Lysyl Oxidase Like 1: Biological roles and regulation

Experimental Eye Research 193 (2020) 107975

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Lysyl Oxidase Like 1: Biological roles and regulation a,∗

a

b

Alison G Greene , Sarah B Eivers , Edward W.J. Dervan , Colm J O'Brien a b

T a,b

, Deborah M Wallace

a

UCD Clinical Research Centre, School of Medicine, University College Dublin, Ireland Dept. of Ophthalmology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, Ireland

A R T I C LE I N FO

A B S T R A C T

Keywords: Lysyl oxidase like 1 Pseudoexfoliation Glaucoma Extracellular matrix Epigenetics

Lysyl Oxidase Like 1 (LOXL1) is a gene that encodes for the LOXL1 enzyme. This enzyme is required for elastin biogenesis and collagen cross-linking, polymerising tropoelastin monomers into elastin polymers. Its main role is in elastin homeostasis and matrix remodelling during injury, fibrosis and cancer development. Because of its vast range of biological functions, abnormalities in LOXL1 underlie many disease processes. Decreased LOXL1 expression is observed in disorders of elastin such as Cutis Laxa and increased expression is reported in fibrotic disease such as Idiopathic Pulmonary Fibrosis. LOXL1 is also downregulated in the lamina cribrosa in pseudoexfoliation glaucoma and genetic variants in the LOXL1 gene have been linked with an increased risk of developing pseudoexfoliation glaucoma and pseudoexfoliation syndrome. However the two major risk alleles are reversed in certain ethnic groups and are present in a large proportion of the normal population, implying complex genetic and environmental regulation is involved in disease pathogenesis. It also appears that the noncoding variants in intron 1 of LOXL1 may be involved in the regulation of LOXL1 expression. Gene alteration may occur via a number of epigenetic and post translational mechanisms such as DNA methylation, long non-coding RNAs and microRNAs. These may represent future therapeutic targets for disease. Environmental factors such as hypoxia, oxidative stress and ultraviolet radiation exposure alter LOXL1 expression, and it is likely a combination of these genetic and environmental factors that influence disease development and progression. In this review, we discuss LOXL1 properties, biological roles and regulation in detail with a focus on pseudoexfoliation syndrome and glaucoma.

1. Introduction 1.1. The lysyl oxidase family The LOXL1 gene is on chromosome 15q24.1 and encodes for the 63kDa LOXL1 enzyme (https://www.uniprot.org/uniprot/Q08397). LOXL1 belongs to the Lysyl oxidase (LOX) family which consists of five members; (LOX and the LOX homologues LOX-like 1 [LOXL1], LOXL2, LOXL3 and LOXL4) (Barker et al., 2012; Csiszar, 2001) (Fig. 1). All the LOX family of enzymes are copper dependent monoamine oxidases with a common conserved C-terminal domain, which contains the active enzyme region, and a variable N-terminal pro-region. The Cterminal contains a conserved tyrosine and lysine residue, which becomes the active Lysyl-tyrosyl-quinone (LTQ) cofactor. The LTQ cofactor is required for enzyme catalytic activity; the oxidation of the amine substrates. The C-terminal domain also contains a copperbinding region and copper binding is required for optimal enzyme activity and LTQ cofactor generation (Gacheru et al., 1990). The amino N-termini vary (Fig. 1). They all contain a signal peptide

∗

when first synthesised as a preproenzyme in the endoplasmic reticulum, this is cleaved during enzymatic activation. LOXL2 – LOXL4 form a subgroup within the family as they all contain 4 scavenger receptor cysteine-rich (SRCR) domains thought to be involved in cell adhesion and protein-protein interactions. LOX and LOXL1 both contain pro-sequences, which enables their secretion as inactive pro-enzymes. These pro-regions interact directly with the extracellular matrix (ECM), directing the deposition of these enzymes onto elastic tissues (Thomassin et al., 2005). LOX and LOXL1 are both involved in collagen and elastin cross-linking. Less is known about the function of the other LOX family members although they likely act in a similar way given the close homology of their catalytic domains (Kagan and Li, 2003; Molnar et al., 2003). Due to the range of LOX family biological functions, abnormalities of LOXL1 expression underlie the development of a number of pathological processes related to an imbalance in ECM synthesis/degradation. The focus of this review is to explore LOXL1, its biological roles and LOXL1 dysfunction in elastosis, fibrosis and autophagy with a particular emphasis on ocular disease. Potential factors of LOXL1

Corresponding author. E-mail address: [email protected] (A.G. Greene).

https://doi.org/10.1016/j.exer.2020.107975 Received 25 September 2019; Received in revised form 12 January 2020; Accepted 13 February 2020 Available online 15 February 2020 0014-4835/ © 2020 Elsevier Ltd. All rights reserved.

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Abbreviations

LTQ MAPK miRNA ncRNA ONH OSA PAI-1 PDR POAG POMP PXF PXFG SNP Smad Sp1 SRCR TAS TGF-β TGF-β1 TGM-2 TIMP-1

ALC Anterior Lens Capsule AP-1 Activator Protein 1 AqH Aqueous Humour BMP-1 Bone morphogenic protein 1 COPD Chronic Obstructive Pulmonary Disease CPAP Continuous Positive Airway Pressure DNMT DNA Methyltransferase ECM Extracellular Matrix FBLN5 Fibulin-5 HIF1α Hypoxia inducible factor 1 alpha HRE Hypoxia Response Element HTF Human Tenon's Fibroblast IOP Intraocular Pressure IPF Idiopathic Pulmonary Fibrosis LTBP-1 Latent TGF binding protein 1 LC Lamina Cribrosa lncRNA Long non-coding RNA LOX Lysyl Oxidase LOXL1 Lysyl Oxidase Like 1 LOXL1-AS1 LOXL1 antisense RNA 1

Lysyl-tyrosyl-quinone Mitogen Activated Protein Kinase microRNA non-coding RNA Optic Nerve Head Obstructive Sleep Apnoea Plasminogen activator inhibitor-1 Proliferative Diabetic Retinopathy Primary Open Angle Glaucoma Proteasome Maturation Protein Pseudoexfoliation syndrome Pseudoexfoliation Glaucoma Single Nucleotide Polymorphism Small Mothers Against Decapentaplegic Specificity protein 1 Scavenger Receptor Cysteine-rich Total Antioxidant Status Transforming Growth Factor beta Transforming Growth Factor beta 1 Transglutaminase 2 Tissue inhibitor of metalloproteinase-1

maturation, although recent research has indicated it may also have a role in type II collagen formation (Alsofi et al., 2016). More specifically, LOXL1 is required for the crosslinking of soluble tropoelastin monomers into growing mature elastin polymers during elastogenesis (Kagan and Li, 2003; Liu et al., 2004). Other matrix-independent roles have also been proposed for LOXL1, such as suppression or promotion of tumorigenesis (Bell et al., 2011; Ji et al., 2007; Tsuchiya et al., 2005; Wu et al., 2007; Zeltz et al., 2019). In vitro studies in rat fetal lung fibroblasts (RFL-6) demonstrated LOXL1 binds to the elastin matrix during elastin deposition (Thomassin et al., 2005). In human glaucoma trabecular meshwork cells, increased LOXL1 activity has been suggested to contribute to increased matrix accumulation (Sethi et al., 2011b). Cultured human Tenon's capsule fibroblasts (HTFs) from pseudoexfoliation syndrome (PXF) have also shown increased expression of elastic proteins in tandem with LOXL1 levels (Zenkel et al., 2011). The main in vitro alterations in LOXL1

dysregulation including genetic variation (SNPs), gene regulation (epigenetics) and environmental factors will be explained (Fig. 2).

