**6. Gelsolin family of actin remodelling proteins**

The dynamic remodelling of the cytoskeleton is facilitated by the gelsolin family of remodelling proteins, which includes gelsolin, villin, adseverin, capG, advillin, supervillin and flightless I [22]. These actin-binding proteins function in the cytoplasm of cells where they control actin organisation by severing pre-existing filaments, capping the fast-growing filament ends and nucleating or bundling actin filaments to enable filament reassembly into new cytoskeletal structures [61–64]. By remaining attached to the "barbed" ends of broken severed actin filament, these remodelling proteins prevent annealing of the broken filaments or addition of new actin monomers. Subsequently, the broken actin filaments are uncapped by interactions with phosphoinositides which results in rapid actin assembly and allows cells to reorientate the cytoskeleton and mediate the changes required for adhesion, motility and contraction [65]. The gelsolin family of actin remodelling proteins has three to six homologous gelsolin-like structural domains known as G1–G6 segmental domains, three actin binding regions and a number of calcium-independent monomer and filament binding domains. Villin, supervillin and Flii have evolved to contain additional domains allowing them to have multiple specific roles and interact with a variety of proteins. Villin contains an additional actin binding domain, termed villin head piece; supervillin contains an N-terminus domain capable of proteinprotein interactions and nuclear localisation, while Flii contains a N-terminus leucine-rich repeat (LRR) domain also capable of multiple protein-protein interactions. In contrast, CapG only contains three gelsolin-like structural domains; however, it still retains full actin severing and capping ability and affects cell migration [8, 66]. There is a high homology in structure of different members of the gelsolin family; however, the differences in structure observed suggest evolutionary changes allowing specific and unique functional properties beyond actin remodelling [62, 67]. Indeed, specific functional roles have been demonstrated in cell motility, apoptosis and gene expression [68]. Several members including Flii, supervillin and gelsolin have roles in as nuclear receptor co-activators regulating gene expression [62, 69], and current studies have identified some of these proteins as new targets for improved healing and reduced scar formation [31].

### **6.1. Gelsolin**

Gelsolin, the most abundant member of this family, is involved in regulating the dynamics of the filamentous actin by binding, severing and capping actin filaments [65]. In resting cells, gelsolin is either inactive or associated with filaments as a capping protein, while stimulation of cells or increased Ca2+ levels lead to an increased gelsolin activity at the plasma membrane and severing and capping of filaments resulting in increased cytoskeletal rearrangements [6]. High gelsolin levels have been associated with stress fibre formation and gelsolin was found to play a role in promoting stress fibre formation and actin stabilisation [70]. Gelsolin is also a secreted protein where its role in plasma is to "clean up" actin filaments that have been released into circulation during burn injury and cell necrosis using its gelsolin domain [27–29]. Plasma gelsolin is able to inactivate pathogen-associated molecular pattern (PAMPs) molecules, like lipopolysaccharides (LPS) and LTA (lipoteichoic acid) resulting in decreased TLR-mediated NF-kB activity [30, 31] suggesting a potential protective role for plasma gelsolin against inflammation. Gelsolin has also been shown to play a role in inflammation with studies suggesting potential clinical applications for plasma gelsolin in diagnosis and disease activity evaluation as patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) who have significantly decreased plasma gelsolin levels compared with healthy controls [23, 24]. A study examining the differential effect of wounding on actin and actin-associated protein in foetal skin explants showed that it is not the migration or proliferation of cells but rather formation of the actin cable that is important in early gestation foetal wound closure [19]. In foetal skin, gelsolin was observed surrounding the actin filaments at embryonic day 19 but not at embryonic day 17 which coincided with its upregulation in embryonic day 19 foetal skin but not embryonic day 17 foetal skin [19]. In adult skin, however, gelsolin is expressed predominantly in suprabasal keratinocytes at the leading edge of migrating epidermis [71] and studies examining the effect of gelsolin on wound repair indicate that increases in cellular gelsolin levels in mouse fibroblasts enhance cellular migration and results in increased rates of wound closure [72]. Moreover, absence of gelsolin in skin fibroblasts results in a variety of actin-related defects, including decreased motility and delayed wound closure potentially due to reduction in the reorganisation of cytoskeletal actin into contractile elements [66].

