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

基本信息

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
项目类别：
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

项目摘要

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，通过1）血管生成增加血管重塑，而2）表型重编程，包括增强的血管舒张。增强的血管舒张反过增强的细胞连接耦合和连接蛋白43细胞 - 细胞通信。失败，被称为前启示性（PE）是由于不适当的“伤害”反应，其中超过生长因子和/或细胞因子的水平关闭细胞 - 细胞连接耦合和连接蛋白的闭合，导致血管舒张的损失和趋势浮肿。我们提出了许多因素通过有限数量的信号传导收敛的许多因素的相互作用然后，途径首先将内皮驱动到抗原呈现状态，然后进入间充质过渡。我们建议针对细胞信号系统似乎是逆转这一点的最佳策略功能障碍。此处发挥的可疑细胞因子与Th1细胞的Th1表型有关主导和TNFα，IL1B，IL6和IL8在体内升高。我们假设这些细胞因子具有多层 SRC和JAK/GP130信号在STAT3和HIF上的收敛性，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 - 细胞通信和内分泌分泌的预期变化，细胞外改变空间（通过MMP）和免疫附着蛋白（ICAM和VCAM）的改变。进一步的分析合并临床表现表明抗原呈递（AP）状态的开始。相同的因素甚至行动更长的可能驱动内皮间充质转变（endomt）。我们建议确定多大程度上响应于TNF和gp130偶联的白细胞介素，AP甚至胚胎细胞脂肪的变化发生了 SRC和JAK信号激酶可能会在转录因子激活中收敛的程度这些结果。这导致了我的目标：F31特定目标1：1a）建立次最大的时间和剂量影响 TNF +-IL6（通过使用ECI来查明最具破坏性的组合。1B）使用在1A中确定的剂量以完成蛋白质分析，以评估是否在酪氨酸在HIF表达之前。 1C）评估TNF +-IL6的效果，单独使用和没有抑制剂PP2 或仅AG490在这些事件中建立SRC和JAK的因果关系。 F31特定目标2：使用通过EAM 1中ECIS确定的相同组合处理，使用P-UAEC中的转录组的变化 RNA-seq，并与细胞态相关的蛋白质表达和功能进行比较。未来方向：使用相同 AIM 2的抗体，我们将开发多维FACS，以检测抗原呈现的表面标记状态或胚胎，然后将相同的面板应用于P-UAEC（被视为AIM 2）和新鲜隔离的HUVEC 使用单细胞sccite-seq来控制对照与PE妊娠。从这个F31的目标中学习经验对于优化治疗条件而言是必要的，让我了解和掌握最先进的sccite- F31培训期间的SEQ技术。有关一年申请的合理性，请参见训练者声明。