136 JOURNAL OF COSMETIC SCIENCE THE EXTRACELLULAR MATRIX- FROM STRUCTURAL RESILIENCE TO MODULATION OF CELL FUNCTIONS Sanford R. Simon, Ph.D. State University of New York at Stony Brook, Stony Brook, NY 11794-8691 The extracellular matrix (ECM) is classically regarded as having a role in maintaining structural integrity of connective tissue. Two components of the interstitial ECM, collagen and elastin, play especially prominent roles in establishing the balance between rigidity and elasticity which characterizes normal connective tissues. When these proteins are serving as structural elements in the interstitium, they exist predominantly as fibers and are characterized by insolubility and resistance to many proteolytic enzymes. Infiltration of the interstitium by inflammatory cells, especially neutrophils and mononuclear phagocytes (monocytes and macrophages) is traditionally implicated as a critical step in establishing a milieu in which specialized proteinases are released, leading to degradation of collagen and elastin fibers. A net loss of elastic fibers due to chronic degradation is considered to be characteristic of normal ageing of skin, while UV damage to the skin is associated with an accumulation of elastin-like material which has been postulated to result from exaggerated biosynthetic activity triggered by peptides released from inflammatory destruction of normal collagen and elastin fibers. Thus, proteolytic degradation of collagen and elastin has been considered to lead to undesirable changes in the characteristics of skin, resulting either in abnormal thinning or thickening. The proteinases implicated in these changes have been purified and extensively studied as soluble enzymes, and considerable efforts have been directed towards characterizing their endogenous inhibitors as well as developing synthetic inhibitors to supplement the endogenous antiproteinases. This model of degradation of purely structural proteins by soluble enzymes released by inflammatory cells has proved to be oversimplified. In this presentation, we consider some of the results which have emerged from many laboratories indicating that the functional roles of compoµents of the ECM go beyond maintenance of rigidity and elasticity. Moreover, inflammatory proteinases may function in highly specialized microenvironments, such as the inflammatory cell surface, and may have physiologically relevant targets beyond the principal structural elements. Thus, attempts to modulate the activity of these proteinases may have consequences beyond changes in collagen and elastin fiber structure. The Roles of ECM-Associated Matricellular Proteins The ECM which separates epithelial and stromal components, as seen at the dermal-epidermal junction, is referred to as basement membrane (BM), and has unique components, including nonfibrillar collagens (types IV, XV, and XVIII) as well as a variety of bridging elements such as laminin, ent':lctin (nidogen), and heparan sulfate proteoglycans. These components interact not only with each other but also with epithelial and stromal cells. More recently, a number of additional proteins have been characterized which appear to function primarily as cell regulatory molecules rather than structural components. These molecules include thrombospondins, SPARC or osteonectin, osteopontin, and tenascins. With the notable exception of osteopontin, which enhances adhesion of cells to BM through integrins, these so-called matricellular proteins typically reduce the adhesion of cells to BM and may facilitate their migration. Moreover, thrombospodin-2 appears to play an important role in clearing the BM of one of the important matrix-degrading proteinases, MMP-2 or gelatinase A, by binding to the enzyme and facilitating its internalization. Several matricellular proteins regulate such cellular events as proliferation and responsiveness to growth factors, apoptosis, and angiogenesis. Virtually all the matricellular proteins are targets of leukocyte elastase, the serine proteinase released from the azurophil granules of neutrophils. In addition, the combined activities of elastase and the matrix metalloproteinases (MMPs) released by neutrophils and macrophages can degrade types IV and XVIII collagen to liberate proteolytic fragments such as endostatin and tumistatin, which bind to integrins on cell surfaces, resulting in significant anti­ angiogenic activities. The consequences of pathologically elevated levels of the inflammatory cell-derived proteinases or of abrogation of proteolytic activity by injudicious use of exogenous antiproteinases on the multiple functional roles of the matricellular proteins have not yet been fully explored. Protei11ase-A11tiprotei11ase Imbalance in ECM Damage One of the more challenging aspects of attempting to intervene in preventing excessive damage to ECM through the use of proteinase inhibitors relates to the interactions between the most common proteinases and their endogenous inhibitors. The serine proteinase, leukocyte elastase, is targeted by the endogenous antiproteinases, alpha-1-Proteinase Inhibitor (alpha-I-PI), secretory leukoprotease inhibitor

