Fibronectin in hyperglycaemia and its potential use in the treatment of diabetic foot ulcers: A review

Abstract Metabolism of fibronectin, the protein that plays a key role in the healing of wounds, is changed in the patients with diabetes mellitus. Fibronectin can interact with other proteins and proteoglycans and organise them to form the extracellular matrix, the basis of the granulation tissue in healing wounds. However, diabetic foot ulcers (DFUs) suffer from inadequate deposition of this protein. Degradation prevails over fibronectin synthesis in the proteolytic inflammatory environment in the ulcers. Because of the lack of fibronectin in the wound bed, the assembly of the extracellular matrix and the deposition of the granulation tissue cannot be started. A number of methods have been designed that prevents fibronectin degradation, replace lacking fibronectin or support its formation in non‐healing wounds in animal models of diabetes. The aim of this article is to review the metabolism of fibronectin in DFUs and to emphasise that it would be useful to pay more attention to fibronectin matrix assembly in the ulcers when laboratory methods are translated to clinical practice.


| INTRODUCTION
Foot ulcers are a major source of morbidity in patients with diabetes. They develop in almost 6% of patients within 3 years after diabetes was diagnosed 1 and in 15% of patients during their lifespan. 2 Diabetic foot ulcer (DFU) is a break of the skin of the foot that involves the epidermis and a part of the dermis. They are often accompanied by neuropathy and/or peripheral arterial disease in the lower extremity. 3 Wound healing is impaired in diabetes. The inflammatory phase in a normal wound is transient and converted to the proliferative phase that leads to the closure of the wound. Inflammation persists in the diabetic wound 2,4 and healing is often complicated by bacterial infection. 5 The typical location of DFUs is on the plantar aspect of the metatarsal heads and the heel and over the dorsal parts of the toes 6 ( Figure 1). Fibronectin (FN) is an extracellular matrix glycoprotein that plays a vital role in all phases of acute wound healing. Its soluble form circulating in the plasma (pFN) is incorporated into the fibrin clot that is formed in the wound immediately after injury. Cellular FN (cFN) is then synthesised by the cells migrating into the clot. 7,8 cFN interacts with cells, collagen and proteoglycans and forms the extracellular matrix (ECM). The provisional matrix is gradually replaced with the granulation tissue 7 (Figure 2A, C, E). FN also supports the epithelialization of the wound and wound angiogenesis. 8 Diabetes increases FN expression in the tissues that are targets of the complications of diabetes. 9 FN is expressed in the dermis of diabetic ulcer area and the expression persists much longer than that in acute wounds. 10 However, the balance between the production and degradation of the ECM is shifted to degradation in DFUs. 4,11 ECM deposition is reduced 12 ; the wound bed in the diabetic wound is necrotic and infiltrated with inflammatory cells (Figure 2B, D, F).
The term DFU covers many aspects of the disease and multiple disciplines are involved in its management. 3,4 FN has a key role in wound healing and deserves more attention in connection with DFUs than it has received until now. The aim of this review is to describe what makes FN important in wound healing, the disturbance of FN metabolism in conditions of hyperglycaemia and the ways that have been proposed to overcome the disorder. The review summarises the results of the studies of FN performed in normal healing wounds and in DFUs and collected in the databases Web of Science, PubMed and Scopus.

