scholarly journals Complement Factor H-Related 3 induces inflammation and complosome activation in human RPE cells

2021 ◽  
Author(s):  
Nicole Schäfer ◽  
Anas Rasras ◽  
Delia Ceteras ◽  
Sabine Amslinger ◽  
Volker Enzmann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Complement Factor ◽  
Rpe Cells ◽  
Age Related ◽  
Anaphylatoxin C3a ◽  
Human Rpe Cells

Abstract Background Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration. The non-canonical local, cellular functions of FHR-3 remained poorly understood.Methods Human retinal pigment epithelium (RPE) cells (ARPE-19 cells and primary human RPE cells (hpRPE)), cultivated in Transwell® inserts, were apically treated with either FHR‑3 alone or with the chimerized monoclonal anti‑FHR-3 antibody RETC-2-ximab, or with FHR-1, FH, Properdin or not treated for 5 – 24 h, respectively. Interaction of FHR-3 with oxidative stress epitopes was determined by ELISA. Internalization studies of FHR-3 or FH by ARPE‑19 cells was determined by immunofluorescence live cell imaging. Impact of FHR-3 on RPE cell-specific complement components and inflammation markers were analyzed on mRNA (RT-qPCR) and on protein level (Western Blot, ELISA, protein secretion assays, immunofluorescence). Results Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by senescent viable RPE cells and modulated time-dependently complement component (C3, CFB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory micro-environment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Additionally, important pattern recognition molecules of the innate immune system, Toll-like receptors 1 and 3, as well as proteasome subunits were impaired in RPE cells after FHR-3 incubation. A chimerized monoclonal anti-FHR-3 antibody, RETC‑2‑ximab, ameliorated the effect of FHR-3 on ARPE-19 cells.Conclusion Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell complosome and as a productive target for a new therapeutic approach using RETC‑2‑ximab for associated degenerative diseases.

scholarly journals Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Nicole Schäfer ◽  
Anas Rasras ◽  
Delia M. Ormenisan ◽  
Sabine Amslinger ◽  
Volker Enzmann ◽  
...  
Keyword(s):  
Complement Activation ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Complement Factor ◽  
Rpe Cells ◽  
Inflammatory Microenvironment ◽  
Age Related ◽  
Anaphylatoxin C3a ◽  
Human Rpe Cells

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell “complosome” and as a putative target for a therapeutic approach for associated degenerative diseases.

scholarly journals Loss of Complement Factor H impairs antioxidant capacity and energy metabolism of human RPE cells

2020 ◽  
Author(s):  
Angela Armento ◽  
Sabina Honisch ◽  
Vasiliki Panagiotakopoulou ◽  
Inga Sonntag ◽  
Anke Jacob ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Energy Metabolism ◽  
Retinal Pigment ◽  
Factor H ◽  
Metabolic Reprogramming ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Complement Factor ◽  
Rpe Cells ◽  
Age Related

AbstractAge-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. About 50% of AMD patients present polymorphisms in the Complement Factor H (CFH) gene, coding for Factor H protein (FH). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. In AMD, retinal homeostasis is compromised due to dysfunction of retinal pigment epithelium (RPE) cells. Whether FH contributes to AMD pathogenesis only via complement system dysregulation remains unclear. To investigate the potential role of FH on energy metabolism and oxidative stress in RPE cells, we silenced CFH in human hTERT-RPE1 cells. FH-deprived RPE cells exposed to oxidative insult, showed altered metabolic homeostasis, including reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation. Our data suggest that FH protects RPE cells from oxidative stress and metabolic reprogramming, highlighting a novel function for FH in AMD pathogenesis.Graphical abstract

scholarly journals FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawshan Choudhury ◽  
Nadhim Bayatti ◽  
Richard Scharff ◽  
Ewa Szula ◽  
Viranga Tilakaratna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Rpe Cells ◽  
Age Related

AbstractRetinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

scholarly journals FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

2020 ◽  
Author(s):  
Rawshan Choudhury ◽  
Nadhim Bayatti ◽  
Richard Scharff ◽  
Ewa Szula ◽  
Viranga Tilakaratna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Rpe Cells ◽  
Age Related

AbstractRetinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. The results obtained from primary RPE cells were replicated using the hTERT-RPE cell line. RNAseq expression analysis of hTERT-RPE cells bound to FHL-1 showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death showed hTERT-RPE cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

scholarly journals Control of Complement Activation by the Long Pentraxin PTX3: Implications in Age-Related Macular Degeneration

2020 ◽  
Vol 11 ◽  
Author(s):  
Matteo Stravalaci ◽  
Francesca Davi ◽  
Raffaella Parente ◽  
Marco Gobbi ◽  
Barbara Bottazzi ◽  
...  
Keyword(s):  
Macular Degeneration ◽  
Complement Activation ◽  
Hot Spot ◽  
Alternative Pathway ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor ◽  
Rpe Cells ◽  
Age Related

Dysregulation of the complement system is central to age-related macular degeneration (AMD), the leading cause of blindness in the developed world. Most of the genetic variation associated with AMD resides in complement genes, with the greatest risk associated with polymorphisms in the complement factor H (CFH) gene; factor H (FH) is the major inhibitor of the alternative pathway (AP) of complement that specifically targets C3b and the AP C3 convertase. Long pentraxin 3 (PTX3) is a soluble pattern recognition molecule that has been proposed to inhibit AP activation via recruitment of FH. Although present in the human retina, if and how PTX3 plays a role in AMD is still unclear. In this work we demonstrated the presence of PTX3 in the human vitreous and studied the PTX3-FH-C3b crosstalk and its effects on complement activation in a model of retinal pigment epithelium (RPE). RPE cells cultured in inflammatory AMD-like conditions overexpressed the PTX3 protein, and up-regulated AP activating genes. PTX3 bound RPE cells in a physiological setting, however this interaction was reduced in inflammatory conditions, whereby PTX3 had no complement-inhibiting activity on inflamed RPE. However, on non-cellular surfaces, PTX3 formed a stable ternary complex with FH and C3b that acted as a “hot spot” for complement inhibition. Our findings suggest a protective role for PTX3 in response to complement dysregulation in AMD and point to a novel mechanism of complement regulation by this pentraxin with potential implications in pathology and pharmacology of AMD.

scholarly journals Complement Factor H Loss in RPE Cells Causes Retinal Degeneration in a Human RPE-Porcine Retinal Explant Co-Culture Model

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1621
Author(s):  
Angela Armento ◽  
Aparna Murali ◽  
Julia Marzi ◽  
Ana C Almansa-Garcia ◽  
Blanca Arango-Gonzalez ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Cell Activation ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Complement Factor ◽  
Rpe Cells ◽  
Age Related ◽  
Culture Model ◽  
Retinal Explants

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

scholarly journals Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Chih-Chao Chang ◽  
Tien-Yi Huang ◽  
Hsin-Yuan Chen ◽  
Tsui-Chin Huang ◽  
Li-Chun Lin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Critical Role ◽  
Age Related Macular Degeneration ◽  
Cell Degeneration ◽  
Rpe Cells ◽  
Age Related ◽  
Human Rpe Cells

Age-related macular degeneration (AMD) affects the retinal macula and results in loss of vision, and AMD is the primary cause of blindness and severe visual impairment among elderly people worldwide. AMD is characterized by the accumulation of drusen in the Bruch’s membrane and dysfunction of retinal pigment epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD remains unclear, and no effective treatment exists. Accumulating evidence indicates that oxidative stress plays a critical role in RPE cell degeneration and AMD. Melatonin is an antioxidant that scavenges free radicals, and it has anti-inflammatory, antitumor, and antiangiogenic effects. This study investigated the antioxidative, antiapoptotic, and autophagic effects of melatonin on oxidative damage to RPE cells. We used hydrogen peroxide (H2O2) to stimulate reactive oxygen species production to cause cell apoptosis in ARPE-19 cell lines. Our findings revealed that treatment with melatonin significantly inhibited H2O2-induced RPE cell damage, decreased the apoptotic rate, increased the mitochondrial membrane potential, and increased the autophagy effect. Furthermore, melatonin reduced the Bax/Bcl-2 ratio and the expression levels of the apoptosis-associated proteins cytochrome c and caspase 7. Additionally, melatonin upregulated the expression of the autophagy-related proteins LC3-II and Beclin-1 and downregulated the expression of p62. Thus, melatonin’s effects on autophagy and apoptosis can protect against H2O2-induced oxidative damage in human RPE cells. Melatonin may have multiple protective effects on human RPE cells against H2O2-induced oxidative damage.

