LAS VICISITUDES DE LA CONCIENCIA HISTÓRICA EN EL SIGLO

Cellular stimuli caused by injury causes the expression of several MMPs at the wound site (Toriseva et al, 2012). MMPs are believed to have a role in all phases of wound healing, contributing to the removal of cellular debris and detachment of keratinocytes from the basement membrane shortly after injury then remodelling of the provisional matrix (Gill & Parks, 2008; Torriseva & Kähäri, 2009;

Mirastschijski, Schnabel & Tomasek, 2010). Proteolytic degradation of the ECM is vital for the efficient healing of cutaneous wounds (Werb, 1997) and the unregulated activity of such proteases is a major pathological mechanism underlying non-healing wounds (Palolhati et al, 1993; Barrick, Campbell & Owen, 1999). It is known that various activated cell types including activated keratinocytes at the wound edge, fibroblasts, and endothelial cells all share a common characteristic of protease expression, however it is the invading neutrophils and macrophages which are considered to be the major source of proteases at the wound site (Eming, Krieg & Davidson, 2007). While fibroblasts are a major source of matrix metalloproteinases (MMPs) in the healing wound, secreting proteases capable of degrading collagens, elastin and proteoglycans, neutrophils and macrophages recruited during the inflammatory phase release elastase and collagenase which degrade damaged extracellular matrix components (Tarnuzzer & Schultz, 1996). Furthermore,

proteinases with broad specificity including elastase, cathepsin G and urokinase may have detrimental effects on growth factor function (Yager et al, 1997; Wlaschek et al, 1997). Neutrophil elastase has also been shown to have antimicrobial activity in the wound environment (Cole et al, 2001).

There are a number of proteolytic enzymes present at the wound site including the serine proteases, such as plasmin and various matrix metalloproteinases (MMPs). These proteases have an important role in the migration of keratinocytes to the wound bed. Immature keratinocytes produce MMPs and plasmin, allowing the keratinocytes to dissociate from the basement membrane thus facilitating migration (Simonetti et al, 2013). MMP-9 has been shown to be upregulated in wounded skin compared with unwounded skin in parallel with an increase in CD44 molecules which are involved in cell-cell and cell-matrix interactions (Simonetti et al, 2013).

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MMP-1 is able to bind to α2β1 integrin within the wound bed, resulting in the migration of keratinocytes which ultimately contact interstitial collagens during epidermal wound healing (Sternlicht & Werb, 2001; Dumin et al, 2001). As studied by Kubo et al (2001), wound edge keratinocytes do not express the receptors required for fibrin/fibrinogen adhesion which is thought to be a mechanism for the removal of fibrin during tissue remodelling (Kubo et al, 2001). MMPs may play a role in this fibrinolytic pathway, allowing the epidermal cells to migrate across the wound bed. Plasminogen also plays a role in keratinocyte migration with one study utilising mice with an impaired plasminogen gene to demonstrate this function (Rømer et al, 1996). The study concluded that there was a reduced migration rate of keratinocytes from the wound margin during re-epithelialisation (Rømer et al, 1996). It is believed that this may be due to the decreased ability of these cells to navigate their way proteolytically through the fibrin-rich ECM in the deep wound tissue (Lund et al, 1999). Lund and colleagues (Lund et al, 1999) have also demonstrated the importance of plasmin and MMPs in wound healing with the residual wound healing capacity observed in the plasminogen-deficient mice possibly being a result of the ability of MMPs to perform alone the functions which they would perform together with plasmin in wild-type mice. However, a direct functional overlap between plasmin and MMPs was not demonstrated.

There is a generally high level of proteolytic activity in chronic wound fluid. Treatment aimed at the re-establishment of the natural balance of proteases as well as cytokines and growth factors and their inhibitors within the wound site, such as the application of combinations of selective inhibitors of metalloproteinases and growth factors may be beneficial (Tarnuzzer & Schultz, 1996). Conversely, it may be that combinations of certain growth factors applied to the wound at precisely timed intervals may be more effective in the promotion of healing (Singer & Clark, 1999).

Protease expression at the periphery of the wound aids leading edge keratinocytes to dissolve the fibrin (in the wound clot) through the fibrinolytic enzyme plasmin (Martin, 1997). This enzyme is derived from plasminogen and is activated by tissue- type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) (Lijnen & Collen, 1987). The role of plasminogen in wound repair has been

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demonstrated in transgenic mice where the plasminogen gene is knocked out and the re-epithelialisation of the wound is almost abolished (Rømer et al, 1996).

Furthermore, key regulators of plasminogen activation are expressed by migrating keratinocytes (Rømer et al, 1996; Morioka et al, 1987; Rømer et al, 1994). Various classes of MMPs are also up-regulated by wound-edge keratinocytes and are involved in cell migration. MMP-9 (gelatinase), for example, has been shown to cleave basal lamina collagen type IV and anchoring fibril collagen type VII, which then releases keratinocytes to the basal lamina (Martin, 1997; Salo et al, 1994). On the contrary, the many experimental studies which have identified MMPs 2 and 9 as type IV collagenases should be interpreted with caution (Rowe & Weiss, 2008). In terms of basement membrane remodelling, the role of MMP2 and MMP9 is

questionable given that the triple helical structure of collagen type IV under in vivo

temperature is resistant to proteolytic breakdown (Rowe & Weiss, 2008). However, evidence has continued to suggest an elevated presence of these MMPs within the basement membrane during tissue repair (Gschwandtner et al, 2008; Matsubara, Zieske & Fini, 1991). In comparison, MMP-1 (interstitial collagenase) is only up- regulated in basal keratinocytes which have already migrated beyond the free edge of the basal lamina, suggesting that cell-matrix interactions control the expression of this MMP (Martin, 1997; Saarialho-Kere et al, 1992). MMP-1 is involved in the migration of keratinocytes on collagen and so is important in the initiation of re- epithelialisation (Pilcher et al, 1997; Toriseva et al, 2012). MMP-10 is also up- regulated by keratinocytes at the margin of the wound and is specifically increased during impaired/prolonged healing (Martin, 1997; Saarialho-Kere et al, 1994).

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