Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate 1 homeostasis : synergistic modulation by cyclic AMP and hyperphosphatemia

14 Vascular calcification is a multifaceted process involving the gain of calcification inducers and the loss of 15 calcification inhibitors. One such inhibitor is pyrophosphate (PPi) and the regulated generation and 16 homeostasis of extracellular PPi is a critical determinant of soft-tissue mineralization. We recently 17 described an autocrine mechanism of extracellular PPi generation in cultured rat aortic vascular smooth 18 muscle cells (VSMC) that involves both ATP release coupled to the ENPP1 ecto19 phosphodiesterase/pyrophosphatase and the efflux of intracellular PPi mediated or regulated by the ANK 20 plasma membrane protein. We now report that increased cAMP signaling and elevated extracellular Pi 21 act synergistically to induce a calcification of these VSMC that is correlated with the progressive 22 reduction in their ability to accumulate extracellular PPi. Attenuated PPi accumulation was mediated in 23 part by a cAMP-dependent decrease in ANK expression coordinated with a cAMP-dependent increase in 24 expression of TNAP, the tissue non-selective alkaline phosphatase that degrades PPi. Stimulation of 25 cAMP signaling did not alter ATP release or ENPP1 expression and the cAMP-induced changes in ANK 26 and TNAP expression were not sufficient to induce calcification. Elevated extracellular phosphate alone 27 elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, when 28 combined with a cAMP stimulus, elevated phosphate induced decreases in the ATP release pathway(s) 29 that supports ENPP1 activity; this resulted in markedly reduced rates of PPi accumulation that facilitated 30 robust calcification. The calcified VSMC were characterized by maintained expression of multiple SMC 31 differentiation marker proteins including SM-α actin, SM22α, and calponin. Notably, the addition of 32 exogenous ATP (or PPi per se) rescued the cAMP plus phosphate-treated VSMC cultures from 33 progression to the calcified state. These observations support a model wherein extracellular PPi 34 generation mediated by both ANKand ATP release-dependent mechanisms serves as a critical regulator 35 of VSMC calcification. 36