ABSTRACT
Alterations in the structure and composition of Bruch's membrane (BrM) and loss of retinal pigment epithelial (RPE) cells are associated with various ocular diseases, notably age-related macular degeneration (AMD) as well as several inherited retinal diseases (IRDs). We explored the influence of stiffness as a major BrM characteristic on the RPE transcriptome and morphology. ARPE-19 cells were plated on soft ( E = 30 kPa ) or stiff ( E = 80 kPa ) polyacrylamide gels (PA gels) or standard tissue culture plastic (TCP). Next-generation sequencing (NGS) data on differentially expressed small RNAs (sRNAs) and messenger RNAs (mRNAs) were validated by qPCR, immunofluorescence or western blotting. The microRNA (miRNA) fraction of sRNAs grew with substrate stiffness and distinct miRNAs such as miR-204 or miR-222 were differentially expressed. mRNA targets of differentially expressed miRNAs were stably expressed, suggesting a homeostatic effect of miRNAs. mRNA transcription patterns were substrate stiffness-dependent, including components of Wnt/beta-catenin signaling, Microphthalmia-Associated Transcription Factor (MITF) and Dicer. These findings highlight the relevance of mechanical properties of the extracellular matrix (ECM) in cell culture experiments, especially those focusing on ECM-related diseases, such as AMD.
Subject(s)
Macular Degeneration , MicroRNAs , Humans , Bruch Membrane , Extracellular Matrix/genetics , Cell Culture Techniques , RNA, Messenger/genetics , MicroRNAs/genetics , Retinal Pigment EpitheliumABSTRACT
Since the groundbreaking approval of the first anti-VEGF therapy in 2004, the retinal therapeutics field has undergone a remarkable transformation, witnessing a surge in novel, disease-modifying therapeutics for a broad spectrum of retinal diseases, extending beyond exudative VEGF-driven conditions. The surge in scientific advancement and the pressing, unmet, medical need have captured the attention of venture capital investors, who have collectively invested close to $10 billion in research and development of new retinal therapeutics between 2004 and 2023. Notably, the field of exudative diseases has gradually shifted away from trying to outcompete anti-VEGF therapeutics towards lowering the overall treatment burden by reducing injection frequency. Simultaneously, a new era has emerged in the non-exudative field, targeting prevalent conditions like dry AMD and rare indications such as Retinitis pigmentosa. This has led to promising drug candidates in development, culminating in the landmark approval of Luxturna for a rare form of Retinitis pigmentosa. The validation of new mechanisms, such as the complement pathway in dry AMD has paved the way for the approvals of Syvovre (Apellis) and Izervay (Iveric/Astellas), marking the first two therapies for this condition. In this comprehensive review, we share our view on the cumulative lessons from the past two decades in developing retinal therapeutics, covering both positive achievements and challenges. We also contextualize the investments, strategic partnering deals, and acquisitions of biotech companies, pharmaceutical companies venture capital investors in retinal therapeutics, respectively. Finally, we provide an outlook and potentially a forward-looking roadmap on novel retinal therapeutics, highlighting the emergence of potential new intervention strategies, such as cell-based therapies, gene editing, and combination therapies. We conclude that upcoming developments have the potential to further stimulate venture capital investments, which ultimately could facilitate the development and delivery of new therapies to patients in need.
Subject(s)
Investments , Retinitis Pigmentosa , HumansABSTRACT
The complement system is involved in the pathogenesis of several ocular diseases, providing a rationale for the investigation of complement-targeting therapeutics for these conditions. Dry age-related macular degeneration, as characterised by geographic atrophy (GA), is currently the most active area of research for complement-targeting therapeutics, with a complement C3 inhibitor approved in the United States earlier this year marking the first approved therapy for GA. This review discusses the role of complement in ocular disease, provides an overview of the complement-targeting agents currently under development for ocular conditions, and reflects on the lessons that can be learned from the preclinical investigations and clinical trials conducted in this field to date.
Subject(s)
Geographic Atrophy , Macular Degeneration , Humans , Macular Degeneration/drug therapy , Eye , Geographic Atrophy/drug therapy , Geographic Atrophy/etiology , Geographic Atrophy/pathologyABSTRACT
Slow-release delivery systems are needed to ensure long-term sustained treatments for retinal diseases such as age-related macular degeneration and diabetic retinopathy, which are currently treated with anti-angiogenic agents that require frequent intraocular injections. These can cause serious co-morbidities for the patients and are far from providing the adequate drug/protein release rates and required pharmacokinetics to sustain prolonged efficacy. This review focuses on the use of hydrogels, particularly on temperature-responsive hydrogels as delivery vehicles for the intravitreal injection of retinal therapies, their advantages and disadvantages for intraocular administration, and the current advances in their use to treat retinal diseases.
