ABSTRACT

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.

摘要

周围动脉疾病（PAD）与心血管事件和死亡的高风险相关，并且被认为是未来的重要社会经济成本。细胞外囊泡（EVs）已成为发现与其蛋白质和核酸相关的新生物标志物的潜在候选者。为了寻找PAD中新的预后和治疗目标，我们确定了循环电动汽车的血栓形成活性，细胞起源和转录组谱。这项前瞻性研究包括对照组和PAD患者。通过流式细胞术在无血小板血浆（n = 45 PAD）中测定凝血时间（Procoag-PPL试剂盒），电动汽车的细胞起源和磷脂酰丝氨酸暴露。使用MARS-Seq RNA-Seq方案（n = 12 /组）生成中型/大型EV的转录组图谱。血清钙卫蛋白（S100A8 / A9）中差异表达基因S100A9的血清浓度通过ELISA在对照组（n = 100）和PAD患者（n = 317）中进行了验证。在人的动脉粥样硬化斑块的电动汽车和组织中也测定了S100A9（n = 3）。PAD患者的循环EV主要来自血小板，主要是膜联蛋白V阳性，并且与无血小板血浆的促凝活性有关。电动汽车的转录组学分析确定了15个差异表达的基因。其中，PAD患者的血清钙卫蛋白升高（主要是膜联蛋白V阳性，并且与无血小板血浆的促凝活性有关。电动汽车的转录组学分析确定了15个差异表达的基因。其中，PAD患者的血清钙卫蛋白升高（主要是膜联蛋白V阳性，并且与无血小板血浆的促凝活性有关。电动汽车的转录组学分析确定了15个差异表达的基因。其中，PAD患者的血清钙卫蛋白升高（p  <0.05）并与协变量调整前后截肢风险增加相关（平均随访3.6年，p  <0.01）。钙卫蛋白与hs-CRP的多变量分析进一步改善了风险分层（p  <0.01）。此外，S100A9也表达于股动脉斑块衍生的电动汽车和组织中。总之，我们发现PAD患者释放的EV主要来自血小板，对AnnexinV呈高度阳性，并富含与血小板生物学和免疫反应有关的转录本。钙卫蛋白可改善截肢风险预测，与hs-CRP联合使用可显着提高截肢风险。我们的结果表明，电动汽车可以作为液体活检以鉴定PAD患者分子特征的有前途的组成部分。