LRG1 promotes metastatic colorectal cancer growth through HER3 signaling.

BACKGROUND AND AIMS: Therapy failure in patients with metastatic colorectal cancer (mCRC, ∼80% occur in the liver) remains an overarching challenge. Preclinical studies demonstrated that HER3 promotes CRC cell survival, but therapies blocking the neuregulin-induced canonical HER3 signaling have made little impact in the clinic. Recent studies suggest that the liver microenvironment promotes CRC growth by activating HER3 in a neuregulin-independent fashion, thus elucidation of these mechanisms may reveal new strategies for treating patients with mCRC. METHODS: Patient-derived primary liver endothelial cells (ECs) were used to interrogate EC-CRC crosstalk. We conducted proteomic analysis to identify EC-secreted factor(s) that triggers non-canonical HER3 activation in CRC, and determined the subsequent effects on mCRC using diverse murine mCRC models. In vitro studies with genetic and pharmacological interventions were used to map the non-canonical HER3 pathway. RESULTS: We demonstrated that EC-secreted leucine-rich alpha-2-glycoprotein 1 (LRG1) directly binds and activates HER3 and promotes CRC growth distinct from neuregulin, the canonical HER3 ligand. Blocking host-derived LRG1 by gene knockout or a neutralizing antibody impaired mCRC outgrowth in the liver and prolonged mouse survival. We identified protein synthesis activated by the PI3K-PDK1-RSK-eIF4B axis as the biologically relevant signaling cascade downstream of the LRG1-HER3 interaction, which was not blocked by conventional HER3-specific antibodies that failed in prior clinical trials. CONCLUSIONS: LRG1 is a novel HER3 ligand and mediates liver-mCRC crosstalk. The LRG1-HER3 signaling axis is distinct from canonical HER3 signaling and represents a new therapeutic opportunity to treat patients with mCRC, and potentially other types of liver metastases.

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.

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.

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 promotes angiogenesis by modulating endothelial TGF-ß signalling.

Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequence of inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-rich alpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growth factor-ß1 (TGF-ß1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGF-ß accessory receptor endoglin, which, in the presence of TGF-ß1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a new regulator of angiogenesis that mediates its effect by modulating TGF-ß signalling.

Retinal Pigment Epithelial Cells Mitigate the Effects of Complement Attack by Endocytosis of C5b-9.

Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration. The alternative pathway of complement activation is strongly implicated in RPE cell dysfunction and loss in age-related macular degeneration; therefore, it is critical that RPE cells use molecular strategies to mitigate the potentially harmful effects of complement attack. We show that the terminal complement complex C5b-9 assembles rapidly on the basal surface of cultured primary porcine RPE cells but disappears over 48 h without any discernable adverse effects on the cells. However, in the presence of the dynamin inhibitor dynasore, C5b-9 was almost completely retained at the cell surface, suggesting that, under normal circumstances, it is eliminated via the endocytic pathway. In support of this idea, we observed that C5b-9 colocalizes with the early endosome marker EEA1 and that, in the presence of protease inhibitors, it can be detected in lysosomes. Preventing the endocytosis of C5b-9 by RPE cells led to structural defects in mitochondrial morphology consistent with cell stress. We conclude that RPE cells use the endocytic pathway to prevent the accumulation of C5b-9 on the cell surface and that processing and destruction of C5b-9 by this route are essential for RPE cell survival.

A new role for annexin A11 in the early secretory pathway via stabilizing Sec31A protein at the endoplasmic reticulum exit sites (ERES).

Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.

High Podocalyxin levels promote cell viability partially through up-regulation of Annexin A2.

Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein that is up-regulated in various types of tumors and whose expression levels positively correlate with tumor grade. We previously found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells (NSPs). Here we investigated the effects of elevated Podxl levels in these cells. NSPs overexpressing Podxl did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumor initiation. However, Podxl overexpression had a positive effect on cell number, sphere formation and cell viability, indicating that it might in this way contribute to the development and/or maintenance of tumors. Proteome analyses of Podxl-overexpressing and control NSPs revealed increased levels of Annexin A2 (ANXA2). We also found increased transcript levels, indicating that PODXL stimulates expression of the Anxa2 gene. Lack of Anxa2 in Podxl-overexpressing NSPs resulted in reduced viability of these cells, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased ANXA2 levels. Finally, our data indicate that Podxl overexpression activates the MAP kinase (MAPK) pathway which in turn up-regulates Anxa2 expression. Our data indicate a novel molecular connection between PODXL and ANXA2: both exert pro-survival effects in NSPs, and PODXL positively regulates ANXA2 expression through the MAPK pathway.

