Ahmad Sarfaraz, Deep Gagan, Punzi Henry A, Su Yixin, Singh Sangeeta, Kumar Ashish, Mishra Shalini, Saha Amit K, Wright Kendra N, VonCannon Jessica L, Dell'Italia Louis J, Meredith Wayne J, Ferrario Carlos M
medRxiv. 2023 Nov 10:2023.11.09.23298324. doi: 10.1101/2023.11.09.23298324.
Extracellular vesicles (EVs) have emerged as a promising liquid biopsy for various diseases. For the first time, using plasma and urinary EVs, we assessed the activity of renin-angiotensin system (RAS), a central regulator of renal, cardiac, and vascular physiology, in patients with control (Group I) or uncontrolled (Group II) primary hypertension.
EVs were isolated from 34 patients with history of hypertension, and characterized for size and concentration by nanoparticle tracking analyses, exosomal biomarkers by immunogold labeling coupled with transmission electron microscopy, flow cytometry and immunoblotting. EVs were analyzed for the hydrolytic activity of chymase, angiotensin converting enzyme (ACE), ACE2, and neprilysin (NEP) by HPLC.
Plasma and urinary EVs were enriched for small EVs and expressed exosomal markers (CD63, CD9, and CD81). The size of urinary EVs (but not plasma EVs) was significantly larger in Group II compared to Group I. Differential activity of RAS enzymes was observed, with significantly higher chymase activity compared to ACE, ACE2, and NEP in plasma EVs. Similarly, urinary EVs exhibited higher chymase and NEP activity compared to ACE and ACE2 activity. Importantly, compared to Group I, significantly higher chymase activity was observed in urinary EVs (p = 0.03) from Group II, while no significant difference in activity was observed for other RAS enzymes.
Bioactive RAS enzymes are present in plasma and urinary EVs. Detecting chymase in plasma and urinary EVs uncovers a novel mechanism of angiotensin II-forming enzyme and could also mediate cell-cell communication and modulate signaling pathways in recipient cells.
细胞外囊泡（EVs）已成为一种有前景的用于多种疾病的液体活检手段。我们首次使用血浆和尿液中的细胞外囊泡，评估了肾素 - 血管紧张素系统（RAS）的活性，该系统是肾脏、心脏和血管生理的核心调节因子，研究对象为血压控制良好（第一组）或未得到控制（第二组）的原发性高血压患者。
从34例高血压病史患者中分离出细胞外囊泡，通过纳米颗粒跟踪分析对其大小和浓度进行表征，通过免疫金标记结合透射电子显微镜、流式细胞术和免疫印迹分析外泌体生物标志物。通过高效液相色谱法分析细胞外囊泡中糜酶、血管紧张素转换酶（ACE）、ACE2和中性内肽酶（NEP）的水解活性。
血浆和尿液中的细胞外囊泡富含小细胞外囊泡，并表达外泌体标志物（CD63、CD9和CD81）。与第一组相比，第二组尿液中细胞外囊泡（而非血浆中细胞外囊泡）的大小显著更大。观察到RAS酶的活性存在差异，血浆细胞外囊泡中糜酶活性显著高于ACE、ACE2和NEP。同样，尿液细胞外囊泡中糜酶和NEP活性高于ACE和ACE2活性。重要的是，与第一组相比，第二组尿液细胞外囊泡中的糜酶活性显著更高（p = 0.03），而其他RAS酶的活性未观察到显著差异。
生物活性RAS酶存在于血浆和尿液细胞外囊泡中。检测血浆和尿液细胞外囊泡中的糜酶揭示了一种生成血管紧张素II的新型酶机制，还可能介导细胞间通讯并调节受体细胞中的信号通路。
