**1. Introduction**

Skin is one of the largest organs in humans. Its three main functions are protection against environmental damage, regulation of body temperature, and perception of environmental

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

change. The skin consists of two distinct layers of tissue, the epidermis and dermis. The epidermis is the outermost layer. The inner layer, dermis, provides cushioning and tensile strength for the skin through the support of the extracellular matrix (ECM) [1]. The ECM—a three-dimensional structure, where cutaneous cells and tissues are embedded—comprises approximately 300 proteins, including collagen, proteoglycans, and glycoproteins [2]. Injury to the skin would account for breaks in these protective layers, which become a cutaneous wound. The wound has to be repaired because of its critical role in prevention of infection, and the well-being of the tissue and the organism. Wounds that undergo a well-coordinated cascade of biochemical events in healing are called acute wounds. On the other hand, wounds that are recalcitrant to healing due to prolonged residency in one of the healing stages are called chronic wounds. For wound healing to progress, the ECM has to be remodeled properly, and endopeptidases such as matrix metalloproteinases (MMPs) contribute to this remodeling process. Whereas these subjects have been reviewed in excellent recent articles [3–6], the emphasis in this chapter focuses on the roles of the family of MMPs that are involved in the wound-healing process.

**Figure 1.** The phases of wound healing. After tissue injury, hemostasis and inflammation start immediately, in which cytokines, growth factors, and ROS are produced to recruit cells to the wound site. The next proliferative phases of wound healing include angiogenesis and re-epithelialization, where new tissue is formed by endothelial cells, fibroblasts, and keratinocytes. In diabetic wound healing, inflammation can be prolonged, causing the wounds to be chronic. The final phase is tissue remodeling. EGF (epidermal growth factor), IL (interleukin), KGF (keratinocyte growth factor), MMPs (matrix metalloproteinases), PDGF (platelet-derived growth factor), ROS (reactive oxygen species), TGF-β (transforming growth factor-beta), TNF (tumour necrosis factor), VEGF (vascular endothelial growth factor). Adapted from Schreml [22].

There are currently at least 24 known MMPs in humans [7]. Not all functions that these enzymes play in humans have been elucidated and concepts in their mechanistic roles in wound healing are emerging only recently. Yet, it is generally appreciated that MMPs play roles in each stage of wound healing, in large measure because of the need for restructuring of the ECM in the process of wound healing. The phases of wound healing consist of (1) hemostasis and inflammation, (2) granulation and angiogenesis, (3) re-epithelialization, and (4) tissue remodeling and are depicted in **Figure 1**. All four phases of wound healing have to be coordinated and integrated properly in a timely and sequential manner for successful healing. The repair processes require the coordination of events involving various cells, the ECM components, growth factors, cytokines, and enzymes. Furthermore, it is increasingly evident that MMPs display a duality of functions in the physiology of the tissue and processes of pathology, as evidenced for chronic wounds, cancers, Parkinson's and Alzheimer's diseases [8–10]. As such, certain MMPs might have a beneficial effect in healing, yet others might exhibit detrimental effect as aberrations in the functions of these enzymes in disease development and progression. The differentiation of these functions—detrimental versus beneficial—has been a challenge. Yet, new tools and capabilities are becoming available to address exactly these issues in various diseases.

### **1.1. Stages of wound healing**

change. The skin consists of two distinct layers of tissue, the epidermis and dermis. The epidermis is the outermost layer. The inner layer, dermis, provides cushioning and tensile strength for the skin through the support of the extracellular matrix (ECM) [1]. The ECM—a three-dimensional structure, where cutaneous cells and tissues are embedded—comprises approximately 300 proteins, including collagen, proteoglycans, and glycoproteins [2]. Injury to the skin would account for breaks in these protective layers, which become a cutaneous wound. The wound has to be repaired because of its critical role in prevention of infection, and the well-being of the tissue and the organism. Wounds that undergo a well-coordinated cascade of biochemical events in healing are called acute wounds. On the other hand, wounds that are recalcitrant to healing due to prolonged residency in one of the healing stages are called chronic wounds. For wound healing to progress, the ECM has to be remodeled properly, and endopeptidases such as matrix metalloproteinases (MMPs) contribute to this remodeling process. Whereas these subjects have been reviewed in excellent recent articles [3–6], the emphasis in this chapter focuses on the roles of the family of MMPs that are involved in the

**Figure 1.** The phases of wound healing. After tissue injury, hemostasis and inflammation start immediately, in which cytokines, growth factors, and ROS are produced to recruit cells to the wound site. The next proliferative phases of wound healing include angiogenesis and re-epithelialization, where new tissue is formed by endothelial cells, fibroblasts, and keratinocytes. In diabetic wound healing, inflammation can be prolonged, causing the wounds to be chronic. The final phase is tissue remodeling. EGF (epidermal growth factor), IL (interleukin), KGF (keratinocyte growth factor), MMPs (matrix metalloproteinases), PDGF (platelet-derived growth factor), ROS (reactive oxygen species), TGF-β (transforming growth factor-beta), TNF (tumour necrosis factor), VEGF (vascular endothelial growth factor).

