The role of integrins in inflammation and angiogenesis

Abstract Integrins are heterodimeric transmembrane cell adhesion molecules made up of alpha (α) and beta (β) subunits arranged in numerous dimeric pairings. These complexes have varying affinities to extracellular ligands. Integrins regulate cellular growth, proliferation, migration, signaling, and cytokine activation and release and thereby play important roles in cell proliferation and migration, apoptosis, tissue repair, as well as in all processes critical to inflammation, infection, and angiogenesis. This review presents current evidence from human and animal studies on integrin structure and molecular signaling, with particular emphasis on signal transduction in infants. We have included evidence from our own laboratory studies and from an extensive literature search in databases PubMed, EMBASE, Scopus, and the electronic archives of abstracts presented at the annual meetings of the Pediatric Academic Societies. To avoid bias in identification of existing studies, key words were short-listed prior to the actual search both from anecdotal experience and from PubMed’s Medical Subject Heading (MeSH) thesaurus. Impact Integrins are a family of ubiquitous αβ heterodimeric receptors that interact with numerous ligands in physiology and disease. Integrins play a key role in cell proliferation, tissue repair, inflammation, infection, and angiogenesis. This review summarizes current evidence from human and animal studies on integrin structure and molecular signaling and promising role in diseases of inflammation, infection, and angiogenesis in infants. This review shows that integrin receptors and ligands are novel therapeutic targets of clinical interest and hold promise as novel therapeutic targets in the management of several neonatal diseases.


INTRODUCTION
Integrins are a family of ubiquitous αβ heterodimeric receptors that exist in multiple conformations and interact with a diverse group of ligands. These molecules mediate interactions between cells and of these cells with the extracellular matrix (ECM) and thereby serve a critical role in signaling and homeostasis. By facilitating dynamic linkages between the intracellular actin cytoskeleton and the ECM, integrins also transduce both external and internal mechanochemical cues and bi-directional signaling across the plasma membrane. 1,2 Integrins are involved in a diverse range of body processes, including cellular survival, inflammation, immunity, infection, thrombosis, angiogenesis, and malignancy. In this review, we highlight the structure and function of integrins; the mechanisms involved in integrin activation and signaling; their role in inflammation, infection, and angiogenesis; and discuss current advances in integrin-targeted therapies. Understanding the factors that regulate integrin structure, function, and signaling would enable us to identify new therapeutic targets.

STRUCTURE OF INTEGRINS
In mammals, the family of integrins is comprised of 24 αβ pairs of heterodimeric transmembrane adhesion receptors and cellsurface proteins. These pairings are known to involve 18 α and 8 β subunits (Fig. 1), 3 and their non-covalent associations involve an α and another β subunit (Fig. 2). 4 The αβ pairings of integrin subunits dictate the specificity of the integrin to a particular ligand, modulate formation of intracellular adhesion complexes, and regulate downstream signaling. 1 Six α (α [1][2][3][4][5][6] ) and seven β (β 1-7 ) subunits are known to form several unique αβ subunit associations (Fig. 1). Interestingly, the earliest discovered integrins, lymphocyte function-associated antigen 1 (integrin α L β 2 ) and macrophage antigen 1 (integrin α M β 2 ), derive their specificity from specific α subunits, but these share the same β subunit. 5 Integrin α subunit family The integrin α subunits carry a 200 amino acid "inserted" domain, the I-domain (αI). When present on an integrin, the αI domain is an exclusive ligand-binding site. αI integrins have 13 extracellular domains in 2 subunits, which interact with a variety of ligands. The I-domains are seen in 6 out of the 15 integrin α subunits.