ABSTRACT
Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of complement factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the predominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro- and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macrophages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-γ or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. Moreover, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture supernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation.
Subject(s)
Complement Activation , Complement Factor H , Humans , Complement Factor H/genetics , Myeloid Cells/metabolism , RNA, Messenger , Muscle Proteins , Intracellular Signaling Peptides and Proteins , LIM Domain ProteinsABSTRACT
Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.
Subject(s)
Cytomegalovirus Infections , DNA, Viral , Humans , Dependovirus/genetics , Genetic Vectors/genetics , Toll-Like Receptor 9/genetics , Immunity, Innate , Dendritic Cells , Deoxyribonucleases/genetics , Transduction, GeneticABSTRACT
Despite comprehensive research efforts over the last decades, the pathomechanisms of age-related macular degeneration (AMD) remain far from being understood. Large-scale genome wide association studies (GWAS) were able to provide a defined set of genetic aberrations which contribute to disease risk, with the strongest contributors mapping to distinct regions on chromosome 1 and 10. While the chromosome 1 locus comprises factors of the complement system with well-known functions, the role of the 10q26-locus in AMD-pathophysiology remains enigmatic. 10q26 harbors a cluster of three functional genes, namely PLEKHA1, ARMS2 and HTRA1, with most of the AMD-associated genetic variants mapping to the latter two genes. High linkage disequilibrium between ARMS2 and HTRA1 has kept association studies from reliably defining the risk-causing gene for long and only very recently the genetic risk region has been narrowed to ARMS2, suggesting that this is the true AMD gene at this locus. However, genetic associations alone do not suffice to prove causality and one or more of the 14 SNPs on this haplotype may be involved in long-range control of gene expression, leaving HTRA1 and PLEKHA1 still suspects in the pathogenic pathway. Both, ARMS2 and HTRA1 have been linked to extracellular matrix homeostasis, yet their exact molecular function as well as their role in AMD pathogenesis remains to be uncovered. The transcriptional regulation of the 10q26 locus adds an additional level of complexity, given, that gene-regulatory as well as epigenetic alterations may influence expression levels from 10q26 in diseased individuals. Here, we provide a comprehensive overview on the 10q26 locus and its three gene products on various levels of biological complexity and discuss current and future research strategies to shed light on one of the remaining enigmatic spots in the AMD landscape.
Subject(s)
Macular Degeneration , Serine Endopeptidases , Humans , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Genome-Wide Association Study , Proteins/genetics , Proteins/metabolism , Macular Degeneration/genetics , Macular Degeneration/metabolism , Gene Expression Regulation , Polymorphism, Single Nucleotide , Genotype , Genetic Predisposition to DiseaseABSTRACT
Age-related macular degeneration (AMD) is a major cause of vision impairment in the Western World, and with the aging world population, its incidence is increasing. As of today, for the majority of patients, no treatment exists. Multiple genetic and biochemical studies have shown a strong association with components in the complement system and AMD, and evidence suggests a major role of remodeling of the extracellular matrix underlying the outer blood/retinal barrier. As part of the innate immune system, the complement cascade acts as a first-line defense against pathogens, and upon activation, its amplification loop ensures a strong, rapid, and sustained response. Excessive activation, however, can lead to host tissue damage and cause complement-associated diseases like AMD. AMD patients present with aberrant activation of the alternative pathway, especially in ocular tissues but also on a systemic level. Here, we review the latest findings of complement activation in AMD, and we will discuss in vivo observations made in human tissue, cellular models, the potential synergy of different AMD-associated pathways, and conclude on current clinical trials and the future outlook.
