Wound healing progresses through a series of processes, which include the formation of granulation tissue, epithelialization and connective tissue remodeling. These events require continuous modification of the complex cellular support matrix. This matrix is comprised of: structural proteins (collagen and elastin); specialized “anchoring” proteins (fibronectin, laminin and fibrillin); and proteoglycans and glycosaminoglycans (GAGs) such as hyaluronic acid, chondroitin sulfate, heparan sulfate, heparin, dermatan sulfate and keratan sulfate. Blood vessels that deliver oxygen and nutrients to the extracellular matrix (ECM) also undergo modification.
Matrix metalloproteinases (MMPs) are neutral endopeptidases. These enzymes modify the ECM in support of wound healing, morphogenesis, tissue resorption and remodelling, nerve growth and hair follicle development. MMPs direct wound healing by controlling platelet aggregation, macrophage and neutrophil function, cell migration and proliferation, angiogenesis and collagen secretion.
MMPs exert their effect by modulating enzyme cascades and by either activating or inactivating matrix proteins, cytokines, growth factors and adhesion molecules. Pathologic MMP expression has been implicated in a variety of disease processes such as chronic wound ulceration, rheumatoid arthritis, osteoarthritis, cancer invasion, cancer metastasis, periodontal diseases, fibrotic diseases, atherosclerosis, epidermolysis bullosa and aortic aneurysm.
MMPs are a gene family that contains zinc2+-binding domain in their active sites and calcium ions to maintain structure. Enzymes are divided into subfamilies of secretory enzymes (collagenases, gelatinases, stromelysins, unclassified) and membrane-bound type enzymes (MT-MMPs) based upon structural characteristics and the substrates they preferentially bind. During normal skin repair, keratinocytes, fibroblasts, macrophages and endothelial cells secrete MMPs and express MT-MMPs on their surfaces.
Multiple factors dictate the type and amount of MMP expression. These factors include available cytokines, growth factors, hormones, oncogenes, changes in cell-cell interactions, cell-matrix interactions as well as feedback from inflammatory mediators. Tumor necrosis factor (TNF), interleukin-1, interleukin-6 and interleukin-8 have been shown to have specific MMP links.
How Do MMPs Make An Impact On Wound Healing?
MMPs exert their effect through several modes of action.
• They change cell adherence to gel, which influences cell migration.
• They promote cellular proliferation apoptosis or morphogenesis, and can dictate the number and type of cellular concentration in the tissue.
• They modulate biological active molecules, such as growth factors (GF) and growth factor receptors (GFR).
MMP proteolytic activity is controlled by three mechanisms:
• regulation of the transcription process;
• enzymes are transcribed as zymogens; and
• interaction with specific tissue inhibitory matrix proteins (TIMPs).
MMPs are synthesized as pre-proenzymes and subsequently bound to the cell surface or secreted into the extracellular space. Interaction of a cysteine amino acid residue with the zinc2+ moiety at the catalytic site maintains the enzyme in a latent state. Zinc2+ has four binding sites and the cysteine-zinc bond produces a fold that conceals the catalytic site. Disruption of the bond exposes the catalytic site and expresses the “active” state. This process is called the cysteine switch. TIMP –1, -2, -3 and –4 inhibit MMP activity by inserting their terminal amino acid residue into the fourth zinc2+ site.
Additionally, medicines are used to inhibit MMPs. Macrolide antibiotics (erythromycin, azithromycin, tetracycline) have been shown to retard tissue breakdown independent of their antibiotic effect. More than thirteen new macrolide-based MMP inhibitors are under investigation. Moreover, some nonsteroidal antiinflammatory drugs (NSAIDs) inhibit MMP activity with transcription repression as the proposed mechanism.
Understanding Key Differences Between Acute And Chronic Wound Fluid
Acute and chronic wounds fluid differs in biochemical content and function. Acute edema fluid activates fibrinogen, initiates pericapillary cuffing and prevents further blood loss. Prolonged cuffing starves injured tissue by reducing the delivery of vital oxygen and nutrients. Fibrin plugs formed during coagulation also block lymphatic drainage and localize the inflammatory reaction to support healing. Inflammatory mediators stimulate MMP transcription and activation. Presumably, the inflammatory mediators vary with differing wound types, the type and extent of colonization and the capability of host modulation.
Postoperative surgical wounds provide a source for acute wound fluid. One can obtain chronic wound fluid from a variety of lower extremity ulcers. Growth factors, MMPs, immune factors, and cytokines are measured. Functional assessments include the ability of the fluids to influence the growth of fibroblasts, endothelial cells and keratinocytes.
Acute wound fluid activates growth factors such as platelet-derived growth factor (PDGF) necessary for fibroblast proliferation, collagen secretion and matrix formation. On the other hand, chronic wound fluid activates MMP-8, -2, and -9 with subsequent degradation of growth factor, reduction of ECM, interference with collagen cross-bridging and decreased deposition of vital stromal structures. In chronic wound fluid samples, epidermal growth factor (EGF) degradation is associated with elevated MMP protease activity. On the other hand, degradation of recombinant vascular endothelial growth factor (rVEGF165) stimulated by chronic leg wound fluid is associated with plasmin.
