Exosomal NEDD4L derived from HG+oxLDL-induced vascular endothelial cells accelerates macrophage M1 polarization and oxLDL uptake by ubiquitinating IκBα and PPARγ.
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引用次数: 0
Abstract
Background: Vascular endothelial cell-derived exosomes are thought to mediate disease progression by regulating macrophage polarization. However, its mechanism in diabetes mellitus (DM)-related atherosclerosis (AS) progress is unclear.
Methods: High-glucose (HG) and oxLDL were used to induce human cardiac microvascular endothelial cells (HCMECs) to mimic DM-related AS model. The conditioned medium (CM) from HG+oxLDL-induced HCMECs was incubated with THP1-M0 monocytes treated with LPS or oxLDL. The mRNA levels of macrophage M1/M2 polarization markers, NEDD4L, IκBα and PPARγ were determined by qRT-PCR. Flow cytometry was used to analyze macrophage marker. Dil-labeled oxLDL and oil red O staining were performed to assess oxLDL uptake by THP1-M0 cells. The levels of inflammatory factors were examined using ELISA. Transmission electron microscope was used for observing foam cell formation and exosome morphology. The protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα, p-P65/P65, anti-lipid metabolism-related markers, and NEDD4L were tested by western blot. The interaction between NEDD4L and IκBα or PPARγ was assessed by Co-IP assay.
Results: The CM of HG+oxLDL-induced HCMECs could promote macrophage M1 polarization, oxLDL uptake and foam cell formation, and exosome inhibiter GW4869 eliminated these effects. NEDD4L was overexpressed in exosomes from HG+oxLDL-induced HCMECs, which could be taken up by THP1-M0 cells. Exosomal NEDD4L knockdown inhibited macrophage M1 polarization, oxLDL uptake and foam cell formation by reducing the protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα and p-P65/P65. NEDD4L could reduce IκBα and PPARγ expression through ubiquitination.
Conclusion: HG+oxLDL-induced HCMECs-derived exosomal NEDD4L could enhance the ubiquitination of IκBα and PPARγ to facilitate macrophage M1 polarization and oxLDL uptake, thus accelerating DM-related AS.
来自HG+oxLDL诱导的血管内皮细胞的外泌体NEDD4L通过泛素化i - κ b α和PPARγ加速巨噬细胞M1极化和oxLDL摄取。
背景：血管内皮细胞来源的外泌体被认为通过调节巨噬细胞极化介导疾病进展。然而，其在糖尿病（DM）相关动脉粥样硬化（AS）进展中的机制尚不清楚。方法：采用高糖（HG）和氧化低密度脂蛋白（oxLDL）诱导人心脏微血管内皮细胞（HCMECs）模拟dm相关AS模型。将HG+oxLDL诱导的hcmes条件培养基（CM）与LPS或oxLDL处理的THP1-M0单核细胞孵育。采用qRT-PCR检测巨噬细胞M1/M2极化标记物NEDD4L、IκBα、PPARγ mRNA表达水平。流式细胞术检测巨噬细胞标志物。采用dil标记oxLDL和油红O染色评估THP1-M0细胞对oxLDL的摄取。采用ELISA法检测炎症因子水平。透射电镜观察泡沫细胞形成和外泌体形态。western blot检测p-Smad1/Smad1、p-Smad2/Smad2、p- κ b α/ i- κ b α、p-P65/P65、抗脂质代谢相关标志物及NEDD4L蛋白水平。采用Co-IP法评估NEDD4L与IκBα或PPARγ的相互作用。结果：HG+oxLDL诱导的HCMECs CM可促进巨噬细胞M1极化、oxLDL摄取和泡沫细胞形成，外泌体抑制剂GW4869可消除这些作用。在HG+ oxldl诱导的hcmes外泌体中，NEDD4L过表达，可被THP1-M0细胞摄取。外泌体NEDD4L敲低通过降低p-Smad1/Smad1、p-Smad2/Smad2、p- κ b α/ i - κ b α和p-P65/P65蛋白水平，抑制巨噬细胞M1极化、oxLDL摄取和泡沫细胞形成。NEDD4L可通过泛素化作用降低i - κ b α和PPARγ的表达。结论：HG+oxLDL诱导的hcmes源性外泌体NEDD4L可增强IκBα和PPARγ的泛素化，促进巨噬细胞M1极化和oxLDL摄取，从而加速dm相关AS。
期刊介绍：
Cell Biology and Toxicology (CBT) is an international journal focused on clinical and translational research with an emphasis on molecular and cell biology, genetic and epigenetic heterogeneity, drug discovery and development, and molecular pharmacology and toxicology. CBT has a disease-specific scope prioritizing publications on gene and protein-based regulation, intracellular signaling pathway dysfunction, cell type-specific function, and systems in biomedicine in drug discovery and development. CBT publishes original articles with outstanding, innovative and significant findings, important reviews on recent research advances and issues of high current interest, opinion articles of leading edge science, and rapid communication or reports, on molecular mechanisms and therapies in diseases.
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