Objectives Angiotensin-(1-7) [Ang-(1-7)] is a new bioactive heptapeptide in the renin-angiotensin system (RAS). It is produced mainly by the activity of angiotensin-converting enzyme 2 (ACE2) and acts through the Mas receptor. Ang-(1-7) has potent protective effects in cardiovascular diseases, opposing many pathogenic actions of angiotensin II (Ang II) mediated by Ang II type 1 (AT1) receptor. However, the role of Ang-(1-7) in VC is still unclear. The axis of ACE2/Ang-(1-7)/Mas is formed, which could counter-regulate the effects of ACE/Ang II/ AT1 axis. And the changes of RAS members during VC also remain elusive. In this study, we tested the levels of major RAS members and investigated the effects of Ang-(1-7) on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. Our aim was to explore the new mechanisms and the actions of novel RAS in VC, and to provide new therapeutic strategies of Ang-(1-7) for VC.
Methods The hemodynamic parameters of rats were detected by the use of cardiac catheter technique. Alkaline phosphatase (ALP) activity and calcium content were determined by colorimetry through a reaction with phenylphosphate disodium substrate method and o-cresolphtalein complexon method, respectively. HE staining and von Kossa staining were used to detect the changes of vascular structure and calcium deposition, respectively. Real-time PCR and Western blot methods were used to test the expression changes of RAS members and phenotypic transformation markers in VSMCs. Radioimmunoassay was used to measure plasma Ang II, and enzyme kinetics method was used to measure the ACE activity.
Results We found that the levels of ACE2 and Mas receptor were significantly increased in calcified aortas by 2.8-fold and 4.5-fold (both P<0.05), respectively, along with the enhanced expression of ACE, AT1, Ang II type 2 (AT2), and angiotensinogen (AGT). Ang-(1-7) treatment led to a reduction in alkaline phosphatase (ALP) activity by 36.9% (P<0.05) and calcium deposition by 49.8% (P<0.01) in VC, alleviated the disorders of hemodynamic parameters, and protected the vascular wall from calcification injury in VC rats. In addition, we observed that Ang-(1-7) prevented the loss of lineage markers of vascular smooth muscle cells (VSMCs) including SM α-actin, SM22α, calponin and smoothlin; attenuated the osteogenic transition of VSMCs by decreasing the expression of bone-associated proteins such as bone morphogenetic protein 2, core binding factor α 1, osteopontin and osteocalcin. Furthermore, Ang-(1-7) reversed the elevated levels of ACE, AT1 and AGT by 61.5% (P<0.01), 72.5% (P<0.05) and 79.2% (P<0.01), respectively; and significantly inhibited the plasma ACE activity in VC rats by 18.6% (P<0.01), which might result in the reduction of circulating Ang II concentration.
Conclusions We provide the first in vivo evidence that Ang-(1-7) can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs, at least in part by decreasing the level and activity of the ACE/Ang II/AT1 axis. The increased expression of ACE2 and Mas receptor in calcified aortas suggests the involvement of ACE2/Ang-(1-7)/Mas axis during VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC, and ACE2/Ang-(1-7)/Mas axis could become a new target for the prevention and therapy of VC.