Targeting RGD-binding integrins as an integrative therapy for diabetic retinopathy and neovascular age-related macular degeneration

Integrins are a class of transmembrane receptors that are involved in a wide range of biological functions. Dysregulation of integrins has been implicated in many pathological processes and consequently, they are attractive therapeutic targets. In the ophthalmology arena, there is extensive evidence suggesting that integrins play an important role in diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, dry eye disease and retinal vein occlusion. For example, there is extensive evidence that arginyl-glycyl-aspartic acid (Arg-Gly-Asp; RGD)-binding integrins are involved in key disease hallmarks of DR and neovascular AMD (nvAMD), specifically inflammation, vascular leakage, angiogenesis and fibrosis. Based on such evidence, drugs that engage integrin-linked pathways have received attention for their potential to block all these vision-threatening pathways. This review focuses on the pathophysiological role that RGD-binding integrins can have in complex multifactorial retinal disorders like DR, diabetic macular edema (DME) and nvAMD, which are leading causes of blindness in developed countries. Special emphasis will be given on how RGD-binding integrins can modulate the intricate molecular pathways and regulate the underlying pathological mechanisms. For instance, the interplay between integrins and key molecular players such as growth factors, cytokines and enzymes will be summarized. In addition, recent clinical advances linked to targeting RGD-binding integrins in the context of DME and nvAMD will be discussed alongside future potential for limiting progression of these diseases.


Introduction
Integrin receptors are transmembrane heterodimeric adhesion proteins that play an essential role in integrating the extracellular to the intracellular environment. In the mid-eighties, the first integrin receptor was discovered based on its engagement with specific motifs on extracellular matrix (ECM) protein fibronectin (Pierschbacher and Ruoslahti, 1984;Tamkun et al., 1986). Since then, a vast number of articles on integrins have been published on a regular basis (on average ~2000 new publications per year and ~27,000 to date) and it is established that these receptors are involved in various cellular processes such as adhesion, differentiation, shape, migration, signalling, invasion, proliferation and survival with clear linkage to pathological processes, including inflammation, vascular leakage, neovascularization and fibrosis Fu et al., 2007;Hammes et al., 1996;Kanda et al., 2012;Koch and Distler, 2007;Santulli et al., 2008;Umeda et al., 2006;Wilkinson-Berka et al., 2006;Zahn et al., 2009). The multi-facetted role integrins play in cell pathophysiology is still being investigated and deciphering the precise nature of integrin-linked molecular mechanisms in health and disease remains a significant and relevant research challenge.
In the last few years, there has been renewed interest in integrins and especially drugs that target arginyl-glycyl-aspartic acid (Arg-Gly-Asp; RGD) -binding integrins in tissues of the eye. This reinvigoration in the area has been driven, at least in part, by recent preclinical and clinical studies which demonstrated promising results in retinal diseases such as diabetic retinopathy (DR), diabetic macular edema (DME) and neovascular age-related macular degeneration (nvAMD) (Askew et al., 2018;Shaw et al., 2020;Tolentino et al., 2016). The main goal of this review is to highlight preclinical as well as clinical knowledge on the role of mainly RGD-binding integrins in DR, DME, proliferative DR (PDR) and nvAMD. In addition, we will endeavour to provide comprehensive information on the intricate cellular and molecular mechanisms of RGD-binding integrin signalling in the main disease hallmarks of these vision-threatening retinal disorders.

Classification and function of integrins
Integrins constitute a family of ubiquitously expressed transmembrane receptors that regulate cell-cell and cell-ECM interactions (Bouvard et al., 2013;LaFoya et al., 2018;Moser et al., 2009;Wu and Reddy, 2012). Integrins are obligate heterodimeric receptors consisting of an αand a β-subunit. Currently, 18 α-subunits and eight β-subunits are known, and various combinations thereof constitute the family of 24 heterodimeric integrin members (Humphries et al., 2006;Pan et al., 2016). The classification of the integrin receptor family into four different classes is based on their structure similarity and ligand recognition pattern: 1) RGD-binding, 2) collagen-binding, 3) leukocyte-specific and 4) laminin-binding integrin receptors (Fig. 1). The first subgroup recognizes the tripeptide sequence RGD in their natural ligands (e.g. fibronectin) and consists of eight different integrins: α v β 1 , α v β 3 , α v β 5 , α v β 6 , α v β 8 , α IIb β 3 , α 5 β 1 and α 8 β 1 . However, it is now known that RGD-binding integrins also bind to many other ECM proteins (e.g. vitronectin, fibrinogen and osteopontin) as well as growth factors, cytokines, enzymes, bacterial proteins and hormones (LaFoya et al., 2018;Ruoslahti, 1996;Wu and Reddy, 2012). Integrin receptors are activated upon binding to their cognate ligands. The affinity and activation state can be influenced by engagement of intracellular proteins, such as talin and kindlin, to the integrin cytoplasmic domains. This outside-in and inside-out signalling enables integrins to 1) carry signals from the extracellular microenvironment and 2) respond to changes from inside the cell to induce a conformational change and modulate their affinity for extracellular ligands (Bouvard et al., 2013;Dalton et al., 2016;Klapholz and Brown, 2017). Integrins exist in a dynamic equilibrium between different conformations with varying ligand affinity: low, intermediate or high affinity status (see Fig. 2). Structural studies have revealed that integrins are inactive when their ectodomains are bent and this low affinity state is induced by intracellular binding of integrin inactivators, such as sharpin or mammary-derived growth inhibitor (MDGI; to the α-subunits) and integrin cytoplasmic domain-associated protein 1 (ICAP-1) or filamin (to the β-subunits). During activation, integrins become extended and ligand binding can promote the transition from a closed intermediate to an open activated conformation of the headpiece. Integrins reach the open high affinity or fully activated state when they are simultaneously bound to the actin cytoskeleton as well as extracellular ligands (Banno and Ginsberg, 2008;Bouvard et al., 2013;Johansson and Mosher, 2013;Moser et al., 2009).

Retinal expression of integrins
A literature overview on the expression of integrin receptors and their subunits in the healthy retina is presented below and summarized in Table 1 and Fig. 3. Overall, these published findings indicate that RGD-binding integrins are expressed in all retinal layers.

Glial cells
In primate eyes, α 2 -, α 3 -, α 6 -, β 1 -and β 4 -integrin subunits were localized to astrocytes within glial columns at the optic nerve head, suggesting participation in attachment of astrocytes to basement membranes (Morrison, 2006). Detailed analysis of integrin α 6 − /− mice, which die at birth, revealed abnormalities in the laminar organization of the developing cortex and retina, indicating the importance of integrin-laminin interactions in proper development of the nervous system (Georges-Labouesse et al., 1998). Cultured human optic nerve head astrocytes were found to express α v -, α 4 -, α 5 -, α 6 -, α 9 -, β 1 -, β 3 -and β 5 -integrin subunits (Neumann et al., 2014). The RGD-binding integrin subunits α v , β 3 , β 5 and β 8 were found to be expressed by cultured human brain astrocytes. Cultured astrocytes derived from the developing murine retina expressed α v and β 8 (Hirota et al., 2011), while in situ hybridization on the postnatal mouse retina revealed β 8 in Müller cells but not in astrocytes (Arnold et al., 2012). As described in human eyes by Brem et al. (1994), α 2 -, α 3 -, β 1 -and β 2 -integrin subunits were detected in the inner limiting membrane, which is formed by Müller cell end feet and astrocytes . The β 1 -integrin subunit was proposed as surface marker for a novel magnetic-activated cell sorting-based approach of Müller cell enrichment from adult murine retinas, as it was expressed at significantly higher levels in Müller cells than retinal neurons (Grosche et al., 2016). Immunocytochemistry revealed expression of α 1 -, α 2 -, α 3 -and β 1 -integrin subunits by Müller cells (Guidry et al., 2003). However, due to trans-differentiation of cultured Müller glia towards a fibroblast-like phenotype, expression of integrin subunits can shift and thereby not represent the healthy situation but rather their potential involvement in the generation of tractional forces (Guidry et al., 2003;Hauck et al., 2003). RGD-binding integrins α v β 5 and α v β 8 and their ligand vitronectin were expressed by astrocytes in vivo and were further upregulated during neurological diseases (Hirota et al., 2011;Milner et al., 1999;Yun et al., 2016). α v β 8 integrin expression in astrocytes was found to be essential for neovascularization in the developing retina.

RGD-binding integrins integrate multiple signalling cascades
Integrins can cross-talk with growth factors and their receptors, which has implications for the diversity of signalling responses occurring during normal physiology and pathogenesis (Eliceiri, 2001). The interaction between RGD-binding integrins and specific growth factors such as VEGF, angiopoietin (Ang), transforming growth factor-β (TGF-β) and basic fibroblast growth factor (bFGF) has been extensively described Table 1 Overview of the integrin receptor (subunit) expression in the retinal cell types and layers.

