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Primacy of Chymase over Angiotensin Converting Enzyme in the Production of Angiotensin II in Rat Bone Marrow Tissue
Author(s) -
Yamashita Tomohisa,
Ahmad Sarfaraz,
Wright Kendra N,
VonCan Jessica L,
Varagic Jasmina,
Sun Xuming,
Wang Hao,
Groban Leanne,
Ferrario Carlos M
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.577.3
Subject(s) - chymase , bone marrow , renin–angiotensin system , angiotensin ii , medicine , endocrinology , chemistry , angiotensin converting enzyme , progenitor cell , biology , stem cell , enzyme , receptor , biochemistry , microbiology and biotechnology , blood pressure
Background The bone marrow is attracting attention as not only the nidus of circulating blood cells but the source of stem and progenitor cell‐based therapy. Renin‐angiotensin system (RAS) and angiotensin II (Ang II) receptor type 1 in bone marrow participate in hematopoiesis, a target in the evolution of inflammatory processes involved in cardiovascular disease pathogenesis. Extending the original demonstration of RAS components in the rat bone marrow (Strawn et al. Br J Haematol. 2004;126:120–6), this study investigated the existence of an Ang II‐forming pathway in which chymase rather than angiotensin‐converting enzyme (ACE) cleaves the peptide from the newly described substrate angiotensin‐(1‐12) [Ang‐(1‐12)] which functions as an endogenous tissue substrate for the direct formation of Ang II by chymase. Methods Bone marrow tissue was obtained from the tibia of twelve weeks‐old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni's fixative. Bone marrow tissues, homogenized with 0.05 M Tris‐HCl and centrifuged at 28,000 g , were subjected to immunoblot and chymase enzymatic activity using high‐performance liquid chromatography (HPLC). Additional samples were prepared for quantitative RT‐PCR analysis normalized to GAPDH. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 considered statistically significant. Results Fluorescent immunohistochemistry showed the existence of immunoreactive (ir‐) Ang‐(1‐12) and ir‐chymase‐positive cells in rat's bone marrow. Immunoblots showed chymase protein expression in cell pellets but not in supernatants. Assessment of 125 I‐Ang‐(1‐12) or 125 I‐Ang I hydrolysis in cell pellets in the presence and absence of specific chymase or ACE inhibitors, revealed that production of Ang II by chymase was two‐thousand fold higher compared to ACE (4,531 ± 137 fmol/mg/min and 2.3 ± 0.3 fmol/mg/min, respectively, P<0.0001, n = 6). ANOVA and post‐hoc Tukey's test showed that gene expression of rat mast cell protease‐5 (rMCP‐5) was at least twice higher than rMCP‐1, rMCP‐2 and rMCP‐4. Conclusion These studies reveal a major role of Ang‐(1‐12) and chymase as the critical pathway for Ang II‐formation in rat's bone marrow. The pleotropic actions of tissue chymase in the degradation of matricellular proteins, activation of matrix metalloproteinases, transforming growth factor‐β, and production of inflammatory cytokines posits this alternate pathway for Ang II‐production as most relevant in understanding and treating cardiovascular disease. Support or Funding Information This study was supported by the National Heart, Blood, Lung Institute of the NIH through HL‐051952 grant. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .