The PI is a physician trained in internal medicine and clinical hematology/oncology. As a research fellow working with the late Dr. Mark Roth, she was introduced to the concept of detection of minimal residual disease in malignancies, and learned to apply molecular biology techniques to studying this problem. She then joined the laboratory of Dr. Bruce Ewenstein in order to learn more about vascular cell biology and signal transduction. This award will help her to develop her skills in critical evaluation of experimental data and biomedical literature, and in experimental design and problem solving, as well as enabling her to gain new technical skills. The PI's ultimate goal is to become an independent investigator, in charge of her own research laboratory at a major biomedical institution. The sponsorships of Dr. Ewenstein and Dr. Fritz Bach along with the guidance from advisors Dr. Robert Handin and Dr. Robert Rosenberg will help her to achieve these goals. The Longwood Medical Area provides outstanding resources that the PI will utilize in her training, including the Harvard Medical School classes and library, and numerous research and educational seminars, allowing interaction with outstanding researchers. In this revised proposal the PI will focus on studying the sorting of proteins into the Weibel-Palade bodies (WPB) in endothelial cells (EC). WPB undergo exocytosis in response to a variety of natural and pharmacologic agonists, such as thrombin and PMA, releasing von Willebrand factor and expressing P-selectin on the cell surface. Increased knowledge of the sorting of proteins into WPB will aid in designing strategies to modulate this process for therapeutic purposes. A recombinant adenoviral vector system will be used for efficient transduction and expression of heterologous proteins in cultures EC. First will be determined whether growth hormone, a secretory protein studied in other cell types, is sorted to WPB when expressed in EC, and whether fusion of growth hormone to proteins not secreted in a regulated manner will direct those proteins to those granules. Next will be determined whether the P-selectin cytoplasmic domain is sufficient for sorting heterologous proteins into WPB in EC, since this domain has been shown to allow for sorting into secretory granules in other cell types. Lastly will be determined whether the P-selectin cytoplasmic domain fused to thrombomodulin, an anti-thrombotic protein, can be targeted to the WPB for rapid expression in response to agonists. The system could theoretically be used therapeutically in a variety of clinical conditions that involve thrombosis and inflammation, such as xenotransplantation and coronary artery disease.
| Datta, Y H; Youssoufian, H; Marks, P W et al. (1999) Targeting of a heterologous protein to a regulated secretion pathway in cultured endothelial cells. Blood 94:2696-703 |
