Wound Healing the presence of more than one bacterial strain. Drug-resistant organisms, which form biofilms, stimulate chronic inflammation. Bacteria have now developed mechanisms by which they can circumvent the effects of antibiotics, so the antibiotics that were effective no longer work, either at all, or as well as they did. Bacteria have become resistant to the drugs that would once have killed them, as they multiply so rapidly that mutations occur, which makes them less susceptible to antibiotics. In addition, the development of new antibiotics, with novel modes of action, is scare, with no new antibiotics developed over the last 25 years, making wound treatment more difficult as infection is detrimental to wound healing. Studies by Margolis and co- workers concluded that ulcer size (>2 cm2), duration (> 2 months) and ulcer depth were the three most important factors determining healing outcomes [11]. The size (area and depth), sepsis, arteriopathy and denervation classification system identifies ulcer size and the presence of arteriopathy as the most important factors associated with DFU healing [12]. In chronic wounds, there is a tendency for the inflammatory response to be altered beyond the normal duration. Pro-inflammatory cytokines, reactive oxygen species and proteolytic enzymes, such as certain MMP, elastases and plasmins, are produced to a greater extent as a result of reduced inhibitor release, such as TIMP. This inactivates growth factors and leads to ECM degradation and alteration of the wound pH, which leads to reduced tissue repair, cellular proliferation and angiogenesis. It has been reported that stress, depression and a hostile marital environment have a possible role in the modulation of MMP and in the expression of TIMP [13, 14], thereby delaying wound healing.
[16]. They play important and beneficial roles in the removal of damaged ECM and bacteria during the inflammation phase, degradation of the capillary basement membrane for angiogenesis and migration of epidermal cells during the proliferation phase, and contraction and remodelling of scar ECM during the remodelling phase.
Table 6.2 lists examples of MMP actions that affect cell migration, differentiation, growth, the inflammatory process, neovascularisation, apoptosis and so on [17]. Their activities are regulated by inhibitors such as TIMP to keep a balance between ECM regeneration and remodelling during normal wound healing. However, in chronic wounds this process is interrupted with increased inflammation and ECM degradation [18]. Regulation of cell growth and differentiation, i.e., altering cell motility, cell-cell interaction, release of growth factors and cytokines that affect cellular proliferation and growth, are other important roles of MMP in addition to ECM remodelling.
Table 6.2 Matrix metalloproteinases and their biological activities
MMP Biological activities Substrate
Collagenases MMP-1 Keratinocyte migration and re-
epithelialization Type I collagen
Cell migration Fibronectin
Platelet aggregation −
Increased bioavailability of IGF-1 and cell
proliferation IGF binding protein-3
Pro-inflammatory Processing IL-1β from the precursor
Anti-inflammatory IL-1β degradation
Anti-inflammatory Monocyte chemoattractant protein-3
PAR-1 activation PAR-1
MMP-8 Tissue remodelling Type I, II and III collagens
MMP-13 Enhanced collagen affinity BM-40 (SPARC/osteonectin)
Release of bFGF Perlecan
Anti-inflammatory Monocyte chemoattractant protein-3 Stromelysins
MMP-3 Cell migration Fibronectin
Mammary epithelial cell apoptosis Basement membrane Mammary epithelial alveolar formation Basement membrane Epithelial-mesenchymal conversion
(mammary epithelial cells) E-cadherin Generation of angiostatin-like fragment Plasminogen
Wound Healing
Enhanced collagen affinity BM-40 (SPARC/osteonectin)
Release of bFGF Perlecan
Increased bioavailability of IGF-1 and cell
proliferation IGF binding protein-3
Pro-inflammatory Processing IL-1β from the precursor Anti-inflammatory Monocyte chemoattractant protein-3 Increased bioavailability of TGF-β Decorin
Disrupted cell aggregation and increased
cell invasion E-cadherin
MMP-10 Tissue remodelling Proteoglycans, fibronectin
MMP-11 Increased bioavailability of IGF-1 and cell
proliferation IGF binding protein-1
Gelatinases
MMP-2 Neutrite outgrowth Chondroitin sulfate proteoglycan
Cell migration Fibronectin
