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

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）从收缩表型到增殖表型的表型转换， 在许多人类闭塞性血管疾病中起着因果作用。为了更好地理解关键的生物学事件 发生在人类血管疾病中，我们分析了来自人类动脉粥样硬化斑块的蛋白质组数据， 与人类冠状动脉疾病相关的基因组数据。这种无偏见的分析表明，许多基因 参与囊泡运输/融合的细胞比例过高。先前的研究表明， PIK 3C 3是囊泡运输/融合的重要调节因子。然而，其在VSMCs中的功能作用仍然存在， 完全未知为了研究PIK 3C 3在VSMC中的作用，我们产生了诱导型SM特异性PIK 3C 3。 Myh 11-CreERT 2转基因驱动的基因敲除（iSM KO）小鼠。出乎意料的是，Pik 3c 3 iSM KO小鼠表现出 Pik 3c 3缺失后4周致死，由于Pik 3c 3缺失导致的假性梗阻性肠 在内脏平滑肌细胞和血管平滑肌细胞中。iSM Pik 3c 3 KO小鼠也表现出血管的显著重塑， 血管壁增厚、扩张和自发性新生内膜。蛋白质组学分析和批量RNA- Pik 3c 3缺陷主动脉的seq显示收缩蛋白的损失，同时炎症表达增加 Hippo-YAP 1通路的基因和靶点，已被证明对VSMC发育至关重要， 表型调节单细胞RNA-seq显示，Pik 3c 3缺陷的主动脉VSMC几乎完全丧失 他们的身份收缩VSMC，同时获得炎症细胞和间充质干细胞的标志物。 这些令人兴奋的数据表明PIK 3C 3在维持SMC身份方面的先前未记录的作用。 从机制上讲，Pik 3c 3失活诱导了YAP 1蛋白表达，沉默Yap 1在很大程度上恢复了YAP 1的表达。 Pik 3c 3缺陷型VSMC的收缩表型。我们假设PIK 3C 3是一个“主”调节器， 通过调节自噬体介导的YAP 1降解来调节VSMC的收缩表型。三个具体 目的是为了检验这一假设。为了避免Myh 11 - 1中观察到的早期致死性内脏表型， 在目的1中，我们将采用新的血管特异性诱导型Itga 8-CreERT 2小鼠来产生CreERT 2转基因。 VSMC特异性Pik 3c 3 KO小鼠。将使用PCSK 9 AAV诱导动脉粥样硬化，并且VSM-1的作用将在2010年12月31日至2011年12月31日期间进行。 将评价Pik 3c 3的特异性缺失对损伤形成的影响。导丝损伤诱导的新生内膜形成将 通过使用这种新的KO小鼠模型也进行了评估。目的2将测试YAP 1是一个关键的介体，赋予 Pik 3c 3缺陷对血管平滑肌细胞的影响。YAP 1将被灭活并遗传失活， 将确定对血管重塑和基因表达的影响。目的3将测试YAP 1蛋白 由Pik 3c 3缺陷诱导的积聚是由于减弱自溶体的自噬通量受损。 介导的YAP 1降解。拟议的研究将确定PIK 3C 3在体内自噬通量中的作用， 泛素和p62/SQSTM 1在体外人VSMCs中PIK 3C 3介导的YAP 1降解中的作用。 这些研究的完成将提供新的见解控制VSMC表型的机制。