Control Of Vegf Binding To Fibronectin By Heparan Sulfate And Hypoxia

Abstract

Purpose: : To investigate the mechanisms and consequences of heparan sulfate-mediated modulation of VEGF binding to fibronectin. To determine how hypoxia influences this process through alterations in heparan sulfate.

Methods: : 125I-VEGF binding to fibronectin was measured with surface coated fibronectin plates. 125I-VEGF binding to cells was measured after subjecting the cells to periods of hypoxia (1% O2). Heparin and heparan sulfate (HS) alterations in fibronectin matrix structure were monitored using quartz crystal microbalance with dissipation technology, and changes in fibronectin fiber structure were investigated using antibody/fibronectin fluorescence ratiometric approaches. HS was isolated from normoxic and hypoxic cells by mild trypsin digestion and ion exchange filtration. The chemical structure of heparan sulfate was determined after lyase digestion with HPLC and MS. HS Domain organization was determined using the chainmaker computer program (ChamP). The function of cell-derived fibronectin matrices was evaluated by measuring the attachment, migration and differentiation of endothelial progenitor cells (EPCs). Differentiation was monitored by real-time PCR of endothelial cell marker mRNA expression.

Results: : Heparin and "active" HS catalytically modify fibronectin to expose binding sites for VEGF. Heparin-modified fibronectin matrices enhanced EPC differentiation compared to fibronectin, and VEGF further enhanced this response. EPC attachment and migration to vascular smooth muscle cell produced fibronectin matrices were increased when smooth muscle cells were subjected to hypoxia during matrix deposition. Hypoxia pre-conditioning led to increased expression and matrix deposition of VEGF, increased syndecan 4 and HS 6-O sulfotransferase-1 expression, and increased binding capacity for VEGF. The effects of hypoxia could be reproduced to some degree by treatment of normoxic cells with heparin. HS from hypoxic cells showed a dramatic increase in the relative ability to catalytically alter fibronectin structure and increase VEGF binding. The change in activity was accompanied by alterations in chemical structure of HS produced by hypoxic cells.

Conclusions: : These data indicate that HS-mediated alterations in fibronectin and VEGF deposition may contribute to generating extracellular matrix that promotes EPC homing and differentiation, which may contribute to hypoxia-induced angiogenesis.

Keywords: vascular endothelial growth factor • extracellular matrix • proteoglycans/glycosaminoglycans