Mesenchymal cell differentiation with
inflammatory phenotype −
Enhanced collagen affinity BM-40 (SPARC/osteonectin) Increased bioavailability of IGF-1 and cell
proliferation IGF binding protein-3
Epithelial cell migration Laminin 5γ2 chain
Anti-inflammatory IL-1β degradation
Anti-inflammatory Monocyte chemoattractant protein-3
Increased bioavailability of TGF-β Decorin Generation of vasoconstrictor Big endothelin Conversion of vasodilator to
vasoconstrictor Adrenomedullin
MMP-9 Generation of angiostatin-like fragment Plasminogen
Enhanced collagen affinity BM-40 (SPARC/osteonectin) Pro-inflammatory Processing IL-1β from the precursor
Anti-inflammatory IL-1β degradation
Reduced IL-2 response IL-2Ra
Bioavailability of TGF-β Precursor of TGF-β Membrane type
MMP-14 Anti-inflammatory Monocyte chemoattractant protein-3
Epilysin
MMP-28 Tissue haemostasis E-cadherin
bFGF: Basic fibroblast growth factor PAR: Protease activated receptor
MMP-1, also known as collagenase-1, is released by keratinocytes and is critical for re-epithelialization by promoting the migration of epithelial cells, fibroblasts and vascular endothelial cells across or through the ECM. The secreted MMP-1 partially digests collagen and weakens the attachment of the cell membrane to the matrix, thereby allowing the cells to migrate across the collagen matrix. MMP-1 is a single polypeptide comprising a major unglycosylated form of 57 kDa, and a minor glycosylated species of 61 kDa. In a recent study it has been shown that MMP-1 is required in the epidermis to facilitate re-epithelialization via remodelling the basement membrane, promoting cell elongation and actin cytoskeletal reorganisation, and activating extracellular signal-regulated kinase signalling. After wounding, MMP- 1 is upregulated by the jun N-terminal kinase pathway and the rate of healing is accelerated in an MMP-1 dependent manner. MMP-1upregulation is triggered by damage rather than by the introduction of pathogens, as shown in Figure 6.2 [19].
MMP-8, known as collagenase-2, can be expressed in a wide variety of cells such as maturing neutrophils, peripheral neutrophils, macrophages, plasma cells, T-cells, bronchial epithelial cells, oral epithelial cells, corneal epithelial cells, colon mucosal cells, keratinocytes, endothelial cells, fibroblasts, colon myofibroblasts, smooth muscle cells, chondrocytes and so on during different inflammatory conditions. Full length MMP-8 is 80 kDa and is upregulated during the remodelling process of wound healing. Activated MMP-8 is more prominent in chronic wounds than in normal healing wounds because of the large number of neutrophils present in chronic wounds.
Similarly, MMP-13 plays an important role in keratinocyte migration, angiogenesis via the digestion of connective tissue growth factor and contraction by activation of latent TGF-β. MMP-8, also called collagenase-3, is expressed in hypertrophic chondrocytes and osteoblasts during human foetal development. Delayed healing and the formation of chronic wounds have been linked to the excessive production of proteolytic enzymes leading to reduced amounts of growth factors and the successive destruction of the ECM. Levels of proteases, such as MMP-13, are found to be profoundly elevated in chronic when compared with acute wound fluids [20].
Gelatinases include two members, namely gelatinase A (MMP-2) and gelatinase B (MMP-9), which have affinity towards denatured collagen. MMP-2 is 62−72 kDa and MMP-9 is 82−92 kDa, and both play a substantial role in the inflammatory process. MMP-2 (type IV collagenase) is expressed by fibroblasts, endothelial and epithelial cells. Increased levels of MMP-2 and MMP-9 have been demonstrated in various chronic wound exudates [21−23], which will degrade matrix proteins and growth factors that are essential for normal healing and leads to a failure of wounds to heal. High wound concentrations of MMP-9 and a high MMP-9 to TIMP-1 ratio predict poor wound healing in DFU.
Wound Healing
Unwounded A
C D
F E
G H
H'
B
B'
Heat Map: αMMP-1Heat Map: αMMP-1Heat Map: αMMP-1Heat Map: αMMP-1
αMMP-1 αFasill DAPI Heat Map: αMMP-1
Heat Map: αMMP-1 αMMP-1 DPPI
256 0
0.5 h 3 h
18 h 5 h
Figure 6.2 MMP-1 is upregulated at the wound site. (A) Heat map showing MMP-1 localisation in an unwounded larval epidermis, pseudocoloured based on pixel intensity, the intensity scale for A−G is displayed at the bottom of the figure.
