Interstitial cells of Cajal in diabetic gastropathy

糖尿病胃病中的卡哈尔间质细胞

基本信息

批准号：
9380395
负责人：
Tamas Ordog
金额：
$ 35.78万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9380395
关键词：
Affect Age Aging Animal Model Binding Biological Markers Biology CRISPR/Cas technology Cell Death Cell Line Cells Chromatin Chronic Clinical Research Clinical Trials DNA Diabetes Mellitus Diabetic mouse Disease Drug usage Enhancers Enteral Epigenetic Process Fibroblasts Flow Cytometry Fluorescence-Activated Cell Sorting Gastric Emptying Gastrointestinal Diseases Gastroparesis Gene Expression Profile Genes Genetic Genetic Transcription Goals Health Healthcare Histone Deacetylase Inhibitor Histones Human IGF1 gene Immunofluorescence Immunologic Impairment In Vitro Insulin Interstitial Cell of Cajal Knowledge Lead Ligands Link Maintenance Mediating Mesenchymal Metabolic Methodology Methods Molecular Conformation Mus Muscle function National Institute of Diabetes and Digestive and Kidney Diseases Neuropathy Operative Surgical Procedures PDGFRA gene Pacemakers Patients Phase Phenocopy Phenotype Physiological Population RNA Interference Receptor Protein-Tyrosine Kinases Regulation Reporting Repression Research Research Personnel Role Signal Transduction Smooth Muscle Stem Cell Factor Stem cells Stomach Succinate Dehydrogenase Succinates System Technology Therapeutic Translations Up-Regulation Western Blotting alpha ketoglutarate base care burden cell type cholinergic cost curative treatments diabetic diabetic gastroparesis disorder control effective therapy epigenetic drug epigenome epigenomics gastrointestinal gene repression genome editing genome-wide human tissue in vivo interstitial cell longitudinal analysis macrophage mitochondrial metabolism motility disorder neuromuscular neuromuscular plasticity neurotransmission novel palliative programs receptor expression self-renewal transcription factor transcriptome sequencing transdifferentiation virtual

项目摘要

Disordered control of smooth muscle function is common in gastrointestinal diseases, which cost billions of dollars in health care spending each year. Gastroparesis is one of the most significant manifestations of gastrointestinal dysmotility, particularly in diabetes mellitus. However, therapeutic options are limited reflecting incomplete understanding of cellular dynamics within the neuromuscular compartment. Previous research has identified interstitial cells of Cajal (ICC) as the cell type most commonly affected in gastroparesis. ICC serve as the physiological pacemaker for phasic contractile activity and mediate cholinergic and nitrergic neuromuscular neurotransmission. Diabetic ICC depletion may arise from reduced insulin/IGF1 signaling and macrophage action, which interfere with expression of the receptor tyrosine kinase Kit. A critical gap in our knowledge is the lack of understanding of the fate of ICC lost from these insults. Cell death has been observed but not consistently, prompting investigators to hypothesize ICC transdifferentiation, which could be reversible. However, evidence of ICC survival following loss of Kit and other biomarkers has been elusive. Therefore, the main objective of this project is to identify ICC fates, their mechanisms and reversibility in diabetes. Our central hypothesis is that depletion of the ICC transcription factor Etv1 from reduced Kit signaling, together with inhibition of “erasers” of repressive epigenetic marks due to disturbed mitochondrial metabolism, lead to decommissioning and eventual silencing of super-enhancers that normally drive the transcription of genes critical for ICC identity including Kit. These changes are accompanied by the upregulation of the closely related gene Pdgfra, conferring on the surviving ICC the identity and function of “fibroblast-like cells” (FLC), which mediate purinergic neuromuscular neurotransmission. Specific Aim 1 is to provide definitive evidence of ICC- to-FLC phenotypic switch using in-vivo genetic lineage tracing and novel cell lines combined with phenotyping by flow cytometry, immunofluorescence, Western blotting and transcriptome sequencing in cell populations purified by fluorescence-activated cell sorting. Specific Aim 2 is to establish the epigenetic mechanisms underlying ICC dedifferentiation and phenotypic conversion into FLC. We will analyze histone and DNA marks, Etv1 binding and chromatin conformation in specific loci and genome-wide. We will perform mechanistic studies using RNA interference (RNAi), as well as in-vitro CRISPR-Cas9-mediated and in-vivo Cre-mediated genome editing combined with longitudinal analysis of gastric emptying. Specific Aim 3 is to determine the role of succinate in the epigenetic repression of ICC genes using RNAi, in-vivo genome editing and the above epigenomic and phenotyping methods. To facilitate translation of our findings, we will validate key mechanisms in human tissues and attempt to restore ICC in mice using epigenetic drugs approved for use in humans. The concept of manipulating interstitial cells via epigenetic reprogramming of cells with persistently altered transcriptional programs will change how we think about neuromuscular plasticity in health and disease.

平滑肌功能控制紊乱在胃肠道疾病中很常见，导致数十亿美元的损失每年的医疗保健支出是胃轻瘫最重要的表现之一。胃肠道运动障碍，特别是糖尿病患者，然而，治疗选择有限。先前的研究对神经肌肉室内的细胞动力学了解不完全。确定 Cajal 间质细胞 (ICC) 是胃轻瘫中最常受影响的细胞类型。用于阶段性收缩活动并介导胆碱能和硝能神经肌肉的生理起搏器糖尿病性 ICC 耗竭可能是由于胰岛素/IGF1 信号传导和巨噬细胞减少所致。我们的知识中的一个关键差距是干扰受体酪氨酸激酶试剂盒的表达。对这些损伤造成的 ICC 命运缺乏了解，已观察到细胞死亡，但尚未观察到。始终如一，促使研究人员解决 ICC 转分化问题，这种转分化是可逆的。然而，Kit 和其他生物标志物丢失后 ICC 存活的证据一直难以捉摸。该项目的主要目标是确定糖尿病中的 ICC 命运、其机制和可逆性。假设是由于 Kit 信号传导减少而导致 ICC 转录因子 Etv1 的缺失，以及由于线粒体代谢紊乱而抑制表观遗传标记的“橡皮擦”，导致通常驱动基因转录的超级增强子的退役和最终沉默对包括 Kit 在内的 ICC 身份至关重要。这些变化伴随着密切相关的上调。基因 Pdgfra，赋予幸存的 ICC“成纤维细胞样细胞”（FLC）的身份和功能，介导嘌呤能神经肌肉神经传递。具体目标 1 是提供 ICC- 的明确证据。使用体内遗传谱系追踪和新型细胞系与表型分析相结合的 to-FLC 表型转换通过流式细胞术、免疫荧光、蛋白质印迹和转录组测序对细胞群进行检测通过荧光激活细胞分选进行纯化具体目标 2 是建立表观遗传机制。 ICC 去分化和表型转化为 FLC 我们将分析组蛋白和 DNA 标记，我们将在特定位点和全基因组范围内进行 Etv1 结合和染色质构象分析。使用 RNA 干扰 (RNAi) 以及体外 CRISPR-Cas9 介导和体内 Cre 介导的研究基因组编辑与胃排空纵向分析相结合具体目标 3 是确定其作用。琥珀酸在使用 RNAi、体内基因组编辑等对 ICC 基因进行表观遗传抑制中的作用表观基因组和表型分析方法为了促进我们的发现的转化，我们将验证关键机制。并尝试使用批准用于人类的表观遗传药物来恢复小鼠的 ICC。通过持续对细胞进行表观遗传重编程来操纵间质细胞的概念转录程序将改变我们对健康和疾病中神经肌肉可塑性的看法。