Disordered wound healing is a significant clinical problem resulting in both the exaggerated healing of post-bum hypertrophic scar and the inadequate healing that results in chronic wounds. We and others have documented significant changes in the relative concentration of three particular cytokines in these clinical situations. These are transforming growth factor-beta, TGF-beta, tumor necrosis factor-alpha, TNF-alpha and interleukin-8, IL-8. These mediators are present in increased amounts in abnormally healing wounds and induce changes in the cells that populate the wounds and the matrix environment in which the cells reside. For example, we have shown increased burn scar production of autocrine and paracrine TGF-beta induces dermal fibrob lasts to synthesize excessive Types I and III collagen and contract matrix at an increased rate. TNF-alpha up-regulates both MMP-2 and -9, two proteases which have been linked to wound chronicity. IL-8 decreases keratinocyte replication and the ability of fibroblasts to contract matrix. The Goal of this proposal is to determine the mechanisms whereby these mediators regulate the interactions between skin cells and matrix. The experimental strategy is based on two primary aims. First, cytokine induced changes in content, synthesis, phosphorylation and organization of cytoskeletal proteins; alpha-smooth muscle actin, talin, vinculin, and alpha2Beta1 integrin in response to cytokine treatment will be characterized using biochemical and molecular biologic approaches. Particular attention will be directed to the role of Rho/GTPases as a critical signaling system in fibroblast reorganization of matrix. Second, the effects of these cytokines on the induction, synthesis and activation of matrix metalloproteases will be determined. We will analyze the promoter regions of MMP-2 and -9 for response elements to cytokines and individual matrix proteins as a specific mechanism for their regulation. Finally, the effects of cytokine-mediated modulation of MMPs on collagen contraction will be documented. These studies will increase our understanding for the mechanism of cytokine-induced abnormalities of wound healing. The long-term goal of this project is to uncover key mechanisms underlying hypertrophic scarring that may lead to improved therapeutic and, perhaps, preventive strategies.