There are currently at least 24 known MMPs in humans [7]. Not all functions that these enzymes play in humans have been elucidated and concepts in their mechanistic roles in wound healing are emerging only recently. Yet, it is generally appreciated that MMPs play roles in each stage of wound healing, in large measure because of the need for restructuring of the ECM in the process of wound healing. The phases of wound healing consist of (1)

wound-healing process.

38 Wound Healing - New insights into Ancient Challenges

Adapted from Schreml [22].

Once injury to the skin takes place, the cutaneous wound immediately enters the first phase of hemostasis (**Figure 1**). The onset of blood vessel constriction prevents excessive bleeding, which is followed by the aggregation of platelets along the damaged endothelium to form a plug. A cascade of events ensues, which leads to the formation of a blood clot. The serineproteinase thrombin cleaves fibrinogen into insoluble fibrin threads that are aggregated with platelets to create the clot. In addition to stopping the bleeding, the blood clot serves as a provisional matrix for cell migration [11]. The surrounding cells of a blood clot also release inflammatory cytokines and growth factors as signaling molecules to attract a variety of cells

**Figure 2.** ECM–growth factor interactions and production of MMPs in wound healing. Monocytes migrate to the wound site and bind to fibronectin released by neutrophils. This interaction causes monocytes to differentiate into macrophages that secrete multiple growth factors. TGF-β1 binds to its receptor on fibroblasts and stimulates the cells to produce ECM components such as collagen, fibronectin, and hyaluronic acid. Neutrophils also produce MMP-8 and -9 in the wound. Binding of AngII to macrophages stimulates the cells to produce ROS and MMPs. MMPs can cleave laminin to release a fragment that binds EGF receptor on fibroblasts and stimulates migration and proliferation of keratinocytes. AngII (angiotensin II), ECM (extracellular matrix), DDR (discoidin domain receptor), EGF (epidermal growth factor), FGF (fibroblast growth factor), MMPs (matrix metalloproteinases), PDGF (platelet-derived growth factor), ROS (reactive oxygen species), TGF-β1 (transforming growth factor-beta 1), VEGF (vascular endothelial growth factor).

to the wound site to initiate the inflammatory phase. These cells include neutrophils, macrophages, and lymphocytes, which defend the site from infectious agents [12]. The earliest arrival of neutrophils takes place only a few hours after injury [13]. Neutrophils are responsible for releasing fibronectin, which has multifunctional roles, including a structural function due to its fibrillary composition, mediating interactions between ECM components and other cells, or serving as a bridge between cells [14, 15]. Fibronectin and fibrin act to provide provisional matrix that promotes cellular migration and adhesion, depending on the wound status. Also, during inflammation, fibronectin and other ECM protein fragments can attract monocytes, a type of white blood cells, to the wound site from the bloodstream. The interactions at the wound site cause monocytes to undergo differentiation into additional macrophages (**Figure 2**) [14]. Macrophages are stimulated by growth factors to produce reactive-oxygen species (ROS), MMPs, and multiple growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) (**Figure 2**) [16]. One factor that can stimulate macrophages is angiotensin II (AngII). The renin–angiotensin system (RAS) is a pathway to regulate angiotensin, a hormone peptide, to eventually produce the primary effector-AngII [17]. This effector is present in macrophages, neutrophils, fibroblasts, and endothelial cells of human skin [18]. With binding and stimulation of AngII, inflammatory cells such as macrophages generate ROS and MMPs that subsequently promote migration and proliferation of keratinocytes. This will be discussed in a later section of this chapter.

