FGFS AND THEIR RECEPTORS, IN-VITRO AND IN-VIVO STUDIES - NEW FGF RECEPTOR IN THE BRAIN, FGF-1 IN MUSCLE, AND THE USE OF FUNCTIONAL ANALOGS OF LOW-AFFINITY HEPARIN-BINDING GROWTH-FACTOR RECEPTORS IN TISSUE-REPAIR

Several heparin-binding growth factors (HBGFs) are thought to play a k ey role in the natural processes of tissue homeostasis, regeneration o r repair. The HBGFs are active upon release from neighbouring inflamma tory or circulating cells, as well as upon release from heparan sulfat e proteoglycosaminoglycans that are associated with the extracellular matrix (ECM). To better understand the physiological role of these HBG Fs, we have focused our effort on studying a subset of HBGFs, namely F GF-1 and FGF-2 and their receptors. We present the purification and ch aracterisation of a new form of heparin-binding FGF receptor from adul t bovine brain (Perderiset et al., 1992). This receptor has now been p urified to homogeneity. Ligand blot and cross-linking experiments perf ormed with labeled FGF-1 or FGF-2 revealed 80-kd and 130-kd bands. Pre liminary sequence information indicates that receptor is different fro m the receptors, FGFR-1 to -4, but it may be related the cysteine-rich -FGF receptor (CFR). We have previously shown that FGF-1, but not FGF- 2, is specifically expressed in myoblastic satellite cells during the proliferating phase preceding myoblast alignment and fusion. We have n ow transfected primary cultures of rat myoblastic satellite cells with FGF1 cDNA and expressed this growth factor constitutively. The transf ected cells were no longer able to form myotubes. Transfection with an tisense FGF-1 induced myotube formation suggesting that endogenous exp ression of FGF-1 is associated with myoblastic cell differentiation. N umerous studies have concluded that the ECM represents a natural reser voir for various HBGFs. The HBGFs are stored through their interaction with matrix-associated heparan sulfates and can become available for stimulating cell migration, multiplication, and differentiation during tissue repair. In order to better understand how the control of the b ioavailability of HBGFs plays a role in wound healing, we have studied the healing effect of various chemically substituted dextrans (CMDBS) selected for their affinity for HBGFs, alone and in association with HBGFs. The CMDBS were obtained by substitution of methyl carboxylic (C M), benzylamide (B), and benzylamine sulfonate (S) groups. We expected that the CMDBS could act in the following ways: (1) to potentiate the biological activity of FGF-1 and FGF-2; (2) to protect FGF-1 and FGF- 2 against thermal or pH inactivation; or (3) to protect FGF-1 and FGF- 2 against proteolytic degradation (Tardieu et al., 1999). Selected CMD BSs were tested alone in cutaneous and flat bone wound-healing models. Rats were skin punched and skin regeneration was studied by morphomet ric and histological analysis. The wounds (6-mm diameter) were filled with collagen plaster alone or soaked with CMDBS. The CMDBS in collage n plaster was able to induce a remarkable effect both on the kinetics and on the quality of the restored skin. We have also looked at the ef fect of CMDBS in the healing of calvarian bone defects. Adult rats wer e trephined (5-mm diameter) and the healing of their defects was deter mined after 35 days. Only those treated with CMDBS show appreciable ne w bone formation. The filling of defects was almost complete, and the response was dose-dependent. Optimal doses were at 50-100 mu g CMDBS, while at 400 mu g CMDBS, less bone was formed. Interestingly, when the defect included bone suture, the suture was restored after CMDBS trea tment. We have measured by biochemical, immunological, and histologica l methods the content and distribution of several HBGFs in the granula tion tissue formed during healing, in the presence or absence of CMDBS . Our results suggest that the endogenous HBGFs naturally released dur ing the regeneration process could be trapped, protected, and released by CMDBS. In conclusion, biopolymers can be designed to mimic some of the mechanisms regulating the availability of growth factors and so c ould be used as wound-healing agents, (C) 1994 Wiley-Liss, Inc.