The role of nitrogen metabolism in smooth muscle cell phenotypic plasticity

氮代谢在平滑肌细胞表型可塑性中的作用

• 批准号: 10535170
• 负责人: Robert Noah Perry
• 金额: $ 3.82万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-19 至 2025-12-18
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535170
• 关键词: ATAC-seq; Address; Algorithms; Antihypertensive Agents; Arteries; Atherosclerosis; Automobile Driving; Bayesian Network; Binding; Biological; Biological Assay; Biology; Blood Vessels; Cause of Death; Cell Culture Techniques; Cell Proliferation; Complex; Coronary Arteriosclerosis; Coronary artery; Data; Diagnosis; Disease; Disease susceptibility; Donor person; Endothelial Cells; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Heart Transplantation; Heritability; Homeostasis; Human; Lead; Lentivirus; Link; Lipids; Measures; Medial; Metabolic Pathway; Metabolic dysfunction; Metabolism; Molecular; Myocardial Infarction; NOS3 gene; Nitric Oxide; Nitric Oxide Synthase; Nitrogen; Pathologic; Pathway Analysis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Process; Production; Recombinants; Regulator Genes; Risk; Risk Factors; Role; Series; Signal Pathway; Smooth Muscle Myocytes; Stimulus; Testing; Time; Transcript; United States; Variant; Vascular Endothelial Growth Factor C; Vascular Smooth Muscle; ascending aorta; biomarker identification; cell type; differential expression; disorder prevention; disorder risk; experimental study; gain of function; gene interaction; gene network; genome wide association study; knock-down; liquid chromatography mass spectrometry; loss of function; macrophage; metabolomics; migration; monocyte; mortality; multi-ethnic; network architecture; new therapeutic target; nitrogen metabolism; novel therapeutic intervention; novel therapeutics; osteogenic; overexpression; particle; preservation; programs; response; statistics; transcription factor; transcriptome sequencing

Coronary artery is the leading cause of death in the US. While lipid-lowering and anti-hypertensive drugs have helped decrease CAD-related mortality by approximately 50% since the 1980s, these therapies only modify CAD risk factors. To date, no approved drug acts at the vascular wall directly against atherosclerosis, the underlying cause of CAD. One opportunity to develop novel therapies is through genetics: CAD is partially heritable, and recent genome-wide association studies identified over 200 loci associated with elevated risk for CAD. While 40% of these CAD loci having established associations with known risk factors, the molecular and cellular mechanisms of the remaining 60% of the CAD loci are unknown. The majority of these unknown loci are predicted to function by regulating gene expression in the vascular wall where the disease develops. Vascular smooth muscle cells (SMCs), which make up the medial layer of arteries, play a critical role in the progression of atherosclerosis, the precursor to coronary artery disease. During initiation and progression of atherosclerosis, SMCs transdifferentiate from a quiescent (healthy) phenotype to a proliferative (pathological) phenotype representative of myogenic, osteochondrogenic, and macrophage-like phenotypes that contribute to plaque build-up. Identifying the molecular mechanisms driving SMC phenotypic plasticity will open up new avenues of treatment for CAD. Preservation analysis of co-expression networks from RNAseq data generated from the ascending aortas of 151 multi-ethnic smooth muscle cell donors cultured in quiescent and proliferative conditions, respectively, revealed phenotype-specific network architecture enriched for nitrogen metabolic processes. Previous studies have shown that metabolic pathways are not only involved in phenotypic changes of other cell types in the vascular wall, but also have the capability to drive them. Therefore, the goal of this proposal is to characterize the role nitrogen metabolism plays in SMC phenotypic plasticity and identify the key regulatory genes driving dysregulation. The project will address this problem through 2 aims. In aim 1, I will characterize the role nitrogen metabolism plays in SMCs during the progression of atherosclerosis using a combined approach of metabolomics, cell type marker identification, and cellular phenotyping assays in response to activation or silencing of the nitrogen metabolism pathway. In aim 2, I will create Bayesian networks (BNs) of genes involved in nitrogen metabolic processes using gene expression data and transcription factor-gene expression relationships generated from time-series experiments linking differentially expressed ATACseq peaks and differentially expressed RNAseq peaks in response to pro-atherogenic stimulus. I will then identify the key driver genes (KDs) of nitrogen metabolic pathways whose expression regulates the changes across the gene expression networks. Gain-of-function and loss-of-function experiments for KDs in SMCs using lentiviral particles will be completed with cellular phenotyping assays to quantify the impact on SMC proliferation, migration, and de-differentiation.

冠状动脉是美国死亡的主要原因。降低脂质和抗高血压药的虽然 自1980年代以来，与CAD相关的死亡率降低了约50％，这些疗法仅修改 CAD风险因素。迄今为止，尚无直接针对动脉粥样硬化的血管壁的批准药物作用， CAD的根本原因。开发新疗法的一个机会是通过遗传学：CAD部分是 可遗传的，最近的全基因组关联研究确定了200多个基因座，与较高的风险相关 卡德。这些CAD基因座中有40％已经建立了与已知危险因素的关联，但分子和 其余60％​​的CAD基因座的细胞机制尚不清楚。这些未知基因座的大多数 预测通过调节疾病发展的血管壁中的基因表达来起作用。 构成动脉内侧的血管平滑肌细胞（SMC），在 动脉粥样硬化的进展，冠状动脉疾病的前体。在开始和进步期间 动脉粥样硬化，SMCS从静止（健康）表型转变为增生（病理） 代表肌原性，骨软骨和巨噬细胞样表型的表型，有助于 牌匾积聚。确定驱动SMC表型可塑性的分子机制将开放新的 CAD治疗途径。从RNASEQ数据生成的共表达网络的保存分析 从151个多种族平滑肌细胞供体的上升主动脉，以静止和增生性培养 分别揭示了富含氮代谢的表型特异性网络结构 过程。先前的研究表明，代谢途径不仅参与表型变化 血管壁中的其他细胞类型，但也有能力驱动它们。因此，目标的目标 建议是表征氮代谢在SMC表型可塑性中的作用并确定关键 调节基因驱动失调。该项目将通过2个目标解决这个问题。在AIM 1中，我会 表征氮代谢在动脉粥样硬化过程中使用A的作用在SMC中的作用 代谢组学，细胞类型标记鉴定和细胞表型测定法的联合方法 对氮代谢途径激活或沉默的反应。在AIM 2中，我将创建贝叶斯 使用基因表达数据和参与氮代谢过程的基因网络（BN） 转录因子 - 基因的表达关系是由时间序列实验产生的，链接差异 表达的Atacseq峰和差异表达的RNASEQ峰响应于促动脉粥样硬化 刺激。然后，我将识别氮代谢途径的关键驱动基因（KD） 调节基因表达网络之间的变化。功能障碍和功能丧失实验 对于使用慢病毒颗粒的SMC中的KD 对SMC增殖，迁移和脱不同的影响。