Raf Kinase and Epigenetic Regulation of Fetal Vascular Development. Project Summary Development of the vasculature is a dynamic process that relies on the coordinated expression of numerous genes. The factors that regulate gene expression during blood vessel development are not well defined, however. Our preliminary data and reports from others of impaired vascular development in B-Raf knockout mice have elucidated the significance of this pathway in the developing vasculature. Importantly, downstream mechanism(s) through which B-Raf regulates vascular development are not known. Based on our preliminary data, we propose that B-Raf kinases via Protein Kinase C (PKC), Signal Transducer and Activator of Transcription 3 (STAT3) and p53 regulate DNA methyl Transferase (DNMT) and Ten-Eleven Translocation Methylcytosine Dioxygenase (TET) gene expression and activities. The altered activities of DNMT and TET lead to changes in promoter DNA methylation resulting in altered expression of genes regulating apoptosis such as Bcl2. These pathways play a crucial role in fetal vascular development. In this project, we will test the hypothesis that B-Raf hypomethylates the Bcl2 promoter to regulate fetal vascular stem cell survival. In the three Specific Aims, we will examine the (1) downstream mediators of B-Raf such as PKC, STAT3, and p53 (2) B-Raf-mediated regulation of DNMT and TET expression and activity, and (3) B-Raf-mediated regulation of promoter DNA methylation of the antiapoptotic gene Bcl2 via DNMT and TET. These studies are highly innovative and significant. Kinase-mediated regulation of epigenetic phenomenon such as DNA methylation is not well studied and is of fundamental importance from a basic science perspective. Moreover, pathways regulating fetal VSC apoptosis are involved in the pathogenesis of several fetal and neonatal disorders such as growth restriction, cerebral palsy, hemangioma, etc. From a clinical perspective, these studies will provide several therapeutic targets to regulate angiogenesis and vascular proliferation in many conditions from organ growth and wound healing to disorders such as myocardial infarction, cerebrovascular accident (stroke), and fetal development in general.
The proposed studies will examine the mechanisms important for proper development of fetal blood vessels. At the cellular level, we will elucidate the pathways and specific molecules which can serve as novel therapeutic targets to treat fetal vascular diseases. Importantly, identified targets may be utilized in adult diseases (such as cancer) which require inhibition of blood supply.