| The research in our laboratory is focused on the response of the retinal pigment epithelium (RPE) to injury and how RPE structure and function are altered in pathologic retinal disorders such as proliferative vitreoretinopathy (PVR). RPE cells regulate the balanced homeostasis of growth factors and extracellular matrix (ECM) proteins. We hypothesized that fibronectin EDA (FN-EDA), an ECM molecule implicated in excessive scar formation and chronic fibrosis, is expressed and regulated by activated RPE cells and thus plays an essential role in PVR pathogenesis.;Using primary cultures of human RPE cells, we detected FN-EDA protein under normal conditions but not in serum-deprived cells. We found that serum increases the content of FN-EDA in these cultures in a dose-dependent manner. Therefore, all subsequent experiments were carried out in the absence of serum to eliminate any serum-related effects on FN-EDA regulation by RPE cells. In frozen sections of normal human retinas, though FN-EDA was present in retinal and choroidal vessels, the RPE monolayer was devoid of FN-EDA. These results suggest that resting RPE cells do not express FN-EDA.;In growth factor-stimulated cultures of RPE cells, we found that FN-EDA mRNA and protein were induced by transforming growth factor-beta2 (TGFbeta2) in a time- and dose-dependent manner but not by connective tissue growth factor (CTGF). By co-stimulating RPE cultures with TGFbeta2 and CTGF, we demonstrated that CTGF, through its N-terminal domain, augments the TGFbeta2-induced expression of FN-EDA at the protein level. Using CTGF domain-specific antibodies, we blocked this synergistic effect. By protein-protein interaction studies, we established that CTGF directly interacts with TGFbeta2 and its receptor TGFbetaRII at its N- and C-terminal domains, respectively. Our results therefore suggest that CTGF modulates TGFbeta2 responses and provide further evidence for CTGF as a down-stream mediator of TGFbeta2.;Finally, we demonstrate that FN-EDA is abundantly expressed in PVR membranes in a pattern that co-localizes with TGFbeta2 and CTGF. FN-EDA upregulates type I collagen in cultures of RPE cells as do TGFbeta2 and CTGF. These findings suggest that FN-EDA is an important mediator of retinal fibrosis, making it a potential target for therapy. |
