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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: J Vasc Surg. 2018 Apr 21;68(6 Suppl):48S–59S.e1. doi: 10.1016/j.jvs.2017.11.091

Fig 2.

Fig 2.

Angiotensin II (Ang II)-induced receptor activator of nuclear factor κB ligand (RANKL) expression in smooth muscle cells. Murine vascular smooth muscle cells (MOVAS cells) were stimulated with Ang II. Expression of RANKL messenger RNA (mRNA) and protein was quantified by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Forskolin (FSK) was used as a positive control. Ang II dose dependently induced Rankl mRNA (A) and RANKL protein expression (B). A time-course experiment showed increased Rankl mRNA (C) and RANKL protein (D) from 24 to 72 hours with a maximum at around 48 hours. Relative expression of Western blots was quantified using ImageJ. E and F, Increased RANKL expression through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway in smooth muscle cells. MOVAS cells were stimulated with 1 μM Ang II. JAK2 and STAT5 activities were assayed in Western blots. E, JAK2 and STAT5 phosphorylation was increased at 12 hours. F, Effect of TG101348, a selective inhibitor of JAK2, on Ang II-induced RANKL expression. TG101348 suppressed Ang II-induced RANKL expression in MOVAS cells. G-I, Analysis of the effects of Ang II and RANKL-neutralizing antibody on MOVAS cells. Cells were cultured in growth medium alone or media containing Ang II (1 μM), RANKL-neutralizing antibody (100 ng/mL), or both for 48 hours. G, Mitochondrial membrane potential of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. H, Proliferation of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. OD, optical density. I, Flow cytometric analysis of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. Cells were analyzed for the expression of tissue inhibitor of metalloproteinase 1 (TIMP1), transforming growth factor β (TGF-β), bone morphogenetic protein 4 (BMP4), matrix metalloproteinase (MMP) 9, and MMP2. Flow cytometric data are represented as the mean fluorescence intensity ± standard deviation of four replicates.

*P < .05. **P < .01.