Rosa M. Lozano

The Activation of Fibroblast Growth Factors by Heparin: Synthesis, Structure, and Biological Activity of Heparin‐Like Oligosaccharides

An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D‐glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right‐hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well‐defined structural models of naturally occurring heparin‐like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half‐maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin‐induced FGF1 dimerization is not an absolute requirement for biological activity.

A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2

Platelet factor 4 (PF‐4) is a CXC‐chemokine with strong anti‐angiogenic properties. We have shown previously that PF‐4 inhibits angiogenesis by associating directly with fibroblast growth factor 2 (FGF‐2), inhibiting its dimerization, and blocking FGF‐2 binding to endothelial cells. We now have characterized a small peptide domain (PF‐447–70) derived from the C‐terminus of PF‐4, which conserves anti‐angiogenic effects of the parent protein. PF‐447‐70 inhibited internalization of 125I‐FGF‐2 by endothelial cells in a time‐dependent manner. The peptide reduced FGF‐2‐stimulated cell migration to control levels in wounded monolayers of bovine capillary endothelial cells. PF‐447–70 also reduced FGF‐2 induced phosphorylation of MAP kinases ERK‐1 and ERK‐2, which are essential for migration and survival of endothelial cells. In a serum‐free exvivo angiogenesis assay, the peptide blocked microvessel outgrowth by 89%. A single amino acid substitution within PF‐447–70 abolished all inhibitory activities. To simulate a real anti‐angiogenic treatment situation, we administered PF‐447–70 systemically to mice implanted subcutaneously with FGF‐2 containing gelatin sponges with the result of sparse, scattered, and immature vessel growth. The small peptide fragment derived from the angioinhibitory CXC‐chemokine PF‐4 might be used as a starting point to develop anti‐angiogenic designer drugs for angiogenesis‐dependent pathologies such as cancer, diabetic retinopathy, and rheumatoid arthritis.

The Activation of Fibroblast Growth Factors (FGFs) by Glycosaminoglycans: Influence of the Sulfation Pattern on the Biological Activity of FGF-1

Six synthetic heparin‐like oligosaccharides have been used to investigate the effect of the oligosaccharide sulfation pattern on the stimulation of acidic fibroblast growth factor (FGF‐1) induced mitogenesis signaling and the biological significance of FGF‐1 trans dimerization in the FGF‐1 activation process. It has been found that some molecules with a sulfation pattern that does not contain the internal trisaccharide motif, which has been proposed for high affinity for FGF‐1, stimulate FGF‐1 more efficiently than those with the structure of the regular region of heparin. In contrast to regular region oligosaccharides, in which the sulfate groups are distributed on both sides of their helical three‐dimensional structures, the molecules containing this particular sulfation pattern display the sulfate groups only on one side of the helix. These results and the fact that these oligosaccharides do not promote FGF‐1 dimerization according to sedimentation‐equilibrium analysis, confirm the importance of negative‐charge distribution in the activation process and strongly suggest that FGF dimerization is not a general and absolute requirement for biological activity.

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors.

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Protection of Rat Myocardium by Mitogenic and Non-Mitogenic Fibroblast Growth Factor During Post-Ischemic Reperfusion

The effects of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) and a non mitogenic form of FGF1 on myocardial ischemia and reperfusion were assessed. Rats underwent 10 minutes of coronary artery occlusion followed by 24 hours of reperfusion. Creatinine kinase content of the affected myocardium showed that both fibroblast growth factors 1 and 2 effectively protected against ischemia reperfusion injury (p < 0.01), and that the vasoactive but nonmitogenic form of the FGF1 was equally protective (p < 0.01 versus control + vehicle). The results were confirmed by light and electron-microscopy histological studies. Histological evaluations after treatment with the non-mitogenic fibroblast growth factor 1 showed that it did not generate the severe hyperplasia and connective tissue disorganization observed with the native mitogenic proteins. The possibility of using a non-mitogenic form of fibroblast growth factor for cardio-protection circumvents many of the potentially undesirable effects that may derive from systemically introducing broad spectrum acting fibroblast growth factors in vivo. This myocardial protection observed 24 hours after the treatment with fibroblast growth factors, and the efficacy of the non-mitogenic form of the protein, also suggest that the protective effect of fibroblast growth factors may be due to the increased blood flow rather than to angiogenesis.

Solution NMR structure of a human FGF-1 monomer, activated by a hexasaccharide heparin-analogue

The 3D structure of a complex formed by the acidic fibroblast growth factor (FGF‐1) and a specifically designed synthetic heparin hexasaccharide has been determined by NMR spectroscopy. This hexasaccharide can substitute natural heparins in FGF‐1 mitogenesis assays, in spite of not inducing any apparent dimerization of the growth factor. The use of this well defined synthetic heparin analogue has allowed us to perform a detailed NMR structural analysis of the heparin–FGF interaction, overcoming the limitations of NMR to deal with the high molecular mass and heterogeneity of the FGF‐1 oligomers formed in the presence of natural heparin fragments. Our results confirm that glycosaminoglycans induced FGF‐1 dimerization either in a cis or trans disposition with respect to the heparin chain is not an absolute requirement for biological activity.

Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity.

Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue C-terminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factor-driven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

Systemic administration of acidic fibroblast growth factor ameliorates the ischemic injury of the retina in rats

The central neuroprotective effects against ischemic injury of fibroblast growth factor (FGF), administered either directly into the central nervous system or systemically, is well documented. Here we show in a rat model of transient retinal ischemia that the neuroprotective effect of systemically administered acidic fibroblast growth factor (aFGF, FGF-1) extends to the retina. Histological findings show a lower decrease of retinal ganglion cells and inner nuclear layer cells (P < 0.0001) in animals receiving FGF-1. These results suggest that FGF may function as a natural protection agent during transient retinal ischemia and further document that an efficient neuroprotection of central nervous tissues can be obtained by systemic administration of this protein. Our data may, thus, contribute to the development of novel and safe therapeutic approach for the treatment of the ischemic injury of the retina.