INTEGRIN-LIGAND BINDING AND CONSEQUENT ACTIVATION
The structure and function of integrins are complex. Integrins bind numerous extracellular ligands, intracellular signaling molecules, and the cytoskeleton in a bivalent-cation-dependent manner with varying specificities. Integrins also have many states with multiple conformations and affinities.
Mechanism of integrin ligand binding and conformational states Integrins bind cell-surface ligands to promote cellular interactions with the ECM and with other cells in the transduction of complex signals that modulate many cellular processes, such as adhesion, migration, and differentiation. These soluble, ECM, or cell surfacebound ligands may include growth factors, structural constituents of the ECM, proteases, cytokines, plasma proteins, microbial pathogens, or receptors specific to immune cells. The affinity and avidity of a ligand may change actively by inside-out signaling in specific pathways. Ligand affinity may vary with the strength of interaction and dissociation of a monovalent protein and its ligand, where ligand avidity refers to its ability to form multiple combinations of bonds. 17 Integrins exist primarily in three conformational states: bent-closed (inactive; the predominant state), extended-closed (active; low affinity or intermediate state), and the extended-open (active; high affinity). 18 The affinity of integrins to various inhibitory and stimulatory ligands is modulated by bivalent cations, which induce a range of conformational changes in integrins ranging from a folded, inactive, and low-affinity state to a high-affinity conformation (Fig. 2). 19 These conformational changes in the extracellular domains of integrins modulate both ligand binding and downstream cellular signaling.

Integrin activation
The activation of integrins increases the affinity of these molecules to extracellular ligands. Integrin tail domains play a critical role in these steps, and any genetic mutations in these parts of integrins can disrupt downstream intracellular signaling. 20 Integrinmediated signaling across cell membranes is typically bidirectional and termed "outside-in" and "inside-out" signaling. 20,21 When integrins interact with ECM ligands, a conformational change allows adherence to downstream adaptor molecules in the cell-membrane plane. 22 Once clustered, integrins are able to recruit and activate kinases such as Src family kinases, focal adhesion and scaffold molecules such as the adaptor protein p130CRK-associated substrate/breast cancer anti-estrogen resistance 1 (p130CAS/BCAR1). 22 These integrin-associated complexes include discrete active and inactive integrin organizations, which can activate unique signaling pathways. 23,24 The extracellular domains of integrins are known to undergo a diverse range of conformational changes that alter the ligandbinding domains. In the cytoplasmic tails of integrins, α-helices are seen as heterodimers, 25 and the β-strands often bind intracellular proteins, such as talin or filamin. 26,27 The cytoplasmic tail may undergo several specific conformational changes to bind a range of other signal transducers. 28,29 Integrin bi-directional inside-out and outside-in signaling Mechanical stress 30 and extracellular chemicals 31 can induce rapid conformational changes to cause inside-out activation of integrins. 32 Integrins display bi-directional signaling across the plasma membrane. Ligand binding induces extracellular-to-cytoplasm signal transduction, and inside-out signaling or priming regulates integrin-ligand binding conformations (Fig. 2). During integrin activation and signaling, the cytoplasmic tail acts as both a receptor and transmitter of signals. Specifically, during inside-out signaling, the activating signals make an impression on the cytoplasmic tail to induce large conformational changes to the extracellular domain, thereby transforming the integrin from a resting to an active state. 33 During outside-in signaling, the binding of a ligand to the extracellular domain of active integrin transmits a conformational change to the cytoplasmic tail, which leads to the activation of kinases and adaptor molecules in the cytosol. 1 In contrast, talin and kindlin interaction with the βcytoplasmic tail can trigger inside-out signaling, leading to integrin activation, clustering, and recruitment of intercellular adaptor proteins to strengthen cellular adhesion. Talin is a large dimeric actin-binding protein and a major regulator of integrin activation, and the regulation of talin-integrin interactions is important in the control of integrin activation and signaling pathways. 33 Direct interactions between the talin head and the short cytoplasmic tails of β integrin subunits disrupt inhibitory interactions between α and β integrin subunits. 33 This leads to conformational changes in the integrin extracellular domains and consequent increase in their ligand affinity. The role of kindlins are not clearly defined, but they are structurally related to the talin head. The synergistic binding of talin and kindlin to β integrin cytoplasmic tails induces integrin activation by disrupting the α-β interactions at the transmembrane and the cytoplasmic domains. 33,34