Angiopoietin (Ang)/Tie pathway
Angiopoietins (Ang-1 and Ang-2) play an essential role in regulating vascular permeability, angiogenesis and inflammation Saharinen et al., 2017). Depending on the context, Ang-2 acts as an agonist or antagonist for the tyrosine kinase with immunoglobulin-like and EGF-like domains 2 or Tie2 receptor (Daly et al., 2006). For instance, a high Ang-2/Ang-1 ratio in combination with VEGF results in reduced vascular stability, leading to increased vascular leakage and neovascularization, while Ang-2 induces EC death and vessel regression in the absence of VEGF (Akwii et al., 2019;Hammes et al., 2011). While initially Ang-2 was reported to act as a competitive antagonist by binding, but not activating, Tie2 on ECs, Tie2 phosphorylation and thus activation by Ang-2 was also described, at least when present at high concentrations (Akwii et al., 2019;Daly et al., 2006). Likewise, Ang-1 fulfils a dual role in neovascularization. Several reports have described an anti-angiogenic and vascular stabilization function of Ang-1 in the eye (Hammes et al., 2011;Lee et al., 2014b;Nambu et al., 2004), whereas other studies reported the stimulating effect of Ang-1 in retinal as well as dermal and cerebral neovascularization (Cho et al., 2006;Hammes et al., 2011;Lee et al., 2013;Pang et al., 2018;Wang et al., 2019). Nevertheless, it is generally accepted that the Ang-2/Ang-1 ratio is augmented under pathological circumstances and results in vascular instability.
The Ang/Tie signalling pathway is modulated by its direct and indirect interactions with RGD-binding integrins. Hakanpaa et al. (2015) demonstrated that Ang-2-dependent activation of β 1 -integrin induced α 5 β 1 translocation into the ends of actin stress fibres in ECs, resulting in vascular endothelium destabilization, which could be blocked by neutralization of β 1 -integrin. In addition, intravitreal (IVT) injection with α 3 -or β 1 -integrin inhibitors protected against Ang-2-dependent pericyte dropout (Park et al., 2014) and IVT treatment with neutralizing antibodies against α v β 5 could attenuate Ang-2-induced astrocyte loss in the diabetic streptozotocin (STZ) mouse (Yun et al., 2016). On the other hand, Ang1 supplementation was suggested as potential therapy for ischemic vascular retinopathies as binding of Ang1 to α v β 5 was shown to induce FAK phosphorylation and fibronectin synthesis in retinal astrocytes, thereby stimulating guided reparative angiogenesis in the retina (Lee et al., 2013).
Both receptor homologues Tie1 and Tie2 can directly associate with the RGD-binding integrins α v β 3 and α 5 β 1 via their extracellular domains, and the integrin/Tie2 co-cluster stabilizes in the presence of fibronectin. Tie2 signalling was significantly sensitized to lower concentrations of Ang-1 following interaction with RGD-binding integrin co-receptors, whereas both a constitutive and transient interaction between Tie2 and α v β 3 following Ang-2 ligand binding was described (Cascone et al., 2005;Dalton et al., 2016;Thomas et al., 2010). Ang-2-induced Tie2/α v β 3 receptor complex formation could recruit and activate FAK, followed by focal adhesion dissociation as well as internalization and lysosomal degradation of integrin α V β 3 , thereby causing EC destabilization (Thomas et al., 2010). Recently, Mirando et al. (2019) indicated that α 5 β 1 integrin heterodimers sequester Tie2 at non-junctional locations in the EC membrane. Upon treatment with AXT107, the heterodimer is disrupted, leading to translocation and complex formation of α 5 and Tie2 at EC-EC junctions and to conversion of Ang2 into a strong Tie2 agonist, whereby EC-EC contacts are reinforced and monolayer permeability is reduced (Mirando et al., 2019). Besides their capacity to bind Tie receptors, direct binding of Ang ligands to RGD-binding integrins has also been demonstrated in Tie2low ECs and several cell types lacking Tie2 such as cancer cells, neurons and cardiomyocytes (Carlson et al., 2001;Dalton et al., 2016;Felcht et al., 2012;Lee et al., 2014b). Although Ang-2 was reported to bind directly to the RGD-binding integrins α v β 3 , α v β 5 and α 5 β 1 by ELISA (albeit with low affinity), thereby promoting Tie2-independent neovascularization (Felcht et al., 2012;Hakanpaa et al., 2015), these results should be interpreted with caution since demonstrating this interaction required the use of a covalent cross-linking agent. While Ang-2 was demonstrated to activate α 5 β 1 -integrin via its N-terminal domain (Hakanpaa et al., 2015) and the Glutamine Gln-362 residue of Ang-2 (Lee et al., 2014a), the fibrinogen-like receptor binding domain of Ang-1 was essential for direct interaction with α v β 3 and α 5 β 1 in a Tie2-independent manner (Dallabrida et al., 2008;Dalton et al., 2016). 4.1.3. Transforming growth factorβ (TGFβ) In physiological conditions, the transforming growth factor-β (TGFβ) subfamily, consisting of TGF-β1, -β2 and -β3, is expressed at low levels and involved in for instance cell growth and matrix synthesis. In contrast, in pathological conditions, these growth factors are expressed at high levels and may cause accumulation of matrix components, fibrosis, inflammation and immune dysregulation (Finnson et al., 2020;Wan and Flavell, 2007). TGF-β is expressed as a full-length protein, containing a large N-terminal domain (referred to as latency-associated peptide LAP-1, 2 or 3) and a smaller C-terminal domain (referred to as mature TGF-β, which can be separated by intracellular proteolysis). A dimer of LAP can form a non-covalent complex with a dimer of mature TGF-β, called LAP-or latent-TGF-β. This complex will remain in the cell until it is bound by latent TGF-β-binding proteins (LTBPs) -1, -2, -3 or -4 to form a large latent complex (LLC). This LLC is secreted from cells and needs further processing to release TGF-β from LAP, which can then bind and activate TGF-β receptors (Annes et al., 2003;Lawrence, 2001;Miyazono et al., 1991;Rifkin, 2005;Taipale et al., 1994;Taylor, 2009).
All α v -containing RGD-binding integrins have been shown in vitro to release and thus activate TGF-β1 or -β3, more specifically via interaction or binding with the RGD motif in the LAP-1 and -3 peptide, respectively (Annes et al., 2003;Asano et al., 2005;Mu et al., 2002;Munger et al., 1999;Reed et al., 2015). The contribution of α v -containing RGD-binding integrins to TGF-β1 activation was further assessed in knock-out animals containing a mutation in the TGF-β1 gene encoding a non-functional or inactive variant of LAP's integrin binding site (RGE instead of RGD). These mice demonstrated the characteristic phenotype of TGF-β1 knock-out animals (e.g. inflammation and vascular defects), while a normal production of latent TGF-β1 was present (Yang et al., 2007b). On the other hand, upon activation and receptor engagement, TGF-β1 or -β3 signalling can also induce the expression of α v -containing integrins (Honda et al., 2010;Zambruno et al., 1995).
The first integrin identified as a LAP-TGF-β activator was α v β 6 (Munger et al., 1999), requiring an intact integrin cytoplasmic domain and the presence of other ECM proteins (e.g. fibronectin) (Guerrero and McCarty, 2018). This RGD-binding integrin can play a very important role in the development of fibrotic conditions (Basta et al., 2020;Horan et al., 2008;Huang et al., 1996;Munger et al., 1999;Puthawala et al., 2008;Wang et al., 2007) and pathological angiogenesis (Guerrero and McCarty, 2018), although its role in the eye remains undefined. Another integrin that was described to activate TGF-β is α v β 8 , expressed by normal epithelial and neuronal cells in vivo (Araya et al., 2006;Mu et al., 2002). In contrast to α v β 6 , activation by α v β 8 does not require the integrin cytoplasmic domain, but rather the presence of membrane type 1-matrix metalloproteinase (MT1-MMP) on the cell surface or in the ECM (Arnold et al., 2012). In the retina, it has been described that deletion of α v β 8 in astrocytes led to impaired blood vessel sprouting and hemorrhages, a phenotype that correlated with reduced α v β 8 -mediated activation of ECM-bound latent TGF-β (Hirota et al., 2011), indicating an essential role for α v β 8 in neovascularization in the developing retina.

Basic fibroblast growth factor (bFGF)
bFGF (also known as FGF2) is a cell signalling protein that is involved in a wide variety of processes and is especially relevant to ocular angiogenesis. Many studies have demonstrated a link between RGD-binding integrins and bFGF-induced neovascularization, from which α v β 3 and α 5 β 1 are the most described. Different studies reported that α v β 3 expression (and not α v β 5 ) in ECs of corneal and chick chorioallantoic membrane (CAM) assays was significantly increased upon induction of angiogenesis using bFGF (Brooks et al., 1994;Friedlander et al., 1995;Koch and Distler, 2007). The exact mechanisms underlying bFGF-induced ocular angiogenesis dependent on integrins are unclear, although one research group reported a direct binding of α v β 3 to FGF1 (Mori et al., 2008). The pro-angiogenic features induced by bFGF could be significantly reduced in vitro or ex vivo by using specific anti-α v β 3 antibodies (Brooks et al., 1994;Rusnati et al., 1997), by a RGD peptidomimetic cyclo[DKP-RGD] 1, which showed low nanomolar affinity for α v β 3 and α v β 5 (Fanelli et al., 2014), by the α 5 β 1 inhibiting molecule JSM6427 (Maier et al., 2007) or by TSRI265, which suppresses α v β 3 -MMP-2 interactions (Silletti et al., 2001), whereas the use of an anti-α v β 5 antibody had no effect (Friedlander et al., 1995;Koch and Distler, 2007). Besides α v β 3 , the integrin α 5 β 1 has also been demonstrated to be highly upregulated in the CAM-model after bFGF-induced angiogenesis (Kim et al., 2000a), which requires interaction of this receptor with fibronectin (Aota et al., 1994). Inhibition of this RGD-binding integrin reduced vascular leakage in a rabbit VEGF/bFGF pellet-induced model of choroidal nevascularization (CNV) . Moreover, it was described that blocking tube formation in an bFGF-induced fibrinous exudate, containing fibrin and fibrinogen, requires the simultaneous inhibition of α v β 3 -and α 5 β 1 -integrins (Laurens et al., 2009). On the other hand, a recent report described that integrin α 5 suppression efficiently prevented the production of TGF-β and bFGF, but not VEGF, showing a direct effect of α 5 inhibition on the expression of bFGF (Lv et al., 2020). The integrins α v β 3 and α 5 β 1 can thus play a key role in bFGF-induced angiogenesis, which is in contrast to VEGF-induced neovascularization, where mainly α v β 5 is involved (Friedlander et al., 1995). Indeed, different integrins are involved in EC-mediated angiogenesis, depending on the specific growth factor that is released by these cells.
Whereas binding of ANGPTL2 to α 5 β 1 was found to promote inflammation (Takano et al., 2019) (see also section 5.1.2), the ANGPTL4 -α v β 3 interaction seemed necessary for inducing protective effects against hypoxia-induced permeability (e.g. increased tight junction integrity) in the retina, more specifically by modulating the Src signalling pathway downstream of VEGFR-2. This observation indicates that the activation of the ANGPTL4 -α v β 3 axis might be a potential protective pharmaceutical intervention in pathological retinal conditions (Gomez et al., 2016).
While interactions between various growth factors and α v β 3 , α v β 5 and α 5 β 1 integrins have been extensively described, their specific role in the eye or retina remains ill-defined. Nevertheless, the strong association between RGD-binding integrins and key growth factors is an essential pathway in pathological angiogenesis, inflammation, vascular leakage and fibrotic reactions and this makes them very relevant for retinal diseases.

