The Role of Collagenase in Wound Healing

Mona Stahle-Backdahl Introduction

Trading Perfection for Promptness-Repair Versus Regeneration

At first glance, the ultimate goal in the process of wound repair would seem to be the complete restoration of injured tissue. However, the capacity to fully regenerate lost tissue has been strongly selected against during evolution, and this ability, with little exception, now remains only in fishes and amphibians. It appears that the transition from aquatic to terrestial life correlates with a decline in regenerative potential. In fact, the ability to rapidly and effectively accomplish wound closure constitutes a critical step in evolution. Achieving full replacement of injured body parts, a time-consuming task, was simply not compatible with a successful struggle for survival in mammals. An important consideration may be that stresses on weight bearing limbs of animals living on dry land make regeneration of appendages impossible. The only mammalian tissue capable of complete regeneration is the deer antler1 and while other related animals developed horns, one can only speculate about the selective advantages of antler evolution.

How does wound healing relate to the phenomenon of epimorphic regeneration? Are they similar processes with tissue repair representing only a blunted, compromised version of complete tissue replacement; or are they biologically distinct? There are important similarities: both processes involve cell migration, proliferation and redifferentiation, however there are also differences, the major one being that epimorphic regeneration entails the impressive capacity to develop entirely new structures such as bone and muscle in their appropriate locations. And yet epimorphic regeneration must be regarded as a luxury in nature, while the universality of wound healing testifies to its critical role in survival.

The Phases of Normal Wound Healing

The purpose of tissue repair is to reinstate the functional and structural integrity of the damaged organ. To this end, normal wound healing follows a sequential pattern involving a series of closely regulated, coordinated, and overlapping events. In response to injury and blood vessel disruption, there is acute inflammation which activates the coagulation cascade and leads to hemostasis. This process releases chemotactic agents which serve as migration signals for adhering neutrophil leukocytes, the key participants in the innate immune system and the first line of defense against potentially harmful microorganisms that

Collagenases, edited by Warren Hoeffler. ©1999 R.G. Landes Company.

may invade the host.2 Although the repair mechanisms are generally applicable to most organs, the present review will focus mainly on skin and epidermal wound closure.

Following after neutrophils, other cells such as monocytes/macrophages and lymphocytes arrive at the site of the wound, starting the complex process of repair and remodeling. A multitude of different growth factors and proteolytic enzymes are released to degrade damaged tissue and debris and stimulate synthesis of new matrix. Granulation tissue is composed of a provisional matrix containing fibronectin, vitronectin, fibrinogen, hyalu-ronic acid and types I and III collagen. Collagen is the major structural component of the skin, and digestion of this protein is critically dependent upon the proteolytic activity of interstitial collagenases produced by resident and migratory cells. In the process of wound repair, fibroblasts proliferate and assume a more contractile phenotype, becoming myofibroblasts to accomplish wound contraction.3 New blood vessels form and sprout through the wound bed.

Shortly after injury, sedentary basal keratinocytes at the wound edge detach from the underlying substratum and assume a migratory cell phenotype. This begins the crucial process of wound re-epithelialization, which forms a protective barrier against the potentially harmful exterior environment. The keratinocytes lose their apical-basal polarity stretch out and move towards the free edge. While initial re-epithelialization begins with cell migration, within 1-2 days epidermal cells behind the advancing front begin to proliferate, generating additional migrating cells. Once the tissue defect is covered and re-epithelialization completed, the keratinocytes resume their normal quiescent phenotype and again firmly anchor to the basement membrane. This apparently programmed sequence of events evidently requires precise spatial and temporal control. Mechanisms regulating this dynamic and important process are the focus of current research in wound healing biology, but are as yet incompletely understood.

The final phase of wound healing involves matrix restructuring. The composition of the connective tissue is gradually altered: fibronectin and type III collagen levels decrease while type I collagen synthesis increases to provide additional tensile strength to the skin. This is a rather slow process, as by three weeks the wound has only an estimated 20% of the strength of the uninjured skin.4 To impart additional strength to the wounded skin, collagen is synthesized at a high rate until it returns to baseline within six to twelve months. Collagen remodeling depends on both increased synthesis and collagen catabolism, controlled by collagenases from granulocytes,5,6 macrophages,7 fibroblasts,8 and epidermal cells.9 The tensile strength of mature scar tissue is estimated at about 70% and thus, once wounded, skin never regains preinjury strength.4

The Collagenases

Within the metalloproteinase (MMP) gene family, the collagenases constitute a distinct subgroup of enzymes sharing the unique ability to specifically cleave interstitial collagen fibers (see chapter 1). Metalloproteinases have somewhat overlapping substrate specificities, with most enzymes capable of digesting fibronectin and gelatin. Unique among these is interstitial collagenase with the exclusive ability to cleave collagen fibers. This specific catalytic event has been studied in greater detail than the other MMP-mediated reac-tions10 and involves cleavage of a single peptide bond.11 At physiologic temperature the two digestion fragments of collagen loosen their triple helical structure and denature into gelatin peptides which are then susceptible to subsequent digestion by a variety of proteases.12 Because it is responsible for performing the initial and rate-limiting step in collagen fiber degradation,13,14 collagenase activity is critical in the remodeling of the dermal matrix.

To date, three different collagenases have been identified: Collagenase-1 (fibroblast collagenase, MMP-1)15,16(see chapter 1), neutrophil collagenase (MMP-8)17,18 (see chapter

2) and, the most recently characterized, collagenase-3 (MMP-13)19(see chapter 3). Neutrophil collagenase has only been detected in neutrophil leukocytes, whereas collagenase-1 is expressed by a variety of cell types other than fibroblasts, such as keratinocytes,20,21 endothelial cells,22 monocytes/macrophages,16,23 chondrocytes and osteoblasts.24,25

The fact that there are at least three distinct collagenases sharing the particular ability to cleave fibrillar collagens appears a luxurious redundancy likely developed to protect a key biological function. However, detailed analysis of the enzymatic properties of the respective enzymes reveals difference in their substrate specificities. Collagenase-1 preferentially cleaves type III collagen, a prominent component of early wound matrix.26 Neutrophil collagenase is more active against type I collagen, and collagenase 3 cleaves type II collagen more efficiently than types I or III.27,28 This pattern of substrate preferences suggests that the collagenases evolved as specialized enzymes to remodel tissues with different collagen composition.

Apart from different substrate specificities, the collagenases also differ profoundly in gene regulation (see chapter 4). Briefly, neutrophil collagenase is transcribed in bone marrow precursor cells and the circulating mature neutrophils store the preformed protein in cytoplasmic granules to be released at the proper signal. On the other hand, collagenase-1 and collagenase-3 are subjected to tight transcriptional regulation and are readily induced by phorbol esters, cytokines and growth factors. These enzymes contain enhancer elements in their promoter regions, like AP-1-binding sites and a PEA3 consensus sequences, which control the responsiveness of these genes to a host of transactivating factors.29-31 The AP-1 site confers inducibility by cytokines and growth factors, and the PEA3 binding site is recognized by the transcription factor c-ets. Interestingly, the stromelysin-1 and the urokinase plasminogen activator genes, both implicated in the activation of procollagenase, share the AP-1/ PEA3 motif,32,33 suggesting that all three genes may be subject to coordinate expression to create a proteolytic cascade operative in vivo.

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