2003 ANNUAL SCIENTIFIC MEETING (SLPI), and elafin. The MMPs are, in tum, targeted by the Tissue Inhibitors of Metalloproteinases (TIMPs). It has been appreciated that a number of the members of the family of MMPs, several of which are classically referred to as collagenases or gelatinases based on their ability to degrade collagen fibers or the unstable fragments formed by limited proteolysis of the fibers, have other physiologically relevant targets. In particular, several MMPs are capable of inactivating alpha-1-PI and SLPI. In fact, alpha-1-PI as well as other members of the so-called class of "serpins," or serine proteinase inhibitors, are the only known targets of one of the MMPs. SLPI and elafin are two-domain molecules, anchored or tightly bound to ECM components. This feature greatly enhances their anti-elastinolytic activity, but when they are cleaved by �MPs, their association with the ECM is disrupted and their potencies as inhibitors of elastase­ mediated ECM degradation are diminished. Meanwhile, unopposed leukocyte elastase can cleave the TIMPs, markedly diminishing their inhibitory activity towards the MMPs. Thus, the two families of proteinases released by inflammatory cells can degrade each other's endogenous antiproteinases. This synergy is suspected as an underlying mechanism for the catastrophic inflammatory injury seen in such conditions as Acute Respiratory Syndrome, or ARDS. However, the cross-inactivation mechanism may be turned to advantage by judicious introduction of exogenous proteinase inhibitors. Rather than attempting to achieve complete inhibition of both classes ofproteinases, it may be adequate to employ exogenous agents fo inhibit the proteinases only to the point at which their unopposed activities are diminished so that they do not destroy each other's endogenous antiproteinases. An example of the use of one such group of synthetic proteinase inhibitors, which are based on the tetracycline ring structure but are distinctive for their lack of antimicrobial activity, will be presented. The most promising of these agents inhibits MMPs as well as leukocyte elastase, blocks neutrophil-mediated ECM degradation, and prevents inactivation of alpha-I-PI.. This so-called "chemically modified tetracycline" also inhibits angiogenic activity associated with cell-B'M interactions, as evidenced by the capacity of the agent to diminish release ofVEGF from monocytes and to' reduce fom1ation of tube-like structures from endothelial cells plated on a reconstituted BM. This anti­ angiogenic activity is currently being evaluated in clinical trials of patients with Kaposi's sarcoma. In animal models of ARDS, the tetracycline derivative reduces morbidity and mortality dramatically. The pleiotropic effects of nonantimicrobial tetracycline derivatives may reflect biological activities beyond direct inhibition of inflammatory proteinases, but may also illustrate how the actions of proteinases on targets (e.g. matricellular proteins) other than structural elements of the ECM may elicit complex physiological responses. Cell Surface-Bound Protei11ases i11 ECM Damage The use of low molecular weight proteinase inhibitors to modulate ECM degradation in an inflammatory milieu has been assumed to confer the advantages of greater bioavailability and stability, but recent studies on the properties of cell-bound proteinases have revealed a somewhat different set of challenges which"may be addressed by even more selective antiproteinase design strategies. While neutrophil elastase and many o� the MMPs are found as soluble enzymes in the extracellular environment, both of these classes of proteinases can also be present at high levels bound to the surfaces of neutrophils and macrophages. �i;:veral forms of metalloproteinases which are true integral membrane proteins have been recently chara'ferized and have been shown to have unique activities associated with their location on the cell surface. In their cell surface-bound state, the serine proteinases and the metalloproteinases display a very different profile of sensitivity to endogenous antiproteinases and exogenous proteinase inhibitors than the soluble enzymes. It has been suggested that surface-bound proteinases on infiltrating inflammatory cells may account for a significant fraction of the ECM damage which occurs even in the presence of high levels of endogenous antiprotelinases. Some examples of markedly different sensitivities of cell-bound and soluble proteinases to natural and synthetic proteinase inhibitors, using low molecular weight substrates as well as experimental ECM models to evaluate inhibitory potencies, will be presented. 137