Sarfaraz Ahmad

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 ¹²⁵I 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. GRAPHICAL

Purpose: Angiotensin-(1–12) [Ang-(1–12)] serves as a primary substrate to generate angiotensin II (Ang II) by angiotensin-converting enzyme and/or chymase suggests it may be an unrecognized source of Ang II-mediated microvascular complication in hypertension-mediated retinopathy. We investigated Ang-(1–12) expression and internalization in adult retinal pigment epithelial-19 (ARPE-19) cultured cells. We performed the internalization of Ang-(1–12) in ARPE-19 cells in the presence of a highly specific monoclonal antibody (mAb) developed against the C-terminal end of the Ang-(1–12) sequence. Methods: All experiments were performed in confluent ARPE-19 cells (passage 28–35). We employed high-performance liquid chromatography to purify radiolabeled, ¹²⁵ I-Ang-(1–12) and immuno-neutralization with Ang-(1–12) mAb to demonstrate Ang-(1–12)'s internalization in ARPE-19 cells. Internalization was also demonstrated by immunofluorescence (IF) method. Results: These procedures revealed internalization of an intact ¹²⁵ I-Ang-(1–12) in ARPE-19 cells. A significant reduction (∼53%, P < 0.0001) in ¹²⁵ I-Ang-(1–12) internalization was detected in APRE-19 cells in the presence of the mAb. IF staining experiments further confirms internalization of Ang-(1–12) into the cells from the extracellular culture medium. No endogenous expression was detected in the ARPE-19 cells. An increased intensity of IF staining was detected in cells exposed to 1.0 μM Ang-(1–12) compared with 0.1 μM. Furthermore, we found hydrolysis of Ang-(1–12) into Ang II by ARPE-19 cells' plasma membranes. Conclusions: Intact Ang-(1–12) peptide is internalized from the extracellular spaces in ARPE-19 cells and metabolized into Ang II. The finding that a selective mAb blocks cellular internalization of Ang-(1–12) suggests alternate therapeutic approaches to prevent/reduce the RPE cells Ang II burden.

There is a critical need to identify new therapeutic targets and treatments, given emerging data documenting the limited effectiveness of current antihypertensive drugs to have a marked impact in preventing target organ damage and cardiovascular death. While monoclonal antibodies (mAb) are revolutionizing the treatment of human diseases, including severe hypercholesterolemia, their applicability to long-term hypertension control remains unexplored. Identification of angiotensin-(1-12) [Ang-(1–12)] as a non-renin-dependent substrate for Ang II generation in rodents and humans led us to engineer a mAb directed against the C-terminus of human Ang-(1–12) [h-Ang-(1-12)] sequence. We utilized a model of hypertension expressing the human angiotensinogen (AGT) gene in the rat genome [TGR(hAGT)L1623 rats] to address the effectiveness of endogenous h-Ang-(1–12) immunoneutralization in controlling the elevated blood pressure and reversing hypertension-induced target organ damage. Acute delivery of the h-Ang-(1–12) mAb (30 mg/Kg, i.v.) in anesthetized hypertensive transgenic rats induced a prompt arterial pressure fall (M ± SE: -18 ± 6/-16 ± 3 mm Hg, n=7; p< 0.001) in the absence of corresponding heart rate changes. The antihypertensive effect of the h-Ang-(1-12) mAb triggered a rise in plasma renin concentrations (from 107 ± 24 to 381 ± 260 ng/mL/h) that was inversely correlated with the fall in mean arterial pressure (r= -0.92, p< 0.001). Prior intravenous delivery of aliskiren fumarate (3-5 mg/Kg, i.v.) while inducing a fall in arterial pressure did not mask the antihypertensive action of the h-Ang-(1-12) mAb. Extension of these acute effects of the h-Ang-(1-12) mAb to awake instrumented transgenic hypertensive rats given consecutive daily doses of the mAb (30 mg/Kg, i.m. x 4 -5 days, n=7) documented a trend for significant statistical drops in daytime blood pressure and no changes in heart rate. We conclude that Ang-(1–12) is an endogenous Ang II forming substrate participating in the control of arterial pressure in a humanized model of high blood pressure and that selective immunoneutralization of this alternate substrate is a promising and novel therapeutic approach to treat hypertension and prevent its adverse cardiovascular sequelae.