**2. Detection of matrix metalloproteinases in tissue**

were analyzed by liquid chromatography—mass spectrometry/mass spectrometry (LC-MS/ MS); the proteinases were identified from the peptides MS/MS data and a protein database search [60] (**Figure 5**). Subsequently, each identified active MMP was quantified using LC-MS/ MS methods and custom-synthesized peptides. This analysis led to the discovery of active MMP-8 and MMP-9 in both diabetic and non-diabetic wounds from mice. The quantification revealed that MMP-9 was elevated at statistically significant levels, whereas levels of MMP-8

**Figure 5.** MMP-inhibitor-tethered affinity resin to identify and quantify active MMPs. Wound tissues are homogenized, and the homogenate is incubated with the MMP-inhibitor-tethered affinity resin, which binds only to active MMPs. The isolated active MMPs are reduced (to reduce disulfide bonds between the thiol groups of cysteine in MMPs), alkylated (to prevent reformation of disulfide bonds), and trypsin digested. The resulting peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified by a protein database. The identified MMPs are quantified using three peptides and three transitions per peptide using LC with multiple-reaction monitoring (MRM). In this highly specific quantitative MS method, the ionized peptide selected in the first quadrupole (Q1) generates to a pool of fragments in the second quadrupole (Q2), where the highest intensity fragment ion is selected for monitoring in the third quadrupole (Q3). This transition from peptide to fragment ion is monitored, and the area under this peak is integrated. Finally, the concentrations of active MMPs in wound samples are quantified by using

Whereas these studies were followed up by investigations of knockout mice as well, the knockout mice do not provide a superior opportunity for elucidation of the functions of MMPs in our opinion. Knockout MMP-9 mice, which survive the embryonic stage, were made diabetic to explore the role of the enzyme. We hasten to add that the compensatory activities of other MMPs in light of the overlapping profiles for the substrates create ambiguity in interpretation of the data. These compensatory activities will be present throughout the embryonic development up to the point in which the experiment is conducted with these mice. The more

peak area ratios relative to the internal standard and calibration curve regression parameters.

were slightly up-regulated after seven days from infliction of the wound [60].

50 Wound Healing - New insights into Ancient Challenges

As indicated above, MMPs are usually not detectable in normal adult tissues, but are upregulated in disease. The tools available for assessment of MMP levels are quantification of mRNA, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, gelatin zymography, *in-situ* gelatin zymography, activity-based enzyme profiling, and TAPI-2 resin [76]. However, these tools generally do not reveal whether the elevated levels of the MMPs that are being monitored are due to the inactive zymogenic forms, the active MMPs, or the MMPs in complex with TIMPs (inactive forms). Quantification of mRNA levels by Northern blot analysis and RT-PCR are limited in that these methods measure mRNA levels and not the amount and activity of the protein. Immunohistochemistry and Western blot require specific antibodies, which usually cannot distinguish between active and TIMP-inhibited MMPs, and might exhibit cross-reactivity. The sensitivity of zymography is not typically high, and this method also detects TIMP-complexed MMPs. *in-situ* zymography is limited by the availability of fluorescent proteinase substrates, which at present can be performed for MMP-1, -2, -3, -7, -8, -9, -12, -13, and -25. This method has limitations for quantitative determinations. Activity-based enzyme profiling of MMPs requires a library of selective MMP-directed probes [76]. A TAPI-2 affinity resin has been reported to identify active MMPs [76]. However, it is very expensive. With the exception of the TAPI-2 resin, the other methods do not identify and quantify the active forms of MMPs. A summary of advantages and limitations of these methods is given in **Table 2**. We add that another complication in these studies is that the active MMPs formed in diseased tissue might be present in minute quantities, such that conventional detection methods at the proteome level might not be able to identify them. We reiterate that of the MMP forms, only the active MMPs in the absence of TIMP complexation would be able to perform its function in manifestation of the disease.

