Our laboratory is currently conducting research into the role essential metals play in regulating proteins during maturation and differentiation of eukaryotic cells. Specifically, we are interested in the role copper plays in regulating angiogenesis and branching morphogenesis of human endothelial cells. It has long been known that copper plays a role in regulating angiogenesis. Copper or copper complexes have been shown to directly stimulate angiogenesis in numerous model systems; moreover, physiological depletion of bioavailable copper has been shown to inhibit angiogenesis in animal and xenograft models. Such findings have led to clinical trials for the treatment of human cancer by the chelation of copper with some efficacy in disease stabilization. While several proteins known to be essential for angiogenesis have been shown to be copper-dependent, the exquisite physiological sensitivity to this metal has as yet been unexplained. We are utilizing an in vitro model of angiogenesis and analyzing this process over time by x-ray fluorescence microprobe (XFM) analysis at the Sector 2 beamlines at the Advanced Photon Source. XFM analysis is based on the ability to focus coherent x-rays using fresnel zone plates to sub-micron beam spots as shown below:

Cells of interest are then raster scanned through this beam spot and the fluorescence that is induced by x-ray excitation is measured using an energy-dispersive detector. By such an approach, a large number of elements (from Z=15-30 by K-line fluorescence, higher Z elements by L-line and M-line) can be both spatially mapped and quantitated in biological specimens. Typical output from scans examining metal content in isolated HMVEC cells are shown below next to a bright-field optical image:

It is our overarching hypothesis that the interaction of a given metal ion with a metalloprotein is a dynamic event and that this interaction provides a type of posttranslational regulation of protein structure, activity, and partner-protein interactions. By analyzing metal content and distribution over time during cellular morphogenic and maturative differentiation, we hope to identify changes that correlate with the essential function of metalloproteins in these processes.