1.2. Lysyl Oxidase Like 1 The LOX family are involved in collagen and elastin cross-linking, and matrix remodelling in development, injury, fibrosis and cancer (Csiszar, 2001; Mäki, 2009). LOXL1 is linked primarily to elastin

Fig. 1. The molecular structure of the Lysyl Oxidase Family of enzymes The lysyl oxidase family shares a common highly conserved C-terminal domain that includes a copper-binding motif, a lysyl-tyrosine-quinone cofactor and a cytokine receptor-like domain. These are necessary for protein conformation and enzymatic activity. LOX and LOXL1 contain pro-sequences, which are cleaved by bone morphogenetic protein 1 before their secretion into the extracellular space. LOXL2 to 4 form a subfamily defined by its scavenger receptor cysteine rich domains which are thought to mediate cell adhesion and proteinprotein interactions. Abbreviations: LOX, Lysyl Oxidase, LOXL1,2,3,4, Lysyl Oxidase like 1,2,3,4, C-terminal, Carboxyl-terminal, N-terminal, Amino-terminal, LTQ, Lysyl-tyrosyl-quinone, SRCR, Scavenger Receptor Cytosine Rich Domain.

Fig. 2. Overview of the regulators of LOXL1 expression and the pathology associated with gene dysregulation Factors such as genetics (LOXL1 SNPs), gene regulation (epigenetics, posttranscriptional regulation, non-coding RNAs and environment (hypoxia, oxidative stress, and geographic location) all regulate LOXL1 expression. Dysregulated LOXL1 expression results in pathology driven by fibrosis, autophagy and elastosis. Abbreviations: SNPs, Single Nucleotide Polymorphisms, LOXL1, Lysyl Oxidase like 1. 2

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2008; Liu et al., 2007, 2004; Wiggs et al., 2009). This supports the hypothesis that LOXL1 is critical in ECM maintenance, particularly in relation to elastin.

activity have been observed in relation to pseudoexfoliation glaucoma (PXFG), which will be discussed in detail later in this review. In vivo, LOXL1 mRNA is most abundant in the aorta, placenta, skeletal muscle, kidney and pancreas, suggesting it's required for the structural integrity of these tissues (Kim et al., 1995). LOXL1 has also been shown to be closely associated with elastic fibres in vivo (Lee et al., 2008; Thomassin et al., 2005). LOXL1 knockdown mice develop multiple abnormalities of elastic tissue that include increased skin laxity, abdominal aortic aneurysms, intestinal diverticula, emphysema and pelvic and rectal prolapse (Alsofi et al., 2016; Drewes et al., 2007; Gauthier and Liu, 2017; Lee et al.,

2. LOXL1 dysfunction 2.1. Elastosis LOXL1 is involved in elastin production and maintenance. Elastin is an extracellular matrix protein, which lends resilience to tissues in the body by allowing them to resume their shape after stretching or

Table 1 LOXL1 expression in disease. Disease (ref)

Ophthalmology Keratoconus (Dudakova et al., 2012)

LOXL1 expression

Mechanism

Therapeutic target

Decreased

Restricted LOXL1 distribution and decreased LOXL1 activity in keratoconic corneas 1. Hypermethylation of CpG islands in LOXL1 promoter (Ye et al., 2015) 2. Reduced binding of the transcription factor RXRα (Berner et al., 2017) 3. Alternative splicing of LOXL1 pre-mRNA(Pasutto et al., 2017) 4. Increased degradation via nonsense-mediated decay (Pasutto et al., 2017) Via stimulation by TGFβ via Smad signalling pathways 1. Following treatment with TGFβ or stimulation by UV/oxidative stress/hypoxia 2. Following treatment with Bafilomycin A1 and Spautin −1 showing LOXL1 may be increased as a result of impaired autophagy (Bernstein et al., 2018) Via stimulation by TGF-β1

β-Aminoproprionitrile

Pseudoexfoliation syndrome and glaucoma (Schlötzer-Schrehardt et al., 2012, 2008; Thorleifsson et al., 2007; Ye et al., 2015)

Decreased

Trabecular meshwork cells (Sethi et al., 2011a,b) Pseudoexfoliation glaucoma - human tenons fibroblasts (Zenkel et al., 2011)

Increased Increased

Pseudoexfoliation glaucoma – aqueous humour (Gayathri et al., 2016) Elastosis/Aging Chronic venous insufficiency (Pascual et al., 2008) Skin aging (Moulin et al., 2017)

Increased

Pelvic organ prolapse (Drewes et al, 2007; Lee et al., 2008; Liu et al., 2007; Wirostko et al., 2016) Cutis Laxa (Debret et al., 2010)

Decreased

Impaired elastin biogenesis; Decreased fibulin-5 along with LOXL1 (Zhou et al., 2013)

Decreased

DNA methylation via reduced Sp1 binding

Decreased

N/A

Decreased

Following exposure to hypoxia. Reduction in HIF1alpha levels leads to reduced protein levels of LOXL1(Öztay et al., 2017) AAA incidence in apo-E knockout mic increased following treatment β-Aminoproprionitrile,

Chronic Obstructive Pulmonary Disease (Öztay et al., 2017; Taylor et al., 2019) Emphysema (Besiktepe et al., 2017)

Abdominal aortic aneurysms (Behmoaras et al., 2008; Remus et al., 2012; Schumacher et al., 2001) Carcinoma Renal cell carcinoma(Tsuchiya et al., 2005) Bladder cancer(Wu et al., 2007)

Decreased Decreased

Decreased

Decreased Decreased

Reduction in total elastin and LOXL1 in saphenous vein segments with increased tropoelastin Increased LOXL1 promoter methylation by DNMT3A

Silenced by promoter methylation; inhibit Ras/ERK signalling pathways

Salivary gland adenoid cystic carcinoma(Bell et al., 2011) Lung adenocarcinoma(Ji et al., 2007)

Increased

Hypomethylated CpG islands

Increased

Prostate cancer (Nilsson et al., 2016)

Increased

LOXL1 expression regulated by Integrin α11 to promote tumorigenicity (Zeltz et al., 2019) Following in vitro exposure to hypoxia

Fibrosis Idiopathic pulmonary fibrosis (Ma et al., 2018; Tjin et al., 2017) (Bellaye et al., 2018) Liver cirrhosis(Zhao et al., 2018a)(Ma et al., 2018) (Zhao et al., 2018b).

Endometriosis(Dentillo et al., 2010)

Increased Increased

LOXL1 deficient mice protected from IPF(Bellaye et al., 2018) Increased elastin cross-linking via TGFβ stimulation

Increased

N/A

3

N/A

Smad inhibitors N/A

N/A

N/A LOXL1 expression restored by DNMT inhibitor 5azacitidine N/A

LOXL1 expression restored following 5-azacitidine treatment. N/A N/A

Strategies to overexpress LOX family enzymes/ support matrix crosslinking and scaffold formation

Reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells decreased colony formation ability. N/A Potential to target α11 β-Aminoproprionitrile reduced tumour growth but only before the cell implantation stage (Nilsson et al., 2016) β-Aminoproprionitrile (pan-LO inhibitor) reduced stiffening in a decellularized model. Fibrosis attenuated following LOXL1 inhibition with AAV2/8-LOXL1-shRNA(Zhao et al., 2018b) Knockdown of LOXL1 inhibited TGFβ related fibrosis. It also reduced expression of pro fibrotic MMPs and type 1 collagen.(Ma et al., 2018) N/A

