Inorganic phosphate-osteogenic induction medium promotes osteogenic differentiation of valvular interstitial cells via the BMP-2/Smad1/5/9 and RhoA/ROCK-1 signaling pathways

Calcific aortic valve disease (CAVD) currently lacks a highly effective in vitro model. The presence of high concentrations of serum inorganic phosphate in patients with end-stage renal disease leads to calcification of vascular and aortic valves. Therefore, we applied inorganic phosphate to induce the osteogenic differentiation of valvular interstitial cells (VICs) and mimic its in vivo pathophysiological effects. Calcification and inflammatory response assays determined that inorganic phosphate-osteogenic induction medium (IP-OIM) was more efficient than classic osteogenic induction medium (OIM) containing organic glycerophosphate. Levels of BMP-2, RhoA, and ROCK-1 were significantly increased in IP-OIM cells. Knockdown efficiency of BMP-2- and RhoA-siRNA in VICs was evaluated, and expression of RhoA and its downstream target ROCK-1 was decreased after BMP-2-siRNA transfection. Moreover, ROCK-1 was significantly downregulated after RhoA knockdown, whereas expression of BMP-2 was unchanged. Interference of BMP-2 had a stronger anti-calcification effect than RhoA, further identifying BMP-2 as an upstream regulator of RhoA/ROCK-1. Stimulation of VICs by IP-OIM led to increased Smad1/5/9 phosphorylation, which peaked at 60 min, while pre-treatment of VICs with the Smad1/5/9 inhibitor Compound C attenuated VICs calcification. These results suggest that IP-OIM induced VICs osteogenic differentiation via Smad1/5/9 signaling. Knockdown of BMP-2 or RhoA also decreased Smad1/5/9 phosphorylation also decreased. We conclude that the RhoA/ROCK-1 axis participates in VICs osteogenic differentiation as a "bypass mediator" of the BMP-2/Smad1/5/9 signaling pathway.