INTEGRINS IN INFLAMMATION AND INFECTION
In the resting state, β 2 integrins are expressed specifically on leucocyte receptors. During inflammation, the inflammatory cytokines activate these integrins and promote cellular adherence to the counter-receptors such as ICAMs and promote phagocytosis and cytotoxic killing. Integrin receptors on leukocytes, such as the macrophage-1 antigen (Mac-1, also known as CR3, α M β 2 , CD11b/CD18) interact with platelet antigens such as the glycoprotein Ibα (GPIbα) during inflammation. Integrins bind to the pro-domain of transforming growth factor (TGF)-β 1 to activate it and promote its secretion. The pro-TGF-βs are biosynthesized and stored in tissues in latent forms, and integrins α V β 6 and α V β 8 can uniquely bind and activate pro-TGF-β 1 and pro-TGF-β 3 . The α V β 6 integrin is known to specifically bind the RGDLXXL/I motif in TGF-β 1 and TGF-β 3 . 35 β 2 integrins promote recruitment of leukocytes to the sites of inflammation by promoting the adhesion of circulating leukocytes to vascular endothelium, transendothelial migration, 36,37 the formation of immunological synapses in leucocytes, 38 and inflammatory signaling in involved cells. 39 Activated β 2 integrins on dendritic cells (DCs) may act as negative regulators of DC migration in certain conditions and may also regulate T cell activation. 40,41 β integrins on the leukocyte surface are also involved in the tethering, rolling, and adhesion of leukocytes to activated endothelial cells. 42 β 2 integrins can also initiate intracellular signaling pathways in macrophages and neutrophils and stimulate cytokine secretion from these cells either directly or in synergy with Toll-like receptors (TLRs). 43 Integrins may also integrate the impact of the epidermal growth factor receptor, platelet-derived growth factor receptor, insulin receptor, met receptor superfamily (hepatocyte growth factor receptor), and the vascular endothelial growth factor receptor (VEGFR) in inflammatory cells. 44 Inactive, low-affinity state  108 The short cytoplasmic tails are composed of 20-70 amino acids and mediate interactions with intracellular cytoskeletal and signaling proteins. 1 In response to intracellular or extracellular stimuli, integrin activation occurs by ligand binding or by the changes on the cytoplasmic domains, resulting in elongation and separation of the legs. Integrins appear in a closed or "bent" conformation on resting cells and display a low binding affinity for ligand rendering them inactive to ligand binding or signal transduction; while once activated, the integrin shape extends to an open conformation leading to a high affinity. 109 In a closed conformation, integrins show low ligand-binding affinity, partly due to the bend in the center of the α and β subunits, which brings the ligand-binding site within 5 nm of the cell surface. 110 However, when the conformation is open, the two subunits straighten with increased integrin affinity for the ligand. 111 The initial binding of extracellular ligand effects separation of the cytoplasmic domains, allowing interaction with signal transduction and cytoskeletal molecules during outside-in signaling, while separation of the cytoplasmic domains by talin and other activators activates the head to enable ligand binding during inside-out signaling. 4 β 2 integrins are important regulators of adhesion, leukocyte recruitment, and immunological signaling. These integrins mediate adhesive interactions between myeloid cells, endothelial cells, antigen-presenting cells, T cells and the ECM. 45 L-selectin, the CCR7 chemokine receptor, interacts with specific carbohydrate epitopes on the endothelium and promotes leukocyte rolling and transmigration through the vascular endothelium. 46 Leukocyte rolling induces a rapid, although transient, increase in the affinity of the β1 and β2 integrins to the endothelial ligands. 47,48 Conformational changes in the structure of the inserted (I) domain of the α L subunit of LFA-1 49 enhance firm leukocyte adhesion under shear flow. 31,49

