Neal S. Fedarko

Bone Sialoprotein Mediates Human Endothelial Cell Attachment and Migration and Promotes Angiogenesis

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via alpha(v)beta(3) integrin receptors. Ligands for alpha(v)beta(3) integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC alpha(v)beta(3) integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-alpha(v)beta(3) but not anti-alpha(v)beta(5) monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-alpha(v)beta(3) antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.

High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures

Four proteoglycans and hyaluronan synthesized by cultured human bone cells were isolated using a two-step high-performance liquid chromatography system involving desalting and buffer exchange with a TSK-GEL HW 40(S) column followed by ion-exchange separation on a Nucleogen 4000-10 DEAE column. The desalting of 4 M guanidinium HCl extracts by a TSK-GEL HW 40(S) column equilibrated in a formamide:KH2PO4 buffer produces greater than 95% recoveries, enables quantitation of label incorporation and requires only 40 min to complete. The Nucleogen 4000-10 DEAE column utilizes the same buffer system and requires only 100 min for the resolution of four distinct types of proteoglycans. The formamide:KH2PO4 buffer system is compatible with a previously developed polyacrylamide gel system for the electrophoretic profiling of proteoglycans. After separation by charge density, proteoglycans were further resolved by size distribution using a calibrated TSK-GEL HW 75(F) column which also enabled the estimation of the apparent Mr of hyaluronan produced by the bone cells. The same TSK-GEL HW 40(S) resin is used to exchange pooled proteoglycans into buffers for analyzing enzyme digests of glycosaminoglycan chains and core proteins. The technique has been applied to the analysis of biosynthetically labeled proteoglycans produced in culture by fetal and adult human bone cells. A distinct pattern of proteoglycan size and secretion for both cell types could be shown using this method. The method of analysis is useful for high yield and rapid screening of various cell types for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.