Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle

Abstract

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.

Fig. 1.

RT-PCR of RNA from aortas. RNA was prepared from freshly isolated aortas as described in the methods. Reaction volumes contained dilutions of reverse transcription products (1:10 to 1:10,000) synthesized from 5 μg of total RNA. ENPP1–3, ectonucleotide pyrophosphatase/phosphodiesterase-1–3; NTPD, nucleotide triphosphate diphosphohydrolase; TNAP, tissue-nonspecific alkaline phosphatase.

Fig. 2.

Synthesis of pyrophosphate (PP_i) from ATP by rat aorta. A: autoradiogram of a representative thin-layer chromatogram of medium after incubation of aortic rings with [γ³²P]ATP in 1 μM ATP with or without 300 μM α,β-methylene-ATP (α,β-meATP), 300 μM β,γ-meATP, or 30 μM MLS38949. B: quantification of radioactivity. Results are normalized to inorganic pyrophosphate (PP_i) production in the absence of aorta and are the means of 3 experiments. *P < 0.001, **P < 0.01 vs. control.

Fig. 3.

Synthesis of PP_i and calcification in aortas from mice lacking NPP1. A: autoradiogram of a representative thin-layer chromatogram of medium after incubation of aortic rings with [γ³²P]ATP in 1 uM ATP with or without 30 μM MLS38949, 300 μM α,β-meATP, or 300 μM β,γ-meATP. B: quantification of PP_i production. Results are the means of 6 experiments. *P < 0.01 vs. control; **P < 0.01 vs. wild-type (WT). C: calcification of aortas from wild-type mice and mice lacking NPP1 (Enpp1^−/−). Symbols represent individual rings and means ± SE. Results are from 1 representative experiment. Similar results were obtained in 2 additional experiments. *P < 0.05.

Fig. 4.

PP transport and calcification in aortas. A: uptake of PP_i in normal and devitalized (frozen and thawed) rat aortas. Results are means ± SE of triplicate measurements; PP_i concentration was 5 μM. B: uptake of PP_i in rat aortas and in aortas from ank/ank mice and wild-type (ANK/ANK) littermates. Results are means ± SE of triplicate measurements for rat aortas and 8 measurements in mouse aortas; time was 180 min.; PP_i concentration was 1 μM. C: calcification of aortas from ank/ank mice and wild-type (ANK/ANK) littermates. Each symbol indicates a separate animal. *P < 0.01.

Fig. 5.

Hydrolysis of PP_i and calcification in aortas. A: hydrolysis of PP_i (1 μM) by rat aorta with or without 30 μM MLS38949, 300 μM α,β-meATP, or 300 μM β,γ-meATP. Results are the means ± SE of 3–5 experiments. *P < 0.001 vs. control; **P < 0.001 vs. MLS38949, by paired analysis. B: calcification of injured aortas cultured without or with 30 μM MLS38949. Results are the means ± SE of 8 aortic rings. *P < 0.005. C: fluorescence microscopy of rat aorta transduced with GFP-containing adenovirus showing expression of GFP in individual cells. Line indicates 500 μm. D: alkaline phosphatase activity in aortas exposed to Ad-TNAP or empty virus. Concentration of levamisole was 1 mM. Results from a representative experiment are shown. E: calcification of aortic rings cultured with Ad-TNAP or empty virus. Symbols represent individual rings and means ± SE. *P < 0.05.

Fig. 6.

Role of NPP1 and NPP3 in PP hydrolysis. A: hydrolysis of PP_i (5 μM) in HEK cells overexpressing ectoenzymes. Results are the means ± SE of 5 experiments, each performed in duplicate or triplicate. *P < 0.02 vs. control, by paired, one-tailed testing. B: hydrolysis of ATP (1 μM) in the same cells. Results are the means ± SE of a single experiment performed in triplicate. Similar results were obtained in an additional experiment. *P < 0.05, by one-tailed t-test. C: hydrolysis of PPi (1 μM) by aortas from Enpp1 mice and wild-type littermates without and with 300 μM β,γ-meATP. Results are the means ± SE of 3–4 different mice, each assayed in duplicate.

Fig. 7.

Diagram of ePP_i metabolism in vascular smooth muscle. PP_i is synthesized by NPP1 and 3 from ATP released from cells. Another source is transport of PP_i across the cell membrane, possibly mediated by ANK, which may also mediate ATP release. PP_i is hydrolyzed by TNAP and NPP3. NTPDs are very active in vascular smooth muscle and could limit availability of ATP for synthesis of PP_i.