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severe in PXFG than in other forms of glaucoma (Grødum et al., 2005) and how the altered biomechanics in the elastic tissue of the ONH can accelerate this damage. Keratoconus is a corneal degeneration in which the ECM of the cornea loses its integrity and there is a breakdown of the cross-linking between the collagen lamellae, changing the cornea from a round shape to a cone shape. Total LOX activity (LOX and LOXL enzymes) was also lower in keratoconic fibroblasts in vitro compared with controls (Dudakova et al., 2012). In vivo, LOXL1 has been found to be a major component of intraocular and extra-ocular PXF fibrils (Schlötzer-Schrehardt et al., 2008; Schlötzer-Schrehardt and Naumann, 2006) along with other elastic fibre components like fibrillin-1 and Latent TGF binding protein 1(LTBP-1). In PXF, a marked decrease in stiffness has been noted in the LC and peripapillary sclera, reflecting an inherent tissue weakness in these eyes leaving them vulnerable to glaucomatous damage (Braunsmann et al., 2012). Patients with PXF also frequently present with lens subluxation or zonular rupture during phacoemulsification (Fontana et al., 2017). It has been previously noted that the zonule microfibrils are crosslinked by LOXL1 (De Maria et al., 2017), so a shortage of LOXL1 may result in degenerative changes to these elastic microfibrils. Defects in elastic fibre assembly have been implicated in many other systemic disease processes (Table 1). LOXL1 downregulation has been associated with chronic venous insufficiency. Decreased LOXL1 expression and increased tropoelastin was observed in patients with varicose veins (Pascual et al., 2008). Emphysematous lung tissues have significantly lower elastin and LOXL1 protein levels compared with unaffected lungs (Besiktepe et al., 2017), The decrease in these proteins may cause the disorganised irregular morphology seen in this disease. Chronic obstructive pulmonary

contracting (Mithieux and Weiss, 2005). LOXL1 deficiency results in elastinopathy and has been observed in ocular and systemic disease. PXF is an age related systemic microfibrillopathy, characterized by the accumulation and deposition of abnormal ECM materials in visceral organs. The gradual deposition of these matrix materials is mainly observed in the anterior segment of the eye. These deposits have also been found in other locations such as the liver, kidneys, heart and lungs (Andrikopoulos et al., 2014; Schlötzer-Schrehardt et al., 2001, 1992; Streeten et al., 1992). PXFG occurs when these fibrillar materials block the trabecular drainage of the eye, leading to an increase in ocular pressure, and development of glaucoma. This subtype of glaucoma represents the most common identifiable cause of open-angle glaucoma worldwide (Ritch, 1994) and is associated with higher rates of intraoperative complications and medication resistance. In the normal mouse eye, scleral elastin is most dense in a ring surrounding the peripapillary optic nerve head (ONH), decreasing with increasing distance from the ONH. The elastin fibres were orientated along the principle stress directions generated from intraocular pressure (IOP) meaning they may help counter damage from elevated ocular pressures. (Gelman et al., 2010). In vitro, decreased LOXL1 expression has been shown in lamina cribrosa (LC) cells from PXFG patients and altered the elastic fibre assembly of optic nerve astrocytes (SchlötzerSchrehardt et al., 2012). LOXL1 dysregulation has been shown to alter the biomechanics of the PXF ONH and was associated with ultrastructural changes in the laminar beams of the elastic fibre network of PXF eyes (Schlötzer-Schrehardt et al., 2012). The main elastic fibre components of PXF material (elastin, fibrillin-1, fibulin-4) were also downregulated along with LOXL1 in the LC. This global reduction in elastic fibre components compromises the LC and ONH structure and may explain why IOP independent optic neuropathy tends to be more

Fig. 3. LOXL1 processing in Elastin biogenesis Intracellularly LOXL1 is secreted as Prepro-LOXL1 which is converted to Pro-LOXL1 following signal peptide cleavage. Extracellularly Tropoelastin attaches to the microfibril scaffold via fibulin-5. Pro-LOXL1 localises to this site and following cleavage of pro-LOXL1 to LOXL1 by BMP-1, tropoelastin monomers cross-link to form mature elastin. LOXL1, Lysyl Oxidase like 1, LOXL1-PP, Lysyl oxidase like 1 propeptide, T, Tropoelastin, N, N-terminal, C, C-terminal, BMP-1, Bone morphogenic protein 1. 4

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Other in vitro studies in a human hepatic stellate cell line (LX-2) demonstrated knockdown of LOXL1 inhibited TGF-β1 induced fibrotic proliferation of these cells. It also suppressed expression of other profibrotic metalloproteinases and type 1 collagen (Ma et al., 2018). Increased LOXL1 expression has also been observed in idiopathic pulmonary fibrosis lung tissues (Tjin et al., 2017). Beta-Aminoproprionitrile, a pan-Lysyl Oxidase inhibitor was found to prevent lung fibrogenesis by blocking TGF-β induced tissue stiffening (Tjin et al., 2017). In transgenic mice, overexpression of LOXL1 causes the formation of protein aggregates in the eye containing LOXL1 similar to those seen in PXF (Zadravec et al., 2019). In animal models of organ fibrosis, inhibition of LOXL1 prevented CCl4 induced liver fibrosis (Zhao et al., 2018a) and LOXL1 knockout mice were protected from an experimental model of TGF-β induced lung fibrosis. Lung stiffness was significantly reduced in these mice compared with controls (Bellaye et al., 2018). Similarly in end stage cirrhosis LOXL1 expression is significantly increased compared with earlier fibrosis (Zhao et al., 2018b). LOXL1 inhibition resulted in a reduction of alpha-Smooth Muscle Actin and elastin with subsequent attenuation of liver fibrosis (Zhao et al., 2018b).

disease (COPD) patients also have increased elastin degradation (Oztay et. al, 2017). PXF itself is associated with many systemic diseases, which may be caused by dysfunctional elastin metabolism. Using the Utah Population Database, PXF was shown to be associated with pelvic organ prolapse (Drewes et al., 2007; Wirostko et al., 2016), inguinal hernias (Besch et al., 2018), atrial fibrillation (Wirostko et al., 2018) and COPD (Taylor et al., 2019). PXF is also associated with abdominal aortic aneurysms (Djordjevic-Jocic et al., 2012; Gonen et al., 2013; Schumacher et al., 2001) and sensorineural hearing loss (Samarai et al., 2012). Aortic wall specimens from PXF patients and animal models (Behmoaras et al., 2008; Remus et al., 2012) showed significantly reduced expression levels of LOXL1 compared with controls and the elastic lamellae in the vessel walls were compromised (Schumacher et al., 2001). PXF patients also have significantly lower ankle branchial indexes compared with controls (Praveen et al., 2011) which may result from their elastotic vascular dysfunction. 2.2. Fibrosis Within the body, there is a normal process of wound healing and scarring, however when this process is allowed to continue unchecked, connective tissue fibrosis occurs. While LOXL1 suppression is associated with increased laxity and impaired elastogenesis, LOXL1 acts as a profibrotic factor when over-activated. (Tjin et al., 2017) (Dentillo et al., 2010) (Table 1). The main activation pathway is via the transforming growth factor beta (TGFβ) family of cytokines (Fig. 3). TGFβ is a family of cytokines known to activate the synthesis of collagen and elastin (Biernacka et al., 2011; Massagué, 2012, 2008). Of the five isoforms, three are present in humans (TGFβ1, 2 and 3). It is well recognised that TGFβ increases ECM deposition and fibrosis (Meng et al., 2016). In healthy eyes, TGF-β1 and TGF-β2 help mediate corneal healing and scar formation in the anterior segment (Pervan, 2017). TGF-β2 is significantly increased in the aqueous humour (AqH) of Primary Open Angle Glaucoma (POAG) patients (Inatani et al., 2001; Lütjen-Drecoll, 2005) while TGF-β1 is significantly increased in the aqueous of PXFG patients only (Schlötzer-Schrehardt et al., 2001). We know in glaucoma that fibrosis occurs due to a build-up and excessive cross-linking of ECM materials in the trabecular meshwork (Acott and Kelley, 2008; Fuchshofer et al., 2006; Sethi et al., 2011b) and lamina cribrosa (Schneider and Fuchshofer, 2015; Wallace et al., 2014). TGF-β increases aqueous humour (AqH) outflow resistance and IOP in perfusion cultured human (Gottanka et al., 2004) and porcine eyes (Bachmann et al., 2006; Fleenor et al., 2006). It now appears that LOXL1 expression is increased by TGF-β1 implicating it in this fibrotic process (Sethi et al., 2011b, 2011a). In vitro studies in trabecular meshwork cells demonstrated that LOXL1 expression and activity was upregulated by TGF-β and blocked by inhibitors of TGF-β and small mothers against decapentaplegic (Smad)(Sethi et al., 2011b). This indicates that LOXL1 expression is stimulated by TGF-β via Smad signalling pathways (Sethi et al., 2011a). Stimulation of Human Tenon's Fibroblasts (HTFs) with TGF- β1 has also been found to increase the expression of LOXL1 and elastic microfibrils which aggregated into PXF-like fibrils (Zenkel and SchlötzerSchrehardt, 2014). In cadaveric ocular tissues the expression of LOXL1 was increased in early stage PXF but decreased in late stage PXF and PXFG (SchlötzerSchrehardt et al., 2008). LOXL1 activation may contribute to the initial abnormal fibrogenesis in PXF. Increased amine oxidase activity has been suggested to contribute to increased matrix accumulation and cross-linking in the trabecular meshwork of glaucoma patients (Sethi et al., 2011b). The above has also been confirmed in vivo (Gayathri et al., 2016) showed that TGF-β1 and TGF-β2 were inversely correlated with LOX activity in the aqueous humour, indicating that the increased level of TGF-β in the aqueous humour of PXF patients is possibly associated with LOX regulation.