Expression of MMPs in normal uninjured skin is generally low. However, their activities are thought to be up-regulated when cutaneous wounds occur. For instance, low RNA expression levels of MMP-2 and MT1-MMP were reported in uninjured murine skin [50]. Once the cutaneous injury happens, up-regulation and expression of many MMPs have been reported, including collagenases (MMP-1 [83], MMP-8 [60, 61], and MMP-13 [70]), gelatinases (MMP-2 [51] and MMP-9 [51, 60]), stromelysins (MMP-3 and MMP-10 [54]), and other MMPs such as MMP-7 [58], MMP-12 [50], and MMP-14 [51]. However, it should be noted that most of these studies employed methods that do not distinguish between the active or inactive forms of MMPs, except the aforementioned TAPI-2 resin and the recent methodology that couples an MMP-inhibitor-tethered affinity resin with mass spectrometry, as mentioned in the previous section [60]. As such, observation of up-regulation or even the expression of a particular MMP does not necessarily imply a role for that MMP in wound healing. Parallel to MMPs, the expression of TIMPs is often increased in order to regulate the proteinase activities [84]. Hence, the biochemical imbalance that leads to aberrant consequence has to be the focus of research in elucidating the mechanistic basis of disease.

### **2.1. Beneficial roles of MMPs in wound repair**

Collagenases have been implicated in wound healing for many years. As the name implies, these proteinases prefer to turn over various types of collagen (types I, II, and III), which is an important process in wound repair (**Table 1**). MMP-1, expressed by keratinocytes about a week after injury occurs, might facilitate keratinocyte migration when these cells come into contact with type I collagen in the early re-epithelization phase [83]. The interaction between keratinocytes at the wound edge and type I collagen in the matrix via the α2β1 integrin receptor enhances the expression of MMP-1 [38]. MMP-1 cleaves type I collagen to generate cleaved fragments, which have been suggested to become a less adhesive binding ligand than the native protein and that loosens the matrix environment for cellular movements. Thus, the complex of MMP-1 and α2β1 promotes migration of keratinocyte on type I collagen in the re-epithelialization phase, as shown in **Figure 6**. However, α2β1 integrin-deficient mice still retain normal re-epithelialization, collagen deposition, and tensile strength, which indicate a possible compensatory mechanism by another integrin receptor [85]. Once the new basement membrane is established after re-epithelialization, the epidermal expression of MMP-1 is terminated by cellular contacts with proteins from the membrane. Specifically, the contact between keratinocytes with laminin-111 (previously called laminin-1) leads to the repression of MMP-1 in the presence of type I collagen [86]. Expression of MMP-1 has also been observed in fibroblasts during granulation and angiogenesis [83], where the enzyme might act to remodel the ECM of the wound [87]. Interestingly, overexpression of human MMP-1 in the epidermis of transgenic mice resulted in delayed wound closure; however, the genomic modification with human DNA in these animals may have resulted in unwanted phenotypes [88].

Roles of Matrix Metalloproteinases in Cutaneous Wound Healing http://dx.doi.org/10.5772/64611 53

such that conventional detection methods at the proteome level might not be able to identify them. We reiterate that of the MMP forms, only the active MMPs in the absence of TIMP

Expression of MMPs in normal uninjured skin is generally low. However, their activities are thought to be up-regulated when cutaneous wounds occur. For instance, low RNA expression levels of MMP-2 and MT1-MMP were reported in uninjured murine skin [50]. Once the cutaneous injury happens, up-regulation and expression of many MMPs have been reported, including collagenases (MMP-1 [83], MMP-8 [60, 61], and MMP-13 [70]), gelatinases (MMP-2 [51] and MMP-9 [51, 60]), stromelysins (MMP-3 and MMP-10 [54]), and other MMPs such as MMP-7 [58], MMP-12 [50], and MMP-14 [51]. However, it should be noted that most of these studies employed methods that do not distinguish between the active or inactive forms of MMPs, except the aforementioned TAPI-2 resin and the recent methodology that couples an MMP-inhibitor-tethered affinity resin with mass spectrometry, as mentioned in the previous section [60]. As such, observation of up-regulation or even the expression of a particular MMP does not necessarily imply a role for that MMP in wound healing. Parallel to MMPs, the expression of TIMPs is often increased in order to regulate the proteinase activities [84]. Hence, the biochemical imbalance that leads to aberrant consequence has to be the focus of research

