Adrenomedullin up-regulates osteopontin and attenuates vascular calcification via the cAMP/PKA signaling pathway

Aim: To determine whether adrenomedullin (ADM) attenuates vascular calcification (VC) by inducing osteopontin (OPN) expression. Methods: A VC model of rat aorta was induced with vitamin D3 plus nicotine (VDN), and vascular smooth muscle cell (VSMC) calcification was induced with beta-glycerophosphate. Von Kossa staining and alizarin red staining were assessed. Alkaline phosphatase (ALP) activity was measured. Immunohistochemical analysis was used to detect alpha-actin, while RT-PCR and Western blot analysis were used to quantify OPN expression. Results: Administration of ADM greatly reduced VC in VDN-treated aortas compared with controls, which was confirmed in calcified VSMCs. The decrease in alpha-actin expression was ameliorated by ADM both in vivo and in vitro. Moreover, mRNA and protein expression levels of OPN were significantly up-regulated in calcified aortas, and ADM increased OPN expression in calcified aortas. Furthermore, ADM up-regulated OPN expression in normal aortas and VSMCs. The ADM-mediated effects were similar to that of forskolin, which activates adenylyl cyclase; additionally, while the PKA inhibitor H89 and Ca2+ chelator Fura-2 blocked the effect of ADM. However, the MEK/ERK inhibitor PD98509 had no effect on ADM induction of OPN mRNA expression. An OPN polyclonal antibody inhibited ADM-mediated attenuation of VC. Conclusion: ADM up-regulates OPN expression and thus attenuates VC via PKA. ADM appears to be an endogenous cardiovascular protective peptide and may represent a new therapeutic target for VC treatment.