### **6.2. Flightless I**

[22]. These actin-binding proteins function in the cytoplasm of cells where they control actin organisation by severing pre-existing filaments, capping the fast-growing filament ends and nucleating or bundling actin filaments to enable filament reassembly into new cytoskeletal structures [61–64]. By remaining attached to the "barbed" ends of broken severed actin filament, these remodelling proteins prevent annealing of the broken filaments or addition of new actin monomers. Subsequently, the broken actin filaments are uncapped by interactions with phosphoinositides which results in rapid actin assembly and allows cells to reorientate the cytoskeleton and mediate the changes required for adhesion, motility and contraction [65]. The gelsolin family of actin remodelling proteins has three to six homologous gelsolin-like structural domains known as G1–G6 segmental domains, three actin binding regions and a number of calcium-independent monomer and filament binding domains. Villin, supervillin and Flii have evolved to contain additional domains allowing them to have multiple specific roles and interact with a variety of proteins. Villin contains an additional actin binding domain, termed villin head piece; supervillin contains an N-terminus domain capable of proteinprotein interactions and nuclear localisation, while Flii contains a N-terminus leucine-rich repeat (LRR) domain also capable of multiple protein-protein interactions. In contrast, CapG only contains three gelsolin-like structural domains; however, it still retains full actin severing and capping ability and affects cell migration [8, 66]. There is a high homology in structure of different members of the gelsolin family; however, the differences in structure observed suggest evolutionary changes allowing specific and unique functional properties beyond actin remodelling [62, 67]. Indeed, specific functional roles have been demonstrated in cell motility, apoptosis and gene expression [68]. Several members including Flii, supervillin and gelsolin have roles in as nuclear receptor co-activators regulating gene expression [62, 69], and current studies have identified some of these proteins as new targets for improved healing and reduced

Gelsolin, the most abundant member of this family, is involved in regulating the dynamics of the filamentous actin by binding, severing and capping actin filaments [65]. In resting cells, gelsolin is either inactive or associated with filaments as a capping protein, while stimulation of cells or increased Ca2+ levels lead to an increased gelsolin activity at the plasma membrane and severing and capping of filaments resulting in increased cytoskeletal rearrangements [6]. High gelsolin levels have been associated with stress fibre formation and gelsolin was found to play a role in promoting stress fibre formation and actin stabilisation [70]. Gelsolin is also a secreted protein where its role in plasma is to "clean up" actin filaments that have been released into circulation during burn injury and cell necrosis using its gelsolin domain [27–29]. Plasma gelsolin is able to inactivate pathogen-associated molecular pattern (PAMPs) molecules, like lipopolysaccharides (LPS) and LTA (lipoteichoic acid) resulting in decreased TLR-mediated NF-kB activity [30, 31] suggesting a potential protective role for plasma gelsolin against inflammation. Gelsolin has also been shown to play a role in inflammation with studies suggesting potential clinical applications for plasma gelsolin in diagnosis and disease activity evaluation as patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) who have significantly decreased plasma gelsolin levels compared with healthy controls [23,

scar formation [31].

144 Wound Healing - New insights into Ancient Challenges

**6.1. Gelsolin**

Flightless I (Flii) is a highly conserved multifunctional protein possessing an unique structure, containing six gelsolin domains and an additional 11 tandem repeats of a 23-amino acid leucine-rich repeat (LRR) motif not present in other family members [5]. The specificity of its structure allows Flii to regulate multiple intracellular and extracellular processes [5]. Flii uses its gelsolin domain to bind and remodel (via severing, capping and bundling) cytoplasmic actin monomers (G-actin) and actin filaments (F-actin) and it possesses F-actin severing ability [67]. Unlike other members of the gelsolin family, which enhance actin polymerisation, Flii inhibits actin polymerisation [73] and associates with focal adhesions inhibiting their turnover in a Rac1-dependant manner [74]. Unique specificity of its LRR domain allows Flii to interact with multiple signalling and structural proteins including paxillin, talin, vinculin, Ras, Cdc42 and LRR Flightless Interacting proteins 1 and 2 [48, 74]. The bipartite domain structure of Flii provides capacity for it to transduce cell signalling events into remodelling of the actin cytoskeleton and Flii has been proposed to be involved in a variety of signalling pathways, many of which are important in wound healing [74–77]. In addition, Flii binds to proteins other than actin, both in the cytoplasm and in the nucleus as well as outside the cell [76, 77]. It is sequestered in the cytosol by the active form of the calmodulin-dependent protein kinase type II (CaMK-II) protein [76]. Within the nucleus, it binds to a variety of coactivator complexes and to nuclear hormone receptor molecules, thereby mediating changes in transcription [76]. Flii may therefore provide a link between cell signalling pathways and actin-dependent morphogenetic processes including proliferation, migration and adhesion [31, 74].

