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

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

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

Chymase and ACE contribution to generate Ang II from Ang-(1-12).

<p>Chymase and ACE enzyme mediated generation of Ang II product from the metabolism of ¹²⁵I-Ang-(1-12) (1 nmol/L) by plasma membrane isolated from human atrial tissues. The contribution of each enzyme (chymase, and ACE) activity (fmol Ang II formation×min⁻¹×mg⁻¹) were calculated based on Ang II product analysis by HPLC in each human samples incubated with or without the chymostatin and lisinopril (each added at a dose 50 µM) for 60 min at 37°C. Data are expressed as mean ± SEM; total n = 9 (4 female).</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

Simple regression analysis for log FABP4 at baseline.

<p>Simple regression analysis for log FABP4 at baseline.</p

Flow chart of study participants.

<p>A total of 44 patients with type 2 diabetes mellitus were recruited, and 39 patients were finally analyzed in the present study.</p

Characteristics of the patients with decreased and increased FABP4 level by canagliflozin.

<p>Characteristics of the patients with decreased and increased FABP4 level by canagliflozin.</p