FAK/Src tyrosine kinases
The tyrosine kinases FAK and Src are important mediators of integrins, regulating cytoskeletal organization and motility in response to cell adhesion (Shattil, 2005). Integrin-mediated adhesion induces autophosphorylation of FAK, thereby creating a binding site for Src, which will lead to phosphorylation of other tyrosine residues in FAK and as such maximizes its kinase activity (Mitra and Schlaepfer, 2006). This active FAK/Src complex can then further activate downstream signalling molecules in a signalling cascade, involving key intermediates such as phosphoinositide 3 kinase (PI3K), Akt, and ERK, and was demonstrated to regulate retinal angiogenesis and edema Kornberg et al., 2004;Scheppke et al., 2008;Seo and Suh, 2017;Sergeys et al., 2020;Toutounchian et al., 2017). In addition, anti-oxidant protection was elicited by the interaction between α v β 5 integrin, FAK and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), activated through the binding of photoreceptor outer segment (POS) to RPE cells in culture (Roggia and Ueta, 2015). Contradictory findings were described on whether or not α v β 5 integrin is involved in the regulation of FAK and receptor tyrosine kinase Mer (MerTK) in the context of RPE cell phagocytosis (Qin and Rodrigues, 2012). These results could suggest that the interaction between α v β 5 integrin/FAK and other factors (e.g. PGC-1α and MerTK) can have important protective functions in RPE cells. A study in the context of DR showed that cysteine rich protein (Cyr61), a secreted, ECM-associated signalling protein of the CTGF, Cyr61 and Nephroblastoma overexpressed gene (CCN) family, induced the expression of MCP-1, leading to an inflammatory response, which seemed to be mediated by the activation of the integrin α ν β 3 , FAK, PI3K/Akt and IκB kinase/NF-κB pathways in chorio-retinal vascular ECs (You et al., 2014), suggesting that these pathways can be considered as a possible inhibitory target for DR. Direct interaction between integrins and Src in the retina was only described during neurite outgrowth on primary retinal neurons, in which activation of Ephrin-A5 was dependent on the activation of β 1 -integrins and various members of the Src family (Davy and Robbins, 2000).

Matrix metalloproteinases (MMPs)
The relationship between integrins and matrix metalloproteinases (MMPs) is extensively described in literature but remains complex and context-dependent. There is evidence that fibronectin, through its interaction with RGD-binding integrin receptors, can regulate the expression and activity of MMPs, especially MMP-2 and MMP-9 (Huhtala et al., 1995;Shibata et al., 1997;Stanton et al., 1998;Xie et al., 1998). Several papers also described a link between increased MMP levels and integrin activation, leading to altered cell adhesion, growth and motility as demonstrated for integrin α v β 3 (Kanda et al., 2000;Cohen et al., 2014;Crisp et al., 2014), for α v β 8 (Mu et al., 2002) and for α 5 β 1 (Bax et al., 2004;Huhtala et al., 1995). In the eye, MMP-2 inhibition (but not MMP-9 inhibition) was shown to reduce axon outgrowth of mouse RGCs via a β 1 -integrin dependent pathway, using ex vivo retinal explant models (Gaublomme et al., 2014). Moreover, increased RPE cell adhesion to collagen matrix (Ulbrich et al., 2011), diabetes-induced pericyte cell death (Yang et al., 2007a) and increased Müller cell migration (Lorenc et al., 2015) were related to increased α 2 -or β 1 -integrin and MMP-2 expression, which are key processes in AMD and DR. On the other hand, there is also evidence that activation of MMPs can lead to loss of integrins. Indeed, in various retinal neurodegeneration models, upregulated MMP-9 levels were associated with decreased RGC expression of β 1 -integrin and FAK and Akt dephosphorylation, all prior RGC apoptosis. Cell death was prevented by maintaining β1-integrin activation with agonistic antibodies (D'Onofrio et al., 2019;Santos et al., 2012). All these results point towards a clear, but complex, link between integrins and MMP. The interaction is context-dependent and will determine whether activated MMPs will lead to either loss or activation of integrins and whether RGD-binding integrin receptors will regulate the expression and activity of MMPs.
Overall, a complex interplay between integrins and cytokines, as well as various enzymes is described mainly in the context of inflammation (e.g. α 5 β 1 and α v β 3 ), angiogenesis/edema (α v β 3 ), RPE cell phagocytosis (α v β 5 ) and/or RGC apoptosis (β 1 -integrins). Although the relevance of these interactions on an ocular level is not described that much in detail, these could possibly play a role in various retinopathies, such as AMD and DR.
To summarize, RGD-binding integrins affect a multitude of diseaserelated proteins (e.g. growth factors, cytokines and enzymes) and their molecular pathways, and are able to modulate growth factor signalling in various ways (see also Fig. 4). Given their potential to integrate multiple cellular signalling networks, RGD-binding integrin antagonists are assumed to exert broader biological effects and target additional points in pathological processes as compared to anti-VEGF therapies.

Role of RGD-binding integrins in disease hallmarks of diabetic retinopathy and neovascular age-related macular degeneration
Although DR and nvAMD are distinct retinal disorders, they share several common disease hallmarks such as chronic inflammation, vascular leakage, neovascularization and fibrosis. In the following paragraphs, we will discuss the molecular interactions and signalling pathways by which RGD-binding integrins regulate these pathological processes. The cellular and molecular machinery by which RGD-binding integrins contribute to these vision-threatening processes has been derived mostly from cell culture observations. However, the importance of RGD-binding integrins has also been studied in experimental animal models for DR and nvAMD and these have proved valuable for advancing our understanding, not least in terms of the potential for therapeutic targeting of these receptors (see also Table 2).

Inflammation
As discussed in section 4.2.1, RGD-binding integrins can regulate inflammatory processes by interacting with several cytokine-mediated signalling pathways. This section will review the current understanding on the role of RGD-binding integrins in leukocyte adhesion, migration and infiltration at the vascular endothelial barrier and in modulating inflammatory immune signalling pathways.

Leukocyte-endothelial interactions
Blocking integrin function or genetic deletion of integrins has been described to interfere with the adhesion and transmigration of circulating leukocytes to/through the ECs of the blood vessel wall, which are critical events during inflammatory reactions and various vascular processes. Leukostasis likely contributes to retinal vascular leakage and thereby enhances the pathogenic effects on the retina during DR. The leukocyte adhesion cascade involves distinct adhesive leukocyte-EC interactions and signalling pathways in a specific spatiotemporal manner. The initial interaction between leukocytes and endothelium appears to be transient, resulting in the rolling of leukocytes along the vessel wall. Subsequently, leukocytes become activated by endothelial factors, leading to their arrest and firm adhesion, and will ultimately, transmigrate through the intercellular junctions into the underlying tissue, also called diapedesis. Once integrins are activated on the leukocyte surface, they mediate strong adhesive interactions with counterreceptors (ligands) on the endothelium, including vascular cell adhesion molecule-1 (VCAM-1 or CD106) and intercellular adhesion molecule-1 (ICAM-1 or CD54), which are highly upregulated in response to inflammatory mediators such as TNF-α (Barouch et al., 2000;Goda et al., 2000;Gustavsson et al., 2010;Jin et al., 2006;Muller, 2003;Weerasinghe et al., 1998).  The leukocyte adhesion receptors such as α L β 2 , α M β 2 and α 4 β 1 integrins are well-known players in this leukostasis process (Mitroulis et al., 2015). Moreover, under inflammatory conditions, antibodies against the RGD-binding integrin α v β 3 were able to inhibit trans-endothelial monocyte migration to a similar extent as anti-β 1 or anti-β 2 antibodies, likely by modulating the α L β 2 integrin-mediated migration of monocytes on ICAM-1 (Weerasinghe et al., 1998). The presence of β 1 -and β 3 -integrins in close association with cell surface tissue transglutaminase on podosome-like adhesive structures of differentiated monocytes further supports the involvement of RGD-binding integrins in extravasation and migration of leukocytes during inflammation (Akimov and Belkin, 2001). The RGD-binding integrin α v β 3 was also described to regulate lymphocyte migration and subsequent extravasation on/through ECs and interstitial tissue, which seems to be modulated by a cross-talk between α v β 3 and α 4 β 1 integrins (Imhof et al., 1997;Lacy-Hulbert et al., 2007). In addition, myeloid cell transmigration can result from engagement of integrin-associated protein (IAP or CD47) -α v β 3 on ECs by leukocyte platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31), leading to increased intracellular Ca 2+ levels, EC retraction and loss of tight junctions (Porter and Hogg, 1998). Adhesion and trans-endothelial leukocyte trafficking are also modulated by α 5 β 1 . This effect was suggested to result from its close physical and functional interaction with endothelial endoglin, enhanced α 5 β 1 -fibronectin binding and/or FAK-extracellular signal-regulated kinase (ERK) signal pathway activation (Labus et al., 2018;Rossi et al., 2013;Yang et al., 2012). Trans-endothelial migration of neutrophils was regulated by combined α v β 3 and α 5 β 1 integrin activity (Gonzalez et al., 2007).
Besides these in vitro studies, there is limited in vivo evidence that points towards a role for RGD-binding integrins in leukocyte-endothelial Table 2 Overview of preclinical studies using RGD integrin receptor antagonists in animal models for DR, DME or nvAMD.
interactions. Subcutaneous administration of the peptidomimetic α vintegrin antagonist S247 (25 mg/kg body weight, twice/day) in an acute kidney allograft rejection model could significantly reduce mononuclear cell (T-cells, monocytes, macrophages) infiltration by approximately 45%, adhesion by 65% and trans-endothelial migration by 60% (Bedke et al., 2007). Concomitant with the impaired adhesion and migration of T-cells in the absence of β 3 in vitro, lymphocyte infiltration was also significantly attenuated (60% reduction for CD4 + T-cells, 36% for CD8 + T-cells) in a model of heart transplant rejection using β 3 knock-out animals (Lacy-Hulbert et al., 2007). On the other hand, systemic treatment with α v β 5 blocking antibodies (4 mg/kg) in a sepsis-induced experimental animal model could decrease, yet not completely prevent, mortality, but could not reduce sepsis-induced cytokine/chemokine serum levels. In addition, thioglyccolate-induced leukocyte migration into the peritoneum could not be inhibited by α v β 5 neutralization (Su et al., 2013). Although this study suggested α v β 5 as a regulator of endothelial permeability by affecting inflammation-induced EC cytoskeletal rearrangement, additional molecular players are likely to contribute to the inflammation-driven processes in these models of severe disease. However, differences in for instance dosages, tissues and thus expression of integrins might explain the discrepancy between these in vivo studies.
The in vivo effect of RGD-binding integrins on leukocyte adhesion in the eye is to our knowledge poorly investigated. While retinal vascular permeability was significantly inhibited, leukocyte adhesion was (notsignificantly) reduced by 48% after oral administration of the α v -integrin antagonist JNJ-26076713 (60 mg/kg body weight, twice/day) in STZ-induced diabetic rats (Santulli et al., 2008). Intravitreal pretreatment with the α v β 3 and α 5 β 1 inhibitor AXT107 (1 μg/eye) in the acute TNF-α-induced mouse model significantly reduced the number of retinal adherent leukocytes by 29% (Mirando et al., 2020). Of note, AXT107 was found to reduce TNF-α-induced VCAM-1 and ICAM-1 levels on HUVECs (Mirando et al., 2020).

Leukocyte activation
Next to their implication in immune cell recruitment, α v β 3 and α 5 β 1 activation also regulates macrophage inflammatory immune signalling pathways at the site of injury. These integrin-mediated processes are mainly driven via NF-κB, a transcription factor of a wide variety of genes coordinating inflammatory responses, including TNF-α, IL-1β and IL-6 (Antonov et al., 2011;Kurihara et al., 2011;Liu et al., 2017) (see also section 4.2 and Fig. 5). As such, α v β 3 was demonstrated to enhance TNF-α-and LPS-induced macrophage-related inflammatory responses by inducing NF-κB activation (Antonov et al., 2011). The PI3K/Akt signalling pathway was postulated as possible mechanism by which α v β 3 activation triggers NF-κB dependent pro-inflammatory gene activation.
Blockage of inflammatory reactions in the eye has been demonstrated after α 5 β 1 neutralization. As such, inhibition of α 5 β 1 by the small peptide ATN-161 could reduce VEGF-induced retinal leakage and neovascularization by suppressing NLRP3 inflammasome signalling and was shown to inhibit NF-κB activation and suppress retinal neovascularization in the mouse retinopathy of prematurity (ROP) model (Sui et al., 2018a(Sui et al., , 2018b. In addition, intravitreal administration of the α 5 β 1 inhibitor JSM6427 in a rabbit model of retinal detachment resulted in a significant inhibition of microglia and macrophage proliferation (Zahn et al., 2010). Subconjunctival or topical application of the small molecule integrin α 5 β 1 inhibitor CLT-28643 significantly reduced leukocyte density in the bleb in a mouse model of glaucoma filtration surgery (Van Bergen et al., 2016).
To conclude, α v β 3 and α 5 β 1 are the RGD-binding integrin receptors which have been linked regularly to inflammatory responses that are central to the pathology of retinal vascular disorders, especially by mediating immune cell interactions with ECs and the ECM and by inducing inflammatory signalling pathways.

Vascular leakage
Several papers have reported on the role of RGD-binding integrins α v β 3 , α v β 5 and α 5 β 1 in vascular leakage, as demonstrated in mice carrying a genetic deletion in an integrin subunit or by functional inhibition of integrins (Hakanpaa et al., 2018;Eliceiri et al., 2002;Robinson et al., 2004;Su et al., 2012). Remarkably, while vascular permeability was reduced in β 1 and β 5 -deficient animals or after α v β 5 and β 1 neutralization, vascular leakage was either unaltered or increased in β 3 knock-out mice or after α v β 3 blockade, indicating distinct regulatory functions for α v β 3 , α 5 β 1 and α v β 5 on endothelial barrier permeability.
In the brain, the RGD-binding integrins α 5 β 1 , α v β 1 , α v β 3 and α v β 5 have been associated with BBB permeability during pathogenesis Lee et al., 2019;Roberts et al., 2017;Shimamura et al., 2006;Wang et al., 2019Wang et al., , 2020. Similarly, in the eye, several publications reported on the role of the RGD-binding integrins α 5 β 1 , α v β 3 and α v β 5 in retinal and choroidal vascular permeability. Inhibition of α 5 β 1 integrin by volociximab or JSM6427 significantly inhibited angiogenesis-driven leakage in the laser-induced CNV cynomolgus monkey model (Ramakrishnan et al., 2006;Zahn et al., 2009). In addition, choroidal vascular leakage was dose-dependently suppressed by JSM6427 in a VEGF/bFGF-induced CNV rabbit model . Treatment with the α 5 β 1 and α v β 3 integrin-binding peptide AXT107 significantly reduced VEGF-induced retinal leakage in the rabbit eye and in rho/VEGF mice, concomitant with disruption of VEGF, hepatocyte growth factor (HGF) and PDGF-BB signalling as demonstrated using cultured human retinal ECs (Silva et al., 2017). More recently, application of AXT107 was shown to suppress Ang-2, LPS and TNF-α-induced vascular leakage in the mouse eye (Mirando et al., 2019(Mirando et al., , 2020. In detail, AXT107 was found to dissociate α 5 from the α 5 β 1 complex at non-junctional sites of ECs, leading to the translocation and complex formation of α 5 and Tie2 at EC junctions, resulting in activation of Akt-mediated survival pathways, reduced myosin regulatory light chain 2 (MLC-2) activity, actin rearrangement and thus stabilization of cell-cell interactions.
Topical ocular application of the α v β 3 antagonist SF0166 dosedependently attenuated retinal vascular permeability in the rabbit VEGF-induced leakage model (Askew et al., 2018). On the contrary, inhibition of hypoxia-induced retinal vascular permeability by ANGPTL4 was found to rely on the binding of ANGPTL4 to α v β 3 , indicative for a protective effect of α v β 3 on permeability. The vaso-protective effects of ANGPTL4/α v β 3 on adherens and tight junctions were ascribed to modulation of the VEGFR-2/Src signalling pathway (Gomez et al., 2016). In addition, vascular leakage was augmented in ANGPTL4 knock-out mice subjected to laser-induced CNV (Gomez et al., 2016).
Blocking α v integrins via cyclic RGD peptide or JNJ-26076713 administration was demonstrated to reduce retinal vascular permeability in the rat laser-induced CNV model and in the rat STZ-induced diabetes model, respectively (Santulli et al., 2008;Yasukawa et al., 2004). Remarkably, enhanced EC monolayer permeability and disrupted VE-cadherin localization at cell junctions were observed in vitro with the α v integrin antagonist cilengitide (Alghisi et al., 2009). Interestingly, α v β 5 , a known receptor of Ang-2, was upregulated in high glucose-treated astrocytes. Ang-2-induced astrocyte apoptosis during vascular leakage in STZ-treated animals was attenuated by α v β 5 blocking antibodies, suggesting Ang-2/α v β 5 integrin signalling as potential therapeutic target during early DR (Yun et al., 2016). Strong anti-leakage effects, comparable to anti-VEGF treatment, were reported for a novel pan RGD integrin antagonist THR-687 in a mouse VEGF-induced permeability model and cynomolgus laser-induced CNV model   (Fig. 6). THR-687 potently inhibits multiple integrin receptors belonging to the RGD class, including α 5 β 1 , α v β 3 , α v β 5 , α v β 6 and α v β 8 with IC 50 values in the low nanomolar range .
Retinal edema and VEGF-induced blood vessel permeability were also suppressed by EGT022, an RGD-containing disintegrin originated from the human-derived protein 'a disintegrin and metalloproteinase 15' (ADAM15), in the ROP model and in a modified Mile's assay, respectively (Jang et al., 2016). Such findings reinforce the concept that RGD integrin antagonism, especially for the integrins α v β 5 and α 5 β 1 , offers promising options for preventing vaso-permeability occurring in retinal vascular disorders. In addition, these studies underpin the integrated nature of RGD-binding integrins in multiple signalling cascades and their interaction with several vascular leakage-associated growth factors including, but not limited to, VEGF, FGF, Ang-2 and ANGPTL4 (see also section 4).

Angiogenesis
As already highlighted in section 4, RGD-binding integrins strongly interact with pro-angiogenic growth factors. This dynamic and highly regulated process of new blood vessel growth consists of ECM remodelling, endothelial cell migration, proliferation, survival and adhesion, sprouting and tube formation. The involvement of RGD-binding integrins in each of these steps will be discussed below and is schematically illustrated in Fig. 7. In relation to the current evidence of RGD-binding integrin receptors as therapeutic targets, there is an extensive literature based on animal models of pathological retinal and choroidal angiogenesis.
In the patient context, it is important to note that integrins α v β 3 , α v β 5 and α 5 β 1 have been observed in surgically removed patient CNV membranes. The integrins α v β 3 and α 5 β 1 were found especially in the early, active stages of disease progression and colocalized with ECs, while α v β 5 expression occurred in the mid and late (fibrotic) stages without colocalization with ECs (Cui et al., 2009). Similarly, Friedlander et al. (1996), observed α v β 3 (not α v β 5 ) on blood vessels in choroidal neovascular membranes from AMD patients, while both α v β 3 and α v β 5 were present in retinal neovascular membranes from patients with active vasoproliferative retinopathy (Friedlander et al., 1996). These findings indicate different pathological pathways driving retinal and choroidal neovascularization and a distinct role of RGD-binding integrins in these processes (Das and McGuire, 2003).