Collagenases have been implicated in wound healing for many years. As the name implies, these proteinases prefer to turn over various types of collagen (types I, II, and III), which is an important process in wound repair (**Table 1**). MMP-1, expressed by keratinocytes about a week after injury occurs, might facilitate keratinocyte migration when these cells come into contact with type I collagen in the early re-epithelization phase [83]. The interaction between keratinocytes at the wound edge and type I collagen in the matrix via the α2β1 integrin receptor enhances the expression of MMP-1 [38]. MMP-1 cleaves type I collagen to generate cleaved fragments, which have been suggested to become a less adhesive binding ligand than the native protein and that loosens the matrix environment for cellular movements. Thus, the complex of MMP-1 and α2β1 promotes migration of keratinocyte on type I collagen in the re-epithelialization phase, as shown in **Figure 6**. However, α2β1 integrin-deficient mice still retain normal re-epithelialization, collagen deposition, and tensile strength, which indicate a possible compensatory mechanism by another integrin receptor [85]. Once the new basement membrane is established after re-epithelialization, the epidermal expression of MMP-1 is terminated by cellular contacts with proteins from the membrane. Specifically, the contact between keratinocytes with laminin-111 (previously called laminin-1) leads to the repression of MMP-1 in the presence of type I collagen [86]. Expression of MMP-1 has also been observed in fibroblasts during granulation and angiogenesis [83], where the enzyme might act to remodel the ECM of the wound [87]. Interestingly, overexpression of human MMP-1 in the epidermis of transgenic mice resulted in delayed wound closure; however, the genomic modification with

human DNA in these animals may have resulted in unwanted phenotypes [88].

complexation would be able to perform its function in manifestation of the disease.

in elucidating the mechanistic basis of disease.

52 Wound Healing - New insights into Ancient Challenges

**2.1. Beneficial roles of MMPs in wound repair**

**Figure 6.** Involvement of MMPs in the wound-healing process. The healed or healthy skin, consisting of ECM and blood vessels, is populated by fibroblasts. Once the skin is damaged by a full-thickness injury, it becomes a cutaneous wound. In the early phase of inflammation, the wound is occupied with fibrin clot to seal the wound, and levels of MMP-2 and MMP-9 are increased. Fibroblasts and macrophages migrate into the wound site, where they are stimulated to release more MMPs to remodel the ECM. The inflammation phase is followed by angiogenesis, in which the upregulation of a variety of MMPs (including MMP-1, MMP-8, MMP-9, and MMP-13) would stimulate epithelial cells (keratinocytes) to proliferate and migrate to re-epithelialize over the wound area. However, prolonged inflammation could cause the wounds to become chronic, as it has been observed in diabetic foot ulcers. During chronic wounds, the irregular up-regulation of MMP-9 has been associated with reduction of MMP-8 and plays a detrimental role in ECM remodeling. Tissue remodeling and expression of MMPs are attenuated when the epithelial cells proliferate and differentiate in order to reform the new epithelium. During this last phase of wound healing, fibroblasts can continue to remodel the underlying dermis over a period of several months.

Expression of human MMP-1 has shown similar patterns to expression of murine MMP-13 in an excisional wound healing model (**Figure 6**) [50, 89]. Both MMP-1 and MMP-13 may share roles in promoting the survival of fibroblasts while remodeling collagen deposition in the wound ECM [69]. Hattori *et al*. have shown that MMP-13 knockout mice had both delayed reepithelialization and wound closure (**Table 3**) [70]. However, in another study, MMP-13 knockout mice showed normal efficiency of re-epithelialization, wound closure, inflammatory response, and unaltered remodeling of the wound matrix [90]. The inconclusive evidence on the role of MMP-13 in wound repair could in principle be explained by the redundancy in functions of proteinases that we mentioned earlier. There was up-regulated expression of MMP-8 (or collagenase-2) in these MMP-13-deficient animals [90], for example. The relevance of MMP-8 in wound repair was unclear until this study indicated that there is enzymatic compensation by MMP-8 to facilitate normal wound healing [90]. There is mounting evidence that MMP-8 plays a beneficial role in repairing the wounds. As MMP-8 is expressed and secreted mostly by neutrophils, its expression peaks after four days in non-diabetic wounds, detected by ELISA [61]. In humans, MMP-8 is the most prevalent collagenase in cutaneous wounds, where it is required for debridement of the wound and for the removal of damaged type I collagen [61]. Yet, it is still challenging to conclude whether this is the active form of MMP-8 by the methods that were used. In mice, the exact levels of active MMP-8 were quantitatively determined by the use of an MMP-inhibitor-tethered affinity resin coupled with mass spectrometry [60]. In this study, Gooyit *et al*. also demonstrated that selective inhibition of MMP-8 caused delayed wound healing and incomplete re-epithelialization in diabetic mice [60]. In addition, MMP-8-deficient mice displayed a significant delay in wound healing, caused by a lag in neutrophil infiltration, persistent inflammation and impaired re-epithelialization (**Table 3**) [62]. However, MMP-8 knockout mice show compensation in MMP-9 [62], making it difficult to separate the roles of MMP-8 and MMP-9 in wound healing. Furthermore, topical application of active recombinant MMP-8 on murine diabetic wounds resulted in a significant acceleration in both wound healing and re-epithelialization [63]. These studies reveal the beneficial role of MMP-8 in wound healing, where the neutrophil-derived MMP-8 can facilitate repair processes by providing debridement for damaged proteins and paving a pathway for the formation of the provisional matrix for keratinocyte migration (**Figure 6**).


