Lysosome Regulation of Exosome Release and Function in Arterial Smooth Muscle

溶酶体对动脉平滑肌中外泌体释放和功能的调节

• 批准号: 10092204
• 负责人: PinLan Li
• 金额: $ 48.84万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092204
• 关键词: Abbreviations; Address; Agonist; Alkaline Phosphatase; Animals; Aorta; Applications Grants; Arterial Fatty Streak; Arteries; Autophagocytosis; Binding; CRISPR/Cas technology; Calcium; Calmodulin; Cell Proliferation; Cell membrane; Cells; Ceramides; Code; Collagen; Coronary; Coronary Arteriosclerosis; Coronary artery; Defect; Deposition; Development; Early Endosome; Enzymes; Excretory function; Extracellular Matrix; Farber' s lipogranulomatosis; Functional disorder; Future; Gene Deletion; Genes; Human; Image; Injury; Knockout Mice; Link; Lipids; Lysosomes; Medial; Mediating; Membrane Proteins; Metabolism; MicroRNAs; Molecular; Multivesicular Body; Mus; Muscle Proteins; Myocardial Infarction; Pathogenesis; Pathogenicity; Pathologic; Phenotype; Physiological; Play; Prevention; Process; Production; Proteins; Recycling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Sphingolipids; Sphingomyelins; Sphingosine; Stimulus; Testing; Transmission Electron Microscopy; Vacuolar Protein Sorting; Vascular calcification; Vesicle; base; calcification; cell dedifferentiation; exosome; extracellular vesicles; galactosylgalactosylglucosylceramidase; insight; interstitial; knockout gene; late endosome; mineralization; nanoparticle; novel therapeutics; osteogenic; patch clamp; prevent; programs; promoter; receptor; sensor; therapeutic target; trafficking; vesicular release

Project Summary Exosomes are from the intraluminal vesicles (ILV) of multivesicular bodies (MVBs) produced via endocytic process. Mature MVBs can fuse with lysosomes to deliver their contents for degradation and recycling or fuse with the plasma membrane where ILVs are released as exosomes. Recent studies have indicated that enhanced exosome excretion in arterial smooth muscle cells (SMCs) is an essential mechanism triggering or promoting calcifying nidus formation and extracellular matrix (ECM) mineralization in the arterial wall under different pathological conditions. However, little is known so far how MVB fate and exosome excretion are controlled by lysosomes in SMCs and whether lysosomal dysfunction increases exosome excretion from SMCs to activate or accelerate vascular calcification process. The present grant proposal will test a central hypothesis that lysosomal acid ceramidase (AC)-mediated sphingolipid metabolism plays a crucial role in the control of lysosome trafficking or fusion to MVBs and subsequent exosome excretion, maintaining the normal phenotype and function of SMCs. AC gene defect or functional deficiency may disturb lysosome degradation of MVBs, increasing exosome excretion and resulting in calcifying nidus formation and ultimate vascular calcification under pathological conditions. To test this hypothesis, three Specific Aims are proposed. Specific Aim 1 will determine whether exosome excretion in arterial SMCs is fine controlled by lysosomal AC activity and whether the deficiency of this AC regulation causes arterial calcification in smooth muscle-specific AC gene knockout mice (Asah1fl/fl/SMcre) with analysis of exosomes, SMC phenotypes, and calcification in the arterial wall and cultured coronary arterial SMCs. Specific Aim 2 attempts to test whether lysosomal AC- mediated sphingolipid signaling regulates lysosome trafficking to and fusion with MVBs to limit exosome excretion from SMCs with gene deletion (Asah1fl/fl/SMcre), CRISPR-Cas9 gene editing, and SM22α promoter- driven gene rescuing. In Specific Aim 3, we will address whether the AC regulation of lysosome trafficking or fusion to MVBs is attributed to its action on lysosomal TRPML1 channel activity and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging with GCaMP3-ML as an indicator. To our knowledge, these proposed studies will represent the first effort to investigate the lysosome regulation of exosome excretion from SMCs and associated pathogenic role in vascular calcification. The findings will provide new insights into the pathogenesis of arterial calcification and identify lysosomal AC as a therapeutic target for prevention or treatment of vascular calcification.