2.3. Autophagy/proteopathy Autophagy is a mechanism, which allows orderly degradation and removal or recycling of cellular waste components. Impaired autophagy is seen in many age related diseases such as age related macular degeneration and Alzheimer's disease (Bordi et al., 2016; Kivinen, 2018; Liu and Li, 2019; Uddin et al., 2018). More specifically in glaucoma, autophagy has been implicated in the trabecular meshwork outflow pathway and in the survival of retinal ganglion cells (Boya et al., 2016; Deng et al., 2013; Hirt and Liton, 2017). Recent evidence suggests that, like in other age-related protein aggregation diseases, there is dysfunctional autophagy in PXF (Bernstein et al., 2018; Wolosin et al., 2018). This may be due to protein folding defects in LOXL1 or due to an inability to degrade the LOXL1 containing protein aggregates produced in PXF. In vitro studies of PXF HTFs showed they were larger than their POAG counterparts and did not acquire the spindle formation normally seen in fibroblast cultures (Aboobakar et al., 2017). Furthermore they accumulated more fibulin-5 (Bernstein et al., 2018; Want et al., 2016). The cells behaviour following serum starvation was also different. Normally, when healthy cells are serum starved, they respond by converging their cell microtubules into a dense juxta-nuclear microtubuleorganising centre (MTOC). The endosomes and lysosomes then coalesce to this area to allow clearing of cellular waste via the autophagosomes. However, on serum starving of PXF HTFs their endosomes and lysosomes did not relocate to the perinucleus and the MTOC was barely distinguishable. Taken together, this indicates that there is an impaired autophagy system in PXF (Want et al., 2016; Wolosin et al., 2018) (Bernstein et al., 2018). As well as impaired autophagy PXF HTFs had a lower autophagic flux rate or rate of cellular degradation and clearance. This was demonstrated by the accumulation of the autophagosome marker Microtubule-associated protein light chain 3 (LC3 II) (Want et al., 2016). LC3-II is incorporated into the autophagosomal membrane and controls late autophagosome transit (Tanida et al., 2008). Increased LC3-II indicates that there is reduced cellular clearance and autophagy dysfunction in PXF, similar to findings in Alzheimer's (Bordi et al., 2016). In addition, there was also reduced autophagy of dead mitochondria which may interfere with the cellular control of reactive oxygen species leading to an increased oxidized protein load (Want et al., 2016). Impaired mitochondrial turnover has the secondary effect of a loss of ATP production and results in a self-perpetuating cycle as the autophagy process depends on ATP for energy (Want et al., 2016). The gene Proteasome Maturation Protein (POMP) is also downregulated 5

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3. Genetics

2007), along with Middle Eastern (Abu-Amero et al., 2011; Shihadeh et al., 2018; Yaz et al., 2018) and Latin/Central American populations (Jaimes et al., 2012) have replicated these findings. Meta-analyses have confirmed the global association of LOXL1 SNPs and PXF/PXFG risk (Chen et al., 2010; Tang et al., 2014; Wang et al., 2016). Large effect sizes have been seen in all ethnic groups with large variations in the odds ratio (OR) for different SNPs based on ethnicity. Oddly, these risk variants are reversed in certain populations, meaning the same variant may be either protective or a risk variant depending on ethnicity. For the SNP rs1048661, the G allele is associated with a decreased risk in Japanese (Fuse et al., 2008; Hayashi et al., 2008; Mabuchi et al., 2008; Mori et al., 2008; Ozaki et al., 2008; Tanito et al., 2008) and Korean (Park et al., 2013; Sagong et al., 2011) populations but an increased risk in all other groups. For the SNP rs3825942, the G allele is associated with decreased risk for PXF in Black South Africans (Rautenbach et al., 2011; Williams et al., 2010) but increased risk in all other populations studied. For rs2165241 the T allele was found to be high risk in the majority of populations but low risk in Japanese (Ozaki et al., 2008; Tanito et al., 2008), Korean (Park et al., 2013; Sagong et al., 2011) and Chinese (Chen et al., 2009) populations. South Asian populations such as Pakistani (Micheal et al., 2012) and Indians (Dubey et al., 2014; Gayathri et al., 2016; Ramprasad et al., 2008) did not demonstrate SNP risk reversal. This “allelic flipping” seems to indicate that R141L and G153D variants may not be solely responsible for PXF development. This has been largely confirmed by functional studies. These coding variants have been found to have no effect on amine oxidase activity, meaning other variants in the LOXL1 genomic non-coding region must confer risk (Kim and Kim, 2012). However a paper by (Sharma et al., 2016) showed that positions 141 and 153 on the LOXL1 N-terminus are possible recognition sites for protein-protein interactions. Immunoprecipitation of these variants showed differences in their processing by BMP-1, which leads to enzymatic activation of LOXL1. The BMP-1 cleavage site at position 134 is located close to the PXF associated SNPs R141L and G153D (Borel et al., 2001) giving credence to the idea that alterations in LOXL1 processing may contribute to PXF development (Sharma et al., 2016).

3.1. Common coding variants

3.2. Common non-coding variants

In 2007, a genome-wide association study performed in a Scandanavian population, identified three single nucleotide polymorphisms (SNPs) in LOXL1 that are strongly associate with developing PXF/PXFG (Thorleifsson et al., 2007). Two coding SNPs are proteincoding variants found in exon 1 of LOXL1 (rs1048661 and rs3825942) while the third SNP is located nearby in intron 1 (rs2165241). These coding variants were present in up to 98% of PXF cases, but were also found in up to 85% of unaffected individuals indicating several genetic and environmental factors are involved in disease pathogenesis. The mechanism whereby these genetic variants contribute to PXF and PXFG is poorly understood. SNPs rs1048661 and rs3825942 lead to amino acid changes Arg141Leu (G > T) and Gly153Asp (G > A) respectively. These SNPs can be carried together and individuals with the high-risk (G-G) haplotype for these coding SNPs were found to have an estimated 700-fold increased risk for PXFG compared to those with the low risk haplotype (Thorleifsson et al., 2007). The association of LOXL1 coding variants rs1048661 (R141L) and rs3825942 (G153D) with PXFG risk has been replicated in European populations, including German (Pasutto et al., 2008; SchlötzerSchrehardt et al., 2008), Italian (Pasutto et al., 2008; SchlötzerSchrehardt et al., 2008), Austrian (Mossböck et al., 2008), Finnish (Lemmelä et al., 2009), Polish (Malukiewicz et al., 2011), Spanish (Álvarez et al., 2015) and Greek (Anastasopoulos et al., 2014; Chiras et al., 2013; Metaxaki et al., 2013) populations among others. Other Caucasian groups such as Australian Caucasians (Hewitt et al., 2007), and US Caucasians (Challa et al., 2008; Fan et al., 2008; Fingert et al.,

