Understanding endothelial cell fate changes as mediators in the pathogenesis of preeclampsia

摘要：在健康妊娠中，血管重塑是通过以下方式实现的：1）血管生成增加，2） 表型重编程包括增强血管舒张。 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 关闭细胞与细胞的连接耦合和连接蛋白关闭，导致血管舒张功能丧失并倾向于 水肿。 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 过渡。我们认为，针对细胞信号系统似乎是扭转这一局面的最佳策略。 功能障碍。 The suspected cytokines at play here are associated with a Th1 phenotype where Th1 cells 占主导地位，TNFα、IL1B、IL6 和 IL8 在体内升高。 We hypothesize these cytokines have a multilayered 由于 Src 和 JAK/GP130 信号在 STAT3 和 HIF 上的融合而对内皮破坏产生影响 活动。经过 20 小时的初步表型功能测定和转录组学数据分析揭示 细胞间通讯以及内分泌分泌的预期变化，细胞外的修饰 空间（通过 MMP）以及免疫附着蛋白（ICAM 和 VCAM）的改变。 Further analysis combined 临床表现表明抗原呈递（AP）状态的开始。 The same factors acting even longer may drive an endothelial mesenchymal transition (EndoMT). We propose to determine the extent to which AP 甚至 EndoMT 细胞命运的变化是对 TNF 和 Gp130 偶联白细胞介素的反应而发生的，并且 extent to which Src and JAK signaling kinases may converge though transcription factor activation to initiate 这些结果。 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 或单独使用 AG490 来确定 Src 和 JAK 在这些事件中的因果关系。 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，并与细胞状态相关的蛋白质表达和功能进行比较。 Future Direction: Using the same 来自 Aim 2 的抗体，我们将开发多维 FACS 来检测抗原呈递的表面标记 state or EndoMT, and then apply this same panel to P-UAEC (treated as in Aim 2) and freshly isolated HUVEC 使用单细胞 scCite-Seq 对比对照妊娠和 PE 妊娠。 The learning experiences from the Aims of this F31 are necessary to optimize treatment conditions and for me to understand and master the cutting edge scCITE- Seq technique in the post F31 training period.请参阅实习生声明以了解一年申请的理由。

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 learning experiences from the Aims of this F31 are necessary to optimize treatment conditions and for me to understand and master the cutting edge scCITE- Seq technique in the post F31 training period. See Trainee statement for justification of one year application.