Abstract: In healthy pregnancy, vascular remodeling is achieved by 1) increased angiogenesis, and 2) phenotypic reprogramming including enhanced vasodilation. Enhanced vasodilation in turn is achieved via enhanced cell junctional coupling and Connexin 43 cell-cell communication. The failure, known as preeclampsia (PE), is due to an inappropriate `wounding' response in which excessive levels of growth factors and/or cytokines shut down cell-cell junctional coupling and Connexin closure, resulting in loss of vasodilation and a tendency to edema. We propose the interactions of a number of factors that converge through a limited number of signaling pathways then drive endothelium first to an antigen presenting state and then possibly into a Mesenchymal Transition. We propose that targeting the cell signaling system appears to be the best strategy in reversing this dysfunction. The suspected cytokines at play here are associated with a Th1 phenotype where Th1 cells dominate and TNFα, IL1B, IL6 and IL8 are elevated in vivo. We hypothesize these cytokines have a multilayered impact on endothelial destruction due to a convergence of Src and JAK/GP130 signaling on STAT3 and HIF activity. Preliminary phenotypic functional assays and transcriptomics data analysis over 20 hours reveals expected changes in cell-cell communication as well as endocrine secretion, modification of the extracellular space (via MMPs), and alterations to immune attachment proteins (ICAM and VCAM). Further analysis combined with clinical presentation suggests the onset of an antigen presenting (AP) state. The same factors acting even longer may drive an endothelial mesenchymal transition (EndoMT). We propose to determine the extent to which AP and even EndoMT cell fate changes occur in response to TNF and the Gp130 coupled interleukins, and the extent to which Src and JAK signaling kinases may converge though transcription factor activation to initiate these outcomes. This leads to my aims: F31 Specific Aim 1: 1A) Establish time and dose effects of submaximal TNF +- IL6 (low through high dose through the use of ECIS to pinpoint the most damaging combination. 1B) Use the doses determined in 1A to complete protein analysis to evaluate if there is parallel STAT phosphorylation on Tyrosine that precedes HIF expression. 1C) Evaluate effects of TNF +- IL6, with and without inhibitors PP2 alone or AG490 alone to establish cause and effect of Src and JAK in these events. F31 Specific Aim 2: Using the same combined treatments determined by ECIS in Aim 1, identify the changes in transcriptome in P-UAEC using RNA-Seq, and compare to cell state related protein expression and function. Future Direction: Using the same antibodies from Aim 2, we will develop multidimensional FACS to detect surface markers for antigen presenting state or EndoMT, and then apply this same panel to P-UAEC (treated as in Aim 2) and freshly isolated HUVEC from control vs PE pregnancies using single cell scCite-Seq. The l...