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.

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。增强的血管舒张又是通过以下方式实现的 增强细胞连接耦合和 Connexin 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“伤害”反应造成的，其中生长因子和/或细胞因子水平过高 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 浮肿。我们提出了通过有限数量的信号汇聚的许多因素的相互作用 然后途径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状态 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。此处发挥作用的可疑细胞因子与 Th1 表型相关，其中 Th1 细胞 占主导地位，TNFα、IL1B、IL6 和 IL8 在体内升高。我们假设这些细胞因子具有多层 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。结合进一步分析 临床表现表明抗原呈递（AP）状态的开始。同样的因素甚至起作用 更长的时间可能会驱动内皮间质转化（EndoMT）。我们建议确定在多大程度上 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 Src 和 JAK 信号激酶通过转录因子激活聚合的程度 这些结果。这导致了我的目标：F31 具体目标 1: 1A) 建立次最大的时间和剂量效应 TNF +- IL6（通过使用 ECIS 从低剂量到高剂量确定最具破坏性的组合。1B） 1A 中确定的剂量以完成蛋白质分析，以评估是否存在平行的 STAT 磷酸化 HIF 表达之前的酪氨酸。 1C) 单独使用或不使用抑制剂 PP2 评估 TNF +- IL6 的效果 或单独使用 AG490 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 目标 1 中由 ECIS 确定的相同组合治疗，使用以下方法识别 P-UAEC 中转录组的变化 RNA-Seq，并与细胞状态相关的蛋白质表达和功能进行比较。未来方向：使用相同的 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 状态或 EndoMT，然后将相同的面板应用于 P-UAEC（如目标 2 中的处理）和新分离的 HUVEC 使用单细胞 scCite-Seq 对比对照妊娠和 PE 妊娠。从这次F31的目标中学习的经验 对于优化治疗条件以及我了解和掌握最先进的 scCITE- 来说是必要的 F31 后训练期的 Seq 技术。请参阅实习生声明以了解一年申请的理由。