Due to the lack of a definitive association with LOXL1 variants in the coding region and functional changes in the enzyme, regulatory regions and non-coding areas of the gene have also been a focus of attention. Early papers looked at the LOXL1 promoter region leading to the discovery of two additional risk variants, rs16958477 and rs12914489 (Fan et al., 2011). rs16958477 is located near the transcription start site and has been shown to influence transcriptional activity, the C allele being associated with increased transcription. The A allele is associated with transcriptional repression and was identified as a risk allele in American Caucasians (Fan et al., 2011). rs16958477 was found to alter LOXL1 expression in vitro (Ferrell et al., 2009). rs12914489 is in the distal promoter region and it's A allele was also identified as a risk allele (Fan et al., 2011) while the G allele was found to be protective. Pasutto et al. recently uncovered a common risk variant (rs11638944 C > G) located in a polymorphic locus spanning introns 1 and 2 of LOXL1 that altered gene expression in Japanese and European populations (Pasutto et al., 2017). They demonstrated that increased transcriptional activity at this locus reduced LOXL1 mRNA expression, via transcriptional, co-transcriptional and post-transcriptional mechanisms. These will be discussed in greater detail in the gene regulation section of this review. At the transcriptional level, the decreased LOXL1 levels were due to reduced binding of the transcription factor RXRα (retinoid X receptor alpha). RXRα is known to be a repressor of transcription in the absence of a ligand (Szanto et al., 2004). At the co-transcriptional level, downregulation was via of alternative splicing of LOXL1 pre-mRNA

in iris and ciliary body samples in PXF. This gene is involved in proteasome maturation and a reduction in POMP suggests a reduction of ubiquitin conjugating enzymes in PXF tissues (Aung et al., 2017). The SNP rs7329408 in POMP was associated with increased PXF risk and the odds ratios for this SNP increased in PXF patients with increasing geographical latitude away from the equator. (Aung et al., 2017). We hypothesise that these environmental risk factors work in tandem with the cellular dysfunction seen in PXF and this will be discussed later in this review. LOXL1 has been identified by proteomic analysis as a component of exfoliation material (Ronci et al., 2013; Sharma et al., 2009) and (Bernstein et al., 2018) have demonstrated that the LOXL1 protein is degraded via the autophagy pathway by treating PXF tenons fibroblasts with Bafilomycin A1, an inhibitor of autophagy and Spautin-1, an inhibitor of autophagy activation. LOXL1 expression increased following both treatments demonstrating that LOXL1 is a target of the autophagy pathway. Next, they examined LOXL1 for any evidence of structural problems within the protein. They found that the LOXL1 N-terminus exists in a highly disordered state, which increases the chance of protein misfolding. This N-terminus instability seemed to only be present in the smaller LOX proteins, with LOXL2-4 having a highly structured N-terminus. The main risk domains for polypeptide misfolding in LOXL1 lie between residues 151 and 170 and the maximum misfolding occurs at position 153, which is the location of the high-risk variant rs3825942 (Gly153Asp) (Bernstein et al., 2018). This risk allele is found in 98% of people with PXF (Thorleifsson et al., 2007). When glycine is replaced by aspartate in this position there is a decrease in PXF risk, 16 fold for those with glycine-aspartate heterozygosity and 26 fold for those homozygous for aspartic acid compared with those who are homozygous for glycine (Bernstein et al., 2018). Further research into understanding the basis of autophagic dysfunction and reducing the magnitude of its functional consequences is important as it may offer a means of future therapeutic options for PXFG or LOXL1 proteopathy in general.

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rs201011613; A > T which had conferred strong protection from PXF. This protection was not absolute as the rare T variant was seen in two PXF patients, but despite this it still conferred a 25-fold resistance to disease. The variant is contained in the catalytic domain of LOXL1 and its protective effect could be due to increased stabilization of the ECM rendering cells more resistant to environmental stressors which aim to destabilize the ECM.

(Pasutto et al., 2017). The alternatively spliced transcript LOXL1-α has a premature stop codon in exon 2 resulting in the formation of an inactive isoform. Increased formation of LOXL1-α has the secondary effect of reducing formation wild type LOXL1. At the post-transcriptional level, the alternatively spliced LOXL1 transcript is associated with nonsense-mediated decay (NMD), a degradation pathway that removes spliced unproductive mRNA to regulate gene expression. Alternative mRNA splicing coupled with NMD is known to regulate gene expression. The combination of reduced RXRα binding, increased splicing and higher levels of NMD resulted in an up to 50% reduction in levels of wild-type LOXL1 mRNA in the cells carrying the risk allele. It was later found that this “alternative splicing coupled to NMD” can be modulated by PXF associated stressors like oxidative stress, caffeine and retinoic acid (Berner et al., 2017). Recently, a non-coding variant rs7173049 A > G, has been associated with a decreased PXF risk (Berner et al., 2019; SchlötzerSchrehardt and Zenkel, 2019). Uniquely, this SNP was consistently associated with a decrease in PXF risk in multiple populations, with no evidence of “allelic flipping”. Binding of the allele by the transcription factor thyroid hormone receptor beta (THRβ) increased expression of immunoglobulin superfamily containing leucine-rich repeat protein 2 (ISLR2) and stimulated by retinoic acid receptor 6 (STRA6) (Berner et al., 2019). ISLR2 is a protein involved in axonal extension and neural development (Abudureyimu et al., 2018) and STRA6 is a receptor that regulates uptake of vitamin A in ocular tissues (Chen et al., 2016). ISLR2 and STRA6 are both reduced in PXF tissues and the protective rs7173049 G allele correlates with increased expression of these genes. Inhibition of the retinoic acid resulted in up-regulation of PXF associated matrix genes in vitro. This indicates that impaired retinoid metabolism may be involved in PXF pathogenesis (Berner et al., 2019). These data lead us to believe that a complex combination of exonic and intronic variants in LOXL1 are involved in PXFG risk and that noncoding variants in the gene can have functional effects on LOXL1 expression conferring both risk and protection from disease development.

4. LOXL1 regulation 4.1. Cellular signalling As mentioned previously, LOXL1 is required for elastin maturation, or more specifically, for the crosslinking of soluble tropoelastin monomers into growing mature elastin polymers during elastogenesis. Elastic fibre formation involves the deposition of tropoelastin onto a microfibril scaffold composed mainly of fibrillin-1, fibrillin-2 and fibronectin. The scaffold helps align the tropoelastin cross-linking domains. Fibulin-5 binds to the N-terminus of fibrillin-1 to co-localise with the microfibrils where it is thought to aid in ‘coacervation’ or aligning of the cross linking domains (Papke and Yanagisawa, 2014). Latent Transforming growth factor beta binding protein 2 (LTBP2) also interacts with fibulin-5 to facilitate elastin deposition (Robertson et al., 2015). Intracellularly LOXL1 is synthesised as the 90 kDa preproenzyme (Fig. 3) (Csiszar, 2001). Following cleavage of its N-terminal signal peptide it is secreted extracellularly as a pro-enzyme. The secreted proLOXL1 localises to the sites of elastogenesis by binding to the C-terminus of both tropoelastin and fibulin-5 through its N-terminus propeptide domain (Thomassin et al., 2005; Trackman, 2018). Fibulin-5 acts to tether pro-LOXL1 to tropoelastin. (Liu et al., 2004; Papke and Yanagisawa, 2014; Thomassin et al., 2005). Fibulin-5 is crucial in elastogenesis and mutations result in Cutis Laxa (Callewaert et al., 2013; Claus et al., 2008) and Age Related Macular Degeneration (Jones et al., 2010; Lotery et al., 2006; Stone et al., 2004). The lysine rich propeptide region of LOXL1 has the highest affinity for tropoelastin and facilitates correct localisation and function of pro-LOXL1 via proteinprotein interactions. (Thomassin et al., 2005; Trackman, 2018). The pro-enzyme is than cleaved by the pro-collagen C proteinase bone morphogenetic protein 1 (BMP-1) for catalytic activation (Borel et al., 2001). The exact cleavage site of BMP-1 remains unknown, however

