RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

OCG with increased RANKL expression was present in Ang II-induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast-related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted.

AAAs were produced in apolipoprotein E knockout mice via the administration of Ang II. Additionally, receptor activator of nuclear factor kB ligand (RANKL)-neutralizing antibody (5 mg/kg) was administered to one group of mice 7 days prior to Ang II infusion. Aneurysmal sections were probed for presence of RANKL and tartrate-resistant acid phosphatase via immunohistochemistry and immunofluorescence staining. Mouse aortas were also examined for RANKL and matrix metalloproteinase 9 expression via Western blot. In vitro murine vascular smooth muscle cells (MOVAS) and murine macrophages (RAW 264.7) were analyzed for the expression of osteogenic factors via Western blot, qPCR, and flow cytometry in response to Ang II or RANKL stimulation. The signaling pathway that mediates Ang II-induced RANKL expression in MOVAS cells was also investigated via application of TG101348, a Janus kinase 2 (JAK2) inhibitor, and Western blot analysis.

Osteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride-induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)-induced dissecting aneurysm (Ang II-induced AAA) in apolipoprotein E knockout mice.

Immunohistochemical staining of Ang II-induced AAA sections revealed OCG as evidenced by increased RANKL and tartrate-resistant acid phosphatase expression compared with control mice. Immunofluorescence staining of AAA sections revealed co-localization of vascular smooth muscle cells and RANKL, revealing vascular smooth muscle cells as one potential source of RANKL. Systemic administration of RANKL-neutralizing antibody suppressed Ang II-induced AAA, with significant reduction of the maximum diameter of the abdominal aorta compared with vehicle controls (1.5 ± 0.4 mm vs 2.2 ± 0.2 mm). Ang II (1 μM) treatment induced a significant increase in RANKL messenger RNA expression levels in MOVAS cells compared with the vehicle control (1.0 ± 0.2 vs 2.8 ± 0.2). The activities of JAK2 and signal transducer and activator of transcription 5 (STAT5) were also significantly increased by Ang II treatment. Inhibition of JAK2/STAT5 suppressed Ang II-induced RANKL expression, suggesting the involvement of the JAK2/STAT5 signaling pathway. Conclusions: OCG with increased RANKL expression was present in Ang II-induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast-related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted.