WNT SIGNALING CAN PLAY AN IMPORTANT ROLE IN VASCULAR CALCIFICATION IN PATIENTS WITH ANKYLOSING SPONDYLITIS

Background: Vascular calcification is highly correlated with atherosclerosis. Ankylosing spondylitis (AS) is associated with a process of accelerated atherosclerosis. Wnt signaling plays an important role in the pathogenesis of vascular calcification. However, there has been no study of the role of Wnt signaling in vascular calcification in patients with AS. Objectives: We investigated the relationship between vascular calcification and Wnt signaling in patients with AS. Methods: Sixteen male patients aged over 20 years with AS were enrolled. They fulfilled the modified New York criteria and each of their ankylosing spondylitis disease activity score was more than 2.1. Sex and age matched nineteen healthy controls were also recruited. Mouse MOVAS vascular smooth muscle cell line (American Type Culture Collection, ATCC® CRL-2797™) were stabilized in maintain media for 24 hours. Then media were exchanged for the 10% serum of patients with AS or controls in maintain media. Cells were stimulated for another 72hours. We exchanged this medium with calcification medium. Cells were cultured until 2 weeks then stained with Alizarin Red S and the optical density (OD) was measured. For Western blotting and RT-qPCR, cells were stabilized for 24 hours and stimulated for another 72 hours through the same procedure as that of Alizarin Red S staining. After cell stimulation, the level of mRNA and protein were measured by RT-qPCR and western blot, respectably. We measure the level of Low-density lipoprotein receptor-related protein (LRP)5, LRP6, Dickkopf-related protein 1, Wnt3a, matrix metalloproteinase-7(MMP-7), beta-catenin for canonical Wnt signaling; Receptor Tyrosine Kinase Like Orphan Receptor 2, Wnt5a, Runt-related transcription factor 2 (RUNX2) for non-canonical Wnt signaling. We also checked the level of Alkaline phosphatase (ALP), IL-17, IL-23 and TNF-a. Results: The level of OD of MOVAS cells treated with serum from AS patients (19.503 ± 6.422, mean ± SD) was significantly higher than that from controls (10.994 ± 4.291) (P=0.000, Mann-Whitney test). The protein level of MMP-7 and beta-catenin of MOVAS cells treated with serum from AS patients (1.881 ± 0.687; 1.301 ± 0.342) was significantly higher than that from controls (0.779 ± 0.48; 0.854 ± 0.285) respectively (P=0.005,: P=0.002, Mann-Whitney test). The mRNA level of RUNX2, ALP, IL-17 and IL-23 of serum from AS patients (2.697 ± 1.46; 2.687 ± 1.753; 2.253 ± 1.128; 2.574 ± 1.142) was significantly higher than that from controls (1.396 ± 0.587; 1.696 ± 0.637; 1.358 ± 0.473; 1.386 ± 0.714) respectively (P=0.000; P=0.037; P=0.044; P=0.007, Mann-Whitney test). There was positive correlation between the mRNA level of WNT5a and RUNX2 (rho=-0.705, p=0.002, Spearman rank correlation coefficient) and the protein level of WNT5a and ALP, MMP-7 and TNF-a, MMP-7 and IL-17, MMP-7 and IL-23 (rho=-0.601, p=0.039; rho=-0.769, p=0.026; rho=-0.828, p=0.011; rho=-0.777, p=0.003), respectively. Conclusion: 1. Vascular smooth muscle cell calcification was increased in patients with ankylosing spondylitis than those of the control group. 2. The level of several molecules(i.e. Beta-catenin, RUNX2, MMP-7) related to Wnt signaling of vascular smooth muscle cells treated with serum of patients with AS was elevated significantly compared to those of controls and positively related. 3. Wnt signaling can play an important role in vascular calcification in patients with ankylosing spondylitis. Acknowledgments: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03030825) Disclosure of Interests: None declared