Tie1 and Tek (also known as Tie2) are endothelial protein receptor tyrosine kinases (RTKs). Along with the vascular endothelial growth factor (VEGF) receptor, these are the only known endothelial cell-specific RTKs. Due to embryonic lethality of panendotheilal deletion of either Tie 1 or Tie2 the exact roles and mechanisms of Tie1 or Tie2 in regulating cardiac development has not been clearly delineated. Furthermore the ligand(s) for Tie1 or the targets of Tie1 activation have not been identified. We hypothesize that in the cardiovascular system Tie1 is not only an inhibitory co-receptor for Tie2 activation, but also capable of signaling autonomously of Tie2. We therefore propose to: 1) Determine the unique functions of Tie1 and Tie2 signaling during early and late stages of cardiac development in vivo: Recently generated mice harboring a floxed allele of Tie1 or Tie2 will be used in conjunction with a novel early endocardial specific (Nfatc1Cre) or a later valvular endocardial Cre line (Nfatc1enCre) to produce stage specific cardiac deletion of Tie1 and Tie2 throughout the continuum of heart development in utero. Morphological and functional analysis will be used to characterize the unique phenotype that results from cardiac specific RTK attenuation. 2) Define the specific Tie1-Tie2 interactions that are required for normal cardiac morphogenesis at both early and late stages of heart formation in vivo. Recombinase mediated cassette exchange (RMCE) will be used to evaluate potential critical domains of Tie1-Tie2 interaction specifically in the endocardium. We will generate a series of cDNA knock-in mice lacking the extracellular domain (ECD), intracellular domain (ICD) as well as point mutations in critical tyrosine kinase domains in the cytoplasmic region of Tie1 (tyrosine to phenylalanine/Y1113F and lysine to alanine/K866A). These animals will be crossed to endocardial specific Cre lines and evaluated for specific defects in cardiac development. 3) Delineate down-stream targets of Tie1 activation in the cardiovascular system. In the absence of a known ligand for Tie1 activation, hemodynamic activation of Tie1 signaling will be used to evaluate the effect of defined deletion / mutations of Tie1. Immortalized endocardial cell lines derived from the conditional Tie1 mutants will be exposed to altered shear stress, turbulence and stretch and evaluated for alterations in previously described signal transduction pathways attributed to Tie activation. The most informative Tie1 mutant animals will be used for RNA-Seq and bioinformatic analysis for construction of signaling networks that will define a unique singling signature for Tie1 and help delineate endothelial cell autonomous and non-autonomous signaling pathways in the heart.
Upon completion, these experiments will have, for the first time, characterized the mechanism of Tie1- Tie 2 interactions in modulating critical events in cardiac ontogeny. The critical importance of the endocardium in regulating cardiac development and effecting cardiac response to injury is not well understood. The information gained by these studies will potentially be exploited for developing new therapeutic strategies derived from the new knowledge of TIE/TEK signaling mechanisms obtained.