**Table 3.** Metalloproteinase gene targeting in mice studies and wound phenotypes.

The underlying cause of diabetic complications leads to up-regulation of MMP-9 compared to MMP-8. For instance, biopsy samples from diabetic patients revealed only two-fold increase in MMP-8, but 14-fold increase in MMP-9 expression when compared to non-diabetic tissues [49]. Although the application of recombinant MMP-8 accelerated healing of full-thickness wounds in diabetic mice, it might have a similar beneficial mechanism as the marketed drug Santyl®, which is indicated for debridement of chronic dermal ulcers and severely burned areas and contains collagenase derived from *Clostridium histolyticum*. Clinical evidence suggests that collagenase treatment expedites the removal of necrotic tissues and enhances keratinocyte migration [91]. When used after debridement, Santyl® promotes wound healing in patients with pressure ulcers, venous leg ulcers, diabetic ulcers, and severely burnt wounds [91]. However, excessive use of active recombinant MMP-8 may affect the formation of new ECM and may not be beneficial in wound healing. A dose-response study with active recombinant MMP-8 topically administered to wounds of diabetic mice showed that higher levels of this proteinase did not accelerate wound healing [63].

of MMP-8 in wound repair was unclear until this study indicated that there is enzymatic compensation by MMP-8 to facilitate normal wound healing [90]. There is mounting evidence that MMP-8 plays a beneficial role in repairing the wounds. As MMP-8 is expressed and secreted mostly by neutrophils, its expression peaks after four days in non-diabetic wounds, detected by ELISA [61]. In humans, MMP-8 is the most prevalent collagenase in cutaneous wounds, where it is required for debridement of the wound and for the removal of damaged type I collagen [61]. Yet, it is still challenging to conclude whether this is the active form of MMP-8 by the methods that were used. In mice, the exact levels of active MMP-8 were quantitatively determined by the use of an MMP-inhibitor-tethered affinity resin coupled with mass spectrometry [60]. In this study, Gooyit *et al*. also demonstrated that selective inhibition of MMP-8 caused delayed wound healing and incomplete re-epithelialization in diabetic mice [60]. In addition, MMP-8-deficient mice displayed a significant delay in wound healing, caused by a lag in neutrophil infiltration, persistent inflammation and impaired re-epithelialization (**Table 3**) [62]. However, MMP-8 knockout mice show compensation in MMP-9 [62], making it difficult to separate the roles of MMP-8 and MMP-9 in wound healing. Furthermore, topical application of active recombinant MMP-8 on murine diabetic wounds resulted in a significant acceleration in both wound healing and re-epithelialization [63]. These studies reveal the beneficial role of MMP-8 in wound healing, where the neutrophil-derived MMP-8 can facilitate repair processes by providing debridement for damaged proteins and paving a pathway for

the formation of the provisional matrix for keratinocyte migration (**Figure 6**).

