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：在健康的妊娠中，血管重塑是通过1)增加血管生成和2) 表型重编程，包括增强的血管扩张。增强的血管扩张又通过以下途径实现 增强了细胞连接偶联和连接蛋白43的细胞间通讯。这种失败被称为先兆子痫 (PE)是由于过度的生长因子和/或细胞因子水平的不适当的“伤害性”反应 关闭细胞-细胞连接偶联和连接蛋白关闭，导致血管扩张丧失，并有 浮肿。我们提出了一些因素的相互作用，这些因素通过有限数量的信号汇聚在一起 然后，通路驱动内皮细胞首先进入抗原提呈状态，然后可能进入间质 过渡。我们认为，以细胞信号系统为目标似乎是扭转这一趋势的最佳策略 功能障碍。这些可疑的细胞因子与Th1表型有关，其中Th1细胞 体内肿瘤坏死因子α、白介素1β、白介素6、白介素8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上的融合对内皮破坏的影响 活动。初步的表型功能分析和超过20小时的转录组数据分析显示 细胞间通讯以及内分泌的预期变化，细胞外的修饰 间隙(通过MMPs)和免疫附着蛋白的改变(ICAM和VCAM)。结合进一步的分析 临床表现提示出现抗原提呈(AP)状态。同样的因素起着同样的作用 较长的时间可能导致内皮细胞间充质转化(EndoMT)。我们建议确定在何种程度上 AP甚至EndoMT细胞命运的改变都是由于肿瘤坏死因子和gp130偶联的白细胞介素所引起的，并且 通过转录因子激活启动Src和JAK信号通路的融合程度 这些结果。这导致了我的目标：F31具体目标1：1a)建立亚极量的时间和剂量效应 肿瘤坏死因子+-白介素6(低剂量到高剂量，通过使用ECIS来确定最具破坏性的组合。1b)使用 在1A中确定的剂量完成蛋白质分析以评估是否存在平行状态的磷酸化 HIF表达之前的酪氨酸。1C)评估单独使用和不使用PP2抑制剂时肿瘤坏死因子+-白介素6的效果 或单独使用AG490来确定在这些事件中Src和JAK的因果关系。F31特定目标2：使用 在AIM 1中由ECIS确定的相同组合处理，利用 RNA-Seq，并比较细胞状态相关蛋白的表达和功能。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志 State或EndoMT，然后将相同的面板应用于P-UAEC(如AIM 2中处理)和新分离的HUVEC 对照和使用单细胞SCCite-Seq的PE妊娠。从F31的目标中学习的经验 是优化治疗条件所必需的，也是我了解和掌握尖端CCITE- F31后训练期的SEQ技术。有关申请一年的理由，请参阅学员声明。