From organic and inorganic phosphates to valvular and vascular calcifications

Abstract

Calcification of the arterial wall and valves is an important part of the pathophysiological process of peripheral and coronary atherosclerosis, aortic stenosis, ageing, diabetes, and chronic kidney disease. This review aims to better understand how extracellular phosphates and their ability to be retained as calcium phosphates on the extracellular matrix initiate the mineralization process of arteries and valves. In this context, the physiological process of bone mineralization remains a human model for pathological soft tissue mineralization. Soluble (ionized) calcium precipitation occurs on extracellular phosphates; either with inorganic or on exposed organic phosphates. Organic phosphates are classified as either structural (phospholipids, nucleic acids) or energetic (corresponding to phosphoryl transfer activities). Extracellular phosphates promote a phenotypic shift in vascular smooth muscle and valvular interstitial cells towards an osteoblast gene expression pattern, which provokes the active phase of mineralization. A line of defense systems protects arterial and valvular tissue calcifications. Given the major roles of phosphate in soft tissue calcification, phosphate mimetics, and/or prevention of phosphate dissipation represent novel potential therapeutic approaches for arterial and valvular calcification.

Keywords: Ageing; Atherosclerosis; Exosomes; Smooth muscle cells; Aortic stenosis.

Figure 1

Sources of phosphate for valvular and vascular mineralization. Phosphates exist in the body both in the chemical form of minerals (inorganic phosphate, Pi) and in biologically active forms integrated in molecules for structural and energy metabolism (organic phosphate). Extracellularly exposed phosphate groups in organic molecules will become hot sports for calcium precipitation and hydroxyapatite crystallization. The organic integration of inorganic phosphate can also take the reverse form, where Pi is released extracellularly from organic sources to directly form mineralization. Photo inset shows: A: Electron microscopic view of SMC phagocytic consequences: 1. Cytosolic microvesiculation, 2. Exosome release.

Figure 2

Calcifying cardiovascular pathologies. Cardiovascular mineralization occurs as atherosclerosis, medial calcification, and valve calcifications. Atherosclerotic plaque calcification is characterized by lipoprotein infiltration, foam cell formation and exosome release from smooth muscle cells, as well as intraplaque haemorrhage, cell death, and DNA retention. In contrast, medial calcification develops in the absence of inflammatory cells and involves degradation of the internal elastic lamina in the extracellular matrix (ECM), DNA damage, exosome release, and calcium phosphate precipitation from circulating inorganic phosphate (Pi). Valve calcification is highly dependent on biomechanical and haemodynamic factors, and has the characteristics of atherosclerotic and medial calcification, with a strong inflammatory component. The initial localizations of calcifications are indicated in blue.

Figure 3

Membranous phospholipids. Phosphate at the polar head of phospholipids in cell membranes, i.e. the plasma membrane, mitochondrial membranes, and the nuclear envelope, as well as in exosomes. Bottom photo panel: The co-localization of oxidized phospholipids stained with the antibody Ab E06 (Sigma) and lipids (oil red O staining), calcification (Alizarin red staining after EDTA chelation of Ca⁺⁺), Hoechst staining at 330 nm (UV), and at 550 nm (red) indicates the presence of phosphates (see text^76,77) in a section of an aortic valve leaflet in the initial calcification step.

Figure 4

Smooth muscle cell homeostasis and endosomal/exosomal turnover. Homeostatic endosomal activity is a highly regulated balance between endosomal entry (phagocytosis, endocytosis, autophagy, heterophagy) and exit by molecular exocytosis and secretion and exosome release. Such release generated by smooth muscle cell membranous phospholipids can drive the development of arterial calcifications.

Figure 5

Nucleic acids. The phosphate group in nucleic acids is attached to a main chain of sugars (ribose or deoxyribose), in which sequential base pairs are anchored. Bottom photo panel of different staining of an aortic valve calcification: (i) Alizarin red staining of calcium before EDTA chelation; (ii) higher magnification; (iii) Hoechst fluorescent staining (DAPI, U.V. wave length: 330 nm) after EDTA chelation of calcium. (iv) Hoechst fluorescent staining (red, wave length: 550 nm). Hoechst binds to the guanine-cytosine pair of intact DNA, but also to phosphate (see text). (v) Anti-phosphatidylcholine antibody (Sigma) stained by E06 Calcification. (vi) TUNEL binds to DNA breakages of fragmented DNA through an enzymatic terminal deoxynucleotidyl transfer of a tagged oligonucleotide. Therefore, this staining shows the presence of damaged DNA in the calcification background after treatment with EDTA. (vii) Immunostaining of calcification by DNA (ab27156, abcam) and histone antibodies (ab AE-4, Santa Cruz Biotech). (viii) Scanning Electron Microscopy of intact calcification.

Figure 6

Purinergic and pyrophosphate metabolism into inorganic phosphate (Pi) and its regulation. Intracellular ATP is transported to the extracellular space by the transporter ABCC6. The hydrolysis of ATP by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) generates inorganic pyrophosphate (PPi), which inhibits the formation of hydroxyapatite (HAP). In contrast, metabolism of ADP to by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) and PPi metabolism by tissue non-specific alkaline phosphatase (TNAP) increase Pi and HAP formation. Pi levels are regulated by calcification inhibitors, including Fetuin-A produced by the liver, which forms colloidal calcium-phosphate complexes called calciprotein particles (CPPs). It also shows the endogenous Pi regulator klotho, a co-receptor for fibroblast growth-factor 23 (FGF23), which decreases renal phosphate reabsorption.

Figure 7

Therapeutic potential of phosphate inhibitors for cardiovascular mineralization. Examples of phosphate binders, bisphosphonates and inositol hexaphosphates and their effects on the absorption and resorption of inorganic phosphate (Pi), as well as on hydroxyapatite (HAP) deposition and growth during valvular and vascular mineralization.