Annexin A6 regulates interleukin-2-mediated T-cell proliferation.

Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4(+) T cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment.

Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Complement Stimulates Retinal Pigment Epithelial Cells to Undergo Pro-Inflammatory Changes.

BACKGROUND/AIMS: We examined the effect of human complement sera (HCS) on retinal pigment epithelial (RPE) cells with respect to pro-inflammatory mediators relevant in early age-related macular degeneration (AMD). METHODS: RPE cells were treated with complement-containing HCS or with heat-inactivated (HI) HCS or C7-deficient HCS as controls. Cells were analysed for C5b-9 using immunocytochemistry and flow cytometry. Interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were quantified by ELISA and RT-PCR. Tumour necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were analysed by Western blotting. The intracellular distribution of nuclear factor (NF)-x03BA;B was investigated by immunofluorescence. RESULTS: A concentration-dependent increased staining for C5b-9 but no influence on cell viability was observed after HCS treatment. ELISA and RT-PCR analysis revealed elevated secretion and expression of IL-6, IL-8, and MCP-1. Western blot analysis showed a concentration-dependent increase in ICAM-1, VCAM-1, and TNF-α in response to HCS, and immunofluorescence staining revealed nuclear translocation of NF-x03BA;B. CONCLUSION: This study suggests that complement stimulates NF-x03BA;B activation in RPE cells that might further create a pro-inflammatory environment. All these factors together may support early AMD development.

Topical delivery of Avastin to the posterior segment of the eye in vivo using annexin A5-associated liposomes.

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.

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.

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes.

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.

Apelin is required for non-neovascular remodeling in the retina.

Retinal pathologies are frequently accompanied by retinal vascular responses, including the formation of new vessels by angiogenesis (neovascularization). Pathological vascular changes may also include less well characterized traits of vascular remodeling that are non-neovascular, such as vessel pruning and the emergence of dilated and tortuous vessel phenotypes (telangiectasis). The molecular mechanisms underlying neovascular growth versus non-neovascular remodeling are poorly understood. We therefore undertook to identify novel regulators of non-neovascular remodeling in the retina by using the dystrophic Royal College of Surgeons (RCS) rat and the retinal dystrophy 1 (RD1) mouse, both of which display pronounced non-neovascular remodeling. Gene expression profiling of isolated retinal vessels from these mutant rodent models and wild-type controls revealed 60 differentially expressed genes. These included the genes for apelin (Apln) and for its receptor (Aplnr), both of which were strongly up-regulated in the mutants. Crossing RD1 mice into an Apln-null background substantially reduced vascular telangiectasia. In contrast, Apln gene deletion had no effect in two models of neovascular pathology [laser-induced choroidal neovascularization and the very low density lipoprotein receptor (Vldlr)-knockout mouse]. These findings suggest that in these models apelin has minimal effect on sprouting retinal angiogenesis, but contributes significantly to pathogenic non-neovascular remodeling.

Genetic ablation of retinal pigment epithelial cells reveals the adaptive response of the epithelium and impact on photoreceptors.

The retinal pigment epithelium (RPE) plays a critical role in the maintenance of the outer retina. RPE cell death or dysfunction drives the pathophysiology of many retinal diseases, but the physiological response of the retina to RPE cell loss is poorly understood, mainly because of the absence of suitable experimental models. Here, we generated a transgenic mouse in which an inducible Cre recombinase is expressed exclusively in the RPE under the control of the monocarboxylate transporter 3 gene promoter (RPE(CreER)). This was crossed with a transgenic mouse harboring a diphtheria toxin A (DTA) chain gene rendered transcriptionally silent by a floxed stop sequence. We show that activation of DTA in the double transgenic mouse (RPE(CreER)/DTA) led to 60-80% RPE cell death, with surviving cells maintaining the integrity of the monolayer by increasing their size. Despite the apparent morphological normality of the enlarged RPE cells in the RPE(CreER)/DTA mice, functional analysis revealed significant deficits on electroretinography, and retinal histopathology showed regions of photoreceptor rosetting and degeneration although with retention of a normal vascular network. Our study reveals that whilst the RPE monolayer has a remarkable intrinsic capacity to cope with cellular attrition, specific aspects of RPE multifunctionality essential for photoreceptor survival are compromised. The RPE(CreER)/DTA mouse offers advantages over models that employ chemical or mechanical strategies to kill RPE cells, and should be useful for the development and evaluation of RPE-based therapies, such as stem cell transplantation.

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.

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.

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.