Integrins in Wound Healing

Abstract

Significance: Regulation of cell adhesions during tissue repair is fundamentally important for cell migration, proliferation, and protein production. All cells interact with extracellular matrix proteins with cell surface integrin receptors that convey signals from the environment into the nucleus, regulating gene expression and cell behavior. Integrins also interact with a variety of other proteins, such as growth factors, their receptors, and proteolytic enzymes. Re-epithelialization and granulation tissue formation are crucially dependent on the temporospatial function of multiple integrins. This review explains how integrins function in wound repair. Recent Advances: Certain integrins can activate latent transforming growth factor beta-1 (TGF-β1) that modulates wound inflammation and granulation tissue formation. Dysregulation of TGF-β1 function is associated with scarring and fibrotic disorders. Therefore, these integrins represent targets for therapeutic intervention in fibrosis. Critical Issues: Integrins have multifaceted functions and extensive crosstalk with other cell surface receptors and molecules. Moreover, in aberrant healing, integrins may assume different functions, further increasing the complexity of their functionality. Discovering and understanding the role that integrins play in wound healing provides an opportunity to identify the mechanisms for medical conditions, such as excessive scarring, chronic wounds, and even cancer. Future Directions: Integrin functions in acute and chronic wounds should be further addressed in models better mimicking human wounds. Application of any products in acute or chronic wounds will potentially alter integrin functions that need to be carefully considered in the design.

Hannu Larjava, DDS, PhD, Dip. Perio.

Figure 1.

Wound healing phases. The approximate timing of coagulation, inflammation, re-epithelialization, angiogenesis as well as granulation tissue formation and remodeling. Adapted from Häkkinen et al.

Figure 2.

Structural and functional domains in fibronectin (FN), tenascin-C, and laminin-332, including major ECM and keratinocyte and fibroblast integrin binding sites. (A) FN consists of two similar subunits that are linked in an antiparallel orientation by two disulphide bridges at their C-termini. It is formed by repeating homologous type I, II, and III units, and it binds to a number of biologically important molecules, including heparin (and heparan sulfate proteoglycans), denatured collagen, fibrin, and tenascin-C (TN-C). FN has three sites of alternative splicing: type III repeats A and B as well as the CSIII segment. The binding site for α₅β₁, α_vβ₁, α₈β₁, and α_vβ₆ integrins is located in module III₁₀. In addition, α₅β₁ integrin interacts with a second site, located in module III₉. Integrins α₄β₁ and α₉β₁ bind to module IIIA. FN is present in blood plasma in a soluble, globular form, and its cell-binding sites are unexposed. The cell-binding sites become exposed when it is absorbed to fibrin and polymerizes into insoluble fibrils. (B) Three tenascin-C monomers are joined together via their N-terminal tenascin-C assembly (TA) domains to form a trimer. Two trimers are further linked together to form a hexamer. Each arm of mammalian tenascin-C consists of EGF-like repeats (EGFL) that are recognized by EGFR, FN type III-like repeats that contain binding sites for FN and heparin, and a C-terminal fibrinogen globe (FBG), also interacting with heparin. Nine additional type III repeats can be included or excluded by alternative RNA splicing (light green). Binding sites for integrins are located in module III₃. (C) Laminin (LM) 332 is a T-shaped molecule consisting of three genetically distinct polypeptide chains, α₃, β₃, and γ₂. The α₃ chain contains five C-terminal globular domains (LG), the β₃ chain an N-terminal globular domain (LN), and the γ₂ chain an interrupting globular domain (L4) in the short arm. Proteolytic cleavage between LG3 and LG4 domains of the α₃ chain results in a functional conversion of laminin-332 from a motility to an adhesion factor. LG3 domain contains the binding site for α₃β₁ and α₆β₄ integrins, whereas LG4 includes a heparin/heparan sulfate proteoglycan-binding site. The β₃ and γ₂ chains can also be cleaved. The γ₂ L4 domain contains binding sites for nidogen and fibulin, which aid the incorporation of laminin-332 to the basement membrane. This arm also contains the binding site for α₂β₁ integrin. The β₃ LN domain contains a binding site for type VII collagen and a laminin-332–laminin-311 interaction site, which facilitate laminin-332 integration to the ECM. Adapted from Larjava et al. ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, EGF receptor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 3.

Integrin heterodimers. Schematic presentation of the 24 integrin receptors. Integrins α₁β₁, α₂β₁, α₁₀β₁, and α₁₁β₁: αI domain-containing collagen receptors; α₅β₁, α₈β₁, α_vβ₁, α_vβ₃, α_vβ₅, α_vβ₆, α_vβ₈, and α_IIbβ₃: RGD-binding integrins; α₃β₁, α₆β₁, α₇β₁, and α₆β₄: laminin receptors; α₄β₁, α₉β₁, and α₄β₇: α₄/α₉β₁ integrin family; α_Dβ₂, α_Lβ₂, α_Mβ₂, α_Xβ₂, and α_Eβ₇: leukocyte integrin subgroup. RGD, arginine-glycine-aspartic acid.

Figure 4.

Basic functions of integrins. (A) Integrins mediate cell adhesion to ECM and form adhesion plaques and intracellular complexes with cytoskeletal and signaling proteins. Most integrins are connected to actin microfilaments, whereas hemidesmosomal α₆β₄ integrin is linked to cytokeratins via plectin protein. (B) Leukocyte integrins can mediate cell–cell adhesion. (C) Integrins also have numerous other functions, such as binding to growth factors. For example, integrins α_vβ₆ and α_vβ₈ are critical for in vivo activation of TGF-β1. TGF, transforming growth factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 5.

