The disruptive role of LRG1 on the vasculature and perivascular microenvironment.

The establishment of new blood vessels, and their subsequent stabilization, is a critical process that facilitates tissue growth and organ development. Once established, vessels need to diversify to meet the specific needs of the local tissue and to maintain homeostasis. These processes are tightly regulated and fundamental to normal vessel and tissue function. The mechanisms that orchestrate angiogenesis and vessel maturation have been widely studied, with signaling crosstalk between endothelium and perivascular cells being identified as an essential component. In disease, however, new vessels develop abnormally, and existing vessels lose their specialization and function, which invariably contributes to disease progression. Despite considerable research into the vasculopathic mechanisms in disease, our knowledge remains incomplete. Accordingly, the identification of angiocrine and angiopathic molecules secreted by cells within the vascular microenvironment, and their effect on vessel behaviour, remains a major research objective. Over the last decade the secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1), has emerged as a significant vasculopathic molecule, stimulating defective angiogenesis, and destabilizing the existing vasculature mainly, but not uniquely, by altering both canonical and non-canonical TGF-ß signaling in a highly cell and context dependent manner. Whilst LRG1 does not possess any overt homeostatic role in vessel development and maintenance, growing evidence provides a compelling case for LRG1 playing a pleiotropic role in disrupting the vasculature in many disease settings. Thus, LRG1 has now been reported to damage vessels in various disorders including cancer, diabetes, chronic kidney disease, ocular disease, and lung disease and the signaling processes that drive this dysfunction are being defined. Moreover, therapeutic targeting of LRG1 has been widely proposed to re-establish a quiescent endothelium and normalized vasculature. In this review, we consider the current status of our understanding of the role of LRG1 in vascular pathology, and its potential as a therapeutic target.

Structural basis of human LRG1 recognition by Magacizumab, a humanized monoclonal antibody with therapeutic potential.

The formation of new dysfunctional blood vessels is a crucial stage in the development of various conditions such as macular degeneration, diabetes, cardiovascular disease, neurological disease and inflammatory disorders, as well as during tumor growth, eventually contributing to metastasis. An important factor involved in pathogenic angiogenesis is leucine-rich α-2-glycoprotein 1 (LRG1), the antibody blockade of which has been shown to lead to a reduction in both choroidal neovascularization and tumor growth in mouse models. In this work, the structural interactions between the LRG1 epitope and the Fab fragment of Magacizumab, a humanized function-blocking IgG4 against LRG1, are analysed, determining its specific binding mode and the key residues involved in LRG1 recognition. Based on these structural findings, a series of mutations are suggested that could be introduced into Magacizumab to increase its affinity for LRG1, as well as a model of the entire Fab-LRG1 complex that could enlighten new strategies to enhance affinity, consequently leading towards an even more efficient therapeutic.

Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway.

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1-/- explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

LRG1 as a novel therapeutic target in eye disease.

Retinal and choroidal diseases are major causes of blindness and visual impairment in the developed world and on the rise due to an ageing population and diabetes epidemic. Standard of care is centred around blockade of vascular endothelial growth factor (VEGF), but despite having halved the number of patients losing sight, a high rate of patient non-response and loss of efficacy over time are key challenges. Dysregulation of vascular homoeostasis, coupled with fibrosis and inflammation, are major culprits driving sight-threatening eye diseases. Improving our knowledge of these pathological processes should inform the development of new drugs to address the current clinical challenges for patients. Leucine-rich α-2 glycoprotein 1 (LRG1) is an emerging key player in vascular dysfunction, inflammation and fibrosis. Under physiological conditions, LRG1 is constitutively expressed by the liver and granulocytes, but little is known about its normal biological function. In pathological scenarios, such as diabetic retinopathy (DR) and neovascular age-related macular degeneration (nvAMD), its expression is ectopically upregulated and it acquires a much better understood pathogenic role. Context-dependent modulation of the transforming growth-factor ß (TGFß) pathway is one of the main activities of LRG1, but additional roles have recently been emerging. This review aims to highlight the clinical and pre-clinical evidence for the pathogenic contribution of LRG1 to vascular retinopathies, as well as extrapolate from other diseases, functions which may be relevant to eye disease. Finally, we will provide a current update on the development of anti-LRG1 therapies for the treatment of nvAMD.