Among many growth factors and cytokines, TGF-βs play critical roles in regulating the development of the ECM. There are three isoforms (TGF-β1–3) in humans, with each playing distinct roles in regulating synthesis of the ECM components, and even cellular proliferation or cellular death [19, 20]. TGF-βs are produced in latent forms that need to be activated by cleavage of their pro-peptides, before exerting their activities on the ECM, which include stimulation of cellular production of ECM components [20]. The most well known is TGF-β1, which can control production and degradation of many constituents involved in wound healing [14]. Once TGF-β1 binds to its receptor, this interaction stimulates the synthesis of ECM components such as collagen, fibronectin, and hyaluronic acid in many types of cells, including fibroblasts [21]. Fibroblasts are cells that synthesize collagen and other constituents deposited on the ECM [14]. Besides monocytes/macrophages, fibroblasts also generate ROS, including peroxide anion, hydroxyl ion, and superoxide anion, which are important in defense against pathogenic microorganisms [22]. ROS, in turn, has the effect of stimulating the production of more cytokines that lead to increased production of proteinases such as MMPs to modify components of the ECM [22]. Dualities of functions reveal themselves in ROS as well. The function against the pathogens is beneficial, but high-level ROS can cause damage to the ECM components [22]. This fine balance for ROS could stimulate complex signal pathways that would lead to up-regulation of MMPs in the wounds. The enhanced presence of ROS and the attendant stimulated turnover of ECM components could cause tissue destruction and hinder the repair processes [23]. This duality of roles for ROS was observed in a murine wound model that documented severe damage to the endothelium in a background that lacked ROSdetoxifying enzymes [24]. In diabetic patients with chronic wounds, the production of ROS has been found to exceed the antioxidant capacity, adding more oxidative stress to the wounds that subsequently increases MMP levels by 60-fold over those in acute wounds [12].

to the wound site to initiate the inflammatory phase. These cells include neutrophils, macrophages, and lymphocytes, which defend the site from infectious agents [12]. The earliest arrival of neutrophils takes place only a few hours after injury [13]. Neutrophils are responsible for releasing fibronectin, which has multifunctional roles, including a structural function due to its fibrillary composition, mediating interactions between ECM components and other cells, or serving as a bridge between cells [14, 15]. Fibronectin and fibrin act to provide provisional matrix that promotes cellular migration and adhesion, depending on the wound status. Also, during inflammation, fibronectin and other ECM protein fragments can attract monocytes, a type of white blood cells, to the wound site from the bloodstream. The interactions at the wound site cause monocytes to undergo differentiation into additional macrophages (**Figure 2**) [14]. Macrophages are stimulated by growth factors to produce reactive-oxygen species (ROS), MMPs, and multiple growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) (**Figure 2**) [16]. One factor that can stimulate macrophages is angiotensin II (AngII). The renin–angiotensin system (RAS) is a pathway to regulate angiotensin, a hormone peptide, to eventually produce the primary effector-AngII [17]. This effector is present in macrophages, neutrophils, fibroblasts, and endothelial cells of human skin [18]. With binding and stimulation of AngII, inflammatory cells such as macrophages generate ROS and MMPs that subsequently promote migration and proliferation of keratino-

Among many growth factors and cytokines, TGF-βs play critical roles in regulating the development of the ECM. There are three isoforms (TGF-β1–3) in humans, with each playing distinct roles in regulating synthesis of the ECM components, and even cellular proliferation or cellular death [19, 20]. TGF-βs are produced in latent forms that need to be activated by cleavage of their pro-peptides, before exerting their activities on the ECM, which include stimulation of cellular production of ECM components [20]. The most well known is TGF-β1, which can control production and degradation of many constituents involved in wound healing [14]. Once TGF-β1 binds to its receptor, this interaction stimulates the synthesis of ECM components such as collagen, fibronectin, and hyaluronic acid in many types of cells, including fibroblasts [21]. Fibroblasts are cells that synthesize collagen and other constituents deposited on the ECM [14]. Besides monocytes/macrophages, fibroblasts also generate ROS, including peroxide anion, hydroxyl ion, and superoxide anion, which are important in defense against pathogenic microorganisms [22]. ROS, in turn, has the effect of stimulating the production of more cytokines that lead to increased production of proteinases such as MMPs to modify components of the ECM [22]. Dualities of functions reveal themselves in ROS as well. The function against the pathogens is beneficial, but high-level ROS can cause damage to the ECM components [22]. This fine balance for ROS could stimulate complex signal pathways that would lead to up-regulation of MMPs in the wounds. The enhanced presence of ROS and the attendant stimulated turnover of ECM components could cause tissue destruction and hinder the repair processes [23]. This duality of roles for ROS was observed in a murine wound model that documented severe damage to the endothelium in a background that lacked ROSdetoxifying enzymes [24]. In diabetic patients with chronic wounds, the production of ROS

cytes. This will be discussed in a later section of this chapter.