| FIBRONECTIN STRUCTURE
FN exists as two isoforms, plasma, and cellular, which is the result of the alternative splicing of FN mRNA. pFN is synthesised in the liver by hepatocytes and circulates in the blood, cFN is synthesised in tissues by various cell types, including macrophages, fibroblasts, and endothelial cells. pFN and cFN are involved in various phases of wound healing. 7 FN molecule is composed of two subunits whose molecular weight varies from 230 to 270 kDa as a consequence of the splicing. The subunits comprise domains that are able to interact with cells and with macromolecular ECM components ( Figure 3). The domains are composed of modules I, II and III that differ in their lengths and amino acid composition. 13,14 The aminoterminal domain of FN binds fibrin and Factor XIII (transglutaminase, TG-2) and can interact with proteins in the cell walls of Staphylococcus aureus and other bacteria. FN monomer also contains binding sites for cellular integrins, collagen, heparin, and heparan sulphate proteoglycans, and various growth factors. 13,14 FN binding sites are distributed along the FN monomer. 14 The central part of the monomer contains extra domains A (EDA, EIIIA) and B (EDB, EIIIB). These extra domains are either included or skipped when the primary gene transcript of FN is spliced. They are found in cFN, not in pFN. 15 The arg-gly-asp (RGD) sequence projecting from the core of the FN molecule 16 is located between EDA and EDB; it is able to bind cellular integrins α5β1. The sequence pro-his-ser-arg-asn (PHSRN), called the synergy site, supports the binding. 13 A large exon codes for the variable (V) region that is necessary for the secretion of FN from the cells and is found both in pFN and cFN. 17 A pair of cysteine residues near the carboxyl terminus can form the FN dimer through disulphide bonds. 13,14 FN differs from other ECM proteins, such as laminins, tenascins, and fibrinogen/fibrin in that it can undergo large conformation changes. There are a few flexible regions in the molecule that allow interactions between modules on the same or on the opposite FN subunit. 18

| EXTRACELLULAR MATRIX ASSEMBLY
As a result of internal interactions between their domains, soluble FN molecules have a closed compact conformation that prevents them from undesirable interactions with other macromolecules and cell receptors. 18 FN matrix assembly starts by a selective binding of FN to the cell surface; the 70 kDa N-terminal region and the RGD loop are involved in the binding to cellular receptors. 13,19 FN adhesion induces a reorganisation of the actin cytoskeleton and receptor clustering. FN molecules are coupled to the contractile cytoskeleton. Cellgenerated force is used to unfold FN molecules partially. The interdomain interactions are disrupted, the molecules become more extended and FN binding domains that were previously sequestered are exposed. 14,20 Different domains in the FN molecule come into contact with a range of domains in neighbouring FN molecules. FN molecules attach laterally and FN fibrils are formed on the cell surface. 13,17 The pool of FN adsorbed to the cell surface is transformed into a detergent-insoluble matrix 14,17 ; the insolubility arises from non-covalent but strong protein-protein interactions. 21 The deposition of collagen in the ECM is dependent on the polymerisation of FN. 22,23 Collagen type I is deposited into fibrillar structures in the ECM when FN fibrils are present. The continued synthesis of FN is necessary to stabilise both FN and collagen fibrils. 24 As collagen type I deposition increases, collagen fibres become primary tensionbearing matrix elements. 23 Continued presence of FN is also critical for the incorporation of latent TGF-β binding protein 1 (LTBP-1) with latent transforming growth factorβ (TGF-β) into the ECM. 25 The presence of EDA domain in FN molecule increases the efficiency of LTBP binding 26 and is permissive for the induction of myofibroblast phenotype in fibroblasts by TGF-β1. 27 Myofibroblasts are the main cells producing FN and other ECM proteins which are needed to restore tissue integrity after injury. 28 The assembly of fibrillins, the major component of microfibrils that are present in elastic and non-elastic extracellular matrices, also requires FN. 29

| FIBRONECTIN IN ACUTE WOUND HEALING