Extracellular matrix remodelling
For angiogenesis to occur, ECs must invade the surrounding ECM, which can be remodelled by many processes, including synthesis, contraction and proteolytic degradation (Larsen et al., 2006). Proteolytic degradation is mediated by enzymes such as MMPs and plasminogen activators (Theocharis et al., 2016). In ECs, a functional cooperation was observed between MT1-MMP and β 1 and α v β 3 integrins during migration (Galvez et al., 2002). Interestingly, preactivated MMP-2 selectively bound α v β 3 , but not α v β 5 integrin, in bovine microvascular ECs and this interaction was inhibited by the cyclic RGD peptide EMD 66203 (Nisato et al., 2005). Blockage of the α v β 3 interaction with MMP-2 via TSRI265 almost completely abolished bFGF-induced angiogenesis in the chick CAM assay (Silletti et al., 2001). Furthermore, the α 5 β 1 antagonist ATN-161 strongly reduced ocular neoascularization, partially by decreasing expression of MMP-2 and MMP-9, in ROP and laser-induced CNV mouse models (Sui et al., 2018b). More information on the link between integrins and MMPs can be found in section 4.2.3.
A close interaction between integrins and the urokinase-type plasminogen activator (uPA) system has also been described during angiogenesis. The latter system includes, amongst others, uPA and glycolipidanchored uPA receptor (uPAR) (Tang and Wei, 2008). uPA accumulation was reduced in EC culture medium after exposure to α v β 3 or α 5 antagonists (Laurens et al., 2009). In HUVECs, uPAR co-clustered with α 5 β 1 and knockdown of uPAR or inhibition of the uPAR-integrin interaction by a blocking peptide, prevented VEGF-induced internalization of α 5 β 1 , which is crucial to support angiogenesis (Alexander et al., 2012;Uhrin and Breuss, 2013). Bifulco et al. (2013) described another uPAR blocking peptide that prevented the recruitment of α v β 3 integrin at focal adhesions in VEGF-stimulated ECs (Bifulco et al., 2013). In VEGF-stimulated human retinal ECs, inhibition of uPAR resulted in decreased activation of ERK, p38, JNK and AKT, which is proposed to be via combined intraperitoneal (IP, 15 mg/kg, 1 h before VEGF) and IVT (50 μg/eye, simultaneous with VEGF) treatment. Aflibercept (IP, 25 mg/kg, 24 h before VEGF injection) was applied as positive control (n = 9-11 animals/condition). (B) CNV-induced vascular leakage in the cynomolgus monkey was measured via fluorescein angiography and expressed as mean percentage grade 4 or 5 lesions at 3 weeks following induction of laser lesions. THR-687 was administered at high (4.5mg/eye), medium (2.25mg/eye) and low (0.45mg/eye) dose levels, once a week for a period of 3 weeks. Lucentis (0.5mg/eye) was applied as positive control, administered once at 1 day after laser treatment (n = 5-6 animals/condition). Both data packages were previously described in Hu et al. (2019). Data are mean ± SEM and analyzed via one-way ANOVA with post hoc Tukey test (GraphPad Software, ***p < 0.001).
Thus, there is evidence to support a role for RGD-binding integrins in ECM remodelling by ECs via MMPs and the uPA system.
It was described that the chemokine (C-X-C motif) ligand (CXCL)-4 can function as a ligand for RGD-binding integrins, such as α v β 3 , α v β 5 and α 5 β 1 . Indeed, it was reported that HUVECs adhere to immobilized CXCL-4 through different RGD-binding integrins, supporting EC spreading and migration in an integrin-dependent manner. Soluble CXCL-4 on the other hand inhibited integrin-dependent EC adhesion and migration (Aidoudi et al., 2008), contributing to its anti-angiogenic effect.

Endothelial cell proliferation and survival
EC proliferation is tightly regulated by cell-ECM and cell-cell adhesion, as well as growth factors, cytokines and hormones. Of these environmental cues, the integrins are the most critical as they can regulate mammalian cell cycle progression through FAK and G1 phase cyclindependent kinases (CDKs). Integrin signalling has been associated with the induction of cyclin D1 and the downregulation of CDK inhibitors, thereby stimulating cell proliferation. In brief, integrins can increase cyclin D1 expression through ERK and PI3K activation, stimulate translation of cyclin D1 mRNA through Rac and stabilize cyclin D1 through PI3K (Moreno-Layseca and Streuli, 2014;Schwartz and Assoian, 2001;Zhao et al., 1998). In addition, FAK participates in multiple growth regulatory events such as JNK-mediated transcription of insulin Fig. 7. Schematic illustration on the potential role of RGD integrin receptors in angiogenic processes. Endothelial cell migration is regulated by α 5 β 1 , α v β 3 and α v β 5 integrins after binding to ECM molecules or CXCL4. Integrin α v β 3 can directly bind to the ECM degradation enzymes MMP-2 and MT1-MMP. Physical association of uPAR with α v β 3 and α 5 β 1 integrins recruits the uPA/uPAR complex to the cell surface where it leads to ECM degradation. Additionally, activation of α v β 3 or α 5 β 1 integrinα 5 β 1 integrin leads to accumulation of uPA in the extracellular space, potentially via transcriptional regulation. uPAR-α 5 β 1 integrin interaction is crucial for VEGF-induced internalization of α 5 β 1 integrin. Integrins can also promote EC proliferation via increased CyclinD1 expression through ERK and PI3K activation, and via FAK regulation of JNK-mediated transcription of IRS-1. Additionally, RGD-binding integrins play a vital role in EC survival, as demonstrated by α 5 β 1 inhibition (via ATN-161) through NF-κB signalling. Activated αvβ3 integrin inhibits the p53-Bax cell death pathways, whereas the cytoplasmic domain of unligated integrin β3 interacts with and activates caspase-8. Integrin α v β 3 binding of osteopontin stimulates EC survival through NF-κB activation via Src and Ras. Likewise, binding of fibronectin to integrin α 5 β 1 can suppress PKA activity, which induces EC apoptosis via caspase-8 activation. (ECM, extracellular matrix; ERK, extracellular signalregulated kinases; FAK, focal adhesion kinase; IRS-1, insulin receptor substrate 1; JNK, c-Jun N-terminal kinases; MMP, matrix metalloproteinase; MT1-MMP; membrane type-I matrix metalloproteinase; OPN, osteopontin; PI3K, phosphatidylinositol 3-kinases; PKA, protein kinase A; uPA, urokinase plasminogen activator; uPAR, uPA receptor).
RGD-binding integrins have also been implicated in EC survival, although the cell signalling pathways are complex. For example, EC adhesion mediated by β 1 -or α v -integrins induced tyrosine phosphorylation and activation of the EGF receptor, and led to MAPK/ERK activation and EC survival (Moro et al., 1998;Perruzzi et al., 2003).
Furthermore, the ligation state of α v β 3 integrin directly influenced p53 activity and bax-triggered cell death pathways. Whereas antagonists for α v β 3 integrin caused activation of p53, agonists suppressed this activity (Stromblad et al., 1996). Furthermore, the RGD-binding integrin inhibitor BCH-15046 was shown to induce apoptosis of ECs plated on vitronectin or type I collagen (Meerovitch et al., 2003). Moreover, the cytoplasmic domain of unligated integrin β 3 interacted with and activated caspase-8 to induce EC death (Stupack et al., 2001). In fact, the anti-angiogenic activity of the α v integrin antagonists cilengitide, AV-38/398 and S 36578-2 was attributed to anoikis, i.e. cell death mediated by cell detachment, through activation of caspase-8 and -9 (Christenheit et al., 2016;Maubant et al., 2006). Furthermore, the RGD integrin inhibitor BCH-15046 was shown to induce apoptosis of ECs plated on vitronectin or type I collagen (Meerovitch et al., 2003).
Additionally, α v β 3 integrin binding of osteopontin and vitronectin stimulated EC survival through NF-κB activation via Src and the small GTP-binding protein Ras (Scatena et al., 1998). Correspondingly, besides inhibiting MMP expression, the α 5 β 1 inhibitor ATN-161 was able to reduce retinal and choroidal neovascularization in ROP and laser-induced CNV mouse models, respectively, by decreasing NF-κB activation and promoting vascular EC apoptosis (Sui et al., 2018b).

Endothelial cell sprouting, tube formation and vessel maturation
In the postnatal mouse retina, angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes that express and assemble fibronectin (Kubota and Suda, 2009;Stenzel et al., 2011).
During the later stages of vascularization, the immature vascular plexus is reorganized into a mature and stable network through a series of maturation steps such as mural cell (vascular smooth muscle cells and pericytes) coverage and production of basement membrane. Beside the important role of β 1 -integrin in EC migration, proliferation and tube formation, it is also indispensable for stable, mature and non-leaky vessels. In the postnatal mouse retina, β 1 -integrin is essential for proper localization of VE-cadherin, which in turn is important for EC maturation, as it suppresses EC sprouting and proliferation (Yamamoto et al., 2015). Integrin α v β 3 also plays a crucial role in embryonic neovascularization and vessel maturation, as shown by treatment with anti-α v β 3 antibodies in the quail early embryo, whereby lumen formation was hampered (Drake et al., 1995). In addition, pharmacological inhibition as wells as genetic ablation of β 3 -integrin resulted in reduced vascular smooth muscle cell coverage in the developing mouse retina (Scheppke et al., 2012). RGD-binding integrins also modulate the Ang/Tie signalling pathway, which plays an essential role in vascular stability. This is described in more detail in section 4.1.2.
Taken together, evidence indicates that RGD-binding integrins are essential for several angiogenesis-related processes, including EC proliferation, migration, sprouting and maturation, indicating RGD-binding integrin antagonism as a promising treatment strategy for pathological angiogenesis.

Integrin antagonists in animal models of retinal and choroidal neovascularization
Previous studies have demonstrated the involvement of RGD-binding integrins in the regulation of EC proliferation, survival and migration during angiogenesis. These integrins include α v β 3 , α v β 5 , α 5 β 1 and α v β 8 (Avraamides et al., 2008;Yue et al., 2012). Systemic or local administration of integrin α 5 β 1 , α v β 3 and α v β 5 antagonists can inhibit retinal and choroidal angiogenesis in animal models of ocular disease, such as the ROP and CNV model. Indeed, topical administration of SF0166 and TAT PTD-endostatin-RGD, which specifically binds α v β 3, significantly reduced pathological neovascularization in the ROP model (Askew et al., 2018;Li et al., 2016). The α v integrin antagonist JNJ-26076713 also dose-dependently inhibited retinal neovascularization in the ROP model after oral administration (Santulli et al., 2008). A reduction in VEGF and VEGFR2 levels, as well as in pathological retinal neovascularization, was observed after intraperitoneal treatment of the α v β 3 and α v β 5 integrin antagonist SB-267268 in the same model (Wilkinson-Berka et al., 2006). Subcutaneous injection of a cyclic α v -integrin antagonist or the RGD-containing disintegrin EGT022 was also able to prevent hypoxia-induced retinal neovascularization in the mouse eye (Hammes et al., 1996) or increase pericyte coverage and thereby stimulating retinal microvessel maturation (Jang et al., 2016), respectively. By blocking the interaction of α 5 β 1 with fibronectin, sustained subcutaneous administration of JSM6427 did not only suppress laser-induced CNV, but could also induced regression of previously established CNV in mice (Umeda et al., 2006).
Beside these studies, showing an anti-angiogenic efficacy via topical, oral, intraperitoneal or subcutaneous delivery, several others confirmed this effect after IVT administration of RGD-binding integrin antagonists in animal models of retinal or choroidal neovascularization. By interacting with α 5 β 1 and α v -containing integrins in an RGD-independent manner, laser-induced CNV prevention and regression were obtained after single IVT administration of lebecetin in mouse eyes (Montassar et al., 2017). This heterodimeric C-type lectin also demonstrated inhibition of retinal neovascularization in the mouse ROP model (Montassar et al., 2017). Single IVT delivery of the α v β 3 and α 5 β 1 integrin antagonist peptide C16Y significantly inhibited laser-induced CNV, which was even more pronounced using a C16Y nanoparticle solution (Kim and Csaky, 2010). OCU200 (Ocugen), a tumstatintransferrin fusion protein, inhibited as well new blood vessel formation after IVT administration in the ROP and laser-induced CNV model by binding to α V β 3 integrins. IVT treatment with the α 5 β 1 RGD integrin antagonist JSM6427, or with the α v β 3 integrin antagonists tetrac and GOPPP, also reduced the number of preretinal nuclei or neovascular tufts in the ROP model (Li et al., 2014;Maier et al., 2007;Santulli et al., 2008;Yoshida et al., 2012). While the avascular area was increased after JSM6427 treatment or remained unaltered after local tetrac administration, vaso-obliteration was significantly decreased by GOPPP, which indicates promotion of normal vessel regrowth. In addition, GOPPP treatment significantly inhibited ERK1/2 phosphorylation and hypoxia-inducible factor 1α (HIF-1α) and VEGF levels in the ROP model. Rat eyes treated IVT with the α v integrin antagonist cyclic RGD peptide showed significant inhibition of laser-induced CNV and IVT administration of the specific α 5 β 1 inhibitor ATN-161 was described to reduce SDF-1-mediated CNV and leakage in the laser model (Lyu et al., 2018;Sui et al., 2018b;Yasukawa et al., 2004). Moreover, combination therapy with ATN-161 and an anti-VEGF antibody in a rat CNV model showed a stronger anti-angiogenic effect as compared to either agent alone . Analogously, combined IVT treatment with the α v β 3 and α 5 β 1 inhibitor AXT107 and aflibercept, a soluble decoy receptor that inhibits both VEGF and PlGF, in a mouse CNV experiment exhibited a synergistic, suppressive effect on subretinal neovascularization (Silva et al., 2017). When AXT107 was applied in the mouse ROP model, it significantly suppressed ischemia-induced retinal neovascularization (Silva et al., 2017). Also ALG-1001 demonstrated a strong reduction in laser-induced CNV by more than 40% and in retinal neovascularization in the mouse ROP model by more than 50%. In addition, combined administration of ALG-1001 and ranibizumab, a recombinant humanized IgG1 monoclonal antibody fragment that binds and inhibits VEGF, resulted in a 35% better performance than either drug alone in reducing neovascularization in a VEGF transgenic mouse model (Boyer, 2012).
As such, a substantial number of preclinical observations demonstrate potent anti-angiogenic and normal vessel stabilizing effects of anti-RGD integrin therapy in animal models of ocular disease. These observations are summarized in Table 2. Interestingly, studies that applied a combination therapy with an anti-VEGF agent demonstrated a stronger reduction of pathological neovascularization than a single treatment.

