Abstract P161: Conversion of Secreted High-Molecular-Weight FGF-2 to an 18 kDa Low-Molecular-Weight FGF-2 by Limited Proteolysis Suppresses the Paracrine Induction of Cardiomyocyte Hypertrophy

Abstract

Background: High molecular weight, 23 kDa rat FGF-2 (Hi-FGF-2) is composed of an N-terminal extension as well as the 18 kDa core sequence present in low molecular weight (Lo-) FGF-2. Hi- and Lo-FGF-2 have distinct biological activities: unlike Lo-FGF-2, Hi-FGF-2, which is secreted by cardiac non-myocytes, promotes cardiomyocytes hypertrophy in vivo and in vitro . We tested if the paracrine pro-hypertrophic effects of secreted Hi-FGF-2 can be blunted by limited proteolysis of its N-terminal extension. Experimental: Heparin-sepharose-bound Hi-FGF-2 present in rat tissue extracts converted to an approximately 18 kDa protein (*Lo-FGF-2) through truncation of its N-terminal extension, in the absence of protease inhibitors. Serine protease inhibitors such as PMSF fully prevented this conversion. Similarly, purified recombinant rat Hi-FGF-2 was converted to *Lo-FGF-2 by thrombin, a serine protease, and the effect was prevented by hirudin, a thrombin specific inhibitor. Conditioned media from unstimulated or angiotensin II-stimulated rat cardiac non-myocytes (fibroblasts) were used to treat neonatal cardiomyocytes in culture, in the absence or presence of thrombin. Conditioned medium from stimulated (but not unstimulated) cells contained Hi-FGF-2, which promoted cardiomyocyte hypertrophy (increase in cell surface area), and the effect was due to secreted FGF-2 because it was prevented by neutralizing anti-FGF-2 antibodies. Thrombin itself had no effect on cell size but prevented the pro-hypertrophic effect of the conditioned media; while converting the secreted Hi-FGF-2 to *Lo-FGF-2. Conclusions: The truncation of the N-terminal extension of secreted Hi-FGF-2 by serine protease(s) can provide a strategy to prevent undesirable paracrine effects of Hi-FGF-2 (such as cardiac hypertrophy), while maintaining potentially beneficial (cytoprotective, angiogenic) effects associated with Lo-FGF-2.