In acute wounds, pFN is deposited in the wound site within seconds after injury as the collagen-binding site interacts with the collagen exposed by the denuding vessel walls. pFN integrates into the fibrin network formed in the wound, enhances mechanical strength of the clot, and promotes platelet aggregation. 30 FN is crosslinked to fibrin by activated Factor XIII 31 and forms a provisional fibrin-FN matrix which is later replaced by the ECM. Rapid formation and deposition of the ECM are required to provide the scaffold for efficient binding of cells and physical support for the expansion of blood vessels in angiogenesis. 7 Macrophages migrate into the clots at early time intervals after wounding; they are the first cells expressing cFN locally. 32 The presence of FN is an absolute requirement for the later migration of fibroblasts into the plasma clot. 33 Fibroblasts that invade the wound become a major source of cFN. 32 cFN forms a dense network around the fibroblasts and regulates the deposition of collagen and other ECM molecules. 7 FN is a reservoir for growth factors important for wound healing, platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor (HGF). The growth factors sequestered in the ECM can be released by controlled enzymatic digestion. 34 FN mRNA in normal skin does not contain EDA or EDB segments but mRNA coding for these domains is found in the cells at base of the acute wound and at the wound edges. 35 Mice without the EDA exon in FN protein display abnormal skin healing; the granulation tissue shows edematous regions and wound re-epithelialization is impaired. 36 Keratinocyte migration and proliferation are crucial for the re-epithelialization of wounds. Epidermal cells expressing α5β1 integrin, the main FN receptor, migrate from the wound edges over the provisional matrix containing fibrin and FN. 37 FN provides a substrate for endothelial cell proliferation and ingrowth. It promotes endothelial cell survival and migration, 38,39 while FN fragments have anti-angiogenic effects. 39 FN scaffold is cleared before tissue regeneration is completed. 40

| EFFECT OF HYPERGLYCAEMIA ON FIBRONECTIN METABOLISM
Diabetes mellitus is a metabolic disorder characterised by chronic hyperglycaemia, which means that glucose concentration in fasting venous blood exceeds 6.1 mmol/L. 41 The high glucose burden perturbs cellular physiology. FN expression is increased in various organs of animal models of diabetes, in the kidneys, 42 heart, 43 colon, 44 and retinas. 45 Plasma levels of cFN are significantly higher in patients with diabetes compared to that in healthy control subjects. 46 Circulating FN may be deposited in tissues. 47 Cultured human dermal fibroblasts respond to high glucose concentration 48 or to advanced glycation end products (AGEs) 49 by increased expression of FN mRNA and protein. Human endothelial cells respond to high glucose in a similar way. 50 The elevation of FN mRNA persists after multiple cell divisions in the absence of high glucose. 9 In normal skin, FN can be detected only in blood vessels. In acute wounds, it is detected in the re-epithelialized area and its expression increases until 3 months after wounding. FN staining disappears completely by 12 months after wounding. At this time FN staining still persists in the ulcer area. 10 Human fibroblasts isolated from DFU and cultured on 3D matrix secrete threefold and fourfold more FN than fibroblasts from non-ulcerated diabetic skin and normal skin fibroblasts, respectively. However, diabetic fibroblasts produce thinner tissue in a 3D ECM tissue model and do not respond to TGF-β1 by increasing FN production as normal human dermal fibroblasts. 51 Impaired migration on FN-coated surfaces was observed in fibroblasts isolated from diabetic mice 52 and in fibroblasts from streptozotocin (STZ)-diabetic rats. 53 The cause of decreased adhesion of fibroblasts to FN and impaired migration may be a reduced expression of α5 integrin subunit, the component of the main FN receptor, in the plasma membrane. The content of αv subunit that might compensate for the loss of α5 is also decreased. 53 Both the glucose aldehydic group and methylglyoxal, the product of glucose metabolism, react with the amino group of amino acids. 54,55 Methylglyoxal, a highly reactive carbonyl compound, is a major precursor of the AGEs. Methylglyoxal reacts with lysine and arginine residues in proteins. 56 A number of targets for this glycating agent are found in FN, including the arginine residue in the RGD cell-binding site. The accumulation of AGEs modifies FN matrix assembly and cell attachment. 55 AGEs accumulating in the neurovascular system cause irreversible structural changes of macromolecules. Nerve fibre damage by AGEs mediated by their receptor (RAGE) results in peripheral sensory neuropathy. 