Raju Sneha, Turner Mandy E, Cao Christian, Abdul-Samad Majed, Punwasi Neil, Blaser Mark C, Cahalane Rachel M E, Botts Steven R, Prajapati Kamalben, Patel Sarvatit, Wu Ruilin, Gustafson Dakota, Galant Natalie J, Fiddes Lindsey, Chemaly Melody, Hedin Ulf, Matic Ljubica, Seidman Michael A, Subasri Vallijah, Singh Sasha A, Aikawa Elena, Fish Jason E, Howe Kathryn L
Toronto General Hospital Research Institute, University Health Network, ON, Toronto, Canada (S.R., S.R.B., K.P., S.P., R.W., D.G., J.E.F., K.L.H.).
Institute of Medical Science, Temerty Faculty of Medicine, University of Toronto, ON, Canada (S.R., S.R.B., J.E.F., K.L.H.).
Arterioscler Thromb Vasc Biol. 2025 Jul;45(7):1277-1305. doi: 10.1161/ATVBAHA.124.322324. Epub 2025 May 29.
Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque.
EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV-cargos were assessed via whole transcriptome microRNA-sequencing and mass spectrometry-based proteomics. EV multiomics was integrated with bulk and single-cell RNA-sequencing datasets to predict EV cellular origin and ligand-receptor interactions, and multimodal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays.
Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-microRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells, smooth muscle cells, and immune cells. Integrated tissue vesiculomics and single-cell RNA-sequencing indicated complex EV-vascular cell communication that changed with disease progression and plaque vulnerability (ie, symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque EVs were predicted to mediate communication with endothelial cells. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization, well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones.
Our findings indicate that EVs may drive dynamic changes in plaques through EV-vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.
颈动脉粥样硬化是由细胞间通讯调控的，这种通讯推动疾病沿着临床连续谱（从无症状到有症状）发展。细胞外囊泡（EVs）是细胞衍生的纳米颗粒，代表了细胞通讯的一种新范式。关于它们的生物载荷、细胞来源/去向以及在人类动脉粥样硬化斑块中的功能作用，我们了解甚少。
通过尺寸排阻色谱法从人类颈动脉内膜切除术样本中富集EVs，这些样本被分为配对的斑块和边缘区（有症状的n = 16，无症状的n = 13）。通过全转录组微小RNA测序和基于质谱的蛋白质组学评估EVs的载荷。将EV多组学与批量和单细胞RNA测序数据集整合，以预测EV的细胞来源和配体-受体相互作用，并完成EV载荷的多模态生物网络整合。通过内皮细胞血管生成试验评估EV的功能影响。
颈动脉斑块中的EVs比相邻的边缘区更多，关键致动脉粥样硬化途径中的EV微小RNA和EV蛋白质有差异富集。EV细胞来源分析表明，组织EV特征起源于内皮细胞、平滑肌细胞和免疫细胞。整合的组织囊泡组学和单细胞RNA测序表明，EV与血管细胞之间存在复杂的通讯，这种通讯随疾病进展和斑块易损性（即有症状疾病）而变化。与边缘区相比，有症状患者的斑块以内皮途径参与增加和更复杂的配体-受体相互作用为特征，而无症状患者的斑块则不然。预测斑块EVs介导与内皮细胞的通讯。通路富集分析描绘了一种在内皮细胞血管生成和新生血管形成中起作用的内皮特征，这是斑块不稳定的众所周知的指标。这在功能上得到了验证，即与匹配的边缘区相比，人类颈动脉有症状斑块的EVs诱导了芽生血管生成。
我们的研究结果表明，EVs可能通过EV与血管细胞的通讯和效应功能驱动斑块的动态变化，这些功能是斑块易破裂的典型特征，从而引发有症状疾病。由EVs介导的内皮定向血管生成过程的发现为动脉粥样硬化创造了新的治疗途径。