3.3. Rare variants A deep re-sequencing of PXF patients following GWAS revealed 63 rare non-synonymous variants in LOXL1 (Aung et al., 2017). They were mainly more common in the control group, suggesting they could offer protection against PXF development. They also identified a rare variant

Fig. 4. Cell signalling pathways in LOXL1 gene expression. The proposed mechanism of TGFβ regulation of LOXL1 via the PI3K/AkT, Smad2/3 and MAPK/JNK1/2 pathways. Abbreviations: TGFβ, Transforming growth factor beta, TGFβR, Transforming growth factor beta receptor, BMP4/7, Bone morphogenic protein 4/7, BMPR1/2, Bone morphogenic protein receptor 1/2, P13K, Phosphoinositide 3-kinase, Smad 2, Mothers against decapentaplegic homolog 2, Smad 3, Mothers against decapentaplegic homolog 3, Smad 4, Mothers against decapentaplegic homolog 4, JNK1/2, c-Jun N-terminal kinase 1/2, ERK1/2, Extracellular signal-regulated kinase 1/2, MAPK, Mitogen Activated Protein Kinase, AP-1, Activator Protein 1, LOXL1, Lysyl Oxidase like 1, BMP-1, Bone morphogenic protein 1.

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pathogenesis. Methylation inhibition represents a current focus in the management of cancer. DNA methyltransferase inhibitors such as 5-azacitidine are currently in use as hypomethylating agents for the treatment of myelodysplastic syndromes (Roboz et al., 2016; Tran et al., 2011). LOXL1 promoter transcriptional activity has been increased using 5azacitidine in skin fibroblasts (Moulin et al., 2017) and human bladder cancer cell lines (Wu et al., 2007). Non-coding genetic material may also regulate LOXL1 gene expression. While these RNAs lack protein-coding capacity, many noncoding RNAs (ncRNAs) play important roles in development and disease pathogenesis. These regulatory ncRNAs are classified on the basis of size into groups shorter than 200 nucleotides (microRNAs [miRNAs], small interfering RNAs) and those longer than 200 nucleotides (long non-coding RNAs [lncRNAs]). microRNAs (miRNA) are small, non-coding RNAs which span 18–24 nucleotides. Microarrays of miRNAs have identified 11 upregulated miRNAs and 18 downregulated miRNAs in the aqueous humour of glaucoma patients (Tanaka et al., 2015), however it remains to be seen whether this has any influence on LOXL1 expression. Drewry et al., identified five miRNAs (miR-122-5p, miR-3144-3p, miR-320a, miR320e and miR-630) which were significantly upregulated in the AqH of PXFG patients compared with control patients. (Drewry et al., 2018). Specifically miR-122-5p may target TGF-β1 genes and be involved in TGF-β signalling, and therefore could influence the ECM deposition patterns seen in PXFG (Drewry et al., 2018). These miRNAs may serve as future biomarkers in disease pathogenesis. Their specific relation to LOXL1 gene regulation has yet to be established.

the putative cleavage site at position 134 is notably near the two PXF associated SNPs R141L and G153D (Borel et al., 2001). Pro-LOXL1 is cleaved into the LOXL1 enzyme (62 kDa) and LOXL1 pro-peptide (28 kDa). LOXL1 deaminates tropoelastin which cross-links form mature elastin polymer (Fig. 3)(Kagan and Li, 2003), with these cross-links conferring mechanical integrity and high durability. There are a number of intracellular signalling pathways, which stimulate LOXL1 secretion. LOXL1 mRNA activity is mainly induced by TGF-β1 and TGF-β2 through three different pathways. The TGF-β ligands bind to the cell surface receptor and phosphorylate the Smad2/ Smad3 complex, which utilizes Smad4 to translocate to the nucleus (Sethi et al., 2013, 2011b). This complex may bind itself or with Activator Protein 1 (AP-1) to the LOXL1 promoter to regulate transcription (Sethi et al., 2011b). TGF-β receptor activation may also activate LOXL1 via MAPK/JNK1/2 pathway, which phosphorylates the c-Jun component of AP-1 (Sethi et al., 2013, 2011b). Thirdly, TGF-β may upregulate LOXL1 via the P13K/Akt pathway (Fig. 4) (Gao et al., 2018). The BMP antagonist gremlin has also been found to indirectly stimulate LOXL1 expression by blocking BMP4 and BMP7, which normally inhibit TGFβ2 induction of LOXL1 (Sethi et al., 2013). There is evidence to support the function of LOXL1 and the above biological pathways. In vitro LOXL1 knockdown inhibits TGF-β1 induced hepatic fibrosis (Ma et al., 2018) while in vivo TGF-β1 promotes LOXL1 expression in fibroblasts of neonatal rat lung (Boak et al., 1994) and rat vascular smooth muscle cells (Shanley et al., 1997). 4.2. Gene regulation Epigenetics is the study of changes in gene function that do not involve changes in the DNA sequence itself (Wu and Morris, 2001) with an example being DNA methylation, which describes the addition of a methyl group to a cytosine base (Jin et al., 2011). The transfer of this methyl group is orchestrated by the DNA methyltransferase (DNMTs) family of enzymes (DNMT1, DNMT3A and DNMT3B). DNMT1 is considered a maintenance enzyme in charge of copying patterns of DNA methylation from the parent to the daughter DNA during replication. DNMT3a and 3b, on the other hand create de novo patterns of methylation (Jin and Robertson, 2013). When this methylation occurs at the gene promoter, it leads to transcriptional repression or ‘gene silencing’ (Jin et al., 2011). Repression of LOXL1 by DNA methylation has been observed in vitro. Aged skin fibroblasts displayed higher binding of DNMT3A at the LOXL1 promoter and this reduced LOXL1 transcription (Moulin et al., 2017). In primary bladder cancer, LOXL1 and 4 are silenced by promoter hypermethylation (Wu et al., 2007). Reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells decreased colony formation ability (Wu et al., 2007). DNA methylation has been implicated in the regulation of fibrotic genes in glaucoma. Our group has previously demonstrated increased global DNA methylation and decreased TGF-β1 promoter methylation in POAG LC cells (McDonnell et al., 2016a,b). In the Uighur population, hypermethylation of CpG islands in the LOXL1 promoter region has been observed in the anterior lens capsules of PXF patients compared to controls (Ye et al., 2015). LOXL1 mRNA levels were reduced in PXF lens capsules, suggesting that hypermethylation downregulates gene expression at this locus. In systemic disease, Debret et al. described a case of autosomal recessive Cutis Laxa in which LOXL1 was silenced via DNA hypermethylation (Debret et al., 2010). This paper also demonstrated that the transcription factor specific protein 1 (Sp1) is the main inducer of LOXL1 promoter activation in healthy fibroblasts (Debret et al., 2010). These Sp1 binding motifs are located in a GC rich area prone to methylation and methylated DNA has been shown to counteract Sp1 binding preventing transcription (Clark et al., 1997). Cutis Laxa is an extracellular matrix disorder characterised by skin elastosis, indicating that the LOXL1 silencing may play a functional role in the disease