57 Reduced ability to sense pain leads to the development of foot trauma and tissue damage. 58 Glycation of FN and laminin impairs neurite outgrowth, which contributes to the failure of axonal regeneration. 59 Microangiopathy may play a role in the pathogenesis of tissue breakdown in the diabetic foot. 58 Hyperglycaemia induces the formation of reactive oxygen species (ROS) in cultured human endothelial cells. Oxidative stress results in their decreased proliferation and tube formation. 60 The interaction of AGEs with their receptor stimulates the generation of ROS which leads to inflammatory changes in vasculature. 61 The lack of EDA-FN exacerbates endothelial dysfunction in diabetes. 62 Deoxyguanosine in genomic DNA may also react with methylglyoxal which causes DNA instability through strand breaks and crosslinks. 56 Hyperglycaemia is associated with increased production of ROS 63 ; their major sources are mitochondrial respiration and NADPH oxidases. 64 Human dermal fibroblasts cultured in the medium containing high glucose express senescenceassociated β-galactosidase, a marker of cellular senescence, produce high amounts of ROS and their migration is impaired. 65 Treatment of fibroblasts with hydrogen peroxide, an important source of ROS, leads to a decreased expression of FN and collagen type I and an increased expression of MMPs. Senescent fibroblasts are resistant to growth factor stimulation. 66 Repeated passaging induces senescence in human dermal fibroblasts. The amount of FN that can be extracted from senescent fibroblasts is markedly decreased when compared to controls. 67 Senescence decreases the level of α5 integrin subunit in human dermal fibroblasts although the expression of α5 mRNA is increased. 68 These events are outlined in Figure 4A.
The inflammatory phase in wound healing is prolonged in diabetes and the transition to the proliferative phase fails. The inflammatory cells, neutrophils, and macrophages, remain in the wound in large numbers and create an environment rich in proinflammatory cytokines and proteinases. 4,69 Leukocyte-derived proteinases degrade FN and other ECM proteins and inactivate physiologic proteinase inhibitors present in the cellular environment. 70 FN found in the wound fluid in DFUs is degraded to a great extent, in contrast to surgical wounds. 71,72 Granzyme B, a product of cytotoxic lymphocytes, is a serine proteinase expressed in the skin wounds in diabetic mice. It cleaves FN, resulting in the release of multiple FN fragments. 73 These fragments reduce endothelial cell adhesion, migration and tube formation. 74 The functions of FN may be significantly altered by the fragmentation of the molecule and the loss of various domains. 75 The behaviour of human fibroblasts is modified in the presence of FN fragments; the cells have different shapes and the efficiency of their attachment to the substrates coated with FN fragments is greatly decreased compared to full-length FN. 72 FN fragments enhance neutrophil migration and macrophage activation 75 and induce the expression of inflammatory cytokines in fibroblasts. 76 Full-length FN inhibits matrix metalloproteinase (MMP) secretion by monocytes but FN fragments have the opposite effect. 77 As a result of these events, the deposition of the ECM in diabetic wounds is diminished and does not support cellular functions and angiogenesis adequately. 78

| TREATMENT OF DIABETIC FOOT ULCERS
High MMP-9 level and high MMP-9/TIMP-1 (tissue inhibitor of metalloproteinases-1) ratio are associated with poor healing of DFU. 79 Wound healing in diabetic mice is accelerated by the application of a specific inhibitor of MMP-9. The inhibitor does not affect MMP-8 that is beneficial for wound healing. 80 A serine proteinase inhibitor that acts on granzyme B and other serine proteinases increases the content of full-length FN in wound tissue, promotes collagen deposition and the reepithelialization of the wounds. 73 Collagen-based wound dressings absorb and inactivate tissue proteinases in human chronic wounds. 81 The healing of excision wounds made in the skin of STZ-diabetic rats is significantly accelerated by a local application of human pFN. The number of fibroblasts infiltrating the wound is enhanced, collagen synthesis and the re-epithelialization of the wounds are increased. 82 pFN also potentiates the favourable effects of erythropoietin 83 and cytokine CXCL11 84 on wound healing in diabetic mice.