项目摘要 外泌体来自通过内吞植物的内部蔬菜（ILV）（MVB） 过程。成熟的MVB可以与溶酶体融合，以递送含量以降解和回收或保险丝 与质膜一起以外泌体释放的ILV。最近的研究表明 动脉平滑肌细胞（SMC）中增强的外泌体排泄是一种基本机制触发或 在动脉壁中促进钙化的Nidus形成和细胞外基质（ECM）矿化 不同的病理条件。但是，到目前为止，鲜为人知的MVB命运和外泌体排泄是如何 由SMC中的溶酶体控制以及溶酶体功能障碍是否增加了SMC的外泌体排泄 激活或加速血管钙化过程。目前的赠款提案将测试中央 假设溶酶体酸神经酶（AC）介导的鞘脂代谢在 控制MVB的溶酶体运输或融合以及随后的外泌体排泄，保持正常 SMC的表型和功能。 AC基因缺陷或功能缺陷可能会干扰溶酶体降解的 MVB，增加外泌体排泄并导致钙化Nidus形成和最终血管 病理条件下的钙化。为了检验这一假设，提出了三个具体目标。具体的 AIM 1将确定动脉SMC中的外泌体排泄是否受溶酶体AC活性的控制 以及该AC调节的缺乏是否导致平滑肌特异性AC中的动脉计算 基因敲除小鼠（ASAH1FL/FL/SMCRE），分析外泌体，SMC表型和钙化 动脉壁和培养的冠状动脉SMC。特定的目标2尝试测试是否溶酶体AC- 介导的鞘脂信号传导调节溶酶体贩运和与MVB的融合以限制外泌体 具有基因缺失（ASAH1FL/FL/SMCRE），CRISPR-CAS9基因编辑和SM22α启动子的SMC的排泄 驱动的基因营救。在特定的目标3中，我们将解决溶酶体贩运的AC法规还是 与MVB的融合归因于其对溶酶体TRPML1通道活动的作用和相关的Ca2+释放 使用分离的溶酶体和溶酶体特异性Ca2+成像的斑块夹具用GCAMP3-ML作为一个 指标。据我们所知，这些拟议的研究将代表研究溶酶体的第一个努力 调节SMC的外泌体排泄和血管钙化中相关的致病作用。这 发现将为动脉计算的发病机理提供新的见解，并将溶酶体AC识别为A 预防或治疗血管钙化的治疗靶标。

项目成果

Concentration-Dependent Diversifcation Effects of Free Cholesterol Loading on Macrophage Viability and Polarization.

• DOI: 10.1159/000430365
• 发表时间: 2015
• 期刊: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
• 作者: Xu X;Zhang A;Li N;Li PL;Zhang F
• 通讯作者: Zhang F

Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

• DOI: 10.1111/jcmm.12326
• 发表时间: 2014-11
• 期刊: Journal of cellular and molecular medicine
• 影响因子: 5.3
• 作者: Xu M;Li XX;Chen Y;Pitzer AL;Zhang Y;Li PL
• 通讯作者: Li PL

Oxidative stress triggers Ca-dependent lysosome trafficking and activation of acid sphingomyelinase.

• DOI: 10.1159/000341460
• 发表时间: 2012
• 期刊: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
• 作者: Li X;Gulbins E;Zhang Y
• 通讯作者: Zhang Y

Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs.

• DOI: 10.2741/4415
• 发表时间: 2016-01-01
• 期刊: Frontiers in bioscience (Landmark edition)
• 作者: Xu M;Zhang Q;Li PL;Nguyen T;Li X;Zhang Y
• 通讯作者: Zhang Y

Regulation of exosome release by lysosomal acid ceramidase in coronary arterial endothelial cells: Role of TRPML1 channel.

• DOI: 10.1016/bs.ctm.2022.09.002
• 发表时间: 2022
• 期刊: CURRENT TOPICS IN MEMBRANES
• 作者: Li, Guangbi;Huang, Dandan;Li, Pengyang;Yuan, Xinxu;Yarotskyy, Viktor;Li, Pin-Lan
• 通讯作者: Li, Pin-Lan