Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

动脉粥样硬化是一种常见的与年龄相关的疾病，其特点是强烈的免疫活性。动脉粥样硬化斑块由血液中浸润的内皮细胞、血管平滑肌细胞（VSMC）、脂质和免疫细胞组成。在疾病进展过程中，斑块内的 VSMC 会发生衰老，并分泌可主动调节斑块微环境的 SASP（衰老相关分泌表型）因子。我们证明衰老的 VSMC 分泌的细胞外囊泡 (senEV) 数量增加。基于对 VMSC 衍生的 EV 和 SASP 可溶部分 (sSASP) 的无偏蛋白质组学分析，在每个 SASP 区室中鉴定出了 900 多种蛋白质。将 VMSC 衍生的 EV 的组成与 SASP 图谱进行比较，发现多种蛋白质，包括丝氨酸蛋白酶抑制剂家族 F 成员 1 (SERPINF1) 和血小板反应蛋白 1 (THBS1)，是衰老 VSMC 和成纤维细胞分泌的 EV 中常见上调的成分。在可溶性 SASP 因子中，只有生长分化因子 15 (GDF15) 在衰老 VSMC、成纤维细胞和上皮细胞的分泌组中普遍增加。EV 蛋白的生物信息学分析区分了参与免疫细胞功能调节的功能组织蛋白网络。因此，衰老VSMC释放的EV诱导T细胞分泌IL-17、INFγ和IL-10以及单核细胞产生的TNFα。此外，senEV 影响单核细胞的分化，有利于具有促炎特征的混合 M1/M2 极化。总而言之，我们的研究提供了对 VSMC SASP 的复杂、公正的分析，并证明源自衰老 VSMC 的 EV 通过调节免疫细胞活性来影响细胞因子环境。我们的结果强化了衰老细胞作为动脉粥样硬化炎症的重要诱导剂的作用。