*mmp-8* Knockout Delayed re-epithelialization, delayed arrival of and prolonged

inflammation

**Table 3.** Metalloproteinase gene targeting in mice studies and wound phenotypes.

*hmmp-1* Overexpression in keratinocytes

54 Wound Healing - New insights into Ancient Challenges

**Gene Modification Wound phenotype Reference**

*mmp-3* Knockout Impaired wound contraction [55]

*mmp-9* Knockout Enhanced re-epithelization, accelerated wound closure [63, 64]

*mmp-10* Overexpression in KCs Unaltered wound closure, scattered epithelialization [67] *mmp-13* Knockout Delayed wound closure, and reduced re-epithelialization [70]

mortality were observed in these mice

The underlying cause of diabetic complications leads to up-regulation of MMP-9 compared to MMP-8. For instance, biopsy samples from diabetic patients revealed only two-fold increase in MMP-8, but 14-fold increase in MMP-9 expression when compared to non-diabetic tissues

*mmp-14* Knockout Unaltered wound closure over 7 days' but premature morbidity and

Delayed re-epithelialization [88]

[62]

[92, 93]

Gelatinases, MMP-2 and MMP-9, are also involved in wound repair. Early expression of both gelatinases is observed in platelets, where MMP-9 is involved in platelet production and MMP-2 mediates platelet adhesion and aggregation [52]. The early expression of gelatinases might contribute to degradation of gelatin matrix in biofilm produced by bacteria [94]. This degradation serves to weaken the attachment of bacterial biofilm to the wound site and might be a strategy in fighting infection [94]. In addition, gelatinases are able to digest various constituents of the wound matrix to initiate angiogenesis in the repair processes. After tissue injury, AngII, which plays roles in inflammation, cell proliferation, and migration, would stimulate macrophages and neutrophils to generate ROS and MMPs to promote cell adhesion and ECM formation [17]. Specifically, AngII has been shown to induce the expression of both gelatinases, MMP-2 [95] and MMP-9 [96, 97]. AngII has been demonstrated to promote angiogenesis via activation of VEGF and endothelial nitric oxide [98], whereas studies with AngII-type 1a receptor knockout mice or with inhibition of AngII receptor have resulted in delayed wound healing with reduced angiogenesis in animals [99]. Given the beneficial role of AngII in angiogenesis of wound healing, it is interesting to note that there is discrepancy in the outcomes of diseases treated with this factor's inhibitors. These inhibitors can either block AngII receptors or inhibit the enzyme that generates factor AngII in the RAS pathway. In cancer, AngII inhibitors have been shown to reduce the tumor-related VEGF expression, angiogenesis, and tumor size [16]. These drugs are also used to treat hypertension, in which dysregulation of RAS causes poor blood flow, inadequate supply of oxygen, and impaired wound healing [12]. When used in anti-hypertensive therapy, drugs such as losartan has been demonstrated to promote wound healing in diabetes-induced mice by improving vascular perfusion, without affecting VEGF expression [100]. To this extent, anti-hypertensive therapy appears to be beneficial for wound healing. Thus, there is a need for further research to elucidate the precise role of these inhibitors in diabetic patients with impaired wound healing.

