A Selective Chymase Inhibitor, TEI-f00806, Eliminates the Ang II Production From Rat Ang-(1-12) in Bone Marrow Tissue

Background: Rat bone marrow (BM)-chimeric experiments reveal a critical role of locally formed angiotensin II (Ang II) in mediating the oxidant-rich inflammatory milieu associated with cardiovascular disease pathogenesis. Extending our first demonstration of renin angiotensin system (RAS) components in the BM, we recently reported a primary role of chymase rather than angiotensin converting enzyme (ACE) in BM generation of Ang II from the novel substrate angiotensin-(1-12) [Ang-(1-12)]. To verify chymase contribution over other proteases such as cathepsins, Ang-(1-12) metabolism studies in rat BM were carried out in the absence and in the presence of a selective orally active chymase inhibitor (TEI-F00806). Methods: Plasma membrane (PM) of BM and left ventricle (LV) tissues were obtained from 12 week-old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni’s fixative. PMs were incubated with 125 I-Ang-(1-12) and the metabolic products during incubation were determined by high-performance liquid chromatography. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 to determine statistical significance. Results: Assessment of 125 I-Ang-(1-12) hydrolysis in the absence of the chymase inhibitor revealed that chymase activity in BM membranes was 280 fold higher than that in LV (4,531 ± 137 fmol/mg/min and 16.3 ± 0.3 fmol/mg/min, respectively, n = 6, P < 0.001). In the presence of TEI-F00806, 96% of the chymase-mediated Ang II production was inhibited in BM. Confocal microscopy showed chymase-positive but tryptase-negative cells as the most dominant in BM, indicating that stem/progenitor cells rather than mature mast cells might be the source of chymase in BM. Conclusion: The current findings demonstrate that BM has tremendously higher chymase-mediated Ang II producing activity from Ang-(1-12) compared to heart tissue. The fact that TEI-00806 inhibited the Ang II producing activity of chymase underscores a tissue-selective enzymatic mechanism for Ang II formation from Ang-(1-12) contributing to the chronic inflammatory processes accompanying the pathogenesis of cardiovascular disease.