Fibrosis
Fibrosis is the result of a wound healing response that follows an acute or chronic injury. It involves excessive cell proliferation, migration and ECM deposition/remodelling and adhesion (Roy et al., 2016). In the retina, fibrotic scarring can compromise vision by causing biological damage and mechanical disruption to the visual axis. This is exemplified by epiretinal fibrotic tissue in DR, subretinal fibrosis in nvAMD and tractional fibrotic membranes in proliferative vitreoretinopathy (Friedlander, 2007).
RGD-binding integrins have been implicated in fibrosis, and modulation of integrins has demonstrated profound effects on fibrosis in multiple organs and disease states (Conroy et al., 2016). For example, β 6 knockout mice showed a delayed onset of fibrosis after corneal incision  and α v β 3 and α 5 β 1 integrins were present in human proliferative vitreoretinopathy tissue (Guenther et al., 2019;Robbins et al., 1994;Zahn et al., 2010). As previously mentioned, the observed α v β 5 integrin immunolabeling on surgically removed human CNV membranes of mid and late stages indicate its involvement during active remodelling and fibrosis, which was much less evident for α v β 3 and α 5 (Cui et al., 2009). The role of RGD-binding integrins in the different processes and their interaction with growth factors associated with fibrosis is discussed in more detail.

Myofibroblast differentiation
The involvement of fibroblasts in fibrotic processes throughout the body is well established. TGF-β is upregulated and activated in fibrotic diseases and is a crucial regulator in the differentiation of myofibroblasts, the key effector cells in fibrotic diseases. TGF-β-stimulated fibroblasts undergo a reorganization of the actin cytoskeleton and upregulation of α smooth muscle actin (αSMA) expression, which is often used as a myofibroblast marker (Biernacka et al., 2011;Evans et al., 2003). However, the retina is devoid of fibroblasts, so other cell types including glial cells, RPE cells, ECs and macrophages may contribute to retinal fibrosis, as their presence in epiretinal membranes has been demonstrated (Bringmann et al., 2009;Ishikawa et al., 2016;Little et al., 2018;Tamiya et al., 2010;Tsotridou et al., 2020). In brief, damaged RPE cells that have lost cell-cell contact have been reported to undergo epithelial-to-mesenchymal transition (EMT) and contribute to fibrotic processes in the retina (Tamiya et al., 2010), whereas ECs in DR and nvAMD may transdifferentiate into myofibroblasts through endothelial-to-mesenchymal transition (EndoMT) (Cao et al., 2014;Sun et al., 2018). Both myofibroblast differentiation via EMT and EndoMT is mediated by TGF-β signalling. The cross-talk between α v -integrins and TGF-β signalling is extensively described in section 4.1.3. Additionally, there is substantial evidence for RGD-binding integrins during EMT and EndoMT. Likewise, EMT in bronchial epithelial cells was mediated by integrin α v β 6 . Furthermore, overexpression of integrin β 3 promoted EndoMT , and knock-down of integrin β 1 suppressed EndoMT in ECs (Shi et al., 2015).

Extracellular matrix deposition and contraction
Fibroblasts respond to injury by synthesizing various ECM components and thereby mediating a reparative process (Evans et al., 2003;Roy et al., 2016). In the eye, multiple (transdifferentiated) cell types contribute to this ECM deposition, such as ECs, RPE cells and macroglia . Altered ECM composition is not only a consequence but also a driver of fibrosis. An early step in pathological ECM accumulation is fibronectin matrix assembly, indicating that RGD-binding integrin-directed therapies likely impact fibrotic diseases by interfering with cell-fibronectin interactions and as such with matrix deposition . Importantly, a key role for fibronectin in complications characteristic of proliferative vitreoretinopathy, including retinal detachment, was demonstrated in rodent models of retinal injury. Administration of nonpeptidic RGD mimetics SF-6.5 and NS-11 inhibited the adhesion of human tenon's capsule fibroblasts to fibronectin in culture (Hershkoviz et al., 1994). In respect of the eye, α 5 β 1 inhibition by JSM6427 was found to concentration-dependently block attachment of PMA-activated ARPE-19 cells to fibronectin, and to inhibit bFGF-stimulated migration of ARPE-19 cells towards fibronectin (Zahn et al., 2010). ARPE-19 adhesion to fibronectin could not be inhibited by α v β 3 or α v β 5 blocking antibodies (Zahn et al., 2010).
ECM stiffness is sensed via inside-out and outside-in signalling across cell adhesions composed of integrins and focal adhesion complexes. Increased matrix stiffness is coupled to elevated levels of several integrins such as integrin β 1 , α v β 3 and α 5 β 1 (Balcioglu et al., 2015;Dong et al., 2019;Yeh et al., 2017). Furthermore, in response to elevated matrix stiffness, cell contractile forces increase and thereby induce a conformational change in the LAP-TGF-β latency complex, resulting in the release of TGF-β and activation of a positive feedback loop of ECM synthesis and stiffening (Wipff et al., 2007). This feedback loop was mediated by TGF-β-SMAD signalling, which induces collagen and fibronectin gene expression (Biernacka et al., 2011) and inhibits MMP-dependent matrix degradation (Edwards et al., 1987). Wipff et al. (2007) indicated that integrin-mediated myofibroblast contraction activates TGF-β, as both RGD peptides and an integrin α v β 5 blocking antibody inhibited latent TGF-β activation. Moreover, treatment of lung fibroblasts with the pan-α v blocking antibody abituzumab reduced αSMA expression, IL-6 production and collagen gel contraction (Samy et al., 2017). Additionally, two disintegrins, echistatin and flavordin, were able to inhibit RPE cell-induced vitreous contraction and tractional retinal detachment in vitro and in the rabbit eye, respectively (Yang et al., 1996). Lygoe et al. (2004) demonstrated that function blocking antibodies against α v or β 1 could suppress TGF-β-induced αSMA expression in three human fibroblast cell lines. Furthermore, antibodies against α v , α v β 3 or α v β 5 could inhibit fibroblast contraction in a collagen gel contraction assay, without affecting adhesion.
These findings are in agreement with the observed anti-fibrotic properties of the pan-RGD-binding integrin antagonist THR-687 (see Fig. 8). THR-687 significantly reduced collagen contraction induced by both human dermal fibroblasts ( Fig. 8a and b) and ARPE19 cells in a concentration-dependent manner ( Fig. 8d and e). Furthermore, THR-687 significantly decreased the expression of αSMA in fibroblasts (52% reduction at 20 μM) and ARPE19 cells (38% reduction at 10 μM) compared to vehicle-treated cells (Fig. 8c, f). Moreover, repeated IVT injections of THR-687 in the cynomolgus laser-induced CNV model dose-dependently inhibited collagen deposition (Sirius Red staining) in the laser spots, although only the highest dose (4.5 mg/eye) induced a significant reduction (Fig. 8g and h).