scholarly journals Interlink between Inflammation and Oxidative Stress in Age-Related Macular Degeneration: Role of Complement Factor H

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 763
Author(s):  
Sara Romero-Vazquez ◽  
Víctor Llorens ◽  
Alba Soler-Boronat ◽  
Marc Figueras-Roca ◽  
Alfredo Adan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Risk Factors ◽  
Macular Degeneration ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Low Grade ◽  
Complement Factor ◽  
Age Related ◽  
And Oxidative Stress

Age-related macular degeneration (AMD) heads the list of legal blindness among the elderly population in developed countries. Due to the complex nature of the retina and the variety of risk factors and mechanisms involved, the molecular pathways underlying AMD are not yet fully defined. Persistent low-grade inflammation and oxidative stress eventually lead to retinal pigment epithelium dysfunction and outer blood–retinal barrier (oBRB) breakdown. The identification of AMD susceptibility genes encoding complement factors, and the presence of inflammatory mediators in drusen, the hallmark deposits of AMD, supports the notion that immune-mediated processes are major drivers of AMD pathobiology. Complement factor H (FH), the main regulator of the alternative pathway of the complement system, may have a key contribution in the pathogenesis of AMD as it is able to regulate both inflammatory and oxidative stress responses in the oBRB. Indeed, genetic variants in the CFH gene account for the strongest genetic risk factors for AMD. In this review, we focus on the roles of inflammation and oxidative stress and their connection with FH and related proteins as regulators of both phenomena in the context of AMD.

scholarly journals Complement factor H deficiency combined with smoking promotes retinal degeneration in a novel mouse model

2021 ◽  
pp. 153537022110522
Author(s):  
Liwen Feng ◽  
Kailai Nie ◽  
Qing Huang ◽  
Wei Fan
Keyword(s):  
Macular Degeneration ◽  
Cigarette Smoke ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Complement Factor H ◽  
Complement Factor ◽  
Wild Type ◽  
Preclinical Research ◽  
Age Related

Age-related macular degeneration is the leading cause of blindness in the elderly. The Y402H polymorphism in complement factor H promotes disease-like pathogenesis, and a Cfh+/− murine model can replicate this phenotype, but only after two years. We reasoned that by combining CFH deficiency with cigarette smoke exposure, we might be able to accelerate disease progression to facilitate preclinical research in this disease. Wild-type and Cfh+/− mice were exposed to nose-only cigarette smoke for three months. Retinal tissue morphology and visual function were evaluated by optical coherence tomography, fundus photography and autofluorescence, and electroretinogram. Retinal pigment epithelial cell phenotype and ultrastructure were evaluated by immunofluorescence staining and transmission electron microscopy. Cfh+/− smoking mice showed a dome-like protruding lesion at the ellipsoid zone (drusen-like deposition), many retinal hyper-autofluorescence spots, and a marked decrease in A- and B-wave amplitudes. Compared with non-smoking mice, wild-type and Cfh+/− smoking mice showed sub-retinal pigment epithelium complement protein 3 deposition, activation of microglia, metabolic waste accumulation, and impairment of tight junctions. Microglia cells migrated into the photoreceptor outer segment layer in Cfh+/− smoking mice showed increased activation. Our results suggest that exposing Cfh+/− mice to smoking leads to earlier onset of age-related macular degeneration than in other animal models, which may facilitate preclinical research into the pathophysiology and treatment of this disease.

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

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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