Expression of MMP-2 is demonstrated to coincide with expression of laminin-332 (also referred to as laminin-5) during enhanced keratinocyte migration in wound healing [101]. Since both MMP-2 and MMP-9 can cleave the gamma-2 chain of laminin-322 [102], they result in a promigratory and EGF-like fragment that binds EGF receptor to trigger cell migration of keratinocytes at the wound matrix [34], demonstrated in **Figure 2**. This cleaved fragment has been found in both tumors and tissues that undergo remodeling, except for intact epidermis [34, 103, 104]. Interestingly, MMP-8 also cleaves laminin-332, which indicates the mechanistic redundancy of the roles of MMPs during wound healing [102]. Furthermore, the two gelatinases might contribute to angiogenesis possibly by activating cytokines such as TNF-α (tumor necrosis factor-alpha) [105] and VEGF [106, 107]. However, cleavage of laminin-332 by MMP-2 has only been shown in tumor cells and normal breast epithelial cells, but not in normal keratinocytes in wound repair. Some studies have implicated MMP-2 in cleaving the latencyassociated peptide (LAP) of pro-TGF-β and latent TGF-β binding protein (LTBP) to release activated TGF-β to bind the ECM [108–110]. Another study has also indicated that the active form of MMP-2 can activate MMP-9 in cell culture [53]. Pro-MMP-2 itself needs to be activated, and it has been shown that this activation requires the cluster of MMP-14, pro-MMP-2, and αVβ3 integrin in a model of breast cancer cells [111]. It is important to note that active MMP-2 was not observed in wounds of diabetic and non-diabetic mice in the studies that used the MMP-inhibitor-tethered affinity resin, described earlier [60]. Therefore, the role of MMP-2 during wound healing has remained obscured with no *in vivo* verification to date. In fact, the study that implicated both active MMP-2 and MMP-9 in human wound healing used gelatin zymography as the tool [112]. However, this method lacks the ability to detect exclusively the active MMPs in the wound tissues, because the denaturation of the TIMP-MMP complex during electrophoresis could also result in an active MMP-2 band (**Table 2**). On the other hand, inhibition of MMP-9 activity with an antibody or MMP-9 ablation has delayed keratinocyte migration *in vitro*, which indicates the necessary involvement of MMP-9 during normal wound closure [65]. Others have also demonstrated *in vitro* that MMP-9 appears to promote keratinocyte migration [113]. Indeed, the study with the MMP-inhibitor-tethered affinity resin revealed that active MMP-9 was essentially undetectable in the intact skin, but it was expressed as early as one day after injury and remained up-regulated throughout the two weeks of study in non-diabetic and diabetic mice [60]. In the case of diabetic mice with delayed wound closure, the analysis showed up-regulation of active MMP-9, which could be detrimental to the repair process [60]. Besides promoting angiogenesis, gelatinases and other MMPs interestingly can inhibit angiogenesis by generating anti-angiogenic peptides from other precursor proteins. For instance, distinct proteinases such as MMP-3, -7, -9, -13, and -20 have been shown to generate active endostatin from human collagen XVIII [114] *in vitro*, whereas MMP-2, -3, -7, -9, and -12 are responsible for generating angiostatin from plasminogen [68, 115].

Expression of MMP-3 and MMP-10 (two stromelysins) has been found in epidermal cells during human and murine wound healing using RNA probes (**Table 1**). MMP-3 is expressed by the basal-proliferating keratinocytes, which are in contact with the intact basement membrane and close to the wound edge [54]. Expression of MMP-3 is also detected in fibroblasts during wound healing [11]. Research has shown that wound closure was delayed in non-diabetic MMP-3 knockout mice due to impaired wound contraction (**Table 3**) [55]. The implicated involvement of MMP-3 in normal wound healing may have resulted from demonstration that MMP-3 could activate MMP-9 [56], the gelatinase that plays roles in keratinocyte migration. However, MMP-2 could also trigger activation of MMP-9 [53], which corroborates the possibility of mechanistic compensation by other MMPs in physiology. Thus, the involvement of MMP-3 in the repair processes of wound healing still remains ambiguous. Nonetheless, it has been demonstrated that MMP-3 can activate several pro-MMPs, digest many ECM components, and increase the availability, as well as the activities, of cytokines and growth factors [116]. These findings disclose roles for MMP-3 in cell migration and proliferation during wound repair.