Gliosis
In the mammalian retina, three main types of glial cellsastrocytes, Müller cells and resident microgliaserve to maintain retinal homeostasis. Reactive gliosis describes the response of astrocytes and Müller cells to injured or diseased tissue in the central nervous system (Fischer and Bongini, 2010). Müller cell gliosis involves cellular hypertrophy, increased proliferation, inflammation as well as tissue and vascular remodelling. Following retinal damage, the expression of vimentin and glial fibrillary acidic protein (GFAP) are dramatically upregulated in Müller cells and astrocytes (Bringmann et al., 2006;Lewis and Fisher, 2003). This increases the stiffness of the cells, thereby discouraging axonal regeneration (Bringmann and Wiedemann, 2012). In severe forms of reactive gliosis, cells can form glial scars that are inhibitory to neuronal regeneration (Sofroniew, 2009). Robel et al. (2009) demonstrated that β 1 -integrin-mediated signalling in astrocytes was required to promote their acquisition of a mature, non-reactive state (Robel et al., 2009). Conditional deletion of β 1 -integrin in glia and neurons resulted in partial reactive gliosis including astrocyte hypertrophy and the upregulation of GFAP and vimentin, yet without proliferation (Robel et al., 2009). Similar results were reported with β 1 -integrin deficient ependymal stem cells, where astrocyte differentiation was suppressed by β 1 -integrin via the integrin-linked kinase (ILK) pathway (Pan et al., 2014). Moreover, the α v β 3 integrin binding agonist, C16, alleviated astrogliosis and demyelination in an acute experimental allergic encephalomyelitis rat model (Han et al., 2013). Contrarily, over-expression of β 3 -integrin in astrocytes was sufficient to induce astrocyte reactivity (Lagos-Cabre et al., 2017), suggesting a delicate balance between the mature and reactive state of glial cells.
In the eye, IVT treatment with the α 5 β 1 small molecule inhibitor JSM6427 was shown to significantly reduce the number of proliferating Müller cells in a rabbit model of retinal detachment, together with a reduction in the number and length of subretinal scars (Zahn et al., 2010). Postnatal mouse retinas with a conditional deletion of α v -or β 8 -integrin in astrocytes and neurons exhibited reactive gliosis in response to haemorrhage (Hirota et al., 2011).
Overall, RGD-binding integrins and especially α v integrins have been positively linked to fibrotic responses such as myofibroblast differentiation and contraction and could be a valuable target to tackle retinal fibrosis. Although some RGD-binding integrins seem crucial for the mature, non-reactive state of glial cells, validation studies with pharmacological inhibitors are needed to confirm these cell type-specific, integrin knockout findings.
As described in these sections and summarized in Fig. 9, there is a growing amount of preclinical evidence indicating an important role for RGD-binding integrins in the pathogenesis of DR and nvAMD, demonstrating clinical promise with RGD-binding integrin intervention in these vision-threatening disorders (Bhatwadekar et al., 2020).

Diabetic retinopathy
DR is the most common microvascular disorder caused by diabetes mellitus and a leading cause of blindness in working-aged people in industrialized countries. DR can be broadly classified into a nonproliferative and a proliferative stage. The first discernible symptoms of non-proliferative DR are microaneurysms and retinal haemorrhages. Proliferative DR occurs with further retinal ischemia and is characterized by the growth of pathological blood vessels on the surface of the retina. These new vessels are unstable and may leak, leading to vitreous haemorrhage. DME is a complication of DR characterized by fluid accumulation in the macula of patients and can occur at any stage of the disease (Ford et al., 2013;Gao et al., 2008;Mohamed et al., 2007). For many years, DR was considered as a vascular eye disorder, but now, it is generally acknowledged that pathological processes such as oxidative stress, neurodegeneration, chronic inflammation, gliosis and fibrosis also play a major role in the pathogenesis of early as well as advanced DR (Brownlee, 2005;Curtis et al., 2009;Stitt et al., 2013Stitt et al., , 2016. The current first-line treatment for centre-involved DME are anti-VEGF agents (i.e. Eylea® or aflibercept, Lucentis® or ranibizumab and the off-label used Avastin® or bevacizumab). Even though anti-VEGF therapy has proven to be effective, various reports have stated that a subset of the DME patients respond sub-optimally to anti-VEGF treatment and display persistent edema with associated visual function loss (Nguyen et al., 2010(Nguyen et al., , 2012. These findings suggest that other pathways, in addition to VEGF, contribute to the development of DME. Alternative therapies of advanced DR consist of IVT corticosteroids injections, retinal laser photocoagulation or surgical vitrectomy. However, all these currently available therapies suffer from potential significant side effects. It has been reported that repeated treatment with VEGF inhibitors can result in increased risk of fibrotic complications and tractional retinal detachment (Moradian et al., 2008) and even signs of neurodegeneration have been reported (Beck et al., 2016). Corticosteroids come with high risk of cataract formation and increased intraocular pressure (Beck et al., 2016;Nurozler and Unlu, 2017;Yamamoto et al., 2003). Hence, numerous pharmaceutical companies endeavour to discover novel, alternative drug candidates for DR and DME with a superior long-term effectiveness and safety profile, and the potential to reduce the treatment burden. Fig. 8. THR-687 can inhibit fibrosis in preclinical models. Human dermal fibroblasts (HDF) or human retinal pigment epithelial cells (ARPE19), seeded in (A-B) or on top (D-E) of collagen gels, respectively, were treated with different concentrations of THR-687. A negative control was taken along to exclude intrinsic contractile properties of the gel (no HDF/no ARPE19). Contraction was monitored during 14 or 4 days for HDF or ARPE19, respectively (A, D) (protocols adapted from (Lygoe et al., 2004;Morales et al., 2007)). Images were quantified by measuring the area of the gel over the area of the well (B, E). Afterwards, cells were extracted from the collagen gels and alpha smooth muscle actin (αSMA) protein levels were measured (n = 3-4 independent experiments) (C, F). Cynomolgus monkeys received 9 laser spots to induce choroidal neovascularization (CNV) and were given either vehicle (3x IVT weekly), Lucentis (0.5mg/eye; 1x IVT) or THR-687 (0.45mg/eye, 2.25mg/eye or 4.5mg/eye; 3x IVT weekly). Animals were sacrificed at day 22 and collagen deposition in the lesion area (red dotted line) was measured by staining ocular paraffin sections with Sirius Red (SR) (G). Quantification was performed by measuring the Sirius Red positive area over the total lesion area (n = 3-6 eyes/condition). (H). Panel A-F represents new data, whereas panel G-H represent a new analysis of samples from a previously published study . Data are mean ± SEM and analyzed via one-way ANOVA with post hoc Bonferroni test (GraphPad Software, *p < 0.05; **p < 0.01; **p < 0.001; ****p < 0.0001).
There is clinical evidence that integrins play an essential role in the pathogenic processes of DR and DME. Immunostainings on human retinal tissues derived from PDR patients revealed that actively proliferating vascular ECs express increased α v β 3 and α v β 5 levels (Friedlander et al., 1996). In general, DR has not been found to be strongly correlated with genetic mutations or polymorphisms. Among the candidate genes are aldose reductase, the receptor for advanced glycation end products, VEGF and interestingly also α 2 β 1 integrin (Cabrera et al., 2020;Priscakova et al., 2016). Indeed, several papers reported that Bgl II polymorphism of the α 2 -subunit of integrin α 2 β 1 is associated with the prevalence of DR in type II diabetes mellitus patients (Azmy et al., 2012;Gong et al., 2015;Matsubara et al., 2000;Midani et al., 2019;Petrovic et al., 2003), although these findings might be population-dependent (Cabrera et al., 2020;Cepeda-Nieto et al., 2015).
Recently, three RGD-binding integrin targeting therapies have been investigated in clinical trials for treatment of DME (see also Table 3). First, risuteganib (ALG-1001; Luminate®; Allegro Ophthalmics, LLC) is a novel, IVT-administered, synthetic RGD oligopeptide that mainly affects the RGD-binding integrins α v β 3 , α v β 5 , α 5 β 1 , but also integrins from other classes, namely α 3 β 1 and α M β 2 (Shaw et al., 2020;Tolentino et al., 2016). Risuteganib has been evaluated in a randomized, prospective, double-masked phase 2b trial for DME patients (NCT02348918, DEL MAR study) where its safety and effectiveness were compared to bevacizumab. In stage 1 of the DEL MAR monotherapy study, 136 subjects were randomized to three risuteganib groups (1.0, 2.0, or 3.0 mg) treated with 3 monthly IVT injections, followed by 3 months off-treatment, and a 1.25 mg bevacizumab arm with 6 monthly IVT injections. This trial met its primary and secondary endpoints of non-inferiority in best corrected visual acuity (BCVA) improvement as well as reduction in central macular thickness (CMT) as compared to bevacizumab. In the stage 2 of the DEL MAR study, 80 participants with DME were randomly assigned to 1 of 5 treatment groups. The control group received monotherapy of five monthly injections of 1.25 mg bevacizumab, while two other groups received sequential therapy of a single IVT injection of 1.25 mg bevacizumab at week 0 followed by three IVT injections with 1.0 or 0.5 mg risuteganib at weeks 1, 4 and 8. The two last groups received combination therapy of 1.25 mg bevacizumab with 1.0 or 0.5 mg risuteganib IVT injected at weeks 1, 4 and 8. The sequential therapy regimen with 1 mg risuteganib was most efficacious and met the primary endpoint of non-inferiority in BCVA gain when Fig. 9. Overview of the most relevant RGD-binding integrins contributing to the pathological processes implicated in retinal vascular disorders.

Table 3
Overview of ongoing and completed clinical trials using RGD integrin receptor antagonists for treatment of DME or nvAMD. compared to bevacizumab, with fewer total injections. A 12-week durability after the last dose of risuteganib was demonstrated with sequential therapy. Remarkably, combination therapy was far inferior to sequential therapy as well as bevacizumab monotherapy. Of note, patients who had been sub-optimal responders to prior anti-VEGF therapy appeared to have a better response to risuteganib treatment (Dugel, 2019;Shaw et al., 2020). Additional (clinical) research is needed to better understand how anti-integrin therapy could fit into the current treatment regimens for DR and DME. Second, SF0166 (OcuTerra Therapeutics, formerly SciFluor Life Sciences, Inc.) is a potent α v β 3 antagonist which was topically administered in a phase ½ study for DME patients (NCT02914613). In this study, safety and preliminary efficacy of SF0166 were investigated in 40 DME patients who were randomized to a dose of 2.5% or 5% eye drop formulation, self-administered twice-a-day for 28 days. The primary endpoint was reached given that no drug-related serious adverse events were observed throughout the study. Both doses of SF0166 exhibited therapeutic efficacy with 53% of the subjects displaying a decline in retinal thickness, and BCVA improvements were also reported. Durability of retinal thickness response to SF0166 treatment was observed during the 28-day follow-up period after discontinuing treatment (Askew et al., 2018;Dugel, 2019). Although the data from the phase ½ study of SF0166 did seem to indicate a biological effect, no further development of this compound has been reported to date. In addition, up to now, no topically administered drug is approved for the treatment of DR or AMD. Third, given the favourable preclinical safety and efficacy profile for the novel small molecule integrin receptor antagonist THR-687 (Oxurion NV) (see section 5) , a clinical phase 1, open label, multicentre study (NCT03666923) was carried out to investigate the safety of a single IVT of THR-687 using three dose levels (0.3, 0.8 and 2.1 mg per eye, expressed as free base) for the treatment of DME (n = 12). THR-687 was found to be safe and well tolerated at all dose levels. Following a single IVT injection of THR-687, a rapid onset of action in mean BCVA was observed as of day 1 with a 3.1 letter gain. The highest impact was observed one month post injection, showing a mean increase of 9.2 letters. The improved vision was maintained up to month 3, with a mean 8.3 letters improvement. BCVA improvement was most pronounced in the high dose group, with a mean BCVA gain of 12.5 letters at month 3. For the high dose the retinal thickness decreased by a mean of 106 μm at day 14 post administration.

