This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cardiovascular disease is the number one cause of death in the United Sates, and atherosclerosis and restenosis are responsible for the majority of these deaths. Neointimal expansion in atherosclerotic blood vessels and during restenosis is characterized by a series of events that reflect an attempt of the resident vascular cells to repair the dysfunctional blood vessel wall. Events such as: phenotypic changes in vascular smooth muscle cells leading to altered production of extracellular matrix, activation and recruitment of inflammatory cells, and recruitment of circulating endothelial progenitor cells reflect an attempt to effectively repair the blood vessel wall by establishing a functioning endothelium. However, in disease these processes may be insufficient or not properly controlled leading to a cascading propagation of the """"""""injured/inflamed"""""""" state and continued vascular dysfunction. At the center of this stimulus-response network is the extracellular matrix (ECM), which both regulates and is regulated by the resident cells in the blood vessel wall. In this project we will define the central role of the ECM as a regulator of vascular growth factor activity. This collaboration involves functional characterization of heparan sulfate (HS) populations that bind fibroblast growth factors (FGFs). The activities of HS oligosaccharides of interest are being probed using a BaF32 cellular assay. The Baf32 cells are deficient in HS synthesis and lack FGF and FGF receptor expression. The cells have been engineered to express FGF receptor and will undergo FGF-mediated mitogenesis only when both FGF and heparin/HS are present. This assay has been scaled down for analysis using a fluorescence multiwall reader. Using this approach, it will be possible to determine the ability of defined HS fractions to induce or inhibit mitogenesis, as appropriate. Progress in 2010: The BaF32 assay was used to demonstrate that HS dp6 that bound to FGF2 showed increased activity in potentiating FGF2 signaling response relative to control dp6 (1). We are now using the glycomics methods developed during previous years to determine the changes to HS expression in aorta during balloon injury. HS was extracted from normal and injured rat aorta tissue. An aliquot of the HS was subjected to disaccharide analysis using a series of lyase enzymes to produce information on HS domain structure. The data will be analyzed using an algorithm """"""""ChamP"""""""" developed by Nugent and co-workers at BUSM. In addition, we are acquiring LC/MS data on HS oligosaccharides corresponding to N-sulfated domains so as to extend the structural detail on HS structure during injury. 1.
N aim y, H., Buczek-Thomas, J. A., Nugent, M. A., Leymarie, N., and Zaia, J. (2011) Highly sulfated non reducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions., J Biol Chem accepted 3/24/11.
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