Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Sequence and expression of complement factor H gene cluster variants and their roles in age-related macular degeneration risk

Purpose: To investigate how potentially functional genetic variants are coinherited on each of four common complement factor H (CFH) and CFH-related gene haplotypes and to measure expression of these genes in eye and liver tissues.

Methods: We sequenced the CFH region in four individuals (one homozygote for each of four common CFH region haplotypes) to identify all genetic variants. We studied associations between the haplotypes and AMD phenotypes in 2157 cases and 1150 controls. We examined RNA-seq profiles in macular and peripheral retina and retinal pigment epithelium/choroid/sclera (RCS) from eight eye donors and three liver samples.

Results: The haplotypic coinheritance of potentially functional variants (including missense variants, novel splice sites, and the CFHR3–CFHR1 deletion) was described for the four common haplotypes. Expression of the short and long CFH transcripts differed markedly between the retina and liver. We found no expression of any of the five CFH-related genes in the retina or RCS, in contrast to the liver, which is the main source of the circulating proteins.

Conclusions: We identified all genetic variants on common CFH region haplotypes and described their coinheritance. Understanding their functional effects will be key to developing and stratifying AMD therapies. The small scale of our expression study prevented us from investigating the relationships between CFH region haplotypes and their expression, and it will take time and collaboration to develop epidemiologic-scale studies. However, the striking difference between systemic and ocular expression of complement regulators shown in this study suggests important implications for the development of intraocular and systemic treatments.

The design and implementation of a study to investigate the effectiveness of community vs hospital eye service follow-up for patients with neovascular age-related macular degeneration with quiescent disease

Introduction
Standard treatment for neovascular age-related macular degeneration (nAMD) is intravitreal injections of anti-VEGF drugs. Following multiple injections, nAMD lesions often become quiescent but there is a high risk of reactivation, and regular review by hospital ophthalmologists is the norm. The present trial examines the feasibility of community optometrists making lesion reactivation decisions.

Methods
The Effectiveness of Community vs Hospital Eye Service (ECHoES) trial is a virtual trial; lesion reactivation decisions were made about vignettes that comprised clinical data, colour fundus photographs, and optical coherence tomograms displayed on a web-based platform. Participants were either hospital ophthalmologists or community optometrists. All participants were provided with webinar training on the disease, its management, and assessment of the retinal imaging outputs. In a balanced design, 96 participants each assessed 42 vignettes; a total of 288 vignettes were assessed seven times by each professional group.The primary outcome is a participant's judgement of lesion reactivation compared with a reference standard. Secondary outcomes are the frequency of sight threatening errors; judgements about specific lesion components; participant-rated confidence in their decisions about the primary outcome; cost effectiveness of follow-up by optometrists rather than ophthalmologists.

Discussion
This trial addresses an important question for the NHS, namely whether, with appropriate training, community optometrists can make retreatment decisions for patients with nAMD to the same standard as hospital ophthalmologists. The trial employed a novel approach as participation was entirely through a web-based application; the trial required very few resources compared with those that would have been needed for a conventional randomised controlled clinical trial.

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo.

PURPOSE: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or "wet" Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. METHODS: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with "wet" AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8(+/-) mice expressing ß-galactosidase. Aged Mfge8(+/-) and Mfge8(-/-) mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. RESULTS: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8(-/-) mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8(-/-) mice as compared to controls. CONCLUSIONS: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8(-/-) mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.

Retinal venular caliber predicts visual outcome after intravitreal ranibizumab injection treatments for neovascular AMD

Purpose

New approaches and potential treatments for dry age-related macular degeneration

Emerging treatments for dry age-related macular degeneration (AMD) and geographi c atrophy focus on two strategies that target components involved in physiopathological pathways: prevention of photoreceptors and retinal pigment epithelium loss (neuroprotection induction, oxidative damage prevention, and visual cycle modification) and suppression of inflammation. Neuroprotective drugs, such as ciliary neurotrophic factor, brimonidine tartrate, tandospirone, and anti-amyloid β antibodies, aim to prevent apoptosis of retinal cells. Oxidative stress and depletion of essential micronutrients are targeted by the Age-Related Eye Disease Study (AREDS) formulation. Visual cycle modulators reduce the activity of the photoreceptors and retinal accumulation of toxic fluorophores and lipofuscin. Eyes with dry age-related macular degeneration present chronic inflammation and potential treatments include corticosteroid and complement inhibition. We review the current concepts and rationale of dry age-related macular degeneration treatment that will most likely include a combination of drugs targeting different pathways involved in the development and progression of age-related macular degeneration.

