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(VSMCs)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。为了更好地了解关键的生物事件 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和 与人类冠状动脉疾病相关的基因组数据。这一不偏不倚的分析揭示了许多基因 参与囊泡运输/融合的比例过高。此前的研究表明，脂蛋白激酶 PIK3C3是囊泡运输/融合的重要调节因子。然而，它在VSMC中的功能作用仍然存在 完全未知。为了研究PIK3C3在VSMC中的作用，我们产生了可诱导的SM特异性Pik3C3 转基因MYH11-CreERT2基因敲除(ISM KO)小鼠。出乎意料的是，Pik3c3 ISM KO小鼠展示了 Pik3c3缺失后4周致死，原因是Pik3c3缺失导致假性肠梗阻 除VSMC外，还存在内脏SMC。ISM Pik3c3KO小鼠也表现出显著的血管重塑 室壁包括增厚、动脉瘤样扩张和自发性新生内膜。蛋白质组学分析和整体RNA- Pik3c3基因缺陷的主动脉SEQ显示收缩蛋白丢失，而炎症表达增加 Hippo-YAP1通路的基因和靶点已被证明对VSMC的发育和 表型调节。单细胞RNA-SEQ显示Pik3c3缺陷的主动脉VSMC几乎完全丢失 它们在获得炎症细胞和间充质干细胞标记物的同时，具有收缩的VSMC的特性。 这些令人兴奋的数据表明，PIK3C3在维持SMC身份方面扮演了以前没有文献记载的角色。 在机制上，Pik3c3失活诱导了YAP1蛋白的表达，沉默YAP1在很大程度上恢复了 Pik3c3缺陷的血管平滑肌细胞的收缩表型。我们假设PIK3C3是PIK3C3的“主控”调节器 通过调节自噬小体介导的YAP1降解来调节VSMC的收缩表型。三个具体的 为了检验这一假说，我们提出了目标。为了避开MYH11的早期致命内脏表型- CreERT2转基因，在目标1中，我们将使用一种新型的血管特异性诱导的Itga8-CreERT2小鼠来产生 VSMC特异性Pik3c3 KO小鼠。用PCSK9 AAV诱导动脉粥样硬化及VSM的作用- 将评估Pik3c3在病变形成中的特定缺失。金属丝损伤诱导的新生内膜形成 使用这种新的KO小鼠模型也进行了评估。目标2将测试YAP1是一个关键的调停者 Pik3c3缺乏对VSMC的影响YAP1将被药物和基因灭活，其 对血管重塑和基因表达的影响将被确定。Aim 3将测试YAP1蛋白 Pik3c3缺乏引起的蓄积是由于自噬通量受损，从而削弱了自溶酶体。 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以及 泛素和p62/SQSTM1在PIK3C3介导的人VSMCs降解YAP1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。