Compensatory regulation of Extracellular Pyrophosphate Metabolism in Vascular Smooth Muscle Cells during Early Calcification

Abstract

Hydroxyapatitedeposition on the medial layer of the aortic walls is the hallmark of vascularcalcification and the most common complication in aging individuals andin patients with diabetes and those undergoing hemodialysis. Hyperphosphatemiais the main risk factor for vascular calcification and extracellularpyrophosphate (ePPi) is a potent physicochemical inhibitor of hydroxyapatitecrystal formation. Most ePPi is degraded to inorganic phosphate (Pi) by tissue non‐specific alkaline phosphatate (TNAP) in the aortic wall. Moreover, ePPiis generated enzymatically by hydrolysis of extracellular ATP by the enzyme ectonucleotidepyrophosphatase/phosphodiesterase 1 (eNPP1). Extracellular ATP is alsohydrolyzed by the ectonucleoside triphosphate diphosphohydrolase 1 (eNPTD1),releasing Pi. This study analyzed changes in extracellularpyrophosphate metabolism during the calcification process.Pi‐induced calcification of ex vivo‐culturedaortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized (118.9 μmolCa2+/gaorta vs. 433 μmolCa2+/g aorta, respectively). Bone morphogenic protein 2 (BMP2)expression was associated with calcium accumulation in Pi‐induced calcificationin cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs);and in calcitriol‐induced calcification in rats. Hydroxyapatite dose‐dependently induced BMP2 overexpression in VSMCs. TNAP mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all three models studied. eNPP1 increased from early to later phases of calcification, whereas eNTPD1 was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all three models studied. Because the rate of pyrophosphate hydrolysis was100 times slower than the rate of ATP hydrolysis (Vmax values of 56.7 and 5016 pmol*mg−1*min−1,respectively, and Km values of 215 μmol/L pyrophosphate and 512 μmol/L ATP, respectively), pyrophosphate synthesis is determined mainly by the ratio ofeNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs.These findings indicate that pyrophosphate synthesis increases in VSMCs during early calcification. Moreover, BMP2 may be a good marker of calcification, especially during early phases. By contrast, TNAP expression is not a good marker of calcification due to its regulation by various stimuli.Support or Funding InformationThis study was supported by grant SAF‐2014‐60699‐JIN from the Spanish Mini sterio deEconomía y Competitividad (MINECO).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.