Neovascular age-related macular degeneration
AMD is the leading cause of blindness in elderly people of industrialized countries and can be divided into a wet/neovascular/exudative (nvAMD) subtype (10-20% of the patients) and a dry/atrophic/nonexudative form (80-90%). According to the severity of the fundus lesions, AMD is currently classified into early, intermediate and advanced stages. Visual acuity is generally unaffected in the early and intermediate stages of the disease, which are primarily characterized by the formation of drusen or pigmentary abnormalities between RPE cells and Bruch's membrane. In the advanced stages, AMD might progress into the currently untreatable geographic atrophy, which is characterized by areas of progressive RPE cell degeneration, subsequent photoreceptor damage and finally irreversible loss of visual function (Bandello et al., 2017;Ferris, III et al., 2013;Gehrs et al., 2006), or into nvAMD, where pathological blood vessels from the choroid invade the macula (Gehrs et al., 2006). CNV is mostly accompanied by subsequent development of subretinal fluid accumulation, haemorrhage, fibrosis and retinal detachment (Little et al., 2018;Ma et al., 2017). Though less prevalent, nvAMD is the leading cause of vision loss as it can lead to legal blindness within six months. Dry AMD is mostly characterized by slow progression, yet, 10-15% of patients with dry AMD progress into nvAMD. AMD is a multifactorial disorder driven by oxidative stress, cell senescence, inflammation, dysregulated lipid metabolism and mitochondrial dysfunction. More information on the mechanisms behind AMD can be found in the reviews of Ambati and Fowler (2012) and Rozing et al. (2020) Rozing et al., 2020). A combination of environmental risk factors such as smoking, hypertension, atherosclerosis, high cholesterol and UV light (Cougnard-Gregoire et al., 2013;Dasari et al., 2011;Garcia-Layana et al., 2017;Lim et al., 2012;Thornton et al., 2005), as well as genetic variants in e.g. complement factors (CFH, CFB, C3), Apolipoprotein E, and HTRA1 McKay et al., 2011;Tzoumas et al., 2021;Yang et al., 2006) contribute to AMD pathology. Although there is currently no treatment for dry AMD patients, prevention of AMD onset is mostly based on supporting/maintaining a healthy lifestyle with regular exercise, no smoking and a nutritious diet rich in green, leafy vegetables. Dietary supplements such as the AREDS2 can slow the progression towards the advanced stages of the disease (Agron et al., 2021).
The ground-breaking introduction of IVT anti-VEGF therapy, such as ranibizumab, aflibercept and bevacizumab, has significantly reduced the incidence of blindness in nvAMD patients. However, the functional outcome following anti-VEGF therapies in people with this advanced form of neovascular AMD is in most cases limited (Ishikawa et al., 2015). Indeed, in comparison to clinical trials, patients are usually undertreated in real-world practice, thereby leading to a decline in visual acuity over time, which might be caused by fluctuations in CST (Avery et al., 2020). Besides encouraging patient compliance and improving patient monitoring, more durable anti-VEGF agents and alternative therapies should be developed. Long-term anti-VEGF treatment in elderly people with nvAMD has also been correlated to potential adverse events, namely enhanced scar formation (Daniel et al., 2014), RGC damage (Beck et al., 2016) and potential development of macular atrophy (Gemenetzi et al., 2017).
Interestingly, it has been reported that high levels of α v β 3 integrin were observed in human neovascular choroidal membranes (Friedlander et al., 1996) and recent clinical trials with compounds targeting RGD-binding integrins have shown promising results for nvAMD patients (see also Table 3). Besides DME, nvAMD was also investigated as an ocular indication for risuteganib (Allegro Ophthalmics) (see also section 6.1). In a phase 1b study, 15 subjects with nvAMD received three monthly IVT injections of risuteganib, which was well tolerated and resulted in a mean improvement in BCVA of five letters as well as a 30% decline in central macular thickness (Shaw et al., 2020). Analogously, a double masked phase ½ study (NCT02914639) evaluated the safety and preliminary efficacy of SF0166 (OcuTerra Therapeutics, formerly Sci-Fluor Life Sciences, Inc. see also section 6.1) in 42 nvAMD patients, who were randomized 1:1 to self-administer a topical formulation of 2.5% or 5% SF0166 twice-a-day for 28 days (Eyewire.news, 2017). The primary endpoint was reached since no drug-related serious adverse events were observed throughout the treatment period of 28 days as well as the 28-day follow-up period. In addition, topical SF0166 treatment led to a clinically significant therapeutic effect in nine out of the 42 patients. A mean improvement in BCVA of approximately five letters and a decrease in central retinal thickness and/or subretinal fluid was observed. To our knowledge, no further development in nvAMD was currently reported for both compounds. Besides these, additional phase 1, open label, dose escalation studies were performed with intravitreal α5β1 inhibitors, i.e. volociximab (Ophthotech, currently IVERIC bio, NCT00782093) and JSM6427 (Jerini Ophthalmic, currently Takeda Pharmaceutical Company, NCT00536016). Despite the preliminary favourable safety profiles and BCVA improvements (Capone et al., 2009;Kuppermann, 2010), no further development of these compounds for nvAMD has been reported to date. A phase ½ clinical trial also recently investigated the safety and efficacy of AS101 (1% oral solution) in patients with nvAMD, but no results are available at the moment (Feramda Biopharmaceutical, NCT03216538). Although AS101 has a wide activity profile, this oral drug is also a functional inhibitor of specific integrins, including α 4 β 7 , α 4 β 1 and the RGD-binding integrin α v β 3 (Dardik et al., 2016;Lee et al., 2014c;Yossipof et al., 2019).

Future directions and conclusions
Recent clinical trials for novel drug candidates developed against RGD-binding integrins have shown promising effectiveness and a favourable safety profile in patients with DME and nvAMD. This has triggered renewed interest in ophthalmic therapies targeting RGDbinding integrins and new inhibitors which have been generated in the last few years. Indeed, while risuteganib, SF0166, and THR-687 already demonstrated biological effects in early clinical trials, promising preclinical efficacy studies with several other anti-RGD integrin drugs including, but not limited to, AXT107 (AsclepiX Therapeutics) and OCU200 (Ocugen) (GlaxoSmithKline), offer further exciting new opportunities. Taken together, there is compelling evidence that RGDbinding integrins are important players in key disease hallmarks of nvAMD and DR, namely chronic inflammation (or para-inflammation), retinal permeability, neovascularization and fibrosis. Moreover, as discussed in this review, RGD-binding integrins, which are present throughout the retina, have the capacity to regulate these visionthreatening processes at multiple levels. They might do so by interacting with various growth factors, cytokines or other stress mediators (e.g. VEGF, Ang-2, TGF-β, bFGF, IL-1β and TNF-α).
Although their specific role in the retina remains incompletely defined, RGD-binding integrins can be considered as functional hubs during pathological signalling and their antagonism could have therapeutic utility for major retinal diseases. Additionally, an RGD-binding integrin inhibitor could have the capacity to act upstream as well as downstream of the VEGF pathway and address VEGF-dependent and VEGF-independent vision-threatening pathological mechanisms, thereby in all likelihood exerting a broader biological effect as compared to anti-VEGF therapy. Moreover, alternative administration routes, such as oral or topical, or optimized dosage forms, such as sustained release formulations, have demonstrated promise in preclinical and/or clinical studies. RGD-binding integrin inhibitors can thus potentially address the treatment burden associated with frequent anti-VEGF injections.
Although RGD-binding integrin inhibitors can have the potential to treat multifactorial and complex eye diseases, possible pitfalls of antiintegrin based therapeutic targets should be taken into consideration since integrins have a wide-ranging impact on many biological functions and fundamental cell signalling cascades. Disruption of normal α v β 5 function can have negative outcomes as demonstrated by β 5 -integrin knock-out mice showing dysregulated photoreceptor outer segment phagocytosis by RPE cells and impaired vision after 1-year (Nandrot et al., 2004;Nandrot and Finnemann, 2006). In addition, in a rat model of retinal ischemia reperfusion injury, it was described that a decrease in β 1 -integrin was associated with RGC death via loss of homeostatic RGC-laminin interaction, implying that laminin-binding integrins are essential for RGC survival (Santos et al., 2012). Therefore, as for any new drug candidate, it is of utmost importance to monitor the possible adverse events of anti-integrin therapeutics and to identify a safe therapeutic window. Encouragingly, the abovementioned RGD-binding integrin inhibiting drug candidates all have shown a favourable safety profile in clinical trials. Nevertheless, given the limited number of subjects and relatively short-term study periods, further assessment is needed.
In summary, an RGD-binding integrin inhibitor can be considered as an integrative therapy given its multi-mechanism targeting properties, which is unique amongst all drug candidates being studied for AMD and DR. As integrin functions are complex and context-dependent, further research is recommended in order to unravel the underlying cellular and molecular mechanisms of action when targeting RGD-binding integrins in the context of DME as well as nvAMD. Importantly, it still also needs to be determined if anti-RGD-binding integrin therapy could be noninferior or superior to anti-VEGF therapy, the current gold-standard of care for DME and nvAMD patients. This would be essential to justify its implementation as a stand-alone therapy and potential first-line treatment. Given the promising results with the sequential therapy regimen in the DEL MAR phase 2b stage 2 clinical study, anti-integrins might also be explored as complementary drugs to anti-VEGF therapy. In addition, it could be useful to evaluate in future clinical studies whether the combination therapy of VEGF and RGD-binding integrin inhibitors could have potential advantages over monotherapy since a synergistic effect has been described before for these two pathways (Silva et al., 2017;Wang et al., 2016). However, as described in section 6.1, a small phase 2b clinical trial in DME patients that received risuteganib and bevacizumab combination therapy was found inferior to bevacizumab monotherapy.
We conclude that it is imperative to continue the search for new and effective drug candidates that could successfully treat all subjects suffering from a complex vision-threatening retinal disorder and we suggest that targeting RGD-binding integrins could be such a nextgeneration therapy which could revolutionize the treatment paradigm for major sight-threatening and life-altering retinal diseases.

Declaration of interest
Inge Van Hove, Tjing-Tjing Hu, Karen Beets, Tine Van Bergen, Isabelle Etienne, Elke Vermassen and Jean H.M. Feyen report direct financial relationship for Oxurion NV. Alan W. Stitt receives remuneration from Oxurion NV for scientific advice.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.