4.3. Post-transcriptional regulation Post-transcriptional LOXL1 gene regulation may also occur via long non-coding RNAs (lncRNA). They can affect target gene regulation by chromatin modification, direct transcriptional regulation, modulation of miRNAs, regulation of genomic imprinting and many other functions at local or distant loci (Patil et al., 2014). Two distinct lncRNAs have been implicated in glaucoma pathogenesis; CDKN2B-AS1 and LOXL1AS1 (Wiggs et al., 2012). CDKN2B-AS1 (also known as ANRIL), alters TGF-β1/Smad signalling resulting in reduced expression of damage response proteins p14, p15 and p16 in oesophageal carcinoma (Chen et al., 2014), thyroid carinoma (Zhao et al., 2016) and non small cell lung carcinoma (Nie et al., 2015). LOXL1–AS1 has been linked to a region of PXF associated variants clustered at the LOXL1 exon 1/intron 1 boundary. This region has been shown to modulate the expression of LOXL1 antisense RNA 1 (LOXL1AS1), a long non-coding RNA (lncRNA) which is encoded on a DNA strand opposite LOXL1 (Hauser et al., 2015). (Hauser et al.) found that a haplotype containing 3 SNPs from intron 1 reduced LOXL1-AS1 promoter activity by 43% in lens epithelial cells. LOXL1-AS1 was also reduced in response to cellular stressors, oxidative stress and cyclical mechanical stress in Schlemm's canal endothelial cells in PXF. This study also identified LOXL1-AS1 in multiple ocular and systemic tissues associated with PXF (Hauser et al., 2015). Although LOXL1-AS1 is not directly involved in the regulation of LOXL1, it may regulate distant genes involved in PXF pathogenesis. LOXL1-AS1 expression has also been found to be upregulated in many cancers stimulating cell proliferation, and invasion via the NF-kB and P13k/Akt pathways (Chen et al., 2019; Gao et al., 2018; Long et al., 2018; Wang et al., 2018) with LOXL1-AS1 knockdown inhibiting tumourogenesis (Gao et al., 2018; Long et al., 2018). The role of this lncRNA in PXF pathophysiology needs to be further delineated. 5. Environment As discussed in Section 3.1, 2 coding variants and 1 non-coding variant were identified in LOXL1. These coding variants formed a high 8

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et al., 2013). Reduced levels of antioxidants, such as ascorbic acid, glutathione and total anti-oxidative capacity in aqueous humour suggest an impaired anti-oxidative defence system in PXF. Concomitantly levels of oxidants such as hydrogen peroxide, nitric oxide and lipid peroxidation products are increased. Total antioxidant status was lower in the plasma of patients with PXFG compared to controls (Abu-Amero et al., 2011). Logistic regression showed that, when other variables are accounted for, Total antioxidant status and a ‘G’ allele in rs3825942 were significant factors for PXFG and may contribute to disease pathogenesis (Abu-Amero et al., 2011). LOXL1 has been shown to be upregulated in an oxidative stress environment. LOXL1-AS1 expression has also been found to be significantly altered in response to oxidative stress in human lens epithelial cells (Hauser et al., 2015). These findings support a functional role for lncRNA LOXL1-AS1 and LOXL1 in the cellular stress response and in PXF pathogenesis. Cellular and dietary antioxidants may represent future treatment strategies for disease.

risk haplotype associated with an increased the risk of developing PXFG (Thorleifsson et al., 2007). However, while these coding variants were present in up to 98% of PXF cases, they were also found in up to 85% of unaffected individuals indicating several environmental factors are involved in disease pathogenesis. These factors will be discussed below. 5.1. Hypoxia Hypoxia describes when insufficient levels of oxygen are supplied to cells or tissues. When cells are exposed to a hypoxic environment there is a reduction Hypoxia inducible factor 1 alpha (HIF1α) oxidation and degradation. This leaves more HIF1α available to move into the cell nucleus. There it binds to the LOXL1 hypoxia response element (HRE) (Dengler et al., 2014) and acts as a transcription factor for LOXL1 mRNA. (Wang and Semenza, 1995; Watson et al., 2010; Ziello et al., 2007). Overall it is known that hypoxia may alter the epigenetic signature (Watson et al., 2010). The glaucomatous environment is a hypoxic one, demonstrated by increased HIF1α in the glaucomatous optic nerve head (Tezel and Wax, 2004). PXFG demonstrates well recognised clinical stigmata related to chronic hypoxia such as anterior chamber hypoxia, iris vasculopathy (Helbig et al., 1994), secondary cataract (Küchle et al., 1997) and reduced ophthalmic artery blood flow with increased vascular resistance (Kocaturk et al., 2016). Our group has previously demonstrated increased DNA methylation, TGF-β1 expression and decreased RAS Protein Activator Like 1 (RASAL1) in Glaucoma Trabecular Meshwork (GTM) cells (McDonnell et al., 2016a,b). These results were replicated in normal trabecular meshwork cells under hypoxic conditions. Culture of Human Tenons Fibroblasts (HTFs) under hypoxic conditions has also been shown to upregulate LOXL1 expression (Zenkel et al., 2011). Of note, basal and stimulated expression of LOXL1 was lower in those carrying the high-risk haplotype, but the difference was not statistically significant. This indicates that LOXL1 gene expression is upregulated in response to hypoxia, and given that glaucoma is a known hypoxic environment, hypoxia could be an initiating environmental factor in disease development. LOXL1 alterations are also observed in other hypoxemic diseases such as lung adenocarcinoma (Ji et al., 2007). In lung cancer patients with chronic obstructive pulmonary disease (COPD), increased HIF1alpha and decreased LOX, LOXL1 and LOXL2 were observed in emphysematous lung tissues when compared with non-emphysematous areas (Öztay et al., 2017). This was accompanied by abnormalities in elastin structure and alveolar wall thinning, likely due to elastosis secondary to reduced LOXL1 crosslinking. Hypoxia from obstructive sleep apnoea (OSA) increases hepatic production of LOXL1 (Mesarwi et al., 2015). A recent study found patients with severe OSA had higher baseline LOX than healthy controls, and serum LOX decreased in patients with OSA on continuous positive airway pressure (CPAP) but not in untreated patients. This suggests that hypoxic stress increases LOX expression and may serve as a biomarker of liver fibrosis in those with OSA and fatty liver disease (Mesarwi et al., 2015). LOXL1 expression was also increased when a prostate cancer cell line was incubated in a hypoxic environment (Nilsson et al., 2016). LOXL1 may play a role in hypoxia driven angiogenesis. Co-culture of adipose derived stromal cells and endothelial cells under hypoxia and normoxia showed angiogenesis was promoted in hypoxia. LOXL1 was also upregulated in these cells (Xie et al., 2017) meaning it may have a role in many hypoxic diseases which display pathogenic angiogenesis.

5.3. Geographical location Chronic ultraviolet radiation (UVR) is linked to LOX family overactivation. UVR upregulates LOXL1, fibrillin-1, elastin and fibulins in PXF human tenon's fibroblasts (Zenkel et al., 2011). UVR is also associated with increased aging in skin and LOX activity was increased in biopsies of the photo-aged forearm, compared with the photo-protected buttock (Langton et al., 2017). Bone Morphogenic Protein 1 (BMP-1) was also increased (Langton et al., 2017). LOXL1 was not significantly increased although increased BMP-1 levels offer the potential of increased LOXL1 activation via pro-enzyme cleavage. Geographically, epidemiological studies have revealed that increasing distance away from the equator is associated with increased risk of PXF (Kang et al., 2012; Pasquale et al., 2014a,b; Stein et al., 2011). This may be due to sunlight being more angulated in the northern hemisphere leading to increased UVR reflectivity and associated damage. In addition to this decreased ambient temperature further away from the equator may lead to PXF formation; cold weather has also been found to lead to aggregation of PXFG material in the aqueous (Lee, 2008; Pasquale et al., 2014a,b). A database in the continental United States showed that each degree higher mean January low temperature was associated with a 3% decreased risk of PXF (Stein et al., 2011). Other factors linked with increased UV exposure such as rural residence (Vijaya et al., 2016), time spent outdoors (particularly in youth) and leisure or work activity over water or snow were all associated with elevated risk of PXF (Kang et al., 2014; Stein et al., 2011).

