Cell-Type-Specific Complement Profiling in the ABCA4−/− Mouse Model of Stargardt Disease

Stargardt macular degeneration is an inherited retinal disease caused by mutations in the ATP-binding cassette subfamily A member 4 (ABCA4) gene. Here, we characterized the complement expression profile in ABCA4−/− retinae and aligned these findings with morphological markers of retinal degeneration. We found an enhanced retinal pigment epithelium (RPE) autofluorescence, cell loss in the inner retina of ABCA4−/− mice and demonstrated age-related differences in complement expression in various retinal cell types irrespective of the genotype. However, 24-week-old ABCA4−/− mice expressed more c3 in the RPE and fewer cfi transcripts in the microglia compared to controls. At the protein level, the decrease of complement inhibitors (complement factor I, CFI) in retinae, as well as an increased C3b/C3 ratio in the RPE/choroid and retinae of ABCA4−/−, mice was confirmed. We showed a corresponding increase of the C3d/C3 ratio in the serum of ABCA4−/− mice, while no changes were observed for CFI. Our findings suggest an overactive complement cascade in the ABCA4−/− retinae that possibly contributes to pathological alterations, including microglial activation and neurodegeneration. Overall, this underpins the importance of well-balanced complement homeostasis to maintain retinal integrity.

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Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

eLife ◽

10.7554/elife.28899 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 88

Author(s):

Mark A Kanow ◽

Michelle M Giarmarco ◽

Connor SR Jankowski ◽

Kristine Tsantilas ◽

Abbi L Engel ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Metabolic Flux ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Disease ◽

Glucose Consumption ◽

Age Related ◽

Underlying Causes ◽

Vertebrate Eye

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.

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Hydroquinone Induces NLRP3-Independent IL-18 Release from ARPE-19 Cells

Cells ◽

10.3390/cells10061405 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1405

Author(s):

Niina Bhattarai ◽

Eveliina Korhonen ◽

Yashavanthi Mysore ◽

Kai Kaarniranta ◽

Anu Kauppinen

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Disease ◽

Oxidase Inhibitor ◽

Age Related Macular Degeneration ◽

Rpe Cells ◽

Pyrin Domain ◽

Age Related ◽

Domain 3 ◽

Human Rpe Cells

Age-related macular degeneration (AMD) is a retinal disease leading to impaired vision. Cigarette smoke increases the risk for developing AMD by causing increased reactive oxygen species (ROS) production and damage in the retinal pigment epithelium (RPE). We have previously shown that the cigarette tar component hydroquinone causes oxidative stress in human RPE cells. In the present study, we investigated the propensity of hydroquinone to induce the secretion of interleukin (IL)-1β and IL-18. The activation of these cytokines is usually regulated by the Nucleotide-binding domain, Leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome. ARPE-19 cells were exposed to hydroquinone, and cell viability was monitored using the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) assays. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of proinflammatory cytokines IL-1β and IL-18 as well as NLRP3, caspase-1, and poly (ADP-ribose) polymerase (PARP). Hydroquinone did not change IL-1β release but significantly increased the secretion of IL-18. Cytoplasmic NLRP3 levels increased after the hydroquinone treatment of IL-1α-primed RPE cells, but IL-18 was equally released from primed and nonprimed cells. Hydroquinone reduced the intracellular levels of PARP, which were restored by treatment with the ROS scavenger N-acetyl-cysteine (NAC). NAC concurrently reduced the NLRP3 levels but had no effect on IL-18 release. In contrast, the NADPH oxidase inhibitor ammonium pyrrolidinedithiocarbamate (APDC) reduced the release of IL-18 but had no effect on the NLRP3 levels. Collectively, hydroquinone caused DNA damage seen as reduced intracellular PARP levels and induced NLRP3-independent IL-18 secretion in human RPE cells.

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Cell type-specific complement expression from healthy and diseased retinae

10.1101/413088 ◽

2018 ◽

Author(s):

Diana Pauly ◽

Nicole Schäfer ◽

Felix Grassmann ◽

Anna M. Pfaller ◽

Tobias Straub ◽

...

Keyword(s):

Complement System ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Müller Cells ◽

Cell Types ◽

Negative Regulator ◽

Retinal Cell ◽

Muller Cells ◽

Complement Components ◽

The Individual

AbstractRetinal degeneration is associated with complement system activation, but retinal sources of complement are unknown. Here, we describe the human and murine complement transcriptomes of Müller cells, microglia/macrophages, vascular cells, neurons and retinal pigment epithelium (RPE) in health and disease. All cell populations expressed c1s, c3, cfb, cfp, cfh and cfi. Murine Müller cells contributed the highest amount of complement activators (c1s, c3, cfb). RPE mainly expressed cfh, while cfi and cfp transcripts were most abundant in neurons. The main complement negative regulator in the human retina was cfi, while cfh dominated in the murine retina. Importantly, the expression of c1s, cfb, cfp, cfi increased and that of cfh decreased with aging. Impaired photoreceptor recycling led to an enhanced c3 expression in RPE and to a reduced cfi expression in microglia/macrophages. Expression of complement components was massively upregulated after transient retinal ischemia in murine microglia, Müller cells and RPE. The individual signature of complement expression in distinct murine and human retinal cell types indicates a local, well-orchestrated regulation of the complement system in both species.