40 Wound Healing - New insights into Ancient Challenges

Granulation and angiogenesis take place in the next phase of wound repair, which is also known as the proliferative phase (**Figure 1**). Granulation tissue is defined as a matrix of collagen, with microscopic blood vessels that are newly formed from preexisting blood vessels in a process called angiogenesis. New blood capillaries supply oxygen to the wound tissue, which is critical for the healing process. Granulation, in the form of a red or pink soft tissue, forms on the surface of the wound. Macrophages are tasked with initiating this phase by degrading the blood clots and by producing a variety of cytokines and chemokines to attract fibroblasts to enter the wound site [22]. The population of fibroblasts at the wound site will expand by both migration and proliferation through dynamic interaction with growth factors and the ECM. This is mediated by integrins, a set of receptors for fibroblast, which consist of an extracellular domain that binds to the ECM, and an intracellular portion that associates with the cytoskeleton for biochemical signaling [25]. Integrins and discoidin domain receptor 2 (DDR2), another receptor for fibroblasts, bind to type I collagen within the ECM. This interaction stimulates the production of MMP-2 to promote migration of fibroblasts to the wound site through the basement membrane during ECM remodeling [26], as indicated in **Figure 2**. In addition, as mentioned earlier, TGF-β1 can stimulate proliferation of fibroblasts. The wound tissue is hypoxic and would require a supply of oxygen for the demands of the biochemical processes of wound healing [22]. Hypoxia stimulates macrophages, keratinocytes, fibroblasts, and endothelial cells to produce more VEGF, which is a cytokine associated with angiogenesis [22]. The enhanced expression of VEGF causes endothelial cells at the wound bed to migrate, proliferate, and form new blood vessels into the wounds to supply oxygen during angiogenesis [27]. In addition, VEGF has been shown to increase expression of the collagen-binding integrin in the dermal microvasculature [28]. These bindings with integrin help cells adhere to the ECM and promote additional growth factor expression. Rossiter et al. have reported that deletion of keratinocyte-specific VEGF impaired angiogenesis and delayed wound healing in a murine model [29]. Other researchers have shown that overexpression of VEGF can lead to enhanced wound healing in murine excisional wounds [30]. Another receptor that plays an important role in cellular migration during angiogenesis is epidermal growth factor (EGF) receptors, which can bind to EGF and laminin to enhance fibroblast migration [31, 32]. Laminin, which is a fibrous constituent of the basement membrane, plays important roles in cell adhesion, migration, and proliferation [33]. At the wound site, cleavage of laminin-332 (also referred to as laminin-5) by MMPs would generate a fragmented laminin peptide that binds to the EGF receptor and enhances the cellular motility of proliferating keratinocytes [34], demonstrated in **Figure 2**. In addition to its role in angiogenesis, fibroblasts are responsible for the production and deposition of immature collagen (type III collagen), which is essential in providing more strength for the wound ECM [22].

The formation of granulation tissue in the last phase provides a support matrix for epithelial cells to migrate across and to cover the wound surface in a process known as re-epithelialization (**Figure 1**). This stage of wound healing mainly involves keratinocytes, which are a predominant cell type in the epidermis of the skin [35]. In fact, to cover the wound surface with a new layer of epithelium, the keratinocytes at the wound edge need to undergo migration, differentiation, and proliferation. In the basal layer of epidermis, keratinocytes have differentiating characteristics, in which they are able to change into a longer and flatter shape before they begin migration [35]. The migrating keratinocytes need to loosen their adhesion to each other before moving away from the wound edge toward the wound's central point to close the open area [35]. It is interesting to note that these cells need to establish adhesion to the new ECM around them via integrins, but at the same time they develop actin filaments to support cellular migration through a wound matrix of necrotic material, clots, and even bacteria [36, 37]. During this process, the continued enhanced expression of MMPs, released by a variety of cells (macrophages, keratinocytes, endothelial cells, and fibroblasts) plays crucial roles in degrading substrates of the provisional wound matrix [38]. After the first layer of cells that cover the wound area, keratinocytes need to proliferate to have adequate depth of cells in the wound. The onset of proliferation is brought about by a variety of factors such as EGF, TGF-α, TGF-β, keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) [39]. The migration and proliferation of these cells require increased supply of oxygen in the wound bed [22]. It is important to note that in chronic wounds, the epidermis fails to re-epithelialize due to non-migratory keratinocytes, compared to acute wounds [35]. Following the robust proliferation and epithelial migration, wound healing enters its last phase of tissue remodeling that could potentially last in the order of years. Type III collagen prevalent during this last phase is gradually replaced by the more stable type I collagen [22]. The collagen fibers at the wound site are rearranged, cross-linked, and aligned to increase the wound's tensile strength [40]. The participation of proteinases is necessary to ensure ECM remodeling, which will bring back normality to the tissue.

