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

了解内皮细胞命运变化作为先兆子痫发病机制的介质

• 批准号: 10462390
• 负责人: Rachel Lee Dahn
• 金额: $ 4.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-06-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462390
• 关键词: Adult; Affect; Agonist; Antibodies; Antigens; Biological Assay; Birth; Blood Pressure; Blood Vessels; Cell Communication; Cells; Clinical; Clinical Trials; Combined Modality Therapy; Connexin 43; Connexins; Coupled; Coupling; Dangerousness; Data; Data Analyses; Diabetes Mellitus; Differentiation Antigens; Disease; Dose; Edema; Endocrine; Endothelial Cells; Endothelium; Event; Extracellular Space; Failure; Functional disorder; Future; Growth Factor; Hour; Hypertension; IL6 gene; IL6ST gene; IL8 gene; Immune; In Vitro; Intercellular Junctions; Interleukins; Lead; Learning; Life; Literature; Matrix Metalloproteinases; Measures; Mediating; Mediator of activation protein; Mesenchymal; Metalloproteases; Methods; Modification; Monitor; Mothers; Obesity; Outcome; Pathogenesis; Phenotype; Phosphorylation; Phosphotransferases; Play; Population; Pre-Eclampsia; Pregnancy; Process; Protein Analysis; Proteins; Publishing; Risk; STAT3 gene; Signal Induction; Signal Pathway; Signal Transduction; Surface; System; TNF gene; Techniques; Technology; Testing; Th1 Cells; Time; Training; Transitional Cell; Tyrosine; Vascular Cell Adhesion Molecule-1; Vascular remodeling; Vasodilation; angiogenesis; cytokine; effective therapy; endothelial dysfunction; experience; healthy pregnancy; in vivo; inhibitor; intercellular cell adhesion molecule; monolayer; novel therapeutics; protein biomarkers; protein expression; protein function; public health relevance; response; single cell analysis; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; treatment optimization; tyrphostin AG-490; unborn child; wound response

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.

翻译后摘要：在健康的怀孕，血管重塑是通过1）增加血管生成，和2） 表型重编程包括增强的血管舒张。增强的血管舒张又通过 增强的细胞连接偶联和连接蛋白43细胞间通讯。这种失败被称为先兆子痫 (PE)，是由于不适当的“创伤”反应，其中过度水平的生长因子和/或细胞因子 关闭细胞间连接偶联和连接蛋白闭合，导致血管舒张功能丧失， 水肿我们提出了一些因素的相互作用，通过有限数量的信号收敛 然后，通路首先驱动内皮细胞进入抗原呈递状态，然后可能进入间充质细胞。 过渡我们认为，靶向细胞信号系统似乎是逆转这种情况的最佳策略。 功能障碍在此起作用的可疑细胞因子与Th 1表型相关，其中Th 1细胞 在体内，TNFα、IL 1B、IL 6和IL 8升高。我们假设这些细胞因子具有多层次的 Src和JAK/GP 130信号对STAT 3和HIF会聚对内皮破坏的影响 活动初步的表型功能测定和转录组学数据分析超过20小时， 细胞间通讯以及内分泌的预期变化，细胞外 间隙（通过MMP），以及免疫附着蛋白（ICAM和VCAM）的改变。进一步分析， 临床表现提示抗原呈递（AP）状态的发作。同样的因素甚至 更长的时间可以驱动内皮间质转化（EndoMT）。我们建议确定 AP甚至EndoMT细胞命运的变化发生在对TNF和Gp 130偶联的白细胞介素的应答中，并且 Src和JAK信号激酶可能通过转录因子激活而会聚以启动 这些结果。这导致了我的目标：F31具体目标1：1A）建立次最大剂量的时间和剂量效应 TNF +-IL 6（低到高剂量，通过使用ECIS来确定最具破坏性的组合。1B）用途 在1A中确定的剂量完成蛋白质分析，以评估是否存在平行的STAT磷酸化。 HIF表达前的酪氨酸。1C）评估TNF +-IL 6与和不与单独的抑制剂PP 2的作用 或单独使用AG 490来确定Src和JAK在这些事件中的因果关系。F31具体目标2：使用 在目标1中通过ECIS确定的相同组合处理，使用 RNA-Seq，并比较与细胞状态相关的蛋白表达和功能。未来方向：使用相同的 从目标2抗体，我们将开发多维流式细胞仪检测抗原呈递的表面标志物 状态或EndoMT，然后将该相同的板应用于P-UAEC（如目标2中处理）和新鲜分离的HUVEC 使用单细胞scCite-Seq.从F31的目标中学习经验 是必要的，以优化治疗条件，并为我了解和掌握尖端scCITE- F31后训练期的Seq技术。一年申请的理由见学员声明。