PIK3C3, a master regulator for smooth muscle identity

PIK3C3，平滑肌特性的主调节器

• 批准号: 10531615
• 负责人: Jiliang Zhou
• 金额: $ 61.99万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10531615
• 关键词: Aorta; Area; Arterial Fatty Streak; Atherosclerosis; Attenuated; Autophagocytosis; Autophagosome; Biological; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cells; Cellularity; Contractile Proteins; Coronary Arteriosclerosis; Data; Dendritic Cells; Development; Disease; Down-Regulation; Elasticity; Endosomes; Event; Exhibits; Flow Cytometry; Fluorescence; Foundations; Gene Expression; Generations; Genes; Genetic; Histology; Homeostasis; Human; Hyperplasia; Impairment; In Vitro; Inflammation; Inflammatory; Injections; Injury; Intestines; Knock-out; Knockout Mice; Lesion; Link; Lipids; Lysosomes; Macrophage; Mediating; Mediator; Membrane; Mesenchymal Stem Cells; Modification; Mus; Pathway interactions; Phenotype; Phosphotransferases; Play; Process; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; Reporter; Role; Severities; Smooth Muscle; Smooth Muscle Myocytes; Synthetic Genes; T-Lymphocyte; Tamoxifen; Testing; Transgenes; Ubiquitin; Vascular Diseases; Vascular Smooth Muscle; Vascular remodeling; Verteporfin; Vesicle; Visceral; Western Blotting; femoral artery; genome wide association study; genomic data; human data; hypercholesterolemia; in vivo; inflammatory marker; inhibitor; insight; mortality; mouse model; novel; pharmacologic; postnatal; preservation; prevent; protein expression; single-cell RNA sequencing; stem cell biomarkers; stem cell genes; trafficking; transcriptome sequencing; vascular smooth muscle cell proliferation; Aorta; Area; Arterial Fatty Streak; Atherosclerosis; Attenuated; Autophagocytosis; Autophagosome; Biological; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cells; Cellularity; Contractile Proteins; Coronary Arteriosclerosis; Data; Dendritic Cells; Development; Disease; Down-Regulation; Elasticity; Endosomes; Event; Exhibits; Flow Cytometry; Fluorescence; Foundations; Gene Expression; Generations; Genes; Genetic; Histology; Homeostasis; Human; Hyperplasia; Impairment; In Vitro; Inflammation; Inflammatory; Injections; Injury; Intestines; Knock-out; Knockout Mice; Lesion; Link; Lipids; Lysosomes; Macrophage; Mediating; Mediator; Membrane; Mesenchymal Stem Cells; Modification; Mus; Pathway interactions; Phenotype; Phosphotransferases; Play; Process; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; Reporter; Role; Severities; Smooth Muscle; Smooth Muscle Myocytes; Synthetic Genes; T-Lymphocyte; Tamoxifen; Testing; Transgenes; Ubiquitin; Vascular Diseases; Vascular Smooth Muscle; Vascular remodeling; Verteporfin; Vesicle; Visceral; Western Blotting; femoral artery; genome wide association study; genomic data; human data; hypercholesterolemia; in vivo; inflammatory marker; inhibitor; insight; mortality; mouse model; novel; pharmacologic; postnatal; preservation; prevent; protein expression; single-cell RNA sequencing; stem cell biomarkers; stem cell genes; trafficking; transcriptome sequencing; vascular smooth muscle cell proliferation

Phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative phenotype, plays a causal role in many human occlusive vascular diseases. To better understand key biological events occurring in human vascular diseases, we analyzed proteomic data from human atherosclerotic plaques and genomic data associated with human coronary artery disease. This unbiased analysis revealed that many genes involved in vesicle trafficking/fusion are over-represented. Previous studies have shown that the lipid kinase PIK3C3 is an essential regulator of vesicle trafficking/fusion. However, its functional role in VSMCs remains completely unknown. To examine the role of PIK3C3 in VSMCs, we generated inducible SM-specific Pik3c3 knockout (iSM KO) mice driven by Myh11-CreERT2 transgene. Unexpectedly, Pik3c3 iSM KO mice exhibited lethality 4 weeks after deletion of Pik3c3, due to a pseudo-obstructive intestine resulting from deletion of Pik3c3 in visceral SMCs in addition to VSMCs. The iSM Pik3c3 KO mice also exhibit dramatic remodeling of the vascular wall including thickening, aneurysmal dilation and spontaneous neointima. Proteomic analysis and bulk RNA- seq of Pik3c3-deficient aorta revealed loss of contractile proteins while increased expression of inflammation genes and targets of the Hippo-YAP1 pathway which has been shown to be critical for VSMC development and phenotypic modulation. Single cell RNA-seq revealed that Pik3c3-deficient aortic VSMCs almost completely lose their identity of contractile VSMCs while acquiring markers of inflammatory cells and mesenchymal stem cells. These exciting data suggest a previously undocumented role for PIK3C3 in maintaining SMC identity. Mechanistically, Pik3c3 inactivation induced YAP1 protein expression and silencing Yap1 largely restored a contractile phenotype in Pik3c3-deficient VSMCs. We hypothesize that PIK3C3 is a “master” regulator of the contractile phenotype of VSMC via regulating autophagosome-mediated degradation of YAP1. Three specific aims are proposed to test this hypothesis. To circumvent the early lethal visceral phenotype seen with Myh11- CreERT2 transgene, in Aim 1 we will employ a novel vascular-specific inducible Itga8-CreERT2 mouse to generate VSMC-specific Pik3c3 KO mice. Atherosclerosis will be induced using PCSK9 AAV and the effects of VSM- specific deletion of Pik3c3 on lesion formation will be evaluated. Wire injury-induced neointimal formation will be assessed as well by using this novel KO mouse model. Aim 2 will test that YAP1 is a critical mediator conferring the effects of Pik3c3 deficiency on VSMCs. YAP1 will be pharmacologically and genetically inactivated, and its effect on vascular remodeling and gene expression will be determined. Aim 3 will test that YAP1 protein accumulation induced by Pik3c3 deficiency is due to the impaired autophagic flux that attenuates autolysosome- mediated YAP1 degradation. Proposed studies will determine the role of PIK3C3 in autophagic flux in vivo and the role of ubiquitin and p62/SQSTM1 in PIK3C3-mediated degradation of YAP1 in human VSMCs in vitro. Completion of these studies will provide novel insights into the mechanism of controlling VSMC phenotype.

血管平滑肌细胞（VSMC）从收缩到增殖物表型的表型转换， 在许多人类闭塞性血管疾病中起因果作用。更好地了解关键的生物学事件 发生在人血管疾病中，我们分析了人类动脉粥样硬化斑块的蛋白质组学数据和 与人冠状动脉疾病有关的基因组数据。这种公正的分析表明许多基因 参与囊泡贩运/融合的代表过多。先前的研究表明脂质激酶 PIK3C3是囊泡运输/融合的重要调节剂。但是，其在VSMC中的功能作用仍然存在 完全未知。为了检查PIK3C3在VSMC中的作用，我们生成了可诱导的SM特异性PIK3C3 由MyH11-Creert2变换驱动的敲除（ISM KO）小鼠。出乎意料的是，PIK3C3 ISM KO小鼠暴露 删除PIK3C3后4周，由于删除Pik3C3导致的伪腹部肠道，导致PIK3C3的杀伤力 在内脏SMC中，除了VSMC。 ISM PIK3C3 KO小鼠还暴露了血管的戏剧性重塑 墙壁，包括增厚，动脉瘤词典和赞助Neintima。蛋白质组学分析和大量RNA- PIK3C3缺乏主动脉的SEQ显示收缩蛋白的损失，而炎症表达增加 Hippo-YAP1途径的基因和靶标被证明对VSMC发展至关重要 表型调制。单细胞RNA-Seq表明PIK3C3缺乏主动脉VSMC几乎完全丢失 它们对收缩VSMC的身份，同时在炎症细胞和间充质干细胞的标记中获得标记。 这些令人兴奋的数据表明，PIK3C3在维持SMC身份方面的作用。 从机械上讲，pik3c3失活诱导YAP1蛋白表达和沉默YAP1在很大程度上恢复了 PIK3C3缺乏率VSMC中的收缩表型。我们假设PIK3C3是 VSMC的收缩表型通过调节自噬体介导的YAP1降解。三个具体 提出了目的来检验这一假设。为了绕过MyH11-看到的早期致命内脏表型 Creert2转换，在AIM 1中，我们将采用一种新型的血管特异性诱导ITGA8-CREERT2小鼠 VSMC特异性PIK3C3 KO小鼠。动脉粥样硬化将使用PCSK9 AAV诱导以及VSM-的影响 将评估PIK3C3在病变形成上的特定缺失。电线损伤引起的新内膜形成将是 也通过使用这种新型KO小鼠模型进行评估。 AIM 2将测试YAP1是一个重要的调解员会议 PIK3C3缺乏对VSMC的影响。 YAP1将在药理和遗传上被灭活，其它将是 将确定对血管重塑和基因表达的影响。 AIM 3将测试YAP1蛋白 PIK3C3缺乏引起的积累是由于自噬的受损而导致的，从而减轻自溶剂体 - 介导的YAP1降解。拟议的研究将确定PIK3C3在体内自噬通量中的作用 泛素和p62/sqSTM1在PIK3C3介导的YAP1降解中的作用在体外人体VSMC中的作用。 这些研究的完成将为控制VSMC表型的机制提供新的见解。