In the repair of acute wounds, interactions between growth factors and the ECM occur in an orchestrated manner, where each phase is allowed to transition properly to the next, resulting in a healed wound. There are numerous factors that contribute to the impairment of wound healing in patients. Some are local factors that directly affect wound closure such as supply of oxygen, infection, venous sufficiency, and imbalance between proteinases or growth factors [12]. Others are underlying conditions that influence the overall health of a patient, including age, diseases, obesity, medications, and an immunocompromised system [12]. For instance, septic conditions have been shown to delay wound healing in mice [41]. In the case of patients affected by hypoproteinemia, their protein deficiency can impair wound healing by affecting capillary formation, cellular proliferation, collagen deposition, and wound remodeling [12]. The most detrimental disease is diabetes, where patients are more prone to develop non-healing ulcers or chronic wounds. In these wounds, the interactions between growth factors and the ECM are disrupted because of biochemical abnormalities of the ECM and aberrantly elevated activities of MMPs [14]. The imbalance between MMPs and their endogenous regulators can cause excessive degradative activities and critical loss of the newly reformed ECM in wound healing.

### **1.2. Matrix metalloproteinases (MMPs): structures and regulation**

wound surface with a new layer of epithelium, the keratinocytes at the wound edge need to undergo migration, differentiation, and proliferation. In the basal layer of epidermis, keratinocytes have differentiating characteristics, in which they are able to change into a longer and flatter shape before they begin migration [35]. The migrating keratinocytes need to loosen their adhesion to each other before moving away from the wound edge toward the wound's central point to close the open area [35]. It is interesting to note that these cells need to establish adhesion to the new ECM around them via integrins, but at the same time they develop actin filaments to support cellular migration through a wound matrix of necrotic material, clots, and even bacteria [36, 37]. During this process, the continued enhanced expression of MMPs, released by a variety of cells (macrophages, keratinocytes, endothelial cells, and fibroblasts) plays crucial roles in degrading substrates of the provisional wound matrix [38]. After the first layer of cells that cover the wound area, keratinocytes need to proliferate to have adequate depth of cells in the wound. The onset of proliferation is brought about by a variety of factors such as EGF, TGF-α, TGF-β, keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) [39]. The migration and proliferation of these cells require increased supply of oxygen in the wound bed [22]. It is important to note that in chronic wounds, the epidermis fails to re-epithelialize due to non-migratory keratinocytes, compared to acute wounds [35]. Following the robust proliferation and epithelial migration, wound healing enters its last phase of tissue remodeling that could potentially last in the order of years. Type III collagen prevalent during this last phase is gradually replaced by the more stable type I collagen [22]. The collagen fibers at the wound site are rearranged, cross-linked, and aligned to increase the wound's tensile strength [40]. The participation of proteinases is necessary to ensure ECM

In the repair of acute wounds, interactions between growth factors and the ECM occur in an orchestrated manner, where each phase is allowed to transition properly to the next, resulting in a healed wound. There are numerous factors that contribute to the impairment of wound healing in patients. Some are local factors that directly affect wound closure such as supply of oxygen, infection, venous sufficiency, and imbalance between proteinases or growth factors [12]. Others are underlying conditions that influence the overall health of a patient, including age, diseases, obesity, medications, and an immunocompromised system [12]. For instance, septic conditions have been shown to delay wound healing in mice [41]. In the case of patients affected by hypoproteinemia, their protein deficiency can impair wound healing by affecting capillary formation, cellular proliferation, collagen deposition, and wound remodeling [12]. The most detrimental disease is diabetes, where patients are more prone to develop non-healing ulcers or chronic wounds. In these wounds, the interactions between growth factors and the ECM are disrupted because of biochemical abnormalities of the ECM and aberrantly elevated activities of MMPs [14]. The imbalance between MMPs and their endogenous regulators can cause excessive degradative activities and critical loss of the newly reformed ECM in wound

remodeling, which will bring back normality to the tissue.

