties together these crucial pathological topics and reflects upon autophagy as a potential therapeutic target in AMD.     

Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization.

High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the development of subretinal choroidal angiogenesis induced by laser photocoagulation. When systemic and local PAI-1 expression was achieved by intravenous injection of a replication-defective adenoviral vector expressing human PAI-1 cDNA, the wild-type pattern of choroidal angiogenesis was restored. These observations demonstrate the proangiogenic activity of PAI-1 not only in tumoral models, but also in choroidal experimental neovascularization sharing similarities with human AMD. They identify therefore PAI-1 as a potential target for therapeutic ocular anti-angiogenic strategies.     

IFN-β signaling dampens microglia reactivity but does not prevent from light-induced retinal degeneration

Chronic activation of microglia is associated with retinal degeneration, which makes them a potential therapeutic target for retinal degenerative diseases including age-related macular degeneration (AMD). Interferon-beta (IFN-β) is a potent immune regulator, commonly used for the treatment of multiple sclerosis patients. We have previously shown that IFN-β prevents microgliosis and choroidal neovascularization in a laser model of wet AMD. Here, we hypothesized that microglia modulation via IFN-β may also dampen mononuclear phagocyte reactivity and thereby protect from retinal degeneration in a light-damage paradigm mimicking some features of dry AMD. BALB/cJ mice received intraperitoneal injections of 10,000 U IFN-β or vehicle every other day; starting at the day of exposure to 15,000 lux white light for 1 h. Systemic treatment with IFN-β partially enhanced IFN-α/β receptor (IFNAR) signaling in the retina and reduced the number of reactivated microglia in the subretinal space. However, four days after light damage neither decreased expression of complement factors nor rescue of retinal thickness was found. We conclude that IFNAR signaling modulate retinal microglia but cannot prevent strong retinal degeneration as elicited by acute white light damage.     

Phosphorylation/inactivation of PTEN by Akt-independent PI3K signaling in retinal pigment epithelium

Retinal pigment epithelium (RPE) plays a critical role in vertebrate vision by providing functional and structural support to the retina. Degeneration of RPE by cumulative oxidative stresses or acute injury frequently results in retinal degenerative diseases, notably age-related macular degeneration (AMD). Moreover, it has been shown that phosphorylation-mediated inactivation of PTEN (phosphatase and tensin homolog) in RPE is closely linked to AMD-like retinal degeneration in mice [1]. In this study, we used AMD mouse models, in which chemokine (C–C motif) ligand 2 (Ccl2) or chemokine (C–C motif) receptor 2 (Ccr2) were genetically ablated, to examine mechanisms linking reactive oxygen species (ROS) to phosphorylation/inactivation of PTEN in RPE. We found that ROS levels were increased in these RPE cells in association with phosphorylation/inactivation of PTEN. Both PTEN phosphorylation/inactivation and consequent Akt activation in the RPE of AMD model mice were inhibited by antioxidant treatment, indicating a functional role for elevated intracellular ROS. We further discovered that PTEN phosphorylation in oxidatively stressed RPE was repressed by a phosphoinositide 3-kinase (PI3K) inhibitor, but not by an Akt inhibitor. Taken together, these results suggest that ROS-activated PI3K potentiates AMD-related RPE pathogenesis through phosphorylation/inactivation of PTEN.     

Treatment with interleukin‐33 is non‐toxic and protects retinal pigment epithelium in an ageing model of outer retinal degeneration

The leading cause of central vision loss, age‐related macular degeneration (AMD), is a degenerative disorder characterized by atrophy of retinal pigment epithelium (RPE) and photoreceptors. For 15% of cases, neovascularization occurs, leading to acute vision loss if left untreated. For the remaining patients, there are currently no treatment options and preventing progressive RPE atrophy remains the main therapeutic goal. Previously, we have shown treatment with interleukin‐33 can reduce choroidal neovascularization and attenuate tissue remodelling. Here, we investigate IL‐33 delivery in aged, high‐fat diet (HFD) fed mice on a wildtype and complement factor H heterozygous knockout background. We characterize the non‐toxic effect following intravitreal injection of IL‐33 and further demonstrate protective effects against RPE cell death with evidence of maintaining metabolic retinal homeostasis of Cfh+/−~HFD mice. Our results further support the potential utility of IL‐33 to prevent AMD progression.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

Oxidative damage-induced inflammation initiates age-related macular degeneration

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.     

Systemic Administration of Abeta mAb Reduces Retinal Deposition of Abeta and Activated Complement C3 in Age-Related Macular Degeneration Mouse Model

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh−/−), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aβ) rich basement membrane deposits associated with activated complement C3. Cfh−/− mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aβ monoclonal antibody (mAb), 6F6, to determine the effect on the cfh−/− retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aβ and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aβ levels after systemic administration of 6F6 show accumulation of Aβ in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aβ mAb treatment can partially prevent or reverse ocular phenotypes of the cfh−/− mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD – Aβ and activated, complement C3.     

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC₅₀ of 6 μM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.     

Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.     

Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

Retinal long-chain PUFAs (LC-PUFAs, C₁₂-C₂₂) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, C_n>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.     

Age-related macular degeneration: a target for nanotechnology derived medicines

Despite the fact that the retina is a fairly accessible portion of the central nervous system, there are virtually no treatments for early age-related macular degeneration (AMD). AMD is a degenerative retinal disease that causes progressive loss of central vision and is the leading cause of irreversible vision loss and legal blindness in individuals over the age of 50. Both environmental and genetic components play a role in its development. AMD is a multifactorial disease with characteristics that include drusen, hyperpigmentation and/or hypopigmentation of the retinal pigment epithelium (RPE), geographic atrophy and, in a subset of patients, late-stage choroidal neovascularization (CNV). Drugs that inhibit vascular endothelial growth factor (VEGF) have proven effective in treating late-stage CNV, but optimal means of drug delivery remains to be determined. Microscopic particles, whose size is on the nanometer scale, show considerable promise for drug delivery to the retina, for gene therapy, and for powering prosthetic "artificial retinas." This article summarizes the pathophysiology of AMD stressing potential applications from nanotechnology.     

Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy‐like retinal degeneration in a mouse model with iron overload

Oxidative stress plays a central role in age‐related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis‐allylic sites of DHA in photoreceptor membranes could prevent iron‐induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation‐prone positions (D‐DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D‐DHA caused a dose‐dependent increase in D‐DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D‐DHA provided nearly complete protection against iron‐induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D‐DHA. Quantitative PCR results were consistent with iron‐induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D‐DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron‐induced retinal degeneration. They also provide preclinical evidence that dosing with D‐DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.     

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.     

Deciphering primate retinal aging at single-cell resolution

Dear Editor, The retina is a light-sensitive highly-organized tissue,which is vulnerable to aging and age-related retinal diseases.Specifically,progressive retinal degeneration leads to visual function deterioration and vision impairment in the elderly(Lin et al.,2016).In diseases such as age-related macular degeneration(AMD),retinitis pigmentosa(RP)and diabetic retinopathy(DR),pathological process lacking effective treatments profoundly and negatively impact on the quality of life in the elderly(Lin et al.,2016;Chen et al.,2019).Thus,an in-depth molecular assessment of the mechanisms driv-ing retinal aging is of urgent scientific and medical importance.     

The 5HT1a receptor agonist 8-Oh DPAT induces protection from lipofuscin accumulation and oxidative stress in the retinal pigment epithelium

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 μM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.