Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

In recent years, the basement membrane (BM)--a specialized form of extracellular matrix (ECM)--has been recognized as an important regulator of cell behaviour, rather than just a structural feature of tissues. The BM mediates tissue compartmentalization and sends signals to epithelial cells about the external microenvironment. The BM is also an important structural and functional component of blood vessels, constituting an extracellular microenvironment sensor for endothelial cells and pericytes. Vascular BM components have recently been found to be involved in the regulation of tumour angiogenesis, making them attractive candidate targets for potential cancer therapies.

Basement membranes: structure, assembly and role in tumour angiogenesis

Basic fibroblast growth factor (bFGF) binds to heparan sulfate proteoglycans at the cell surface and to receptors with tyrosine kinase activity. Prevention of binding between cell surface heparan sulfate and bFGF (i) substantially reduces binding of fibroblast growth factor to its cell-surface receptors, (ii) blocks the ability of bFGF to support the growth of Swiss 3T3 fibroblasts, and (iii) induces terminal differentiation of MM14 skeletal muscle cells, which is normally repressed by fibroblast growth factor. These results indicate that cell surface heparan sulfate is directly involved in bFGF cell signaling.

https://science.sciencemag.org/content/sci/252/5013/1705.full.pdf

Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation.

Summary Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force-sensing has been garnered from studying cells cultured on two-dimensional (2D) glass or plastic surfaces. However, more recently the cell biology field has come to appreciate the dissimilarity between these flat surfaces and the topographically complex, three-dimensional (3D) extracellular environments in which cells routinely operate in vivo. This has spurred substantial efforts towards the development of in vitro 3D biomimetic environments and has encouraged much cross-disciplinary work among biologists, material scientists and tissue engineers. As we move towards more-physiological culture systems for studying fundamental cellular processes, it is crucial to define exactly which factors are operative in 3D microenvironments. Thus, the focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which – in turn – regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats. Additionally, we will describe experimental scenarios in which 3D culture is particularly relevant, highlight recent advances in materials engineering for studying cell biology, and discuss examples where studying cells in a 3D context provided insights that would not have been observed in traditional 2D systems.

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Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues

Fibroblast growth factors (FGFs) are small polypeptide growth factors, all of whom share in common certain structural characteristics, and most of whom bind heparin avidly. Many FGFs contain signal peptides for secretion and are secreted into the extracellular environment, where theycan bind to the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM). From this reservoir, FGFs mayact directlyon target cells, or theycan be released through digestion of the ECM or the activityof a carrier protein, a secreted FGF binding protein. FGFs bind specific receptor tyrosine kinases in the context of HLGAGs and this binding induces receptor dimerization and activation, ultimatelyresulting in the activation of various signal transduction cascades. Some FGFs are potent angiogenic factors and most playimportant roles in embry onic development and wound healing. FGF signaling also appears to playa role in tumor growth and angiogenesis, and autocrine FGF signaling maybe particularlyimportant in the progression of steroid hormone-dependent cancers to a hormone-independent state.

/pdf/fibroblast-growth-factors-their-receptors-and-signaling-4rbv35g0yr.pdf

Fibroblast growth factors, their receptors and signaling.

Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of size and sulfation of heparin in release of basic fibroblast growth factor from the vascular endothelium and extracellular matrix.

The invention relates to new heparin derivatives from bovine or porcine heparin which have a molecular weight equal to or larger than the standard heparin, show a sulfur content which is equal to or higher than that of the starting heparin or at least 13 % w/w, have an anticoagulant activity in the anti-FXa assay of less than 10 % of the standard heparin it was made from, show a ratio of APTT activity over anti-FXa activity of 3-35, show a reduced prolongation of bleeding time compared to the standard heparin it was made from as measured in the rat tail after i.v. administration and show enhancement of the rate of development of coronary collaterals in dogs equal to or better than clinically used heparin. The invention also relates to processes for preparation of the new bovine and porcine heparin derivatives. The use of this product for the manufacturing of a medicament for treatment of ischemic heart disease and related vascular disorders and for the enhancement of the rate of development of coronary collateral perfusion is disclosed.

Novel heparin derivatives and process for the preparation thereof

The invention relates to new heparin derivatives from bovine or porcine heparin which have a molecular weight equal to or larger than the standard heparin, show a sulfur content which is equal to or higher than that of the starting heparin or at least 13% w/w, have an anticoagulant activity in the anti-FXa assay of less than 10% of the standard heparin it was made from , show a ratio of APTT activity over anti-FXa activity of 3-35, show a reduced prolongation of bleeding time compared to the standard heparin it was made from as measured in the rat tail after i.v. administration and show enhancement of the rate of development of coronary collaterals in dogs equal to or better than clinically used heparin. The invention also relates to processes for preparation of the new bovine and porcine heparin derivatives. The use of this product for the manufacturing of a medicament for treatment of ischemic heart disease and related vascular disorders and for the enhancement of the rate of development of coronary collateral perfusion is disclosed.

Carl-Magnus Svahn

Papers

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.

Importance of size and sulfation of heparin in release of basic fibroblast growth factor from the vascular endothelium and extracellular matrix.

Novel heparin derivatives and process for the preparation thereof

Heparin derivatives for treatment of angina pectoris