Research Article: Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

Date Published: March 31, 2017

Publisher: Public Library of Science

Author(s): Ricardo Villa-Bellosta, Magda R. Hamczyk, Vicente Andrés, Elena Aikawa.


Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues.

High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate.

We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification.

Partial Text

Phosphorus is an essential nutrient involved in many biological processes, including cell signaling, nucleic acid synthesis, energy metabolism, membrane function, and bone mineralization. Phosphorus is found in hard tissues (~85%), soft tissues (~14%), and extracellular fluids (~1%)[1]. The majority of phosphorus present in extracellular fluids exists in solution as a free phosphate ion, called inorganic phosphate (Pi). Plasma Pi has a concentration of 0.8–1.5 mmol/L, and exists in three forms: bound to proteins, complexed to cations, and ionized. Serum phosphate concentration is the outcome of the balance among phosphate absorption from the intestine, mobilization from bone, and renal excretion in urine [2]. Phosphate homeostasis is mainly regulated by phosphatonins, including vitamin D, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23)[3].

CPD, mainly in the form of hydroxyapatite or whitlockite, is the hallmark of calcification. Physiological CPD occurs in specific sites of hard tissues such as bone, antlers, and dentine. Soft tissues are not normally mineralized, but aging and several pathological conditions are associated with soft tissue calcification, which increases morbidity and mortality[2]. Elevated serum Pi is a key risk factor for pathological and ectopic soft tissue calcification[6]. Calcification is associated with the presence of macrophages, but our recent work showed that alternatively activated macrophages have an anti-calcifying effect[24]. We hypothesized that macrophages activated by high Pi concentration might influence ectopic calcification. In this study, we show for the first time that mouse macrophages are activated by Pi in a dose-dependent manner (Fig 1). Remarkably, this activation takes at least four days to become manifest, thereby simulating a subacute hyperphosphatemia. Pi-dependent activation of M0φs is mainly characterized by the elevated expression and activity of Arginase 1, a marker of mouse M2φs that is not expressed in M1φs[18]. Moreover, MPiφs showed higher mRNA expression of HIF-1α and PGC-1β, known markers of glycolysis and β-oxidation, respectively[33,34], and this was associated with increased amounts of intracellular and extracellular ATP (Figs 3C and 5B), as also occurs in M2φs[24].




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