Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle

Abstract

Villa-Bellosta R, Wang X, Millán JL, Dubyak GR, O'Neill WC. Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle. Am J Physiol Heart Circ Physiol 301: H61-H68, 2011. First published April 13, 2011; doi:10.1152/ajpheart.01020.2010 is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PPi metabolism controls calcification. To determine the role of ePP i in vascular smooth muscle, we identified the pathways responsible for ePPi 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 PPi from ATP in aortas was blocked by ␤,␥-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1 Ϫ/Ϫ ) did not synthesize PPi from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PPi 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 PPi was reduced 25% by ␤,␥-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 Ϫ/Ϫ 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 ePPi, 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. medial calcification; alkaline phosphatase; ectonucleotide pyrophosphorylase; adenosine 5=-triphosphate; ankyrin EXTRACELLULAR INORGANIC PYROPHOSPHATE (ePP i ) is a potent suppressor of vascular calcification both in vitro (8) and in vivo (13, 18) through its ability to inhibit hydroxyapatite formation (10). The importance of endogenous ePP i homeostasis in human cardiovascular function is demonstrated by the severe arterial calcification that occurs in the absence of a synthetic enzyme (17). Cells contain ectoenzymes capable of synthesizing and hydrolyzing ePP i , and our previous studies in cultured vascular smooth muscle cells have implicated them in the control of ePP i levels (15). However, it is not clear whether ePP i levels in vessels in vivo are controlled by local activity of these enzymes rather than by systemic activity via circulating ePP i levels and whether this affects vascular calcification. Although circulating PP i levels are inversely proportional to arterial calcification in patients with advanced chronic kidney disease (14), the correlation is weak and the role of PP i metabolism in the arterial...

Cellular function and molecular structure of ecto-nucleotidases

“…Nucleosides as final hydrolysis products can in turn be salvaged by cellular reuptake via specific transporters and rephosphorylation inside the cell [5]. In addition, the production of extracellular pyrophosphate from ATP is of major relevance in the control of bone mineralization [6] and vascular smooth muscle calcification [7]. In the digestive system, hydrolysis of extracellular nucleotides may furthermore serve in the absorption of their molecular constituents.…”

“…NPP1 is a marker of late stage in the differentiation of antibody-producing B cells [324,325], it is expressed in T cells [326], and in astrocytic brain tumors [327]. A major function of NPP1 is in bone mineralization and also in vascular smooth muscle cell calcification [6,7,328]. By hydrolyzing extracellular ATP to AMP, it contributes to the formation of extracellular PP i together with the inorganic pyrophosphate transporter ANK-mediated PP i efflux.…”

“…It can serve as a marker for the estimation of hypersensitivity to allergens [339][340][341][342]. Recent experiments implicate a dual role of NPP3 in vascular smooth muscle calcification by producing (together with NPP1) PP i from ATP on the one hand and subsequently hydrolyzing (together with TNAP) the PP i produced [7].…”

“…Recently, it has been demonstrated that soluble heterologously expressed murine NPP1 [362,363] or NPP3 [7] hydrolyzes (at pH 7.4) PP i in addition to ATP. This result corroborates earlier observations that NPP2 cleaves PP i into P i [364].…”

Pyrophosphate: a key inhibitor of mineralisation

“…Sufferers of this condition usually die in infancy because of substantial vascular calcification. Consistent with an inhibitory role, Enpp1 -/-mice exhibit significant vascular calcification in vivo and Enpp1 -/-VSMCs have a reduced ability to generate pyrophosphate from ATP leading to increased calcification in vitro [45,87]. Enpp1 -/-VSMCs also display higher expression of chondrogenic, osteoblastic and osteocytic markers [88].…”