What The Literature Reveals
For chronic venous leg ulcers, studies show a 30-fold elevation in MMP activity when compared to acute wounds. MMP-9 activity exceeds MMP-2 activity. In a pressure ulcer model (decubitus ulcers), the levels of MMP-2 and MMP-9 are elevated more than 10-fold and 25-fold respectively. In pressure ulcers, MMP-8 collagenase activity is also elevated. The diabetic foot ulcer model shows a 65-fold increase in MMP-1 collagenase activity, a three-fold increase in MMP-2 proenzyme activity, a six-fold increase in activated MMP-2, a two-fold increase in MMP-8 activity and a 14-fold increase in MMP-9 activity.
The MMP proteolytic activity in acute wounds is not as pronounced. Acute mastectomy wound fluid shows an increased gelatinase activity for MMP-9 and MMP-2. However, these increases were only five to ten-fold. It is also important to note that the inactive zymogen forms tended to persist in acute wound fluid. These results suggest that the pro-inflammatory environment of non-healing ulcers supports high levels of gelatinase activation.
TIMP activity appears to be inversely related to the chronicity of the wound. In the diabetic foot model, TIMP-2 activity is decreased. In the venous ulcer model, TIMP-1 activity is depressed. Likewise, the pressure ulcer model shows lower TIMP activity secondary to complexes of TIMP and activated MMP.
Proteolytic activity normalizes as the wound healing progresses. Studies comparing protease activity in venous leg ulcers showed a reduction in MMP-2 and MMP-9 levels as the wounds healed over a two-week period following treatment with bed rest and leg elevation. Pro-inflammatory cytokines IL-1, IL-6 and TNF-alpha in wound fluid from previously non-healing lower extremity wounds also decrease during healing.
Platelet derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-basic) and transforming growth factor-beta (TGF-ß) did not significantly change as wounds healed. These results suggest that the impaired healing response seen in chronic wounds may be more dependent upon inflammatory mediators and MMP activation than upon a deficit in growth factors.
Controlled proteolysis is needed for cell migration, angiogenesis and matrix remodeling. Collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) are expressed in keratinocytes bordering both acute and chronic wounds. Differential collagenase expression suggests various MMPs serve different functions during the wound healing process. The presence of collagenase-1 (MMP-1) in keratinocytes appears to be important for cellular migration whereas collagenase-3 (MMP-13), which present exclusively in fibroblasts embedded deep within the chronic ulcer wound bed, probably functions more in matrix remodelling.
TIMP-1 associated with keratinocytes bordering healing wounds at the basement membrane is not expressed in the epidermis of chronic wounds. TIMP-3 expression is conserved in both acute and chronic wounds, localized in the stromal fibroblast and macrophage-like cells surrounding vessels and sweat glands.
Researchers report no qualitative differences in the expression of MMPs -1, -3, and -10 in the epidermis of chronic wounds when they compared them to normally healing wounds. However, the overall number of stromal cells expressing MMP-1 and MMP-3 are greater in chronic wounds. MMP-10 has not been detected in the dermis of chronic wounds.
These studies outline the need for further research to clarify the connection between cellular expression, concentration and function of the MMPs as balanced with TIMPs. At this time, it is difficult to drawn a causal relationship between wound healing and the presence or absence of a given MMP.
Dr. Smith is the Medical Director of the Wound Care Center at the Texas Diabetes Institute in San Antonio, Texas. She is an Assistant Professor in the Department of Rehabilitative Medicine at the University of Texas Health Science Center, is board-certified in emergency medicine and fellowship-trained in hyperbaric medicine.
Dr. Steinberg (pictured) is an Assistant Professor in the Department of Orthopaedics/Podiatry Service at the University of Texas Health Science Center.
1. Vu TH, Werb Z. “Matrix metalloproteinases: effectors of development and normal physiology.” Genes and Development. Vol. 14, No. 17, pp. 2123-2133, Sep 1, 2000.
2. Cullen B. “Part 2: The Role of Oxidized Regenerated Cellulose/Collagen in Chronic Wound Repair” in Matrix Metalloprotease Modulation and Growth Factor Protection. http://www.podiatrytoday.com/WNDS/matrix/pt.cfm .
3. Stacey, Woosey, Wallace. “The Influence of Micro-Environment And Cell Phenotype On Venous Ulcer Healing.” Abstract. European Tissue Repair Society (ETRS). Annual Conference 2001. http://www.etrs.org/bulletin8_3/page7b.html .
4. Rogers, Jude, Oyibo. “Matrix Metalloproteinase (MMP) And Tissue Inhibitor Of Metalloproteinase (TIMP) Expression In Diabetic And Venous Ulcers.” Abstract. European Tissue Repair Society (ETRS). Annual Conference 2001. http://www.etrs.org/bulletin8_3/page7b.html .
5. Ergul et.al. “Evidence for a matrix metalloproteinase Induction/activation system in arterial vasculature and decreased synthesis and activity in diabetes”. American Diabetes Association, Oct 2002. http://www.heart1.com/news/newsfeed.cfm/1280/1 .
6. Renò, et. al “Release and Activation of Matrix Metalloproteinase-9 During In Vitro Mechanical Compression in Hypertrophic Scars”. Arch Dermatol. 2002;138:475-478
7. Cook, et al., J Invest Dermatol 2000; 115: 225–233.
8. Herouy Y, et. al., “Lipodermatosclerosis is characterized by elevated expression and activation of matrix metalloproteinase: Implications for venous ulcer formation: J Invest Dermatol 1988; 111:822-27.
9. Abstract and commentary by: Fedor Lurie, MD, PhD. Volume 8, Number 12 www.venousdigest.com December 2001.