Abstract. Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. 130:393-400). The actual requirement of ECMassociated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.ROLE for cell-matrix interactions in the control of cell proliferation and differentiation has been demonstrated both in vivo and in vitro (6,19,21,23,44). The extracellular matrix (ECM) ~ deposited by cultured bovine corneal endothelial cells (ECs) stimulates proliferation of vascular ECs (17) and neurite extension in the sympathetic cell line PC12 (10, 42). Both these activities are also induced by basic fibroblast growth factor (bFGF) when added to the tissue culture medium (36, 41). Recently, we have extracted bFGF-like growth factors from the subendothelial ECM produced by cultured corneal ECs (39) and from Descemet's membranes of bovine cornea (8). We have also demonstrated that the ECM-associated bFGF is bound to heparan sulfate proteoglycans (HSPGs) (2), suggesting that HSPGs provide a natural storage depot for bFGF and possibly other heparinbinding growth factors (32). However, in view of the molecular complexity of ECM and basement membranes, we could 1. Abbreviations used in this paper: bFGF, basic fibroblast growth factor; CE-ECM, corneal endothelial cell extracellular matrix; ECs, endothelial cells; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycan; NGE nerve growth factor. not ascribe a specific biological activity to the ECM-bound bFGEThe present study was undertaken to investigate the involvement of the ECM-associated bFGF in its induction of EC proliferation and PC12 differentiation. For this purpose we applied two approaches: (a) use of anti-bFGF antibodies which inhibit the activity of soluble bFGF (26); and (b) comparison of the ECM produced by PF-HR-9 mouse endodermal carcinoma cells (3, 25), which do not synthesize bFGE and by PF-HR-9...

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Human urine contains a specific inhibitor of interleukin 1 (IL 1) that is found in increased amounts during fever. This inhibitor was purified by using a sequence of ammonium sulfate fractionation, DEAE cellulose ion-exchange chromatography, molecular sieve chromatography on Sephacryl S-200, affinity chromatography on concanavalin A (Con A) Sepharose, and polyacrylamide gel electrophoresis. Two peaks of IL 1 inhibitory material were eluted from the polyacrylamide gels. One peak contained three proteins of 29, 32, and 67 kd, respectively, which could be visualized by silver staining. The second peak contained only a small amount of the 67 kd protein. A partially purified inhibitor fraction was found to cross-react with antisera directed against two low m.w. urine trypsin inhibitors that are cleavage products of the serum inter-alpha-trypsin inhibitor. Although these findings suggest that the urine IL 1 inhibitor may be related to the inter-alpha-trypsin inhibitor, a more exact identification will require either a homogeneous inhibitor preparation or a monospecific antiserum.

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