Destruction of basement membranes and neovascularization are critical processes tumor progression which often coincide upon the transition of an in situ carcinoma from the prevascular to the vascularized state. The long term objective of the proposed research is to elucidate the involvement of heparanase and extracellular matrix (ECM)-bound endothelial cell (EC) growth factors in tumor metastasis and angiogenesis. We have previously demonstrated that the capacity of various normal and malignant cells to extravasate correlates with heparanase mediated degradation of heparan sulfate (HS) in subendothelial ECM. It was found that basement membranes contain and sequester angiogenic EC growth factors that are structurally related to basic fibroblast growth factor (bFGF) and are released upon degradation of the ECM HS by heparanase. The proposed research is presented in two interrelated major goals: 1) Characterization of normal and malignant cell heparanases and their involvement in cell invasion and tumor metastasis; and II) Characterization and physiological significance of ECM storage and release of FGF.
The specific aims of the first series of experiments are; 1) purification and characterization of heparanases from a human hepatoma cell line (SK-hep_1 ) and normal human neutrophils; 2) preparation of anti-heparanase antibodies for the purpose of detection, quantitation and localization of heparanase in blood samples, malignant effusions and biopsy specimens and for studies on the involvement of heparanase in tumor metastasis; and 3) investigation of heparanase expression in normal T-lymphocytes and its involvement in cell traffic.
Specific aims of part II are to study: 1) involvement of ECM HS-bound FGF in EC proliferation: 2) deposition interaction with HS, and in-vivo localization of ECM-bound FGF; and 3) involvement of heparanase and heparin-like molecules in FGF release from basement membranes: possible role in neovascularization. The methodology for achieving these goals includes advanced techniques for EC culture and preparation of ECM; biochemical and immunological methods for characterization, detection and localization of heparanase and EC growth factors and use of PF-HR9 cells transfected with various FGF expression constructs to study deposition and function of ECM-bound bFGF. Some of the results are expected to have potential clinical applications in cancer metastasis and vascular diseases.
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