Chronic wounds are a major health care problem in the United States affecting an estimated 2.0 to 4.5 million persons at an annual cost exceeding $9 billion. Most of these wounds are either pressure sores or venous leg ulcers and are a direct result of tissue trauma or trauma related injuries. A majority of these wounds are cared for by nurses. Studies on normal wound healing and the basic pathophysiologic mechanisms underlying chronic wounds have been conducted. However, little is known about the interactions between cells and extracellular matrix resulting in the tissue erosion seen in chronic wounds. The long term objective of this study is to determine/characterize the cellular and biochemical mechanisms resulting in defective re-epithelization and proteolytic degradation of extracellular matrix in chronic wounds. The results of this study will uncover fundamental details of the biological events occurring in human chronic wounds and may lead to the development of new treatment modalities and better care. During the five years of the proposed study, research will focus on chronic cutaneous wound fluid and its interactions with extracellular matrix and cells using acute wound fluid as a control. The following specific aims will be systematically studied: I. Fluids from pressure sores and venous leg ulcers will be examined for intact or degraded fibronectin and active fibronectin degrading proteases. We have demonstrated that fibronectin is degraded in venous leg ulcers using immunoblotting. We now propose to use antibodies recognizing both intact FN and FN fragments to examine fluid from pressure sores for fibronectin degradation. Exogenously added FN will be used to demonstrate the presence of active proteases. II. The class, level, and form of metalloproteinases in acute and chronic wound fluids and the proteases which degrade fibronectin will be studied. Immunoblotting with antibodies will be used to further identify the metalloproteinses. To determine which proteases degrade fibronectin we will use fibronectin zymograms. Protease inhibitors and antibodies will be used to determine the class of proteases found in the fibronectin substrate gels. Levels of proteases will be determined using either scanning densitometry or ELISA. Activators, reducing agents, and antibodies will be used to study the form of the proteases. III. Acute and chronic wound fluids will be examined for the presence and level of normally occurring serum and tissue proteinase inhibitors. Normal serum protease inhibitors, alpha-2 macroglobulin, alpha- 1 protease inhibitor, PAI- 1, PAI-2, and tissue inhibitors, TIMP- 1 and TIMP-2 will be assayed using thermolysin, trypsin or elastase assays, ELISA, antisera, and antibodies. IV. Cultured cells will be used to study the effect of chronic wound fluid on cell adhesion, spreading, migration and FN synthesis. Cells cultured in tissue culture flasks will be exposed to wound fluids, purified metalloproteinases, FN degrading proteases, inhibitors and TGF-beta to study effects on cells, cell-matrix interactions and matrix synthesis.
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