103, 104]. Interestingly, MMP-8 also cleaves laminin-332, which indicates the mechanistic redundancy of the roles of MMPs during wound healing [102]. Furthermore, the two gelatinases might contribute to angiogenesis possibly by activating cytokines such as TNF-α (tumor necrosis factor-alpha) [105] and VEGF [106, 107]. However, cleavage of laminin-332 by MMP-2 has only been shown in tumor cells and normal breast epithelial cells, but not in normal keratinocytes in wound repair. Some studies have implicated MMP-2 in cleaving the latencyassociated peptide (LAP) of pro-TGF-β and latent TGF-β binding protein (LTBP) to release activated TGF-β to bind the ECM [108–110]. Another study has also indicated that the active form of MMP-2 can activate MMP-9 in cell culture [53]. Pro-MMP-2 itself needs to be activated, and it has been shown that this activation requires the cluster of MMP-14, pro-MMP-2, and αVβ3 integrin in a model of breast cancer cells [111]. It is important to note that active MMP-2 was not observed in wounds of diabetic and non-diabetic mice in the studies that used the MMP-inhibitor-tethered affinity resin, described earlier [60]. Therefore, the role of MMP-2 during wound healing has remained obscured with no *in vivo* verification to date. In fact, the study that implicated both active MMP-2 and MMP-9 in human wound healing used gelatin zymography as the tool [112]. However, this method lacks the ability to detect exclusively the active MMPs in the wound tissues, because the denaturation of the TIMP-MMP complex during electrophoresis could also result in an active MMP-2 band (**Table 2**). On the other hand, inhibition of MMP-9 activity with an antibody or MMP-9 ablation has delayed keratinocyte migration *in vitro*, which indicates the necessary involvement of MMP-9 during normal wound closure [65]. Others have also demonstrated *in vitro* that MMP-9 appears to promote keratinocyte migration [113]. Indeed, the study with the MMP-inhibitor-tethered affinity resin revealed that active MMP-9 was essentially undetectable in the intact skin, but it was expressed as early as one day after injury and remained up-regulated throughout the two weeks of study in non-diabetic and diabetic mice [60]. In the case of diabetic mice with delayed wound closure, the analysis showed up-regulation of active MMP-9, which could be detrimental to the repair process [60]. Besides promoting angiogenesis, gelatinases and other MMPs interestingly can inhibit angiogenesis by generating anti-angiogenic peptides from other precursor proteins. For instance, distinct proteinases such as MMP-3, -7, -9, -13, and -20 have been shown to generate active endostatin from human collagen XVIII [114] *in vitro*, whereas MMP-2, -3, -7, -9, and -12

56 Wound Healing - New insights into Ancient Challenges

are responsible for generating angiostatin from plasminogen [68, 115].

Expression of MMP-3 and MMP-10 (two stromelysins) has been found in epidermal cells during human and murine wound healing using RNA probes (**Table 1**). MMP-3 is expressed by the basal-proliferating keratinocytes, which are in contact with the intact basement membrane and close to the wound edge [54]. Expression of MMP-3 is also detected in fibroblasts during wound healing [11]. Research has shown that wound closure was delayed in non-diabetic MMP-3 knockout mice due to impaired wound contraction (**Table 3**) [55]. The implicated involvement of MMP-3 in normal wound healing may have resulted from demonstration that MMP-3 could activate MMP-9 [56], the gelatinase that plays roles in keratinocyte migration. However, MMP-2 could also trigger activation of MMP-9 [53], which corroborates the possibility of mechanistic compensation by other MMPs in physiology. Thus, the involvement of MMP-3 in the repair processes of wound healing still remains ambiguous. Nonetheless, it has been demonstrated that MMP-3 can activate several pro-MMPs, digest many ECM Meanwhile, MMP-10 (stromelysin-2), is expressed with a different pattern even though both MMP-3 and MMP-10 can degrade several collagens and non-collagenous connective tissue substrates, including proteoglycans, gelatin, fibronectin, and laminin [117], as indicated in **Table 1**. Human MMP-10 is expressed by epidermal cells about three days post-wounding, where its regulation seems to depend on EGF, TGF-β, and TNF-α cytokines [66]. The role of MMP-10 in wound repair was investigated by overexpressing a constitutively active MMP-10 mutant in keratinocytes, which resulted in normal wound-healing architecture and normal wound-healing rate in these transgenic mice [67]. However, the epidermal histology was demonstrated to have a disorganized migrating epithelium, composed of degradation in the newly formed matrix via laminin-332, abnormal cell-to-cell contacts of keratinocytes, and finally an increased rate of apoptosis of keratinocytes [67]. These findings indicate that levels of MMP-10 require a tightly regulated expression to facilitate keratinocyte migration during wound healing. Although both stromelysins would appear to be players, more investigations are needed to ascertain the roles of active MMP-3 and MMP-10 in the physiology of wound repair.