42 Wound Healing - New insights into Ancient Challenges

healing.

Matrix metalloproteinases are a group of 24 enzymes in humans—there are a total of 28 MMPs known to date, including enzymes from other organisms—which are expressed as zymogenic inactive proteins [7]. These enzymes are highly regulated and one level of regulation is exerted in their proteolytic activation by other proteinases, including by other MMPs [42, 43]. As the pro-domain of the zymogens are removed, the active sites become available for catalysis. Tissue inhibitors of matrix metalloproteinases (TIMPs) are protein inhibitors of these enzymes that form non-covalent complexes with the catalytic domain. The activation events and the inhibition by TIMPs account for various steps in the regulation process, which we will expand on in the following sections. These events are graphically depicted in **Figure 3** for MMP-2.

**Figure 3.** MMPs, as exemplified in this figure by MMP-2, exist in three forms: pro-MMPs (inactive), active MMPs, and TIMP-complexed MMPs (inactive). MMPs are first produced as latent pro-MMPs (1) with a pro-domain (shown in red) blocking the active site (shown in yellow). The removal of the pro-domain is required to activate MMPs by revealing the zinc ion in the catalytic site (2). Active MMPs are then able to cleave substrates. The activity of MMPs is regulated by interaction with TIMPs (shown in purple), which inactivate the MMPs (3).

MMPs are zinc-dependent endopeptidases. They are either secreted into the ECM or are membrane-anchored on the surface of the cell [9, 44, 45]. The most basic components of all MMPs consist of three domains: a signal sequence at the N-terminus, a pro-domain that caps the active site, and a catalytic domain, as depicted in **Figure 4**. This minimal domain organization is present in MMP-7 and MMP-26, also known as the matrilysins. The catalytic domain is characterized by the zinc-binding HExxHxxGxxH motif, containing three conserved histidines [46]. Several MMPs have an additional domain referred to as the hemopexin-like domain, which is linked at the C-terminus of the aforementioned basic sequence. The hemopexin-like domain is believed to play a role in substrate recognition. This organization of domains for MMPs is seen in MMP-3 and -10 (also known as stromelysin-1 and-2), MMP-1, -8, and -13 (also known as collagenases), MMP-12 (metalloelastase), MMP-20 (enamelysin), and MMP-22 and -27 [47] (**Figure 4**). MMP-2 and MMP-9 (or gelatinases) have more complicated structures by having fibronectin repeats inserted into the side of the catalytic sites [47] (**Figure 4**). The membrane-bound MMPs have two types of membrane anchors. One is a transmembrane peptide domain and another is the GPI anchor (**Figure 4**). There are a few other variations, which are summarized in **Figure 4** graphically. The structural similarities among these MMPs are high. Certainly, individual domains are highly similar in both sequence and three-dimensional structures. As a consequence, these enzymes share significant overlap in their substrate preferences, which is likely a reflection of the fact that the functions of disparate MMPs in the physiology of the organism are critical and they exhibit some redundancy in their turnover of the substrates as a consequence. As it pertains to wound healing, important MMPs and their known substrates are listed in **Table 1**.

**Figure 4.** Structures of the MMP family. MMPs are divided into eight subgroups based on structural similarities. Pre: signal sequence, Pro: pro-peptide, Zn2+: zinc-binding site, Catalytic: catalytic domain, F: repeats of fibronectin, Fu: furin-like serine proteinases, Vn: vitronectin-like insert, TM: transmembrane domain, GPI: glycosylphosphatidylinositol, SA: N-terminal signal anchor, CA: cysteine array, Ig-Like: immunoglobulin-like.

As indicated earlier, the functions of these enzymes are highly regulated. This regulation manifests itself at the transcriptional level as well as at the proteome level. Production of MMPs is stimulated in a variety of cells such as keratinocytes, fibroblasts, endothelial cells, and inflammatory cells during wound healing. These cells can be transcriptionally activated by a wide range of cytokines and growth factors including EGF, HGF, FGF, TGF-β, VEGF, PDGF, and KGF, as well as by interleukins and interferons [74]. Since there are many cytokines with the ability to activate transcription to produce the zymogens, there are numerous signaling pathways implicated in the control of proteinase expression. These pathways include, for instance, mitogen-activated protein kinase (MAPK), or growth factor-dependent pathways of Smad, NF-kB, activation of focal adhesion kinase (FAK) by integrin activation, or Wnt cascade [11]. The highly regulated process is critical for the physiological roles. When the regulation goes awry, these enzymes cause pathological consequences. The pathological outcomes of MMP dysregulation have been the subject of many review articles [8, 10, 75, 76].