5.4. Dietary factors Caffeine consumption has been linked with an increased risk of PXF/PXFG (Pasquale et al., 2014a,b; Pasquale et al., 2012). Caffeine increases homocysteine levels (Christensen et al., 2001; Grubben et al., 2000) and homocysteine levels are higher in the plasma (Puustjärvi et al., 2004), aqueous (Puustjärvi et al., 2004)(Bleich et al., 2004) and tears (Roedl et al., 2007) of those with PXF. Low serum folate levels can also contribute to elevated homocysteine levels and is associated with an increased risk of PXF (Xu et al., 2012). Elevated homocysteine may enhance development of PXF material by contributing to vascular damage, oxidative stress and ECM alterations (Pasquale et al., 2014a,b; Pasquale et al., 2012). Reducing caffeine consumption and increasing dietary folate could represent future lifestyle modifications in PXFG management. There is no link between vitamin B6 and B12 and PXFG risk, however increasing intake of these vitamins could also aid in reducing plasma homocysteine levels (Xu et al., 2012). No links between caffeine, folate and LOXL1 specifically have been investigated as of yet.

5.2. Oxidative stress Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defences leading to free radical damage and lipid peroxidation (Burton and Jauniaux, 2011). Evidence suggests that the oxidative/anti-oxidative balance is disturbed in patients with POAG and PXFG (Abu-Amero et al., 2011; Nucci 9

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6. Conclusion

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LOXL1 is an important gene with wide and varied roles in cellular homeostasis. It is dysregulated in many systemic conditions, cancers and fibrotic diseases. While the genetic and environmental regulations of this gene are complex, the emerging understanding of LOXL1 gene regulation reveals many potential therapeutic targets for disease modifying treatments. Development of pseudoexfoliation syndrome and glaucoma, in particular, appears to be linked to LOXL1 risk variants and it will be exciting to see if the increasing knowledge of LOXL1 regulation will improve the management of this condition. Funding This work was supported by the Health Research Board, Ireland [grant number ILP-POR-2017-031]. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exer.2020.107975. References Aboobakar, I.F., Johnson, W.M., Stamer, W.D., Hauser, M.A., Allingham, R.R., 2017. Major review: exfoliation syndrome; advances in disease genetics, molecular biology, and epidemiology. Exp. Eye Res. 154, 88–103. https://doi.org/10.1016/j.exer.2016. 11.011. Abu-Amero, K.K., Kondkar, A.A., Mousa, A., Osman, E.A., Al-Obeidan, S.A., 2011. Decreased total antioxidants status in the plasma of patients with pseudoexfoliation glaucoma. Mol. Vis. 17, 2769–2775. Abudureyimu, S., Asai, N., Enomoto, A., Weng, L., Kobayashi, H., Wang, X., Chen, C., Mii, S., Takahashi, M., 2018. Essential role of Linx/Islr2 in the development of the forebrain anterior commissure. Sci. Rep. 8, 7292. https://doi.org/10.1038/s41598-01824064-0. Acott, T.S., Kelley, M.J., 2008. Extracellular matrix in the trabecular meshwork. Exp. Eye Res. 86, 543–561. https://doi.org/10.1016/j.exer.2008.01.013. Alsofi, L., Daley, E., Hornstra, I., Morgan, E.F., Mason, Z.D., Acevedo, J.F., Word, R.A., Gerstenfeld, L.C., Trackman, P.C., 2016. Sex-linked skeletal phenotype of lysyl oxidase like-1 mutant mice. Calcif. Tissue Int. 98, 172–185. https://doi.org/10.1007/ s00223-015-0076-4. Álvarez, L., García, M., González-Iglesias, H., Escribano, J., Rodríguez-Calvo, P.P., Fernández-Vega, L., Coca-Prados, M., 2015. LOXL1 gene variants and their association with pseudoexfoliation glaucoma (XFG) in Spanish patients. BMC Med. Genet. 16, 72. https://doi.org/10.1186/s12881-015-0221-y. Anastasopoulos, E., Coleman, A.L., Wilson, M.R., Sinsheimer, J.S., Yu, F., Katafigiotis, S., Founti, P., Salonikiou, A., Pappas, T., Koskosas, A., Katopodi, T., Lambropoulos, A., Topouzis, F., 2014. Association of LOXL1 polymorphisms with pseudoexfoliation, glaucoma, intraocular pressure, and systemic diseases in a Greek population. The thessaloniki eye study. Invest. Opthalmol. Vis. Sci. 55, 4238. https://doi.org/10. 1167/iovs.14-13991. Andrikopoulos, G.K., Alexopoulos, D.K., Gartaganis, S.P., 2014. Pseudoexfoliation syndrome and cardiovascular diseases. World J. Cardiol. 6, 847–854. https://doi.org/10. 4330/wjc.v6.i8.847. Aung, T., Ozaki, M., Lee, M.C., Schlötzer-Schrehardt, U., Thorleifsson, G., Mizoguchi, T., Igo, R.P., Haripriya, A., Williams, S.E., Astakhov, Y.S., Orr, A.C., Burdon, K.P., Nakano, S., Mori, K., Abu-Amero, K., Hauser, M., Li, Z., Prakadeeswari, G., Bailey, J.N.C., Cherecheanu, A.P., Kang, J.H., Nelson, S., Hayashi, K., Manabe, S.-I., Kazama, S., Zarnowski, T., Inoue, K., Irkec, M., Coca-Prados, M., Sugiyama, K., Järvelä, I., Schlottmann, P., Lerner, S.F., Lamari, H., Nilgün, Y., Bikbov, M., Park, K.H., Cha, S.C., Yamashiro, K., Zenteno, J.C., Jonas, J.B., Kumar, R.S., Perera, S.A., Chan, A.S.Y., Kobakhidze, N., George, R., Vijaya, L., Do, T., Edward, D.P., de Juan Marcos, L., Pakravan, M., Moghimi, S., Ideta, R., Bach-Holm, D., Kappelgaard, P., Wirostko, B., Thomas, S., Gaston, D., Bedard, K., Greer, W.L., Yang, Z., Chen, X., Huang, L., Sang, J., Jia, H., Jia, L., Qiao, C., Zhang, H., Liu, X., Zhao, B., Wang, Y.-X., Xu, L., Leruez, S., Reynier, P., Chichua, G., Tabagari, S., Uebe, S., Zenkel, M., Berner, D., Mossböck, G., Weisschuh, N., Hoja, U., Welge-Luessen, U.-C., Mardin, C., Founti, P., Chatzikyriakidou, A., Pappas, T., Anastasopoulos, E., Lambropoulos, A., Ghosh, A., Shetty, R., Porporato, N., Saravanan, V., Venkatesh, R., Shivkumar, C., Kalpana, N., Sarangapani, S., Kanavi, M.R., Beni, A.N., Yazdani, S., Lashay, A., Naderifar, H., Khatibi, N., Fea, A., Lavia, C., Dallorto, L., Rolle, T., Frezzotti, P., Paoli, D., Salvi, E., Manunta, P., Mori, Y., Miyata, K., Higashide, T., Chihara, E., Ishiko, S., Yoshida, A., Yanagi, M., Kiuchi, Y., Ohashi, T., Sakurai, T., Sugimoto, T., Chuman, H., Aihara, M., Inatani, M., Miyake, M., Gotoh, N., Matsuda, F., Yoshimura, N., Ikeda, Y., Ueno, M., Sotozono, C., Jeoung, J.W., Sagong, M., Park, K.H., Ahn, J., Cruz-Aguilar, M., Ezzouhairi, S.M., Rafei, A., Chong, Y.F., Ng, X.Y., Goh, S.R., Chen, Y., Yong, V.H.K., Khan, M.I., Olawoye, O.O., Ashaye, A.O., Ugbede, I., Onakoya, A., Kizor-Akaraiwe, N., Teekhasaenee, C., Suwan, Y., Supakontanasan, W., Okeke, S., Uche, N.J.,

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