ROLE OF INTEGRINS IN NEONATAL ORGANS DURING NORMAL DEVELOPMENT AND INFLAMMATION
Integrins in the lung during normal development and in inflammation Integrins and receptor tyrosine kinases act with cytokine and growth factors to modulate the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways during regeneration, inflammation, developmental, and pathological processes in the developing lung. 2,44,50,51 The ECM in the lung contains collagen, fibronectin, laminin, and entactin, 52 and alterations in the formation and structure of the ECM during normal development, healing from injury, or in chronic lung disease could lead to profound alterations in the lung structure. 53,54 For instance, fibronectin in the ECM promotes integrin-mediated cellular migration and differentiation of cells during lung development. 55 β 1 integrin activates several signaling pathways, particularly the PI3K/AKT pathway activated during wound healing in the presence of collage VI in the lung ECM. 56 β 1 integrins play a critical role in alveolar homeostasis, as seen in chronic lung disease depicted in β 1 integrin-deficient mice. 57 In addition, β 1 integrin-deficient alveolar epithelial cells produce excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting that β 1 integrins may be involved in alveolar homeostasis. 58 In murine models of bronchopulmonary dysplasia, perinatal exposure to lipopolysaccharide and increased expression of interleukin-33 may activate neutrophils and promote fibronectin degradation in alveolar epithelial cells. 59 Other studies have noted increased expression of integrin α 2 β 1 on mast cells and activation/release of inflammatory cytokines. 60,61 Similar findings have been noted in murine models with Listeria monocytogenes infections. 62 Mice deficient in integrin α 2 63 and integrin α IIb 64 show defective platelet interaction with collagen. α 2 β 1 integrin-null mice have normal angiogenesis but may have altered angiogenic responses during injury repair. 65 In contrast, integrin β 1 knockout mice may have altered development and are not viable, indicating an essential role of β 1 during development. Table 1 outlines murine models of integrins, their target tissues, and signaling.

