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Cytokines and growth factors are important regulators of wound resolution and tissue repair. The cytokine that is central to both initiation and termination of tissue repair is TGF-β. In mammals, TGF-β exists in three isoforms with almost identical biological properties and targets. Encoded by different genes, the three isoforms: TGF-β1, TGF-β2, and TGF- β3, activate similar receptors and intracellular signalling pathways [178-180]. Although important for successful wound healing, persistent expression of TGF-β1 can promote the development of tissue fibrosis and progression. TGF-β1 is the isoform most extensively studied and has the highest expression in fibrosis, correlating with the severity of progressive fibrosis [178].

TGF-β1, a member of the transforming growth factor-β super-family is a secreted protein that performs many cellular functions, including the control of cell growth, proliferation and differentiation. In humans, the TGF-β1 gene encodes TGF-β1. Dysregulation of TGF-β1 activation and signalling may result in apoptosis [181]. Many cells synthesise TGF-β1 and have specific receptors on their cell surface for this peptide. TGF-β1 was first identified in human platelets as a protein with a molecular mass of 25 kilodaltons (kDa), and an important role in wound healing. It was later characterised as a large protein, synthesised as an inactive 390 amino acid precursor that is secreted as a large latent complex, covalently bound with latency associated peptide (LAP); and stored in an inactive form in the matrix. The inactive

form of TGF-β1 is proteolytically processed to produce a bio-active mature peptide of 112 amino acids [179].

TGF-β binds to at least three membrane receptor proteins, TGF-β receptor (TGF-βR) I, II, and III. They can exist in homo- or heterodimers. The type I and II receptors are transmembrane serine-threonine kinases that typically interact with one another to form a heterodimer in order to facilitate signalling [182]. The type III receptor, also known as betaglycan, is a membrane anchored PG with no signalling structure but can act to present TGF-β to the other receptors on the cell surface [183]. When TGF-β interacts with these receptors, a cascade of intracellular signalling results in altered gene transcription profiles. The principal route of activation is through the Smad signal transduction pathway [184]. However, TGF-β has also been described to recruit alternative pathways including the epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 and 2 (MAPK; ERK1/2), Rho-GTPase, protein kinase B, and the Akt/mTOR pathway [160, 185-187]. TGF-β1 shows a diverse range of different activities on different types of cell depending on biological context, or cells at different developmental stages. Many cells secrete TGF-β1 including fibroblasts, myofibroblasts and most immune cells (leukocytes) [188, 189]. The TGF-β family also play important roles in controlling the immune system, the inflammatory response and foetal development [190, 191]. Specifically TGF-β1 is a known promoter of fibroblast terminal differentiation and EMT; upregulating α-SMA expression in vitro and in vivo [192]. The production and secretion of fibronectin, collagens, and matrix turnover proteins (MMPs and TIMPs) are also processes regulated by TGF-β1 signalling [193].

The traditional TGF-β signalling cascade involves the binding of TGF-β1 to TGF-βRI otherwise known as Alk5, which upon activation forms an active heterodimer with TGF-βRII [182]. The subsequent phosphorylation of targets follows a well-documented cascade event,

first through the phosphorylation of Smad2 and Smad3. When bound to co-Smad4, this Smad-signalling complex translocates to the nucleus where in it acts as a transcription regulator, binding target sites on gene promoters and either enhancing or suppressing gene transcription [184]. A classic gene target of TGF-β1 signalling includes SERPINE1, which encodes for the protein plasminogen activation inhibitor 1 (PAI-1), the expression of which is often used as a marker of TGF-β1 activity [194].

TGF-β-Smad signalling is an essential phase in the differentiation of fibroblasts to myofibroblasts. The signalling mechanism underlying the genesis of the myofibroblast is complex; with respect to the TGF-β1-Smad signalling pathway, the presence of the Smad3- binding element is important for myofibroblast differentiation and oral mucosal fibroblast proliferation [195, 196]. Despite shared expression, the regulation of the α-SMA gene in fibroblasts is more complex and in many respects different from that in SMCs [197]. In addition to transcriptional inducers, suppressors may have a higher basal activity in fibroblasts, compared to SMCs; and serve to keep the fibroblast in an undifferentiated state under normal homeostasis.

Dysregulation of TGF-β1 action has been implicated in a variety of pathological processes, including cancer and autoimmune diseases; and its involvement in progressive tissue fibrosis has been confirmed in human disease and in numerous models of animal disease [72, 73, 178, 198]. Its fibrotic actions have been attributed to its combined abilities to induce fibroblast differentiation, epithelial cell trans-differentiation and to promote accumulation of matrix proteins, including collagens, fibronectin and PGs. As discussed above, TGF-β1 also induces production of TIMPs and inhibits some MMPs and plasmin protease generation, thereby preventing matrix degradation and contributing to the fibrotic process [168]. Furthermore, it modulates expression of FA molecules that contribute to intracellular signalling,

mechanotransduction, stress generation; and facilitate cell-matrix adhesion and matrix deposition [77, 119].

Additional evidence supporting the pro-fibrotic role of TGF-β1 was shown in vivo, where treatment of foetal wounds with exogenous TGF-β1 promoted scarring [131, 145]. Additionally, incisional rat wounds treated with anti-TGF-β1 antibodies or anti-sense nucleotides were shown to suppress ECM synthesis and scarring [199]. However, TGF-β1 knock-out mice models have been shown to have a high rate of intra-uterine death [200], indicating the important role of TGF-β1 in development and cell survival. The few that survived to birth later died of a multi-focal inflammatory syndrome [201], signifying a TGF- β1 role in modulating immune and inflammatory cell types. Furthermore, mice heterozygous for TGF-β1 deletion, although demonstrated a normal phenotype at birth had a greatly increased frequency of carcinoma formation, when challenged with carcinogens [202]. These studies highlighted the potential challenges relating to the direct targeting of TGF-β1 in anti- fibrotic treatments. Therefore, further detailed characterisations of responses involved in the modulation of TGF-β1 actions are necessary to develop more selective, clinically appropriate, anti-fibrotic therapies.