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A Novel HDL-Mimetic Peptide HM-10/10 Protects RPE and Photoreceptors in Murine Models of Retinal Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms20194807 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4807 ◽

Cited By ~ 1

Author(s):

Feng Su ◽

Christine Spee ◽

Eduardo Araujo ◽

Eric Barron ◽

Mo Wang ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Retinal Degeneration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Disease ◽

Geographic Atrophy ◽

Age Related Macular Degeneration ◽

Mimetic Peptide ◽

Age Related

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.

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Mitochondrial Dysfunction in the Aging Retina

Biology ◽

10.3390/biology8020031 ◽

2019 ◽

Vol 8 (2) ◽

pp. 31 ◽

Cited By ~ 11

Author(s):

Janis T. Eells

Keyword(s):

Mitochondrial Dysfunction ◽

Cell Function ◽

Pigment Epithelium ◽

Retinal Pigment ◽

High Energy ◽

Age Related Macular Degeneration ◽

Retinal Cell ◽

Ros Generation ◽

Mtdna Mutations ◽

Age Related

Mitochondria are central in retinal cell function and survival and they perform functions that are critical to cell function. Retinal neurons have high energy requirements, since large amounts of ATP are needed to generate membrane potentials and power membrane pumps. Mitochondria over the course of aging undergo a number of changes. Aged mitochondria exhibit decreased rates of oxidative phosphorylation, increased reactive oxygen species (ROS) generation and increased numbers of mtDNA mutations. Mitochondria in the neural retina and the retinal pigment epithelium are particularly susceptible to oxidative damage with aging. Many age-related retinal diseases, including glaucoma and age-related macular degeneration, have been associated with mitochondrial dysfunction. Therefore, mitochondria are a promising therapeutic target for the treatment of retinal disease.

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Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

10.1101/143347 ◽

2017 ◽

Cited By ~ 1

Author(s):

Mark A. Kanow ◽

Michelle M. Giarmarco ◽

Connor Jankowski ◽

Kristine Tsantilas ◽

Abbi L. Engel ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Metabolic Flux ◽

Vision Loss ◽

Aerobic Glycolysis ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Disease ◽

Age Related ◽

Underlying Causes ◽

Vertebrate Eye

ABSTRACTHere we report multiple lines of evidence for a comprehensive model for retinal energy metabolism. Metabolic flux, locations of key enzymes and our finding that glucose enters the neural retina almost entirely through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood supply passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. A key feature of this model is that aerobic glycolysis in photoreceptors produces lactate to suppress glycolysis in the neighboring retinal pigment epithelium. That enhances the flow of glucose to the retina by minimizing consumption of glucose within the retinal pigment epithelium. This framework for metabolic relationships in retina provides new insights into the underlying causes of retinal disease, age-related vision loss and metabolism-based therapies.

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CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22168727 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8727

Author(s):

Angela Armento ◽

Tiziana L. Schmidt ◽

Inga Sonntag ◽

David A. Merle ◽

Mohamed Ali Jarboui ◽

...

Keyword(s):

Complement System ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Factor H ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Major Pathway ◽

Rpe Cells ◽

Age Related

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

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Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1413347112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1565-1570 ◽

Cited By ~ 51

Author(s):

Richard B. Thompson ◽

Valentina Reffatto ◽

Jacob G. Bundy ◽

Elod Kortvely ◽

Jane M. Flinn ◽

...

Keyword(s):

Fluorescent Dyes ◽

Secondary Ion Mass Spectrometry ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Factor H ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Bruch's Membrane ◽

Bruch’S Membrane ◽

Age Related

Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5–20 μm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch’s membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch’s membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.

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Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

10.1101/2020.07.27.215103 ◽

2020 ◽

Author(s):

Ivo Ngundu Woogeng ◽

Imad Abugessaisa ◽

Akihiro Tachibana ◽

Yoshiki Sahara ◽

Chung-Chau Hon ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Regenerative Medicine ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Human Fibroblasts ◽

Age Related Macular Degeneration ◽

Retinal Cell ◽

Medicine Research ◽

Age Related

SUMMARYRegenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

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CFH loss in human RPE cells leads to inflammation and complement system dysregulation via the NF-ƙB pathway

10.1101/2021.04.19.440385 ◽

2021 ◽

Author(s):

Angela Armento ◽

Tiziana Luisa Schmidt ◽

Inga Sonntag ◽

David Merle ◽

Mohamed-ali Jarboui ◽

...

Keyword(s):

Complement System ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Factor H ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Major Pathway ◽

Rpe Cells ◽

Age Related

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease and chronic inflammatory processes may be involved. Besides ageing and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism by which CFH dysregulation confers such a great risk for AMD and its role in RPE cells homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g. IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g. C3, CFB upregulation and C5 downregulation) that are known to play a role in AMD. Moreover, we identified the NF-ƙB pathway as the major pathway involved in the regulation of these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-ƙB pathway work in synergy to maintain inflammatory and complement balance and in case either one of them is dysregulated, the RPE microenvironment changes towards a pro-inflammatory AMD-like phenotype.

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