(**Figure 4**). The membrane-bound MMPs have two types of membrane anchors. One is a transmembrane peptide domain and another is the GPI anchor (**Figure 4**). There are a few other variations, which are summarized in **Figure 4** graphically. The structural similarities among these MMPs are high. Certainly, individual domains are highly similar in both sequence and three-dimensional structures. As a consequence, these enzymes share significant overlap in their substrate preferences, which is likely a reflection of the fact that the functions of disparate MMPs in the physiology of the organism are critical and they exhibit some redundancy in their turnover of the substrates as a consequence. As it pertains to wound healing,

**Figure 4.** Structures of the MMP family. MMPs are divided into eight subgroups based on structural similarities. Pre: signal sequence, Pro: pro-peptide, Zn2+: zinc-binding site, Catalytic: catalytic domain, F: repeats of fibronectin, Fu: furin-like serine proteinases, Vn: vitronectin-like insert, TM: transmembrane domain, GPI: glycosylphosphatidylinositol,

As indicated earlier, the functions of these enzymes are highly regulated. This regulation manifests itself at the transcriptional level as well as at the proteome level. Production of MMPs is stimulated in a variety of cells such as keratinocytes, fibroblasts, endothelial cells, and inflammatory cells during wound healing. These cells can be transcriptionally activated by a wide range of cytokines and growth factors including EGF, HGF, FGF, TGF-β, VEGF, PDGF, and KGF, as well as by interleukins and interferons [74]. Since there are many cytokines with

SA: N-terminal signal anchor, CA: cysteine array, Ig-Like: immunoglobulin-like.

important MMPs and their known substrates are listed in **Table 1**.

44 Wound Healing - New insights into Ancient Challenges




**MMP Preferred**

MMP-7 (matrilysin)

MMP-8 (collagenase-2) **Substrates** 

46 Wound Healing - New insights into Ancient Challenges

aggrecan; nidogen; fibrillin; E-cadherin

Elastin; fibronectin; laminin; nidogen; collagen IV; tenascin: versican; α1-proteinase inhibitor; E-cadherin; tumour necrosis

factor

Collagen I, II, and III; aggrecan, serpins; 2-MG

**Roles in Wound Healing Cell**

• Affects wound contraction and delayed healing [55]

• Required for reepithelialization of mucosal wounds [58]

• Activates MMP-9 [56] X

• Re-epithelialization of mucosal tissue is impaired in MMP-7 knockout mice [58]

• Promotes cutaneous diabetic

• Most prevalent collagenase in

• MMP-8 knockout mice show delayed wound closure [62]

• Selective inhibition of MMP-8 delays murine diabetic wound

• Topical application of active MMP-8 accelerates murine diabetic wound healing [63]

• Found to be elevated in diabetic foot ulcer patients [49]

• Mainly expressed by neutrophils [3]

wound healing [60]

wounds [61]

healing [60]

**culture**

**Human (H) or Mouse (M) wounds**

M

M

H

M

H

M

**Detection Method**

M Immunohistochemistry

Western blot [62]

M MMP-inhibitor-tethered affinity resin [60]

M *In-situ* zymography [63]

[59]


**Table 1.** Mammalian MMPs: enzymatic substrates and roles in wound healing.

The complex orchestration of events that we outlined in Section 1.1 on wound healing involves important roles by MMPs. However, since MMPs are highly regulated at the proteome level, the transcriptional regulation is not the full picture. Yet, the transcriptional regulation of MMPs is the most studied, as the tools for it are readily available. For example, the increased transcription leads to higher translation to the inactive MMP zymogens, which have to experience proteolytic activation. This activation may only require disruption of the interaction between the active-site zinc ion and the conserved cysteine residue from the sequence …PRCGVPD… of the pro-domain to give rise to the active MMPs [3]. During physiological processes, pro-MMP activation can be achieved either by serine proteinases or by other MMPs [6]. In particular, membrane-type MMPs have been shown to be capable of activating other pro-MMPs, both directly and indirectly. For instance, MMP-14 (or MT1-MMP) is involved in regulating activation of pro-MMP-9 in osteoclast migration [77]. Activation of MMPs by serine proteinases is regulated by inhibition of plasma proteinase inhibitors, including α1-proteinase and α2-macroglobulin or thrombospondin-1 and thrombospondin-2 [3]. The activity of MMPs is primarily regulated *in vivo* by endogenous tissue inhibitors of metalloproteinases (TIMPs) (**Figure 3**). In mammals, there are four TIMPs (TIMP-1, -2, -3, and -4) that bind specifically to inhibit MMPs [78]. The dysregulation such as imbalance between MMPs and TIMPs ratio leads to up-regulation of proteinase activity and damage to the ECM.