Subject(s)
Complement System Proteins , Macular Degeneration , Humans , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/therapy , Complement Activation , Aging , Immune System/metabolismABSTRACT
The SARS-CoV-2 virus continues to cause significant morbidity and mortality worldwide from COVID-19. One of the major challenges of patient management is the broad range of symptoms observed. While the majority of individuals experience relatively mild disease, a significant minority of patients require hospitalisation, with COVID-19 still proving fatal for some. As such, there remains a desperate need to better understand what drives this severe disease, both in terms of the underlying biology, but also to potentially predict at diagnosis which patients are likely to require further interventions, thus enabling better outcomes for both patients and healthcare systems. Several lines of evidence have pointed to dysregulation of the complement cascade as a major factor in severe COVID-19 outcomes. How this is underpinned mechanistically is not known. Here, we have focussed on the role of the soluble complement regulators Complement Factor H (FH), its splice variant Factor H-like 1 (FHL-1) and five Factor H-Related proteins (FHR1-5). Using a targeted mass spectrometry approach, we quantified these proteins in a cohort of 188 plasma samples from controls and SARS-CoV-2 patients taken at diagnosis. This analysis revealed significant elevations in all FHR proteins, but not FH, in patients with more severe disease, particularly FHR2 and FHR5 (FHR2: 1.97-fold, p<0.0001; FHR5: 2.4-fold, p<0.0001). Furthermore, for a subset of 77 SARS-CoV-2 +ve patients we also analysed time course samples taken approximately 28 days post-diagnosis. Here, we see complement regulator levels drop in all individuals with asymptomatic or mild disease, but regulators remain high in those with more severe outcomes, with elevations in FHR2 over baseline levels in this group. These data support the hypothesis that elevation of circulating levels of the FHR family of proteins could predict disease severity in COVID-19 patients, and that the duration of elevation (or lack of immune activation resolution) may be partly responsible for driving poor outcomes in COVID-19.
Subject(s)
COVID-19 , Complement Factor H , Humans , SARS-CoV-2 , Complement Activation , Immunologic FactorsABSTRACT
Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometrybased quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cellspecific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from lowgenetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.
Subject(s)
Chromosomes, Human, Pair 10 , Chromosomes, Human, Pair 1 , Macular Degeneration , Mast Cells , Peptide Hydrolases , Alleles , Choroid/enzymology , Choroid/pathology , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 10/genetics , Humans , Macular Degeneration/genetics , Macular Degeneration/pathology , Mast Cells/pathology , Peptide Hydrolases/genetics , Proteomics , Risk , Tryptases/metabolismABSTRACT
Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.
ABSTRACT
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.
Subject(s)
Complement Factor H , Animals , Macular Degeneration , Retinal Degeneration , SwineABSTRACT
PURPOSE: Glioblastoma (GBM) is an incurable primary brain tumor that has not benefited from immunotherapy to date. More than 90% of GBM expresses the tryptophan (Trp) metabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO). This observation supported the historical hypothesis that IDO suppresses the antitumor immune response solely through a mechanism that requires intratumoral Trp depletion. However, recent findings led us to investigate the alternative hypothesis that IDO suppresses the anti-GBM immune response independent of its association with Trp metabolism. EXPERIMENTAL DESIGN: IDO-deficient GBM cell lines reconstituted with IDO wild-type or IDO enzyme-null cDNA were created and validated in vitro and in vivo. Microarray analysis was conducted to search for genes that IDO regulates, followed by the analysis of human GBM cell lines, patient GBM and plasma, and The Cancer Genome Atlas (TCGA) database. Ex vivo cell coculture assays, syngeneic and humanized mouse GBM models, were used to test the alternative hypothesis. RESULTS: Nonenzymic tumor cell IDO activity decreased the survival of experimental animals and increased the expression of complement factor H (CFH) and its isoform, factor H like protein 1 (FHL-1) in human GBM. Tumor cell IDO increased CFH and FHL-1 expression independent of Trp metabolism. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among patients with glioma. Similar to IDO effects, GBM cell FHL-1 expression increased intratumoral regulatory T cells (Treg) and myeloid-derived suppressor cells while it decreased overall survival in mice with GBM. CONCLUSIONS: Our study reveals a nonmetabolic IDO-mediated enhancement of CFH expression and provides a new therapeutic target for patients with GBM.
Subject(s)
Glioblastoma , Glioma , Animals , Glioma/drug therapy , Humans , Immunosuppression Therapy , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Tryptophan/pharmacologyABSTRACT
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.
