Neuromedin B modulates phosphate-induced vascular calcification

Abstract

Vascular calcification is the heterotopic accumulation of calcium phosphate salts in the vascular tissue and is highly correlated with increased cardiovascular morbidity and mortality. In this study, we found that the expression of neuromedin B (NMB) and NMB receptor is upregulated in phosphate-induced calcification of vascular smooth muscle cells (VSMCs). Silencing of NMB or treatment with NMB receptor antagonist, PD168368, inhibited the phosphate-induced osteogenic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling and VSMC apoptosis. PD168368 also attenuated the arterial calcification in cultured aortic rings and in a rat model of chronic kidney disease. The results of this study suggest that NMB-NMB receptor axis may have potential therapeutic value in the diagnosis and treatment of vascular calcification. [BMB Reports 2021; 54(11): 569-574].

Fig. 1

Expression of NMB and NMB receptor in the phosphate-induced calcification of VSMC. VSMCs were cultured in growth medium or calcification medium for 9 days. (A) Calcified nodules were detected by ARS staining; nodules were photographed with a digital camera (upper) and observed using a phase contrast microscope (lower). Scale bar: 50 μm. The absorbance of the released ARS was measured to evaluate the degree of mineralization (right). (B and C) Total calcium content and ALP activity was measured. (D) Total RNA was isolated and analyzed using quantitative real-time RT-PCR with the specific primers for NMB and NMB-receptors. (E) The expression of NMB and NMB receptor protein was examined using western blotting. (F) The secreted NMB protein in the cell culture medium was measured using ELISA. *P < 0.05; **P < 0.01 vs. control.

Fig. 2

Effect of NMB silencing and PD168368 treatment on the phosphate-induced phenotype switching of VSMCs. After transfection with siRNA for NMB or negative control, VSMCs were incubated in calcification medium. VSMCs were also cultured in calcification medium with or without PD168368 (100 nM). After 7 days, calcified nodules were detected by ARS staining and then absorbance was measured to evaluate the degree of mineralization (A). (B and C) Total calcium content and ALP activity was measured. (D) Changes in the levels of Runx2, calponin, Wnt3a, phospho-β-catenin (Ser33/37/Thr41), phospho-β-catenin (Ser675), β-catenin, phospho-GSK-3β (Ser9), and GSK-3β proteins were evaluated using western blotting. (E) Im-munocytochemical staining of Runx2 (red) and calponin (green) were visualized under a fluorescence microscope. Scale bar: 50 μm. (F) The mRNA levels of Runx2 and calponin were determined using quantitative real-time RT-PCR. *P < 0.05; **P < 0.01 vs. control or control siRNA, ^#P < 0.05; ^##P < 0.01 vs. Pi.

Fig. 3

Effect of NMB silencing and PD168368 treatment on phosphate-induced apoptosis of VSMCs. After transfection with siRNA for NMB or negative control, VSMCs were incubated with calcification medium with or without PD168368 (100 nM). (A) Apoptosis was detected using flow cytometry with PI staining. (B) The apoptotic bodies in VSMCs (green) were determined using TUNEL staining. DAPI (blue) stains nuclear DNA. Scale bar: 50 μm. (C) Changes in total/cleaved-caspase-3, Bcl2, and Bad were estimated using western blotting. (D) Using quantitative real-time RT-PCR, the expression levels of Bcl2 and Bad mRNA were quantified. *P < 0.01 vs. control or control siRNA, ^#P < 0.01 vs. 2.6 mM Pi.

Fig. 4

Effect of PD168368 on the aortic calcification in ex vivo and in vivo. Pieces of rat aortas were cultured in calcification medium without or with 100 nM PD168368. (A) The calcified lesions were stained with von Kossa staining. Scale bar: 50 μm. Percentage of the calcified area was calculated using calcification analyzer. (B and C) Expression of NMB, NMB receptor, Runx2, calponin, Bcl2 and Bad protein were examined using western blotting. The CKD rats were fed a diet supplemented with adenine. For PD168368 treatment, CKD rats were injected intraperitoneally with PD168368 or vehicle. (D) Calcified lesions in aortas of sham and CKD rats were assessed using von Kossa staining. Scale bar: 50 μm. The percentage calcified area was calculated using calcification analyzer. (E) NMB protein levels were assessed using ELISA. (F) Western blots were individually probed with antibodies against Runx2, calponin, Bcl2 and Bad. *P < 0.05; **P < 0.01 vs. control or sham, ^#P < 0.05; ^##P < 0.01 vs. 2.6 mM Pi or vehicle.