The Thermodynamics of Medial Vascular Calcification

Abstract

Medial vascular calcification (MVC) is a degenerative process that involves the deposition of calcium in the arteries, with a high prevalence in chronic kidney disease (CKD), diabetes, and aging. Calcification is the process of precipitation largely of calcium phosphate, governed by the laws of thermodynamics that should be acknowledged in studies of this disease. Amorphous calcium phosphate (ACP) is the key constituent of early calcifications, mainly composed of Ca²⁺ and PO₄ ^3- ions, which over time transform into hydroxyapatite (HAP) crystals. The supersaturation of ACP related to Ca²⁺ and PO₄ ^3- activities establishes the risk of MVC, which can be modulated by the presence of promoter and inhibitor biomolecules. According to the thermodynamic parameters, the process of MVC implies: (i) an increase in Ca²⁺ and PO₄ ^3- activities (rather than concentrations) exceeding the solubility product at the precipitating sites in the media; (ii) focally impaired equilibrium between promoter and inhibitor biomolecules; and (iii) the progression of HAP crystallization associated with nominal irreversibility of the process, even when the levels of Ca²⁺ and PO₄ ^3- ions return to normal. Thus, physical-chemical processes in the media are fundamental to understanding MVC and represent the most critical factor for treatments' considerations. Any pathogenetical proposal must therefore comply with the laws of thermodynamics and their expression within the medial layer.

Keywords: calcium and phosphate homeostasis; ectopic calcification; medial vascular calcifications; mineralization; thermodynamics.

FIGURE 1

Parameters affecting precipitation. (A) La Mer plots representing the different stages of precipitation from solution in relation to supersaturation under different conditions are shown. Initial conditions (left panel): the supersaturation limit value (Virchow, 1858) and a metastable range of ion activities in which the solution does not precipitate, despite being supersaturated, are depicted. This metastable region represents an energy barrier for nucleation. Once a critical concentration is reached (Sc), homogeneous precipitation will take place in the solution. The limits can be reduced in the presence of nucleation promoters (central panel). Promoters in solution have a high affinity for any of the crystal component ions and can induce nucleation even below the critical supersaturation level through a process called heterogeneous nucleation. On the other hand, other molecules, termed inhibitors of nucleation, have the capacity to increase the energy barrier for nucleation, thereby increasing both the critical supersaturation value and the induction time for nucleation (right panel). (B) Effect of pH on the formation of the different species of phosphate and carbonate. Left, molar ratio of the phosphate species H₂PO₄^– (orange), HPO₄^2– (green), and PO₄^3– (violet) in solution as a function of pH, within the pH range of interest: 5.5–8.5. Right, variation of the concentrations of H₂CO₃ (orange), HCO₃^– (green), and CO₃^2– (violet) within the same pH range in DMEM medium. The shaded area corresponds to the expected physiological pH. (C) Effect of pH on the supersaturation of ACP and HAP in blood at the lowest and highest concentrations of Ca and phosphate (Pi), at normal concentration limits and under hyperphosphatemic conditions. (D) Effect of the total concentration of phosphate (Pi) on the supersaturation of ACP and HAP in blood, at pH = 7.4. The lower limit corresponds to the Ca and Pi concentrations of 1.02 and 1.00 mM, respectively; the upper limit corresponds to 1.23 and 1.5 mM; and hyperphosphatemia corresponds to 1.23 and 2.00 mM.

FIGURE 2

Promoters of CaP mineralization. (A) Structure of major biomolecules that as promoters of calcification: phosphorylated proteins, sulfated glycosaminoglycans, carboxyglutamic proteins and phospholipid membranes. (B) CaP mineralization in the presence of nucleation promoters: accumulation of Ca by absorption on promoters, precipitation of ACP, and crystallization of HAP.

FIGURE 3

Hypothetical molecular processes in calcium phosphate medial vascular mineralization. At pH up to 7.40 Ca²⁺ ions are hydrated, and the majority of the phosphate molecules are in the form of HPO4-²- ions. The precipitation is triggered by a local increase of pH to above 7.90, associated with a marked increase in PO₄^3– ions; the main component in ACP and HAP. Ca²⁺ and PO₄^3– ions form complexes of increasing coordination number, eventually forming multinuclear clusters, which contain nine Ca²⁺ ions and six PO₄^3– ions. Subsequently, promoter macromolecules with charged Ca²⁺-ligand groups (phosphate, carboxylate, and/or sulfate) produce a local accumulation of Posner clusters and the appearance of ACP precipitates. Finally, as the process progresses, the precipitate clusters rearrange into dense crystalline HAP nanoparticles.

FIGURE 4

Promoters and inhibitors of CaP mineralization. (A) Structure of major biomolecules that as promoters of calcification: phosphorylated proteins, sulfated glycosaminoglycans, carboxyglutamic proteins and phospholipid membranes. (B) CaP mineralization in the presence of nucleation promoters: accumulation of Ca by absorption on promoters, precipitation of ACP, and crystallization of HAP. (C) Mechanism of crystal growth inhibition. (a) Representation of a Kossel crystal, with the different positions of adatoms on the crystal surface: flat site (surface nucleation) (Virchow, 1858), step site (Niskanen et al., 1994), kink site (Lehto et al., 1996), inhibitor molecule blocking a kink site (I); (b) a screw dislocation, and blocking of face growth by an inhibitor molecule.