The Mentored Career Development Award will give me the opportunity to receive the additional training and research experience necessary to achieve my long-term goal of obtaining an independent faculty position at a research-oriented university, and contribute to the field of Cardiovascular Biology. The proposed project is a valuable tool to extent my knowledge and expertise in complex research methods, including mouse and zebrafish genetics/molecular biology, that are applied in the cardiovascular field. The University of California, San Diego (UCSD) School of Medicine conducts research in a multidisciplinary and highly collaborative environment by interacting with colleagues that span clinical medicine to structural and molecular biology. Moreover, the Ginsberg laboratory is well equipped with cellular, proteomic, and molecular biology tools to understand cellular and molecular biology of the protein products of genes implicated in cerebral cavernous malformations (CCM) (KRIT1, Krev- Interaction Trapped-1, CCM2, and PDCD10). In order to identify the major molecular processes involved in loss of Krit1-induced altered endothelial phenotype and function, a cell culture model to delete Krit1 in mouse endothelium in a time-controlled manner was established. To this end, transgenic mice bearing floxed alleles of Krit1 (Krit1fl/fl) and an endothelial-specific tamoxifen-regulated Cre recombinase (Pdgfb-iCreERT2) were used. In preliminary studies, an increase in expression of Kruppel-like factor 2 (KLF2), a transcription factor implicated in the effects of Krit1 on zebrafish heart development, and a decrease in expression of Thrombospondin 1(TSP1), an anti-angiogenic protein that antagonizes VEGF signaling, was observed. Gain-of-function experiments will be performed to assess whether re-expression of TSP1 can reverse loss of KRIT1 affects endothelial phenotype and function. Moreover, this will be extended by investigating whether TSP1 gene transcription is suppressed as a consequence of deletion of Krit1 in cultured cells. Since SP1/KLF binding sites in human and mouse TSP1 promoter region were identified, the role of KLF2 in downregulation of TSP1 in response to genetic inactivation of Krit1 will be investigated. In addition, the zebrafish CCM model will be used to assess the impact of loss of Tsp1 during cardiovascular malformations in vivo. The studies described in this proposal and the environment at UCSD will complete my training in complex research methods and provide insight into fundamental questions about the function of the KRIT1 gene at the endothelial cell and organism level.
Cerebral cavernous malformations (CCM) are vascular malformations that affect ~ 0.5% of the general population and leads to significant morbidity and mortality in Americans. Currently, the only treatment for this disease involves invasive surgery, and the biological mechanisms of this vascular abnormality are only starting to emerge. This proposal is designed to better understand the biology of CCM and ultimately aid in the development of non-invasive therapies to prevent and/or ameliorate CCM.