Many biological activities of fibrillar FN may be mimicked by recombinant FN peptides containing sites that initiate cell and tissue responses. A chimeric FN fragment constructed by inserting RGD peptide into the heparinbinding region of FN promotes fibroblast proliferation in vitro, increases the deposition of granulation tissue, and accelerates wound closure in the wounds in diabetic mice. 85 The PHSRN (pro-his-ser-arg-asn) sequence that functions in FN as a second cell-binding site besides RGD, stimulates the migration of keratinocytes and fibroblasts into the excision skin wounds in obese diabetic mice and accelerates the closure of the wounds. 86 FNderived peptides containing the repeats that promiscuously bind growth factors can be used to deliver VEGF and PDGF-BB to the skin wounds in diabetic mice. This treatment supports the formation of granulation tissue and wound closure. 87 Connective tissue growth factor (CTGF, CCN2) is a cytokine that stimulates FN mRNA and protein expression in human dermal fibroblasts. 88 The induction of FN mRNA and protein in these cells by glucose treatment is F I G U R E 4 A, Increased metabolism of glucose in diabetes mellitus leads to the overproduction of reactive oxygen species (ROS) that damage fibronectin-producing cells. ROS and advanced glycation end products (AGEs) cause enhanced and prolonged inflammation in the diabetic wound. B, Various ways to replace lacking fibronectin (FN) in diabetic foot ulcers partially mediated by CTGF. 48 CTGF is deficient in diabetic wound tissue. When acellular dermal matrix (ADM) soaked with CTGF is applied on excision skin wounds in STZ-diabetic mice, FN and collagen expression are enhanced and wound closure is accelerated. 89 ADM grafts are able to promote the healing of DFUs and reduce large wound areas. 90 ADMs are of human 90 or animal 91 origin, they are devoid of cellular DNA but contain ECM components including FN. 91 The matrix provides a structure that can be repopulated with the patient's cells. The cell-binding activity of FN in the ADM is important for the matrix function. 92 ADM also induces the expression of FN in the wound tissue. 93 Stem cell therapy is proposed for the treatment of DFUs with the aim to promote angiogenesis and ECM remodelling and improve healing. 94 Adipose-derived stem cells (ASCs) isolated from adipose tissue of non-diabetic patients produce FN and other ECM components when they are cultured in vitro [95][96][97] and accelerate wound healing when they are administered to excisional skin wounds in diabetic mice. 95,98 FN present in the culture medium supports ASCs proliferation and can be used to produce ASC sheets. 98 Cultured human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) secrete FN and other ECM components as well as neurotrophic factors that promote nerve regeneration. 99 The conditioned medium obtained from hUC-MSCs cultures enhances FN expression by human skin fibroblasts, while MMP-1 secretion is decreased. 100 When hUC-MSCs are transplanted intravenously to STZdiabetic rats, a portion of the cells settles in the excisional wounds made in the rat skin and stimulates wound repair. 101 hUC-MSC secrete exosomes, small vesicles rich in proteins and nucleic acids that enter endothelial cells by endocytosis. Exosomes decrease oxidative stress in the cells, promote angiogenesis and accelerate wound healing in diabetic mice. 60 The exosomes isolated from the serum of healthy mice enhance the expression of FN and collagen in fibroblasts and promote granulation tissue formation in the wounds of diabetic mice. 102 Human mesenchymal stem cells secrete exosomes that induce proliferation of neuroblast-like cells. FN is the most abundant protein in the exosomes and mediates the mitogenic properties of exosomes. 103 Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in the pathology of diabetes. 104 The overexpression of lncRNA H19 in DFUs stimulates the synthesis of FN and collagen type I and accelerates wound closure. 105 The negative-pressure wound therapy, that is, the application of subatmospheric pressure to the wound, is beneficial for the healing of DFUs. The effect of the treatment may be partially attributed to enhanced cFN synthesis in the granulation tissue forming in the patients' wounds. 106 The overview of various treatments that increase the level of FN and improve the healing of DFUs in patients or in animal models is given in Table 1.  MMPs secreted into the wound fluid by inflammatory cells abundant in DFUs. 73,80 Moreover, chronic wounds are often contaminated by bacteria. Staphylococcus aureus, one of the most common pathogenic species identified in DFUs, 5 contains FN-binding proteins in the cell wall that facilitate the binding of bacteria to FN in the open wound. 