(B, B′) X−Z images of the epidermis showing antiMMP-1 staining (heat map in B′), the cell border marker FasIII and DAPI. Apical is up. (C−F) Heat maps showing antiMMP-1 staining after wounding. The white, dashed lines in C−E outline the wound bed, and the white arrow in F indicates the closed wound. Comparison of MMP-1 intensity levels can be made between A and C−F, as the images were taken
at matched exposure settings. (C) MMP-1, 0.5 h post-wounding.
(D) MMP-1, 3 h post-wounding. (E) MMP-1, 5 h post-wounding. (F) MMP-1, 18 h post-wounding. (G) Close-up of epidermal cells near the leading edge 5 h post-wounding (white box in E) with white arrows pointing to the distal-edge accumulation of MMP-1. (H, H′) X−Z images of MMP-1 (heat map in H′) and
DAPI in two epidermal cells at the leading edge of a 5-h wound. Apical is up.
The white asterisk indicates the wound bed. The rightmost arrows designate proximal MMP-1 accumulation around the leading edge and the leftmost arrows
indicate distal MMP-1 accumulation. All scale bars are 20 μm. The scale bar in A is also for C–F. DAPI: 4',6-diamidino-2-phenylindole and FasIII: fasciclin III.
Reproduced with permission from, L.J. Stevens and A. Page-McCaw, Molecular Biology of the Cell, 2012, 23, 6, 1068. ©2012, American Society for Cell Biology
[19]
MMP-3, MMP-10 and MMP-11 are stromelysins that have a domain arrangement similar to that of collagenases, but they do not cleave interstitial collagens. MMP-3 is a 51 kDa enzyme which is expressed by keratinocytes, fibroblasts and chondrocytes, and its main role is the activation of proMMP during extracellular turnover by activating collagenases, matrilysin and gelatinase B. Fully active MMP-1 is generated by the action of MMP-3 on proMMP-1 and is unable to degrade type I collagen, however it can degrade type IV, V, IX and X collagens, proteoglycans, gelatin, fibronectin, laminin and so on. MMP-3 may play an important role in the acute inflammatory reaction, regeneration of parenchyma cells, cell migration and proliferation, angiogenesis, and contraction and tissue remodelling. MMP-10 is similar to MMP-3 and is a 53 kDa proenzyme, which participates in proMMP activation, similar to MMP-3, however its catalytic activity towards type IV and type V collagens are comparatively weak.
MMP-11 is expressed in normal and pathological remodelling processes.
MMP-14 is a membrane-type MMP which has intrinsic proteolytic capabilities and can induce its effects by activating MMP-2 and MMP-13. MMP-14 contributes to re-epithelialization. MMP-28 is a 59 kDa protein which is expressed in basal keratinocytes at the edge of the wound. It has been shown that MMP-28 is spatially and temporally regulated, with a strong upregulation of MMP-28 occurring in the mitotic cells of wounded skin, which suggests its requirement in restructuring the basement membrane or degrading the adhesive proteins between the keratinocytes in order to supply new cells for the migrating front [24, 25].
TIMP are endogenous inhibitors of MMP and contain four members, TIMP-1, TIMP-2, TIMP-3 and TIMP-4, which are involved in the degradation of the ECM. MMP are inhibited by the interaction of the N-terminal domain of the TIMP molecule with the active site of the MMP. TIMP-1 is a glycoprotein and a natural inhibitor of MMP. In addition to its inhibitory role against most of the known MMP, this encoded protein promotes cell proliferation and also has an antiapoptotic function. TIMP-1 has been shown to exert cell growth promoting activity on human keratinocytes. Similarly, TIMP-2 also inhibits MMP and also has the ability to directly suppress the proliferation of endothelial cells. It is also involved in the maintenance of tissue homeostasis by suppressing the proliferation of quiescent tissues in response to angiogenic factors, and by inhibiting protease activity in tissues undergoing remodelling of the ECM.
TIMP-3 binds tightly to the ECM and is a good inhibitor of the TNF-a converting enzyme. TIMP-4 is involved in the regulation of platelet aggregation, recruitment, hormonal regulation and endometrial tissue remodelling.
Wound Healing