Selective retina therapy (SRT) in patients with geographic atrophy due to age-related macular degeneration

For geographic atrophy (GA) due to age-related macular degeneration (AMD) there is so far no approved treatment option. Usually, increased autofluorescence (AF) levels of different patterns adjacent to the atrophic area indicate lipofuscin-laden retinal pigment epithelium (RPE) cells at a high risk for apoptosis. Herein, SRT was used to selectively treat these cells to stimulate RPE proliferation, in order to reduce or ideally stop further growth of the atrophic area.

Small dense particles in the retina observable by spectral-domain optical coherence tomography in age-related macular degeneration

To observe detailed changes in neurosensory retinal structure after anti-VEGF upload in age-related macular degeneration (AMD), by using spectral domain optical coherence tomography (SD-OCT).

Hip-implant related chorio-retinal cobalt toxicity

A 39-year-old female with elevated serum cobalt levels from her bilateral hip prostheses presented with a 3-week history of blurred vision in her left eye. Optical coherence tomography revealed patchy degeneration of the photoreceptor-retinal pigment epithelium (RPE) complex. The lesions were hypofluorescent on indocyanine green angiography. We postulate that this is a case of implant-related chorio-retinal cobalt toxicity.

Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage

Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Same-day administration of verteporfin and ranibizumab 0.5 mg in patients with choroidal neovascularisation due to age-related macular degeneration

OBJECTIVE: To evaluate safety of same-day administration of verteporfin and ranibizumab. METHODS: Prospective, open-label, multicentre study; patients with predominantly classic (n = 13) or occult (n = 19) choroidal neovascularisation secondary to age-related macular degeneration received standard-fluence verteporfin at baseline and months 3, 6 and 9, based on fluorescein angiography (FA). Ranibizumab 0.5 mg was administered at baseline and months 1, 2 and 3. MAIN OUTCOME MEASURE: The incidence of severe vision loss (best-corrected visual acuity (BCVA) loss > or = 30 letters; primary safety assessment). RESULTS: No severe vision loss due to ocular inflammation or uveitis occurred. One patient had moderate vision loss (BCVA loss > or = 15 letters). Three patients had mild/moderate uveitis. Two serious ocular adverse events occurred (retinal pigment epithelial tear and moderate BCVA decrease). No systemic adverse events occurred. At 9 months, all lesions were inactive with no recurrent leakage on FA and optical coherence tomography; macular oedema and subretinal fluid resolved. The mean BCVA measured at 2 m improved by 6.9 letters at 4 months and 2.4 letters at 9 months. CONCLUSIONS/APPLICATION TO CLINICAL PRACTICE: Same-day verteporfin and ranibizumab was safe and not associated with severe vision loss or severe ocular inflammation. Lesions stabilized, with minimal treatment required after month 3.

BIOCHEMICAL AND CELLULAR MECHANISMS OF RETINA AND RETINAL PIGMENT EPITHELIUM APOPTOSIS

Oxidative stress, intense light exposure and oxygen imbalances such as hypoxic or hyperoxic conditions perturb mitochondria, nuclear function and further lead to cellular damage of retina and retinal pigment epithelial (RPE) cells. Our major aim is to understand the various biochemical and proteomic events that occur during the progression of retina and RPE cell death. The comprehensive objectives of this dissertation are to understand the functional aspects of protein expression, posttranslational modifications, protein or lipid binding changes, phenotypic, morphological alterations and their regulation during the retina and RPE apoptosis under oxidative stress. The entire study is divided into four chapters Chapter 1 contains introduction and background on apoptotic signaling in retina and RPE cells. In chapter 2, we demonstrated that the oxidative stress biomarker prohibitin shuttles between mitochondria and nucleus as an anti-apoptotic molecule and acts as a transcriptional regulator by altering its lipid binding affinity and by posttranslational modifications during oxidative damage to the retina and RPE. In chapter 3, we demonstrated that oxidative and photo-oxidative stress induced nitric oxide regulates the RPE apoptosis by altering serine/threonine protein phosphatase 2A (PP2A) catalytic subunit, vimentin phosphorylation and Bcl xL expression regulation in the RPE cells in vitro. In chapter 4, we further analyzed the differential expression of prohibitin in the retina and RPE during oxidative stress, diabetic retinopathy (DR) and age-related macular degeneration (AMD) condition. Our analysis of postmortem retinas reveals that prohibitin is significantly increased in aged and AMD retina, and decreased in retinas of human diabetic retinopathy and RPE of AMD. Our study demonstrates that prohibitin levels determine the apoptotic signaling in the retina and RPE during retinal degenerative disease progression.