1.18 Proposed aetiopathogenic mechanisms of keloid formation
1.18.1 Growth factor implication in the aetiopathogenesis of keloids
Aberrant wound healing in keloids and hypertrophic scars may be caused to some extent by abnormal growth factor activity. A major aetiological factor in the pathogenesis of keloids is the production of increased or decreased amounts of the various growth factors causing prolonged or abnormal cell signalling mechanisms.
1.18.1.1 Transforming growth factor-beta
One of the most important and well-researched cytokines implicated in the pathogenesis of excessive scar formation is TGF-β. This cytokine has a pleiotropic role that influences cell growth, differentiation, matrix production and apoptosis (90).
Transforming growth factor β is secreted by many cell types, including fibroblasts, Transforming growth factor-β has an important regulatory function in tissue repair where, its prolonged production and action leads to the growth of fibrous tissue (91). At the site of injury TGF-β stimulates the production of new ECM proteins such as collagens, fibronectin and proteoglycans; and inhibits the synthesis of proteases, but stimulates the production of protease inhibitors.
Transforming growth factor-β also increases the expression of cell surface integrin to enable cell-matrix interaction and matrix assembly (92). The effects of TGF-β are enhanced by the autocrine induction of its production (93).
lymphocytes, platelets,and activated macrophages. It has 3 isoforms: TGF-β1, TGF-β2, and TGF-β3 which have superimposing functions (94). Transforming growth factors β1 and β2 are overproduced by keloid fibroblasts compared with foetal and normal skin fibroblasts (95).
TGF-β1 and TGF-β2 are reported to be profibrotic, whereas, TGF-β3 is reported to have anti- fibrotic functions. When treated with exogenous TGF-β1, keloid fibroblasts produced 12 times more collagen than normal fibroblasts and 4 times more than hypertrophic scar
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fibroblasts(95). Transforming growth factor-β1 promoted fibroblast proliferation by
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blocking fas-mediated apoptosis, preventing the activation of caspases-3, -8, and –9, creating an obstruction in the apoptotic pathway( ). This anti-apoptotic outcome of TGF-β1 was not demonstrated in hypertrophic scars or normal fibroblasts (96). In addition to increasing the proliferation of keloid fibroblasts TGF-β1 also stimulated the synthesis of extracellular matrix components such as elastin, fibronectin, and types 1 and 3 collagen (97), (98), (99) (100). Another effect of TGF-β1 was the enhancement of
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contractile activity in keloid fibroblasts ( ) and the increased synthetic activity of myofibroblasts, resulting in the production of exuberant stromal tissue (102). Transforming growth factor-β2 specifically activated fibroblasts to synthesize and secrete collagen and the TGF-β2
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levels were elevated in both keloids and hypertrophic scars ( ). In in vitro experiments the increased synthetic and contractile activity of keloid fibroblasts; was reversed by the addition of TGF-β2 antibody (104).
The binding of TGF-β to proteoglycans close to the cell surface or in the matrix was proposed to be the signal that terminated its production after the repair process was complete (94).
Thus fibroblasts stimulated by TGF-β are the main perpetrators of scar formation. In foetal wounds which heal without scarring, messenger RNA expression of acidic and basic fibroblast growth factors and TGF-β
Recent research describes that the mechanism of action of TGF-β1 is by constant activation or reduced inhibition of signalling pathways involving intracellular effectors such as mitogen-activated protein kinases (MAPKs) and Sma- and Mad- related proteins (Smads), leading to a persistent autocrine-positive feedback loop affecting an overproduction of extra cellular matrix proteins and ensuing fibrosis (105).
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was decreased in fibroblasts compared with adult fibroblasts ( ). Despite strong evidence supporting the increased production of TGF-β1 in
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cultured keloid fibroblasts compared with cultured normal skin fibroblasts, in the in vivo situation, the plasma of keloid patients did not reflect this and no statistically significant difference was found in the plasma levels of TGF-β1 between keloid and control groups (106).
The key role of TGF-β in stimulating fibroblast proliferation and synthetic activity has attracted many research groups to investigate the modulation of TGF-β signalling mediators or the use of TGF-βinhibitors as a means of treating keloids (105). Ashcroft et al, (107) reported that normal skin and scar fibroblasts treated with conditioned media from keloid fibroblasts showed higher expression of fibrosis- linked molecular markers (including TGF-β) and increased synthetic and mitogenic activity. They indicated that keloid fibroblasts alter the proliferation rate and cellular activities of surrounding normal fibroblasts by their secretions through paracrine effects, promoting lesional growth.
