ACE2 Mediated Cardiac Fibrosis Alleviation Through Angiotensin‐(1‐7)/Mas Axis Regulation of the Renin‐Angiotensin System/Rac Signaling Pathway

This study aimed at investigating whether Angiotensin-(1-7) [Ang-(1-7)] could alleviate cardiac fibrosis by modulating the expression of Rac1 and Rad GTPase and evaluating the specific mechanism in rat cardiac fibroblasts (CFs) and in canine atrial rapid pacing models. Fresh CFs were isolated from ventricles of 1 ~ 3 day-old Sprague-Dawley rats, and were randomly divided into control group, LPS group, Ang-(1-7) group and LPS+Ang-(1-7) group. CCK8 was used to detect cell proliferation. qRT-PCR was to detect the mRNA expression of ACE2. Mongrel dogs were randomized into four groups: Sham-operated (Sham), AF-Control, AF-EGFP and AF-ACE2 groups. Immunoperoxidase staining was used to test Rac1 and Gem expression. Collagen fibers in heart were stained a red color and extent of cardiac fibrosis was evaluated. Histopathology was to observe actin,α-smooth. Enzyme-Linked Immunosorbent Assay was to detect Ang II and Ang-(1-7) expression in canine atrial tissue. And western blot analysis was to detect the protein expression of iNOS, COX2, α-SMA, Rac1, ACE, and AT1R in cells and tissues. Cellular experiments showed LPS promoted proliferation of fibroblasts, which could be inhibited by Ang-(1-7) (p < 0.05). Compared with control group, LPS-induced CFs showed a significant increase in protein expression of iNOS, COX2, and α-SMA, which promoted cell proliferation. However, the application of Ang-(1-7) reversed these effects and suppressed the ACE/AT1R signaling pathway in LPS-induced CFs. In animal experiments, ACE2 over-expression would up-regulate atrial Ang-(1-7) and AT2R and reduce atrial angiotensin II (Ang II) and AT1R expressions (p < 0.05). In AF-ACE2 group, Rac1 was down-regulated and Rad GTPase was up-regulated, in comparison with AF-Control and AF-EGFP groups, associated with reductions of CTGF, fibrosis-related factors α-SMA and N-cadherin. ACE2 was found to alleviate cardiac fibrosis through the Ang(1-7)/Mas signaling pathway in animal experiments.