Flii expression is increased in response to tissue injury in fibroblasts and LPS activation in macrophages [31, 77]. Flii is found in the nucleus, cytosol, lysosomes and like gelsolin is also a secreted protein by both fibroblasts and macrophages through a late endosome/lysosome pathway regulated by Rab7 and Stx11 [5, 77, 78]. Secreted Flii has been detected in human plasma [77], and acute and chronic human wound fluids [78, 79] and this secretion allows it to affect both intracellular and extracellular TLR-mediated signalling and subsequent production of pro-inflammatory cytokines important during wound repair [77]. Like gelsolin, secreted Flii has been shown to inactivate LPS, resulting in decreased TLR activation and downstream inflammation‐mediated signalling [77]. In addition, Flii has been shown to control inflamma‐ some activation by way of direct blocking of caspase‐1 and caspase‐11 and by modulating their subcellular localisation [80]. These findings suggest that Flii upregulation in response to wounding may be directed towards regulating inflammation with unfortunate consequences on healing of wounded area.

Complete knockout of Flii leads to gastrulation failure and embryonic lethality [81], while Flii heterozygous and transgenic mice appear phenotypically normal [82] suggesting an important role in development. In foetal skin, Flii is transiently increased in E17 but not E19 mice skin; however, its expression is downregulated in the E17 keratinocytes immediately adjacent to the wound margin [83] suggesting that temporal regulation of Flii during healing may influence wound repair outcomes. In addition, Flii interaction with tight junction proteins Cld‐4 and ZO‐2 is instrumental in development of skin barrier function and recovery following injury [84]. Wound healing studies using Flii heterozygous and transgenic mice have demonstrated that reduced Flii expression results in improved rate of healing via effects on cellular migration, adhesion and proliferation [31, 48]. In contrast, Flii transgenic mice have thinner more fragile skin, reduced number of hemidesmosomes and impaired cellular migration and adhesion leading to delayed healing [31, 48]. In addition, studies using mice with an inducible fibroblast specific Flii overexpression have shown inhibited wound healing with larger wounds than non‐induced controls, suggesting that fibroblast‐derived Flii may have an important role during wound repair [85].

Flii impairs the turnover of focal adhesions via a Rac1‐dependant mechanism and Flii inter‐ action with Rac1‐interacting proteins may be crucial to its effects on cell migration [74, 86]. In addition, Flii inhibits actin polymerisation [73] and this delicate balance of actin monomers and polymers can be altered using Flii neutralising antibodies (FnAb) raised against LRR domain of Flii [84] affecting collagen contraction, angiogenesis and wound healing outcomes [31, 87, 88].

Topical application of FnAb to wounds in preclinical models of wound repair results in a decreased wound area, a quicker rate of healing and decreased early scar formation [31, 88, 89] (**Figure 6**). Supporting these findings, both in vitro and in vivo studies have demonstrated that Flii plays a role in tissue scarring, collagen deposition and contraction [89, 90]. Using a preclinical model of porcine wound healing, studies have shown that Flii affects collagen I to collagen III ratio, impairs healing and contributes to the formation of early scars [89]. In addition, in vivo studies using human studies and animal models of bleomycin‐induced hypertrophic scaring show that Flii‐deficient mice exhibit reduced scarring in response to bleomycin as evident by decreased dermal thickness, smaller cross‐sectional scar areas, fewer myofibroblast numbers and increased collagen I to collagen III ratios [91]. Use of FnAb in porcine models of wound healing is the first example of using antibodies in large animal in vivo to modify the regulators of actin cytoskeleton that lead to improved wound healing outcomes. No side effects, complications or contraindications were observed when FnAb was administered locally to mouse or pigs suggesting the potential for the development of this therapy for human use. Application of such approaches to regulate different modulators of actin cytoskeleton may therefore lead to novel therapies aimed at optimal tissue regeneration and decreased scar formation following injury.

**Figure 6.** Treatment of excisional wounds with an FnAb improves wound healing and early scar appearance. Representative macroscopic images of wounds treated with either FnAb or a dose-matched IgG control on days 0, 5, 15, 21 and 35. These images were all taken from the same distance. The FnAb- and IgG control-treated wounds are from the same animal and the same position on either flank. Figure adapted from [89] and modified.
