Chymase-Dependent Generation of Angiotensin II from Angiotensin-(1-12) in Human Atrial Tissue

Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified 125I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. 125I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, 125I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of 125I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of 125I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that 125I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min−1×mg−1, n = 9) from 125I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min−1×mg−1). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12)

A Case of Crescentic Glomerulonephritis Complicated with Hypocomplementemic Urticarial Vasculitis Syndrome and ANCA-Associated Vasculitis

Systemic urticaria in a 64-year-old woman was diagnosed as leukocytoclastic vasculitis by a punch biopsy of the skin. Her physical findings improved after prescription of prednisolone at a dose of 20 mg/day, but the skin rash relapsed with renal dysfunction, proteinuria, and hematuria when the dose of prednisolone was reduced over a period of 9 months to 1 mg/day. She was admitted to our institute for further examination, when urinary protein and plasma creatinine levels were 0.8 g/day and 1.7 mg/dL, respectively. Complement analysis showed that levels of total hemolytic component, component C3 fraction, and component C4 fraction were 30∼60% of normal values and the titer of anti-neutrophil cytoplasmic antibody for myeloperoxidase (MPO-ANCA) was 89 EU (normal range, <10 EU), though there were no immunologic disorders such as systemic lupus erythematosus. Cellular crescentic glomerulonephritis was observed by light microscopy, and immunofluorescent studies showed positive staining for IgG, IgM, C3, C4, and C1q. Electron microscopy showed mesangial and subendothelial deposits with circumferential mesangial interposition. She fulfilled the diagnostic criteria for hypocomplementemic urticarial vasculitis syndrome (HUV), and ANCA-associated vasculitis (AAV) was also indicated by small vessel vasculitis and positive MPO-ANCA. Steroid pulse therapy with methylprednisolone followed by oral prednisolone improved her general condition and hypocomplementemia, and MPO-ANCA became negative. HUV and AAV are distinct clinical disorders, though both affect small blood vessels. Here we report a case of AAV-complicated HUV with crescentic glomerulonephritis

Immunohistochemistry of human atrial tissue for chymase.

<p>Immunostaining of human atrial tissue using an Anti-Mast Cell chymase antibody (Abcam Inc., Cambridge, MA; Cat# ab2377) revealed high expression of chymase within atrial cardiac myocytes (A). Negative control without primary antibody shows no staining for chymase (B). <i>(Magnification 400; scale bar is 50 µm)</i>.</p

Diagnosis, drug treatment and clinical statement of human heart patients.

<p>Abbreviation: MV, mitral valve; AF, atrial fibrillation; CAD, Coronary artery disease; ASD, Atrial septal defect; CBS, Cardiac bypass surgery; ACE, angiotensin converting enzyme, and ARB, angiotensin receptor blocker.</p

Comparative Effects of Selective Enzyme Inhibition on ¹²⁵I-Ang-(1-12) Metabolism by plasma membrane isolated from human atrial appendage tissues.

<p>HPLC of human ¹²⁵I-Ang-(1-12) metabolic products generated by plasma membrane (50 µg) isolated from human atrial appendage incubated with or without the presence of RAS inhibitors at 37°C for 60 min. Values are expressed as % (Mean ± SEM) of Ang peptides generated from ¹²⁵I-Ang-(1-12). <i>No RAS inhibitors group</i>: Only aminopeptidases inhibitors (amastatin & bestatin), carboxypeptidases inhibitor (benzyl succinate) and PCMB; <i>All RAS inhibitors group</i>: Above inhibitors+RAS inhibitors (lisinopril, SCH39370, MLN-4760 & chymostatin); <i>Minus RAS inhibitor group</i>: One of the RAS inhibitor (lisinopril or chymostatin) omitted at a time form the <i>All RAS inhibitors group</i>.. Results are the average of nine human samples (n = 9).</p><p>*Percent of ¹²⁵I-Ang-(1-12) parent control remained unmetabolized after 60 min incubated with plasma membrane (50 µg) at 37°C. ND = Not detected;</p>a<p>Significantly different (<i>P</i><0.05) vs. corresponding group of <i>All RAS inhibitors</i>.</p

Outline of enzyme inhibitors employed in the experiments.

<p>Outline of enzyme inhibitors employed in the experiments.</p

Localization of Ang-(1-12) in human atrial tissue.

<p>Comparative adjacent sections of Ang-(1-12) immunoreactivity obtained from human atrial tissue with protein A purified polyclonal antibody produced by AnaSpec. A) Antibody (1∶2,000 dilution) blocked with 100 µmol/L of human Ang-(1-12) peptide, and B) Unblocked antibody (1∶2,000 dilution). <i>(Magnification 400; scale bar is 50 µm)</i>.</p

Ang-(1-12) metabolism by human atrial tissue.

<p>The metabolism of ¹²⁵I-Ang-(1-12) by plasma membrane isolated from human atrial tissues was analyzed by HPLC coupled to an inline BioScan γ-detector. The ¹²⁵I-Ang-(1-12) was incubated with human plasma membrane for 60 min at 37°C with or without the inhibitor cocktail and the metabolites were separated by HPLC. <i>A</i>: Chromatograms represent the hydrolysis of ¹²⁵I-Ang-(1-12) in the presence of all RAS inhibitors (<i>All RAS inhibitor group</i> containing lisinopril, SCH39370, MLN-4760, chymostatin, bestatin, amastatin, benzyl succinate, and PCMB). <i>B</i>: Hydrolysis of ¹²⁵I-Ang-(1-12) in the absence of all RAS inhibitors cocktail (<i>No RAS inhibitors group</i> containing only bestatin, amastatin, benzyl succinate, and PCMB). <i>C</i>: Hydrolysis of ¹²⁵I-Ang-(1-12) in the presence of the inhibitor cocktail that lacks only Lisinopril (<i>No lisinopril inhibitor group</i> containing all inhibitors as described in “<i>A</i>” except ACE inhibitor). <i>D</i>: Hydrolysis of ¹²⁵I-Ang-(1-12) in the presence of inhibitor cocktail that lacks only chymostatin (<i>No chymostatin group</i> containing all inhibitors as described in “<i>A</i>” except chymase inhibitor). Before adding the ¹²⁵I-Ang-(1-12), the plasma membrane was pre-incubated with inhibitors (each added at a dose of 50 µM) for 15 min at 37°C. The arrow indicates the retention time of ¹²⁵I-Ang-(1-12) and its metabolic products. HPLC results are representative of three or more separate metabolism experiments for each human sample.</p