Chymase Activity in Plasma and Urine Extracellular Vesicles in Primary Hypertension.

KEY POINTS: Blood and urine extracellular vesicles isolated from hypertensive patients possess high chymase enzymatic activity. Chymase activity was significantly higher in small extracellular vesicles obtained from hypertensive patients with suboptimal BP control. BACKGROUND: Circulating extracellular vesicles (EVs) carry protected cargoes of nucleic acids, proteins, and metabolites. In this study, we identified and validated the surface proteins and enzymatic activity of chymase, angiotensin converting enzymes 1 (ACE) and 2 (ACE2), and neprilysin (NEP) in EVs isolated from the blood and urine of primary hypertensive patients. METHODS: Peripheral venous blood and spot urine from 34 hypertensive patients were processed to isolate plasma and urinary EVs. Immunogold labeling and transmission electron microscopy validated the presence of the exosomal marker protein CD63 on the surface of plasma and urinary EVs. Flow cytometry characterized plasma and urinary EVs for CD63, CD9, and CD81 surface markers. In addition, exosomal CD63, TSG101, and Alix were analyzed in urine by western blotting. Urinary EVs did not express the endoplasmic reticulum protein calnexin and Golgi protein GM130. Chymase, ACE, ACE2, and NEP activities on (125)I substrates—angiotensin-(1–12) (Ang-[1–12]) and angiotensin II—(1 nmol/L each) were quantified by HPLC. Data were analyzed based on whether the patient's BP was controlled (group 1: <140/80 mm Hg) or noncontrolled (group 2: ≥140/80 mm Hg). RESULTS: Chymase activity on Ang-(1–12) was significantly higher in plasma and urinary EVs than in ACE, ACE2, and NEP. In addition, chymase activity in urine EVs was more than three-fold higher than in plasma EVs. Chymase activity increased in plasma and urine EVs retrieved from group 2 patients. No comparable differences were found in the enzymatic activities of ACE, ACE2, and NEP urinary EVs between group 1 and group 2. CONCLUSIONS: These studies reveal a differential enzymatic activity of renin angiotensin system enzymes in plasma and urine EVs isolated from hypertensive patients. Demonstrating a comparatively high chymase enzymatic activity in EVs expands a previously documented finding of increased plasma Ang-(1–12) in hypertensive patients.