It was reported that keloid fibroblasts have an uncontrolled or different response to TGF-β signalling pathways and demonstrate higher collagen I expression compared to normal fibroblasts (107). TGFβ-1 also stimulated the production of fibronectin (FN), α- smooth muscle actin-(SMA), plasminogen activator inhibitor 1 (PAI-1), connective tissue growth factor (CTGF) and TGFβ. Ashcroft et al (107) attributed the fervent keloid fibroblastic response to autocrine, paracrine, genetic and epigenetic effects as well as to the paracrine effects of secretions of inflammatory cells and re-epithelialising keratinocytes.
Exogenous TGF β1 upregulates the expression of platelet derived growth factor (PDGF) α receptor in keloid-derived fibroblast cells but not in normal dermal fibroblasts.
(105). It has also been shown that TGF β stimulates the expression of vascular endothelial growth factor (VEGF) in keloid fibroblasts (105).
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TGF-β moderates the expression of matrix metalloproteinase (MMPs), which can cleave all extra cellular matrix and basement membrane constituents. Expression of MMP-1 (interstitial collagenase) and MMP-2 (gelatinase-A) in keloids is increased and is associated with enhanced migration and invasion by lesional fibroblasts (58).
1.18.1.2 Platelet derived growth factor and insulin-like growth factor
The increased quantities of profibrotic cytokines such as platelet derived growth factor (PDGF), TGF-β, and insulin-like growth factor-1 (IGF-1), produced during the
exaggerated inflammatory phase and often observed in excessive scarring, were proposed as aetiological factors contributing to keloid formation (108). These authors also reported that the excess of platelets produced during the amplified inflammatory phase in keloids, were a source of the fibrosis-promoting cytokines, TGF-β and IGF-1. Many IGF-binding and IGF-related proteins were reported to be over-expressed in cultured keloid fibroblasts although IGF-1 effects on keloid fibroblasts were shown to be non-stimulatory. It was suggested that the action of IGF-1 was by augmenting paracrine TGFβ- scar forming effects through the increased expression of collagen I, fibronectin and PAI-1 by these fibroblasts (107).
1.18.1.3 Role of interferon, tumour necrosis factor, interleukins and other cytokines in the aetiopathogenesis of keloids
The deficiency of interferon-alpha (IFN- α), which has a down-regulatory effect on collagen synthesis, (75), (109) and reduced levels of interferon-gamma (IFN-γ) (110) which stimulates collagenase activity (109), (111) were other suggested causal factors implicated in the
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aetiopathogenesis of keloids. A study on the bloods from keloid patients showed increased amounts of tumour necrosis factor-alpha (TNF-α), beta-interferon (IFN-β), and markedly reduced levels of IFN-α, IFN-γ and tumour necrosis factor-beta (TNF-β). The net effects of these cytokines (at the increased or decreased concentrations), were reported to be related to elevated quantities of collagen, glycosaminoglycans and decreased collagenase levels in keloidal specimens (75), (109)
One of the most basic inflammatory cytokines, IL-6, was proposed to participate in the pathogenesis of keloids. Apart from promoting the inflammatory processes, it also actively stimulated proliferation of fibroblasts. In normal dermal fibroblasts IL-6 favours secretion of metalloproteinases, whereas in keloid fibroblasts, this process does not occur.
Metalloproteinases are endopeptidases that cleave a wide range of extracellular, bioactive substrates, and regulate the activity of these extracellular proteins (112). Some of the main non-catabolic functions of MMPs include the release of growth factors from the cell membrane or ECM, cleavage of growth factor receptors from the cell surface, shedding of cell adhesion molecules, and activation of other MMPs (113). Experiments where
exogenous IL-6 was administered to cultured fibroblasts showed that interleukin 6 increased the expression of type I collagen and adding an inhibitor for IL-6 to the culture abrogated this result (114). Immunohistochemical studies on keloid fibroblasts showed an increase in the expression of interleukin-6
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a receptor protein (IL-6aR) and other proteins viz., JAK1, STAT3, RAF, ELK and gp130, which are second messengers in the
signalling pathway of this cytokine ( ). The increased expression of IL-6
75 genes in keloid fibroblasts when compared with control fibroblasts, and the higher levels of IL-6 in peripheral blood mononuclear cell (PBMC) -fractions of black patients with keloids, supported the proposition that IL-6 may play a role in pathogenesis of keloid ( ), (115).
Other cytokines such as epidermal growth factor (EGF) and connective tissue growth
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factor (CTGF) were indirectly associated with the pathogenesis of keloids after activation by TGF-β (116), (117).
1.18.2 Essential fatty acid deficiency, nitric oxide and p53 gene mutations involved in the