In addition to gelatinases and collagenases, other MMPs might have roles in wound healing as well, even though the data are not conclusive. For instance, MMP-7 (matrilysin) has been shown to be expressed in injured epithelia of various mucosal tissues, including lung, kidney, cornea, and gut [58, 118–120], even though MMP-7 is not expressed in epidermal wounds. In the lungs, MMP-7 has been demonstrated to play a role in inducing epithelial migration by facilitating the shedding of syndecan-1, a transmembrane heparin sulfate proteoglycan [58]. In the same study, MMP-7 knockout mice displayed impaired re-epithelialization in the mucosal tissue [58]. Also in the lungs, MMP-7 has been shown to cleave E-cadherin in the process of facilitating cell migration away from the edge of the injured wound [118]. On the other hand, another study has shown that MMP-7 and MMP-13 are expressed at the invasive edge of tumors [121]. Another proteinase that might be important for the wound-repair process is MMP-12 (metalloelastase), which is expressed by macrophages surrounding blood vessels in acute murine excisional wounds [50]. Even though MMP-12 was not detected in either acuteor chronic-wound tissues in the presence of macrophages, this proteinase expression was found to be abundant in different human cutaneous granulomas [122]. In addition to its ability to degrade fibrinogen interfering with blood clotting [123], MMP-12 is a potential regulator of angiogenesis, since it was demonstrated to be most efficient at producing angiostatin [68]. Membrane-type MMPs might also be necessary for wound healing, more specifically MMP-14, which is the most extensively studied to date (**Table 1**). The pivotal role of MMP-14 in angiogenesis of wound healing may be attributed to the enzyme's fibrinolytic and collagenolytic activity that is necessary for cell migration [71]. In addition, MMP-14 is needed for TIMP-2 mediated activation of pro-MMP-2, a process that is coordinated by two MMP-14 molecules and TIMP-2 [73]. Although MMP-14-deficient mice display abnormalities in bone development, impaired angiogenesis, and defective type I collagen [93, 124], wound closure in these 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, supporting the concept of proteinase redundancy among MMPs.

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

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 method is the use of MMP knockout animals, which may provide further insights into the roles of MMPs in wound healing (**Table 3**). However, the drawback of knockout animals is the possibility of mechanistic compensation by other MMPs in the absence of the ablated MMP, as discussed earlier. For instance, it has been shown that levels of MMP-9 are increased when MMP-2 or MMP-8 are ablated [62, 134]. Also, many MMPs share the same substrates, indicating the redundancy in the proteinase functions of MMPs [3].

We described earlier the MMP-inhibitor-tethered affinity resin that Gooyit *et al*. used to identify and accurately measure the levels of active MMP-9, which was found to be up-regulated in diabetic mice with delayed wound healing [60]. The dual roles of MMPs are exhibited in this case of MMP-9 up-regulation, which was demonstrated to be detrimental to diabetic wound repair by topical treatment with two distinct selective MMP-9 inhibitors (ND-322 and ND-336) [60, 63]. Inhibition of MMP-9 accelerated wound healing and promoted re-epithelialization. It has been shown that MMP-9 inhibits cell replication during epithelial migration; thus, MMP-9-deficient mice, both diabetic and non-diabetic, display a better rate of wound closure [63, 64]. Similar to MMP-2, MMP-9 can also activate pro-TGF-β and release it from LTBP [108, 109], while TGF-β has been shown to induce pro-MMP-9 in human skin [135]. Since TGF-β1 is a cytokine that elicits recruitment of inflammatory cells during wound healing [136], its up-regulation can regulate wound repair [137]. Interestingly, prolonged elevation in levels of inflammatory cytokines, such as TGF-β, can lead to a prolonged inflammation phase and consequentially delayed wound closure in diabetic mice [138]. However, it has also been shown that non-diabetic MMP-9 knockout mice had delayed wound closure [70]. The apparent conflicting role of MMP-9 may be explained by compensation of other MMPs, such as increased expression of MMP-3 and MMP-13 in MMP-9 knockout animals [139]. In addition, the redundancy of MMP substrates allow other MMP(s) to fulfill the same role, for instance MMP-1, MMP-2, MMP-9, and MMP-13 have a role in keratinocyte migration and can replace MMP-9 during normal wound healing [38, 70, 113, 140]. To date, topical application with a selective MMP-9 inhibitor, by itself or in combination with recombinant MMP-8, has shown therapeutic potential in accelerating murine diabetic wound healing [63]. These treatments improve diabetic wound healing by increasing angiogenesis and restoring levels of inflammatory cytokines, including TGF-β1 [63].
