Fibroblast growth factor-2 (FGF-2) is a member of the FGF family that, like FGF-1 and FGF-9, lacks a signal sequence. These proteins therefore are not secreted through the endoplasmic reticulum and Golgi apparatus. Yet these factors act outside the cell by binding to cell surface GF receptors that mediate diverse biological effects including angiogenesis, tumor growth, bone formation, mitogenesis, migration, wound healing, and neuronal survival. Currently the mechanisms leading to the cellular release of these signal sequence-deficient FGFs are not well understood very little is known about the interaction of released FGF-2 with other proteins in vivo. We have previously demonstrated that released FGF-2 is a key player in mediating responses of the vessel wall to stress and injury which are involved in vascular repair and vascular disease. We have used heparin Sepharose chromatography to purify heparin-binding proteins from plasma and serum. High molecular weight bands (>50 kDa) resistant to denaturing and reducing conditions were detected with 3 different monoclonal antibodies against FGF-2 immuno reactive bands contained plasminogen and fibrinogen. MALDI-TOF mass spectrometry revealed that the FGF-2 immuno reactive bands contained plasminogen and fibrinogen. In a 3T3 cell assay, mitogenic activity susceptible to neutralizing anti-FGF-2 antibodies was associated with the heparin Sepharose purified plasma and serum fractions. Using bovine aortic endothelial (BAE) cells we demonstrate cross-linking of endogenous FGF-2 to added plasminogen. The cross-linking of released FGF-2 to plasma proteins such as plasminogen fragments (angiostatin) could be an important event in regulating the availability of FGF-2 by affecting its half life and localization and thereby controlling processes such as angiogenesis , wound healing and tumor growth. The goals of the present application therefore are to carry out the biochemical characterization of the enzyme that cross-links FGF-2 and explore the mechanism that lead to cellular FGF-2 release. In vivo models in transgenic and gene targeted mice will be used to demonstrate the physiologic significance of these mechanisms.
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