Heparin Mimics Research Articles

Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor

Molecules that mimic the sulfated glycosaminoglycan heparin and bind to heparin-binding growth factors would serve as important building blocks for synthetic biomaterials, e.g. to create a growth factor reservoir within a matrix. Peptide-based heparin mimetics would be particularly attractive, given the ease of peptide synthesis and modification. A sulfated tetrapeptide that fits this description and binds to vascular endothelial growth factor (VEGF) was discovered using a rationally-designed combinatorial approach. A ∼6600 member library of tetrapeptides, designed to include heparin functionality, was synthesized by solid-phase Fmoc chemistry. The library was analyzed on-resin for VEGF binding using a fluorescence assay that employed a 7-amino-4-methylcoumarin-modified VEGF 165. The beads were ranked according to fluorescent signal and SY(SO 3)DY(SO 3) was identified as the top binder. The binding affinity of the peptide for VEGF 165 was ascertained by surface plasmon resonance and compared with the heparin mimic suramin; the peptide binds to VEGF 165 100-fold stronger than the sulfonated compound. These results suggest that the identified peptide may be useful in biomaterial applications where binding of VEGF is desired.

Interactions of Multiple Heparin Binding Growth Factors with Neuropilin-1 and Potentiation of the Activity of Fibroblast Growth Factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.

Metachromatic activity of β-cyclodextrin sulfates as heparin mimics

Heparin is a versatile biologically active substance which has been reported to have an inhibitory effect on angiogenesis if administered together with hydrocortisone. Since very little is known about the mechanism of this activity, β-cyclodextrin sulfates were prepared to mimic heparin. The sulfate groups were introduced into β-cyclodextrin regioselectivly using protecting groups. The obtained polyanions were tested for their complex binding properties by mixing them with cationic dyes and measuring the metachromatic response which proved to be a very useful tool to evaluate the biological activity of these compounds. The results reveal that the activity depends largely upon the charge density at the surface of the β-cyclodextrin sulfates: a large number of sulfate groups or anionic groups relatively close to each other display high activity, whereas molecules with fewer sulfate groups or with them more distant from each other exhibit smaller activities.

In vitro selection of phosphorothiolated aptamers

A pool of RNA molecules that contained exclusively phosphorothioate internucleoside linkages was used as a starting point for the selection of aptamers that bind to basic fibroblast growth factor (bFGF), and appear to act as heparin mimics.

Inhibition of smooth muscle cell proliferation and experimental angioplasty restenosis by beta-cyclodextrin tetradecasulfate.

Heparin inhibits smooth muscle cell proliferation in vitro, a property that makes it potentially useful in preventing restenosis after angioplasty. Its utility in this setting is limited by the inability to use high doses (secondary to anticoagulant effects) and the need for subcutaneous administration. We tested the ability of beta-cyclodextrin tetradecasulfate (CDT), a nonanticoagulant synthetic heparin mimic, to inhibit smooth muscle cell proliferation in vitro and tested its efficacy when orally administered for the prevention of angioplasty restenosis in a rabbit atherosclerosis model. Vascular smooth muscle cells were cultured from rabbit aortas by the explant technique. Passaged cells were plated at low density in microtiter plates in the presence or absence of varying concentrations of heparin or CDT in culture medium containing 10% fetal calf serum. Using both 3H-thymidine incorporation and total protein assays, both heparin and CDT caused a similar dose-dependent inhibition of proliferation. We next tested the effect of orally administered CDT in the prevention of restenosis in focal femoral artery arteriosclerotic lesions created in hypercholesterolemic New Zealand White rabbits by air-dessication endothelial injury and subsequent peripheral angioplasty. Animals were followed up for 1 month and were fed normal chow supplemented by tap water with or without CDT. In animals receiving the highest concentration of CDT (2 mg/mL drinking water), the percentage of arterial cross-sectional area with intimal hyperplasia decreased from 50.5 +/- 1.7% (control) to 26.9 +/- 2.2% (p < 0.001), with the intimal/medial ratio being decreased from 1.4 +/- 0.4 to 0.5 +/- 0.2 (p = 0.056).(ABSTRACT TRUNCATED AT 250 WORDS)