LRG1: an emerging player in disease pathogenesis.

The secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1) was first described as a key player in pathogenic ocular neovascularization almost a decade ago. Since then, an increasing number of publications have reported the involvement of LRG1 in multiple human conditions including cancer, diabetes, cardiovascular disease, neurological disease, and inflammatory disorders. The purpose of this review is to provide, for the first time, a comprehensive overview of the LRG1 literature considering its role in health and disease. Although LRG1 is constitutively expressed by hepatocytes and neutrophils, Lrg1-/- mice show no overt phenotypic abnormality suggesting that LRG1 is essentially redundant in development and homeostasis. However, emerging data are challenging this view by suggesting a novel role for LRG1 in innate immunity and preservation of tissue integrity. While our understanding of beneficial LRG1 functions in physiology remains limited, a consistent body of evidence shows that, in response to various inflammatory stimuli, LRG1 expression is induced and directly contributes to disease pathogenesis. Its potential role as a biomarker for the diagnosis, prognosis and monitoring of multiple conditions is widely discussed while dissecting the mechanisms underlying LRG1 pathogenic functions. Emphasis is given to the role that LRG1 plays as a vasculopathic factor where it disrupts the cellular interactions normally required for the formation and maintenance of mature vessels, thereby indirectly contributing to the establishment of a highly hypoxic and immunosuppressive microenvironment. In addition, LRG1 has also been reported to affect other cell types (including epithelial, immune, mesenchymal and cancer cells) mostly by modulating the TGFß signalling pathway in a context-dependent manner. Crucially, animal studies have shown that LRG1 inhibition, through gene deletion or a function-blocking antibody, is sufficient to attenuate disease progression. In view of this, and taking into consideration its role as an upstream modifier of TGFß signalling, LRG1 is suggested as a potentially important therapeutic target. While further investigations are needed to fill gaps in our current understanding of LRG1 function, the studies reviewed here confirm LRG1 as a pleiotropic and pathogenic signalling molecule providing a strong rationale for its use in the clinic as a biomarker and therapeutic target.

Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis.

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body's vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor­ß (TGFß) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)­dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) as a novel ADC target.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) is present abundantly in the microenvironment of many tumours where it contributes to vascular dysfunction, which impedes the delivery of therapeutics. In this work we demonstrate that LRG1 is predominantly a non-internalising protein. We report the development of a novel antibody-drug conjugate (ADC) comprising the anti-LRG1 hinge-stabilised IgG4 monoclonal antibody Magacizumab coupled to the anti-mitotic payload monomethyl auristatin E (MMAE) via a cleavable dipeptide linker using the site-selective disulfide rebridging dibromopyridazinedione (diBrPD) scaffold. It is demonstrated that this ADC retains binding post-modification, is stable in serum and effective in in vitro cell studies. We show that the extracellular LRG1-targeting ADC provides an increase in survival in vivo when compared against antibody alone and similar anti-tumour activity when compared against standard chemotherapy, but without undesired side-effects. LRG1 targeting through this ADC presents a novel and effective proof-of-concept en route to improving the efficacy of cancer therapeutics.

LRG1 Expression Is Elevated in the Eyes of Patients with Neovascular Age-Related Macular Degeneration.

Leucine-rich a-2-glycoprotein 1 (LRG1) is a candidate therapeutic target for treating the neovascular form of age-related macular degeneration (nvAMD). In this study we examined the expression of LRG1 in eyes of nvAMD patients. Choroidal neovascular membranes (CNVMs) from patients who underwent submacular surgery for retinal pigment epithelium-choroid graft transplantation were collected from 5 nvAMD patients without any prior intravitreal anti-VEGF injection, and from six patients who received intravitreal anti-VEGF injections before surgery. As controls free of nvAMD, retina sections were obtained from the eyes resected from a patient with lacrimal sac tumor and from a patient with neuroblastoma. CNVMs were immunostained for CD34, LRG1, and α-smooth muscle actin (α-SMA). Aqueous humor samples were collected from 58 untreated-naïve nvAMD patients prior to the intravitreal injection of anti-VEGF and 51 age-matched cataract control patients, and LRG1 concentration was measured by ELISA. The level of LRG1 immunostaining is frequently high in both the endothelial cells of the blood vessels, and myofibroblasts in the surrounding tissue of CNVMs of treatment-naïve nvAMD patients. Furthermore, the average concentration of LRG1 was significantly higher in the aqueous humor of nvAMD patients than in controls. These observations provide a strong experimental basis and scientific rationale for the progression of a therapeutic anti-LRG1 monoclonal antibody into clinical trials with patients with nvAMD.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency.

