DESCRIPTION: Retrocorneal fibrous membrane (RCFM) occurs in various corneal diseases or after damage to the corneal endothelium. RCFM of Descemet's membrane is believed to represent an end-stage disease process of the corneal endothelium and can result in corneal opacity, fibrosis and subsequent blindness. The long-term goal of this project is to characterize the mechanism involved in the endothelial mesenchymal transformation (EMT) observed in RCFM. During EMT, three major phenotypes are altered in corneal endothelial cells (CECs): cell proliferation is markedly stimulated; cell shape is changed and contact-inhibited phenotypes are lost; and fibrillar extracellular matrix is produced. During the current funding period, we demonstrated that FGF-2 is the direct mediator for EMT and that PI 3-kinase is a major signaling molecule for both mitogenic and morphogenetic pathways. PI 3-kinase is involved in Gl/S transition as it regulates the expression of Cdk4 and p27, while PI 3-kinase regulates reorganization of actin cytoskeleton. Using a proteomic approach, we identified the protein factor released by inflammatory cells: the cytokine turned out to be interleukin-1beta (IL-1beta). Our pilot studies demonstrate two additional important results: 1) IL-1beta markedly induces FGF-2 synthesis; 2) FGF-2 greatly stimulates transcription of alpha1(I) collagen RNA, suggesting that transcription of alpha1(I) collagen RNA is the rate limiting step for type I collagen production. Our working hypothesis is that when cornea is injured, the infiltrating inflammatory cells release IL-1beta, which in turn greatly induces FGF-2. As a direct mediator of EMT, FGF-2 modulates type IV collagen-synthesizing polygonal CECs to type I collagen-synthesizing fibroblasts through the action of PI 3-kinase. We will test the hypothesis using our in vitro model of EMT induced by FGF-2; 1) we will investigate the mechanism by which FGF-2 enhances synthesis of type I collagen leading to secretion of the molecule; 2) we will focus on the mechanism of p27 degradation, for the activity of p27 is mostly controlled by its concentration; 3) we will investigate how Rho, Rac and Cdc42, and their cross-talks modulate CECs to fibroblastic cells; 4) we will study the role of IL-1beta during EMT; 5) we will attempt to block EMT by antagonizing the specific target proteins (FGF-2, PI 3-kinase, p27 and IL-1beta); and 6) we will test whether metabolic inhibitors that block EMT in the in vitro study will also block RCFM formation in vivo. Thus, our understanding of the whole spectrum of EMT will lead us to clinical applications for blocking corneal fibrosis observed in corneal endothelium-Descemet's membrane.