Integrins in intestinal inflammation and in necrotizing enterocolitis (NEC)
The regulation of intestinal leukocyte responses is vital to maintaining immune homeostasis and prevention of intestinal inflammatory conditions. Integrin α v β 5 is expressed on neonatal intestinal macrophages; the expression is developmentally regulated and is not dependent on microbial colonization. These integrins bind different ECM components, such as laminins, collagens, and fibronectin, and are known to coordinate epithelial cell adhesion and movement. 4,66 These integrins recognize the RGD tri-peptide sequence present in ECM proteins, such as fibronectin and vitronectin. 67,68 The integrin α V β 5 can be found in both focal adhesions and in clathrin-coated membrane domains. 69,70 Integrin α v β 8 plays an important role in epithelial homeostasis and is a major activator of TGF-β expression. 71 α 3 and β 1 integrins, which are known to increase epithelial migration, are upregulated by bacterial products during NEC. 72,73 TLR4 signaling on enterocytes promotes the efflux of β 1 integrins from the cytoplasm toward the cell membrane and enhances cell-matrix contacts that limit cellular movement. 74 In other studies, Besner and colleagues have examined the role of E-cadherin and integrins in NEC and showed that the growth factor, heparinbound epidermal growth factor, can promote intestinal restitution in NEC through its effects on integrin-ECM interaction and intercellular adhesions. 75 Intestinal epithelial cells also express α 3 β 1 , another set of integrins of translational importance. In NEC, increased epithelial expression of α 3 β 1 may impair the migration of epithelial cells needed for mucosal wound healing. 74 However, the same α 3 integrins are also required for morphologic differentiation of the intestinal epithelium in the developing intestine. 76 Despite the physiological needs of the β 1 integrins, therapeutic targeting of these molecules may still be possible with information on the best timing and the possibility of regionally focused intervention.
Integrins in the developing eye and in retinopathy of prematurity In the developing eye, disruption of the oxygen supply to the retina can disrupt neuronal dysfunction needed for transduction and transmission of photosensitive visual signals to the occipital lobe and other cognitive centers. Integrin α 2 β 1 and VEGF interact closely in several intracellular angiogenic signaling (Fig. 3). 77,78 Cyclic peptides selectively inhibit α V β 3 and α V β 5 , and are potent inhibitors of endothelial cell invasion and differentiation induced by VEGF-A or fibroblast growth factor-2. 78 Integrin α V β 3 works Fibroblasts, epithelial cells Accelerated re-epithelialization, enhanced TGF-β signaling, dermal fibroblast infiltration β 3 -deficient mice (genetic knockdown) 119 The role of integrins in inflammation and angiogenesis OJ Mezu-Ndubuisi and A Maheshwari synergistically with VEGF to activate angiogenesis in endothelial cells via VEGFR-2 phosphorylation. 79 Endothelial cells are the primary cells expressing both VEGFR-2 and α 2 β 1 integrin. 80 Proteoglycans such as decorin and perlecan in the ECM of the eye can modulate α 2 β 1 and play a vital role in angiogenesis. 80 The Cterminal fragment of perlecan, known as endorepellin, has an opposite effect and blocks angiogenesis through antagonism of VEGFR-2 and α 2 β 1 integrin on endothelial cells. 80 Retinal pigment epithelial cells express beta-8 integrin at the surface, and the knockdown of beta-8 integrin significantly decreased retinal pigment epithelial cell migration in wound-healing assays. 81 The retinal tissue has one of the body's highest metabolic demands, placing it at risk of injury from oxidative stress, metabolic derangements, and consequent pathologic neovascularization seen in retinopathy of prematurity (ROP) and other proliferative retinal vitreoretinopathies. ROP is a bi-phasic disease of retinal vascular development due to dysregulation of VEGF. 82,83 In phase 1, VEGF is downregulated during exposure to hyperoxia, while in phase 2, VEGF is upregulated in relative/true hypoxia. VEGF is known to have several isoforms; VEGFA165 is the predominant isoform in the eye with multiple pro-and antiangiogenic splice variants. 84 In a newborn mouse model of oxygen-induced retinopathy (OIR), oxidative stress from fluctuating hyperoxia and hypoxia leads to altered vascular development with tortuous arteries, dilated veins, and capillary attrition, akin to human ROP. 82,85 These changes persist in adult mice with longterm abnormalities in vascularization, structure, and function both in vivo and histologically. 86,87 Integrin-targeted therapy holds promise in ROP. Targeting α 2 β 1 integrin expression on endothelial cells mitigates OIR, 88 and the administration of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride, a novel non-peptide αvβ3 antagonist, can inhibit retinal neovascularization. 89 There are exciting possibilities that endothelial α 2 β 1 may be therapeutic target in pathological angiogenesis.

INTEGRINS IN THROMBOSIS AND FIBROSIS
Platelet adhesion and signaling play key roles in hemostasis and thrombosis. Two platelet receptors, integrin α IIb β 3 and GPIbα, mediate the early and mid-stages of platelet adhesion in the vascular environment. 90 GPIbα is a key part of the receptor for VWF, and its binding to VWF enables platelet rolling during the formation of thrombotic plugs at the sites of vascular injury. [91][92][93] αIIbβ3 is expressed on both platelets and the endothelium, and upon activation, it promotes platelet adhesion and aggregation by cross-linking with soluble fibrinogen, fibronectin, and VWF.
In alloimmune thrombocytopenia, autoantibodies are frequently seen against integrin β 3 and GPIbα. 94,95 Intracranial hemorrhages may be seen more frequently in infants with antiβ 3 integrin antibodies than in those with antibodies against GPIbα. 96 Existing in vitro and in vivo data suggest that the β 3 integrin may bind a wider range of ligands, including fibrinogen and VWF, and autoantibodies that block its function may induce a deeper functional deficit than the anti-GPIbα antibodies. 97

