No abstract

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A165 and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A165 binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A165 showed that heparin can also modulate the conformation and secondary structure of the protein.

In vitro devices in combination with cultured cells have been used to study the relationship between shear stress and endothelial injury. Almost exclusively, these investigations have used confluent monolayers and conventional culture media as perfusates and reported little cell loss over a wide range of shear stress conditions. In this investigation when subconfluent endothelial cells were exposed to 22 and 88 dyn/cm2 for 2, 8, and 24 h in a perfusate of medium and 5% serum, a progressive cell loss was observed. Lower cell densities were a product of decreased cell proliferation as measured by bromodeoxyuridine (BrdU) incorporation and loss of the initial cell population. Video recordings indicated that cells characteristically detached by proximal cell peeling from the substrate and an aneurysmal rupture of the cell membrane. Cell retention was increased by including 250 and 475 microM neutral dextran (70 kDa) in perfusates. Experimental evidence suggests dextran does not directly stimulate proliferation or correct an osmotic imbalance. This investigation has substantiated that fluid-generated shear stress can cause endothelial denudation and that conditions (subconfluency, time, and perfusate supplementation) under which shear stress is applied are as important for cell survival as shear stress magnitude.