107 The bacteria secrete proteolytic enzymes that activate host MMPs. 108 Research in this field may bring drugs that inhibit selectively both proteinases accompanying inflammation and those produced by infecting bacteria resistant to antibiotics. 73,80 Numerous methods have been developed to replace missing FN in the diabetic wound ( Figure 4B). The application of pFN on the wound may compensate for the loss of FN caused by proteolysis but the use of this method in medicine is limited by the ability of FN to bind various pathogens. 109 The enhancement of FN synthesis in situ may help to evade the problem. 106,110 Well-defined portions of FN molecule contain amino acid sequences required for the interaction of FN with cells. In the absence of fibrillar FN, recombinant FN peptides provide signals to fibroblasts enhancing their adhesive activity, proliferation and ECM deposition. They enhance keratinocyte migration and wound epithelialization. Revascularization of the wound and granulation tissue deposition is stimulated. FN peptides may be used for a supplemental treatment of chronic diabetic wounds. 85,86,111 ADM acts as a scaffold supporting cellular growth and granulation tissue formation. Proliferation of human dermal fibroblast is different on various commercial ADM preparations and may be influenced by the content of FN which is variable. 91 However, the correlation between FN content in the ADM and the ability of the graft to support fibroblast migration and proliferation has not been studied. Both recombinant FN fragments and ADM can be used to deliver growth factors to poorly healing wounds. 87,89 As a result of DNA damage, caused mainly by oxidative stress, the cells involved in wound healing, dermal fibroblasts, endothelial cells, and keratinocytes become senescent. Macrophages are stalled in the proinflammatory phenotype. 112,113 Senescent fibroblasts may express less FN and collagen but more MMPs when compared to normal fibroblasts. 66 The structural organisation of FN fibres produced by fibroblasts changes with cell aging. 114 Other fibroblast activities may also be disturbed. TGF-β1 is a cytokine involved in all phases of wound healing. It stimulates ECM protein production and inhibits MMPs activity. 115 However, DFU-derived fibroblasts respond differently to TGF-β1 than normal human dermal fibroblasts. 51 It has been shown recently that human DFU fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) that further differentiate to fibroblasts. iPSC-derived fibroblasts produce more FN and collagen than parental cells. 116,117 The matrix produced by the reprogrammed cells can be processed into a scaffold that accelerates the closure of wounds in diabetic mice when it is applied on the wound. 116 The use of reprogrammed patient-specific cells or 3D tissues derived from them may become the future way of DFU treatment. At present, stem cells are proposed as a therapy to treat diabetic foot. They have a high potential for self-renewal and differentiation into specific types of cells. 118 Transplantation of mesenchymal stem cells isolated from bone marrow or adipose tissue to patients with DFU leads to a decrease in wound size, improvement of leg perfusion and a decrease in the frequency of leg amputations. 119 Fibroblasts differentiated from ASC produce more FN, collagen type I and elastin than primary fibroblasts. 120 The direct use of stem cells for therapeutic purposes is limited by risk factors, such as tumour formation, thrombosis, and unwanted immune responses. These risks may be avoided by the use of exosomes, the extracellular vesicles that have a role in intercellular communication. 121 Exosomes from human cord blood plasma promote fibroblast functions, endothelial cell tube formation, and macrophage differentiation to non-inflammatory phenotype. Artificial exosomes that have even better effects on wound healing than naturally occurring exosomes can be constructed. 122 Exosomes derived from mesenchymal stem cells promote nerve growth in the excision wounds in the skin of diabetic rats. 123 Negative pressure wound therapy 106 and ADM application 124 are clinically tested therapies for DFUs that result in the enhancement of granulation tissue formation and accelerated wound healing. Many questions need to be answered before these therapies become a routine in clinical application. They affect FN metabolism; as the outcome of wound healing depends on FN synthesis to a great extent 7 it would be useful to define the relationship between the FN enhancing treatments and DFU healing in more precise terms.

DATA AVAILABILITY STATEMENT
Data derived from public domain recourses.