This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is known that the action of vascular endothelial growth factor (VEGF) is regulated through its interactions with components of the extracellular matrix, such as fibronectin and heparan sulfate. One of the mechanisms by which fibronectin exerts its regulatory activity could be by directly binding to VEGF. We conducted a detailed study of the interactions between VEGF and fibronectin and investigated how heparin modulates this interaction using a combination of in vitro binding assays and atomic force microscopy. Plasma fibronectin was adsorbed either on hydrophobic or hydrophilic surfaces in the presence and absence of heparin and equilibrium binding of soluble 125I-VEGF165 was measured. The fibronectin bound VEGF was released into two successive fractions by incubations with a high ionic strength solution and a NaOH solution respectively, which represent distinct populations of VEGF binding sites on fibronectin. We found two VEGF binding sites per fibronectin dimer, mainly ionic in nature, with a Kd value in the range of 1-6*10-7 M. One of these binding sites is pH sensitive and interacts with VEGF only at low pH, a condition encountered in hypoxic tissues. The pH sensitive binding of VEGF to fibronectin may be reflective of a regulatory mechanism by which fibronectin directs VEGF to hypoxic areas in order to promote angiogenesis. The other binding site is not sensitive to pH, but is highly affected by fibronectin conformation in that it becomes available only under conditions where fibronectin adopts an extended conformation. Heparin stabilizes the extended conformation and consequently increases VEGF binding to this site on fibronectin. The effect of heparin potentially reflects a process whereby heparan sulfate within the extracellular matrix can act as a docking site for fibronectin to coordinate its interactions with VEGF. Consistent with this possible role, adsorption of fibronectin to hydrophilic surfaces, which also favors the extended conformation, increased VEGF binding. The effects of heparin on fibronectin conformation were studied by atomic force microscopy with fibronectin adsorbed on mica in the presence or absence of heparin. Under these conditions, three structural populations of fibronectin molecules were identified: compact molecules of an ellipsoid shape and molecules of a more extended configuration, containing either two or three distinct domains. The length of the molecules belonging to the two- and three-domain categories was significantly increased in the presence of heparin. Changes in the diameter and height of the individual domains that accompany this increase in length suggest that heparin triggers a partial unfolding of fibronectin, exposing various binding sites on the surface of the molecule. This effect is dependent on the particular chemical composition and size of heparin, as other glycosaminoglycans and certain chemical derivatives and fragments of heparin do not act in a similar way. Our data suggest a potential role for heparan sulfate proteoglycans to regulate angiogenesis by modulating the interactions between VEGF and fibronectin within the extracellular matri
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