INTEGRIN-TARGETED THERAPIES
Integrin dysregulation is implicated in the pathogenesis of numerous diseases with altered angiogenesis, inflammation, or in infectious diseases. In these conditions, therapeutic strategies may either directly target integrins or their ligands. Out of the 24 known human integrins, many have already been identified as therapeutic targets for monoclonal antibodies, peptides, and/or small molecules. In adult subjects, efforts are ongoing to target platelet integrin α IIb β 3 to prevent thrombotic complications after percutaneous vascular interventions, lymphocyte α 4 β 1 and α 4 β 7 integrins in the treatment of multiple sclerosis, and β 7 integrins (α 4 β 7 and α E β 7 integrins) in inflammatory bowel disease. 98 Specifically, a humanized anti-α4 antibody (Natalizumab) works in reduction of inflammation in multiple sclerosis by blocking the α 4 β 1 -vascular cell adhesion molecule interaction or the α 4 β 7 -mucosal addressin cell adhesion molecule interaction on mucosal endothelium and blocking leukocyte trafficking across the blood-brain barrier. 99 In another study, a micellar delivery vehicle decorated with an anti-angiogenic peptide has been shown to inhibit α V β 3 -mediated neovascularization in endothelial cells. 100 Several anticancer drugs have also been developed against integrin ligands or by using integrin-targeted encapsulated nanoparticles as vehicles to unload drugs into the vasculature of several tumors. 101 In a mouse model of hepatic fibrosis, cyclic peptide-guided liposomes preferentially targeted the activated hepatic stellate cells (not quiescent ones) to treat the fibrotic phenotype. 102 α V β 3 antagonists are being tried for the inhibition of retinal neovascularization and may have therapeutic value in ROP. 89 In a mouse model of laser-induced choroidal neovascularization, intravenous injection of irradiated nanoparticles loaded with doxorubicin allowed nanoparticle accumulation in the neovascular lesions and reduced the size of neovascular lesions. 103 Integrin antagonists may also be used in fibrotic diseases; IDL-2965 is being studied as a selective, highly potent, anti-fibrotic integrin antagonist in idiopathic pulmonary fibrosis. 104 Small molecule pure antagonists, TDI-4161 and TDI-3761, have been designed to inhibit α V β 3 -mediated cell adhesion to α V β 3 ligands. 105 Further studies are needed to improve the specificity of anti-integrin drugs to improve both the safety profile and therapeutic success of these agents. The schematic shows the interaction between the signaling pathways regulated by αβ integrins and the VEGF receptor. VEGF-A promotes angiogenesis through VEGF receptor-2 (VEGFR2), a tyrosine kinase receptor expressed by endothelial cells. 112 When VEGF-A binds to VEGFR2, numerous intracellular signaling pathways are activated, such as phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (Erk), focal adhesion kinase (FAK), c-Src family, and paxillin, a signal transduction adaptor protein associated with focal adhesion. 113,114 Specifically, FAK phosphorylates its substrate, paxillin, which activates ERK signaling. 114 When integrins activate the tyrosine phosphorylation of FAK, it binds to signaling structural proteins, PI3K, and paxillin. 115 Src family kinases (SFKs) play a critical role in cell adhesion, survival, and angiogenesis, interact with VEGF receptor, regulate gene expression of angiogenic growth factors, modulate cell proliferation via the mitogen-activated protein kinases (MAPK)-ERK pathway, and interact with integrins to regulate cell adhesion and migration. ECM extracellular matrix, VEGF vascular endothelial growth factor.

CONCLUSIONS
Enhanced understanding of integrin ligand interactions will enable development of therapies targeting specific receptors in order to modulate angiogenic, thrombotic, infections, and inflammatory disorders. Although numerous animal studies have shown promise in the clinical use of integrins as therapeutic targets, there is a need for clinical studies to confirm efficacy and safety in neonates and young infants. In this review, we have summarized and outlined the roles of integrins in inflammation, angiogenesis, and infectious conditions. Therapies could be targeted specifically to alpha subunits, but their overlapping roles are a critical factor to be considered. Further studies are needed both on molecular signaling and regulatory mechanisms of integrin function and the safety and efficacy in clinical settings.

AUTHOR CONTRIBUTIONS
Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content; and final approval of the version to be published: both authors.

ADDITIONAL INFORMATION
Competing interests: The authors declare no competing interests.
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