Cytoplasmic integrin signaling complexes. Binding of talin to the cytoplasmic tail of integrin β subunit causes a conformational change in the receptor that allows for binding of intracellular signaling molecules, such as tyrosine kinases FAK, Src, and p130Cas as well as other structural proteins, such as vinculin, which mediate integrin interaction with the actin cytoskeleton. These intracellular protein complexes allow for the translation of the integrin-ECM interaction to a change in cell shape and behavior (e.g., motility). FAK, focal adhesion kinase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 6.

A schematic presentation of KC integrin expression and ECM molecule distribution during human oral mucosal healing. (A) The contact with the ECM molecules and pro-migratory growth factors (e.g., EGF) present in the wound clot activate the wound edge basal KCs. They dissolve their hemidesmosomal contacts with the BM and extend into the wound clot. As they migrate, wound KCs interact with the provisional BM they deposit underneath themselves. In this provisional matrix, they interact with EDA FN via α₅β₁, α₉β₁, and α_vβ₁ integrins, with TN-C via α₉β₁ integrin and with laminin-332 via α₂β₁, α₃β₁, and α₆β₄ integrins. KC migration is sustained by their autocrine expression of HB-EGF. (B) After wound edges have joined, BM is regenerated, and hemidesmosome re-assembly is initiated. At this point, α_vβ₆ integrin expression is induced in the wound KCs. It potentially interacts with and activates latent, ECM-bound TGF-β1 to regulate KC proliferation, inflammation, and granulation tissue remodeling. Migrating wound KCs express α₂β₁, α₃β₁, α₅β₁, α₉β₁, α_vβ₁, and α₆β₄ integrins; suprabasal early-wound KCs express β₁ integrins; late-wound basal KCs express mainly α₂β₁, α₃β₁, α₉β₁, α_vβ₆, and α₆β₄ integrins, whereas the expression of α₅β₁ and α_vβ₁ integrins is downregulated; late wound suprabasal KCs express α_vβ₆ integrin. Mature BM consists mainly of laminin-332, other laminins, collagen types IV and VII, and tenascin-C; provisional BM consists mainly of laminin-332, EDA FN, and tenascin-C. Connective tissue (CT) contains type I collagen, other collagens, and FN. Wound clot (FC) contains fibrin, FN, and vitronectin. Granulation tissue (GT) contains EDA and EDB FNs, collagen types I and III, other collagens, tenascin-C, fibrin, vitronectin. KC, keratinocyte; BM, basement membrane; EDA/B, extra domain A/B; HB-EGF, heparin-binding EGF-like growth factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 7.

Active re-epithelialization of a 3-day-old human palatal mucosal wound. (A) Epithelial keratinocytes migrate into the wound provisional matrix (HE staining). Scale bar: 200 μm. (B) Higher magnification image illustrating fibrin (arrow) between the migrating cells and the CT collagen (Mallory's phosphotungstic acid hematoxylin staining). Scale bar: 50 μm. (C) High expression of α₆β₄ integrin at the leading keratinocytes (indirect immunofluorescence image). (D) High expression of α₃β₁ integrin at the leading keratinocytes (indirect immunofluorescence image). See Larjava et al. for experimental procedures. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 8.

Schematic presentation illustrating how integrins could activate latent TGF-β1 during wound healing. In wounds, macrophages are the main source of TGF-β1, although other cells produce it as well. TGF-β1 is produced in a latent form, in which active TGF-β1 (red circle) is confined inside latency-associated peptide (LAP) that renders it inactive. LAP contains the RGD recognition signal for integrins. LAP further binds latent TGF-β1-binding protein (LTBP) to create a “large latent complex” that subsequently binds to ECM, specifically to FN. Target cells with LAP RGD-recognizing integrins (keratinocytes with αvβ6 integrin and fibroblasts with αvβ5 integrin) attach to the large complex, create a tractional force with their cytoskeleton (red actin filaments), and pull, leading to a conformational change in the complex and release of the active TGF-β1 that can now bind to its receptor complex (TGFβR). Active TGF-β1 signals via smad proteins and stimulate keratinocyte migration and ECM production but inhibit their proliferation. In pericytes and fibroblasts, active TGF-β1 stimulates cell differentiation to myofibroblasts and their ECM production. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 9.

A simplified illustration depicting how pericytes differentiate to matrix-producing myofibroblasts. Chemotactic PDGF, TGF-β1, and CCN2/CTGF signaling by platelets and macrophages causes the pericytes to detach from the vessel wall and produce EDA FN and collagen. Full differentiation of pericytes into myofibroblasts, evidenced by αSMA expression, depends on three essential elements, namely TGF-β1, EDA FN, and tension that can be created in the matrix produced by the pericytes. Myofibroblasts interact with EDA FN via α₅β₁, α_vβ₃, and α_vβ₅ integrins and with collagen via α₂β₁ and α₁₁β₁ integrins. Latent TGF-β1 complex is bound into the FN matrix and possibly activated via α_vβ₅ integrin and by other mechanisms. Whether the wound heals scarless, with scars, or becomes fibrotic depends on both the presence of myofibroblasts and macrophage-derived TGF-β1. αSMA, alpha–smooth muscle actin; CCN, Cyr61-CTGF-Nov; CTGF, connective tissue growth factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound