The goal of this work is to correlate heparan sulfate structure with angiogenic function in vascular tissue. Both normal and pathological angiogenesis are mediated through growth factor stimuli that depend on the structures of cell surface and extracellular matrix heparan sulfate (HS) chains for receptor activation. HS serves as both a spatial and temporal regulator of growth factor activity during angiogenesis. The diversity of HS biological activities arises through their non-template driven biosynthesis. A series of modifying enzymes act upon nascent HS chains to produce mature molecules with characteristic domains of high and low sulfation. Chain lengths and degree of sulfation are heterogeneous and reflect the responses of cells to their growth environment. Variation in expression of HS chain structure is a mechanism whereby cells modulate their responses to growth factor stimuli. In this sense, the diversity of HS chains on cell surfaces and secreted proteins is a means of elaborating the functions of a limited array of growth factors and growth factor receptors. In vascular tissue, homeostasais and mitogenesis are controlled through growth factor signalling cascades. Fibroblast growth factors (FGFs), and their receptors bind HS chains on the cell surface and the extracellular matrix. At the present time, it is clear that both high and low affinity HS domains exist and play important roles in modulating angiogenic responses. Although it is also clear that such subsequences may either potentiate or inhibit growth factor, depending on the context, there is little information concerning their structures. In order to address these questions, new methods will be developed in Aim 1 to enable sequencing of HS compatible with on-line liquid chromatography-tandem mass spectrometry.
In Aim 2, libraries of organ-specific HS will be prepared using two dimensional chromatography. The structures will be determined using on-line tandem mass spectrometry and correlated with function using cell free growth factor binding and cellular mitogenesis assays.
Aim 3 is to determine the manner in which extracellular enzymes (mammalian endo- sulfatases and heparanase) remodel HS chains in biological systems.
In vascular tissue, both normal and pathological cell growth related to angiogenesis is regulated through growth factor signaling cascades. Cells modulate their responses to growth factor signaling by altering the structures of heparan sulfate chains expressed on their surfaces and secreted into the extracellular matrix. This research will explore the structure-function relationships of vascular heparan sulfates in order to inform efforts to develop protein binding microarrays, drugs and therapeutics.
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