**3. Conclusions**

animals remains surprisingly unaffected (**Table 3**) [92]. However, MMP-14 has been demonstrated to regulate cell proliferation by altering the expression of the KGF receptor during wound healing in acute airway injury [72]. The overlapping functions of other membrane-type MMPs or other MMPs may compensate for the absence of MMP-14 in these animals, support-

Cutaneous injuries that are recalcitrant to healing will become chronic wounds. In addition to delayed wound closure, chronic wounds are characterized by excessive proteolysis, prolonged inflammation, and failure in re-epithelialization [125]. Although MMPs play important roles in restructuring the ECM and repairing the wounds, high levels of MMPs can be blamed for increased proteolysis that leads to excessive degradation of ECM constituents and disruptions of cell migration. These unwarranted events cause the wounds to enter a prolonged inflammation. ELISA was used to document 65-fold higher levels of MMP-1, twofold higher of MMP-8, and twofold lower of TIMP-2, whereas gelatin zymography showed 14-fold higher levels of MMP-9, and sixfold higher of MMP-2 in diabetic foot ulcers than in non-diabetic patients with acute wounds [49]. Up-regulation of MMPs hinders wound repair by degrading ECM components and growth factors excessively [126]. As the wounds stay open too long, the invading bacteria might also release bacterial proteinases to cause rapid degradation of growth factors [94]. In order to defend the wounds against the invading microbes, the body will secrete more ROS and inflammatory factors. High levels of ROS such as hydrogen peroxide cause tissue damage [22], and high levels of inflammatory factors can lead to elevated expression of MMPs, as discussed earlier. The delaying mechanism of this vicious cycle keeps the patient's wound in a chronic stage [127]. Most studies emphasize MMP-9 up-regulation, which is associated with poor wound healing in diabetic foot ulcers and chronic wounds (**Figure 6**). When high levels of exogenous MMP-9, parallel to human chronic wounds, was applied to non-diabetic mice, this treatment delayed wound healing of the animals [128]. In one study, up-regulated levels of MMP-9 were found in wound fluid from patients with unhealed diabetic foot ulcers when compared with healed ulcers, as determined by gelatin zymography [129]. Also, in this same study, the researchers found decreased levels of TGF-β1 and TIMP-1 using ELISA [129]. In another study of patients with diabetic foot ulcer, levels of MMP-9 were measured by Western blot with MMP-9 antibody and were higher in patients with high risk of developing foot ulcers [130]. Expression of this proteinase was detected in migrating epithelial cells by ELISA [50, 51] and in inflammatory cells including T cells and neutrophils by gelatin zymography [131, 132]. Nevertheless, it should be noted that increased levels of MMPs, specifically that of MMP-9, as determined by ELISA, Western blot or gelatin zymography do not necessarily imply that it is active or has any role in the pathology of chronic wounds. ELISA and Western blot depend on the specificity of the antibodies, which likely immunoreact with pro-MMPs, active MMPs, and TIMP-complexed MMPs. Similarly, the active MMP-2 and MMP-9 bands detected by gelatin zymography could be from the TIMPcomplexed gelatinases that dissociate during electrophoresis [133]. Therefore, the expression of MMP-9 found in many studies cannot be established conclusively as active MMP-9, the only form of the proteinase that can modify substrates catalytically. Another common research

ing the concept of proteinase redundancy among MMPs.

58 Wound Healing - New insights into Ancient Challenges

**2.2. Roles of MMPs in the pathology of chronic wounds**

MMPs exist in three forms―pro-MMPs, active MMPs, and TIMP-complexed MMPs―of which only the active MMPs play a role in the pathology or repair of acute and chronic wounds. Current methodologies do not distinguish between the three forms of MMPs. Thus, the roles of MMPs in acute and chronic wounds are still not Qualitative and well-characterized quantitative profiling of only the active form of MMPs is necessary for investigating the critical roles of MMPs in remodeling the ECM during wound repair. We used a novel MMP-inhibitortethered affinity resin that binds only the active form of MMPs, from which we identified and quantified active MMP-8 and active MMP-9 in a murine diabetic model with delayed wound healing [60]. We showed that up-regulation of active MMP-9 plays a detrimental role whereas active MMP-8 is involved in repairing the wound in diabetic mice [60, 63]. These studies identified MMP-9 as a novel target for therapeutic intervention in the treatment of chronic wounds. A selective inhibitor of MMP-9 that leaves MMP-8 unaffected would provide the most effective therapy and represents a promising strategy for therapeutic intervention in the treatment of diabetic foot ulcers.