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

A Multifunctional Role of Leucine-Rich α-2-Glycoprotein 1 in Cutaneous Wound Healing Under Normal and Diabetic Conditions.

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFß type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.

Functional Evaluation of AMD-Associated Risk Variants of Complement Factor B.

Purpose: The 32W and 32Q variants of complement factor B (CFB) are associated with reduced risk of developing neovascular age-related macular degeneration (AMD) compared with the common 32R allele. The objective of this study was to determine if the most protective R32Q variant affects the neovascular process in a manner consistent with the reported reduced disease association. Methods: The 32R, 32W, and 32Q human CFB variants were expressed in human embryonic kidney 293T cells and purified from culture supernatant. The ex vivo mouse fetal metatarsal explant model was used to investigate the effect of these three human CFB variants on angiogenesis. Metatarsal bones were isolated from mouse embryos and cultured in the presence of the three CFB variants, and angiogenesis was measured following immunostaining of fixed samples. ELISAs were used to quantify C3 and VEGF protein levels in metatarsal culture and quantitative PCR to measure Cfb, C3, and Vegf expression. Results: We show here that the three CFB variants have different biological activities in the mouse metatarsal assay, with CFBR32 exhibiting significantly greater angiogenic activity than CFBQ32 or CFBW32, which were broadly similar. We also observed differences in macrophage phenotype with these two variants that may contribute to their activities in this experimental model. Conclusions: We have demonstrated that the biological activities of CFBR32, CFBW32, and CFBQ32 are consistent with their AMD risk association, and we provide functional evidence of roles for these variants in angiogenesis that may be relevant to the pathogenesis of the neovascular form of AMD.

Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells.

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and ß-catenin, which was reduced in the presence of activated TGF-ß1. TGF-ß1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced ß-catenin translocation and GSK-3ß phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active ß-catenin and GSK-3ß phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration.

Inhibition of somatosensory mechanotransduction by annexin A6.

Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8.

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

Regulation of C3 Activation by the Alternative Complement Pathway in the Mouse Retina.

The purpose of this study was to examine the retinas of mice carrying hemizygous and null double deletions of Cfb-/- and Cfh-/-, and to compare these with the single knockouts of Cfb, Cfh and Cfd. Retinas were isolated from wild type (WT), Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfh-/-/Cfb+/-, Cfb-/-, Cfh-/- Cfd-/-, and Cfd+/- mice. Complement proteins were evaluated by western blotting, ELISA and immunocytochemistry, and retinal morphology was assessed using toluidine blue stained semi-thin sections. WT mice showed staining for C3 and its breakdown products in the retinal vasculature and the basal surface of the retinal pigment epithelium (RPE). Cfb-/- mice exhibited a similar C3 staining pattern to WT in the retinal vessels but a decrease in C3 and its breakdown products at the basal surface of the RPE. Deletion of both Cfb and Cfh restored C3 to levels similar to those observed in WT mice, however this reversal of phenotype was not observed in Cfh-/-/Cfb+/- or Cfb-/-/Cfh+/- mice. Loss of CFD caused an increase in C3 and a decrease in C3 breakdown products along the basal surface of the RPE. Overall the retinal morphology and retinal vasculature did not appear different across the various genotypes. We observed that C3 accumulates at the basal RPE in Cfb-/-, Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfd-/- and WT mice, but is absent in Cfh-/- and Cfh-/-/Cfb+/- mice, consistent with its consumption in the serum of mice lacking CFH when CFB is present. C3 breakdown products along the surface of the RPE were either decreased or absent when CFB, CFH or CFD was deleted or partially deleted.