**Table 2.** Profiling methods for MMPs.

**MMP Preferred**

MMP-13 (collagnase-3)

MMP-14 (MT1- MMP)

**Substrates** 

48 Wound Healing - New insights into Ancient Challenges

Collagen I, II, III, IV, IX, X and XIV; gelatin; fibronectin; laminin; tenascin; aggrecan; fibrillin;

serpins

Collagen I,II, and III; gelatin; fibronectin; laminin; vitronectin; aggrecan; tenascin; nidogen; perlecan; fibrillin; α1 proteinase inhibitor, α2-macroglobulin

Adapted from Martins and Caley [3].

**Roles in Wound Healing Cell**

• Promotes re-epithelialization indirectly by affecting wound

• MMP-13 knockout mice have reduced re-epithelialization and delayed wound closure [70]

• Involved in KGFR expression, and can regulate epithelial cell

• Activates MMP-2 [73] X

The complex orchestration of events that we outlined in Section 1.1 on wound healing involves important roles by MMPs. However, since MMPs are highly regulated at the proteome level, the transcriptional regulation is not the full picture. Yet, the transcriptional regulation of MMPs is the most studied, as the tools for it are readily available. For example, the increased transcription leads to higher translation to the inactive MMP zymogens, which have to experience proteolytic activation. This activation may only require disruption of the interaction between the active-site zinc ion and the conserved cysteine residue from the sequence …PRCGVPD… of the pro-domain to give rise to the active MMPs [3]. During physiological processes, pro-MMP activation can be achieved either by serine proteinases or by other MMPs [6]. In

proliferation [72]

**Table 1.** Mammalian MMPs: enzymatic substrates and roles in wound healing.

• Promotes keratinocyte migration and invasion [71]

• Keratinocyte migration [70] M

contraction [69]

**culture**

**Human (H) or Mouse (M) wounds**

X mRNA and

M

M

**Detection Method**

immunohistochemistry [70]

35S-labeled antisense RNA

probes [72]

Once activated, the only MMP form that is not complexed by TIMPs would have catalytic competence. Hence, tools are needed for analysis at the protein level in the afflicted/diseased tissue. Many current methodologies to profile MMPs are limited because they are unable to detect active MMPs (summarized in **Table 2**). We have applied unique tools to this end in both diabetic and non-diabetic wounds. An MMP-inhibitor-tethered affinity resin that binds exclusively to the active forms of MMPs was used to fish out activated MMPs that exist in wound tissues [60]. Once bound, the active MMPs were digested with trypsin and the peptides 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 were slightly up-regulated after seven days from infliction of the wound [60].

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

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 superior approach, in our opinion, in elucidating the roles of the two enzymes (MMP-8 and MMP-9) is the use of selective pharmacological agents that afford total temporal control of abrogation of activity within the wounds in the time course of the experiments. Highly selective or specific inhibitors for the given enzyme are critical for the success of these studies. These investigations indeed revealed the duality of MMP functions, beneficial and detrimental, in diabetic wounds [60, 63]. It was documented that MMP-8 had a beneficial role in wound healing, as it might be the body's response to the healing process. On the other hand, MMP-9 was shown to serve a detrimental role in diabetic wound healing; hence, an aberration in the regulatory events in diabetic animals led to its formation with detrimental consequences. Indeed, pharmacological intervention by selective MMP-9 inhibitors with no activity toward MMP-8 would appear to be a promising approach to speed up healing of diabetic wounds. As the non-healing wounds remain open for a long period of time, they face the fatal threat of infections with methicillin-resistant *Staphylococcus aureus* [79, 80] that lead to amputations like in the case of diabetic foot infections [81]. There is a serious need to develop new approaches to facilitate healing in chronic wounds since current treatments have not been proven effective. The only FDA approved drug Regranex™ (becaplermin), a platelet-derived growth factor, is associated with malignancies and increased risk of death [82]. In addition, the effectiveness of negative-pressure wound therapy is still unclear, stem cell therapies do not clear the infection, or topical antibiotics, and antimicrobial dressings induce antibiotic resistance [81].