Subject(s)
Cytokines/metabolism , Gene Silencing , Macular Degeneration/genetics , Retinal Pigment Epithelium/immunology , Cell Line , Complement Factor H/genetics , Complement System Proteins/metabolism , Cytokines/genetics , Gene Expression Regulation , Humans , Macular Degeneration/immunology , Models, Biological , NF-kappa B/genetics , NF-kappa B/metabolism , Retinal Pigment Epithelium/metabolism , Signal TransductionABSTRACT
Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10-6), FHR-2 (p = 1.47 × 10-4), FHR-3 (p = 1.05 × 10-5) and FHR-4A (p = 1.22 × 10-2) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10-17), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10-3 and p = 2.81 × 10-6, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10-16). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
Subject(s)
Complement C3b Inactivator Proteins/metabolism , Complement Factor H/genetics , Complement System Proteins/metabolism , Genetic Predisposition to Disease , Haplotypes , Macular Degeneration/pathology , Polymorphism, Single Nucleotide , Cohort Studies , Complement C3b Inactivator Proteins/genetics , Complement System Proteins/genetics , Genome-Wide Association Study , Humans , Macular Degeneration/etiology , Macular Degeneration/metabolismABSTRACT
Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.
Subject(s)
Complement C3b Inactivator Proteins/metabolism , Complement Factor H/genetics , Genetic Predisposition to Disease , Macular Degeneration/blood , Polymorphism, Single Nucleotide , Aged , Case-Control Studies , Complement C3b Inactivator Proteins/genetics , Female , Humans , Macular Degeneration/genetics , Macular Degeneration/pathology , Male , Risk FactorsABSTRACT
Retinal 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 ß1 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.
Subject(s)
Integrin alpha5beta1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/metabolism , Muscle Proteins/metabolism , Oxidative Stress , Retinal Pigment Epithelium/metabolism , Cell Communication , Cell Death , Cells, Cultured , Extracellular Matrix/metabolism , Gene Expression Regulation , Humans , Immobilized Proteins/metabolism , Protein Binding , Telomerase/metabolismABSTRACT
Age-related macular degeneration (AMD) is a chronic and progressive degenerative disease of the retina, which culminates in blindness and affects mainly the elderly population. AMD pathogenesis and pathophysiology are incredibly complex due to the structural and cellular complexity of the retina, and the variety of risk factors and molecular mechanisms that contribute to disease onset and progression. AMD is driven by a combination of genetic predisposition, natural ageing changes and lifestyle factors, such as smoking or nutritional intake. The mechanism by which these risk factors interact and converge towards AMD are not fully understood and therefore drug discovery is challenging, where no therapeutic attempt has been fully effective thus far. Genetic and molecular studies have identified the complement system as an important player in AMD. Indeed, many of the genetic risk variants cluster in genes of the alternative pathway of the complement system and complement activation products are elevated in AMD patients. Nevertheless, attempts in treating AMD via complement regulators have not yet been successful, suggesting a level of complexity that could not be predicted only from a genetic point of view. In this review, we will explore the role of complement system in AMD development and in the main molecular and cellular features of AMD, including complement activation itself, inflammation, ECM stability, energy metabolism and oxidative stress.
Subject(s)
Aging , Complement Activation , Complement System Proteins/metabolism , Inflammation/physiopathology , Macular Degeneration/pathology , Oxidative Stress , Animals , Genetic Predisposition to Disease , Humans , Macular Degeneration/immunology , Macular Degeneration/metabolism , Risk FactorsABSTRACT
OBJECTIVE: To quantitatively analyse the number of doctors leaving the paediatric specialty training (ST) programme in the UK, to assist with evidence-based workforce planning. DESIGN: Data were sought on those leaving the UK paediatrics training programme between 2014 and 2019 from Heads of Schools of Paediatrics and Freedom of Information Act requests. SETTING: Retrospective data analysis. OUTCOME MEASURES: Overall attrition rate, attrition rate across level of training, attrition rate across geographical area, recorded reason for leaving. RESULTS: All results must be interpreted with caution due to limitations in record keeping and analysis. The annual attrition rate across all ST levels between 2014 and 2019 is estimated at 3.7%-4.2% (ie, 749-845 trainees may have left the paediatric training programme over 2014-2019). No reason for leaving was recorded for three-quarters of individuals, around 630 doctors. Of those leaving paediatrics, significantly more (χ², p=0.015) did so at ST3 (20.3%) versus the next highest training year, ST2 (13.6%). CONCLUSIONS: This project seems to demonstrate worryingly poor record-keeping of the true attrition rate of paediatric trainees by organisations responsible for workforce planning, including Health Education England, the Royal College of Paediatrics and Child Health and individual paediatric schools across the UK. To allow evidence-based workforce planning for the benefit of UK children, it is vital that accurate records on trainees who leave the training programme are kept and shared across the UK.