Alteration of Extracellular Nucleotide Metabolism in Pseudoxanthoma Elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is a rare genetic condition primarily caused by hepatic ABCC6 transporter dysfunction. Most clinical manifestations of PXE are due to premature calcification of elastic fibers. However, the vascular impact of PXE is pleiotropic and remains ill defined. ABCC6 expression has recently been associated with cellular nucleotide export. We studied the impact of ABCC6 deficiency on blood levels of adenosine triphosphate and related metabolites and on soluble nucleotidase activities in PXE patients and Abcc6^-/- mice. In addition, we investigated the expression of genes encoding ectocellular purinergic signaling proteins in mouse liver and aorta. Plasma adenosine triphosphate and pyrophosphate levels were significantly reduced in PXE patients and in Abcc6^-/- mice, whereas adenosine concentration was not modified. Moreover, 5'-nucleotidase/CD73 activity was increased in the serum of PXE patients and Abcc6^-/- mice. Consistent with alterations of purinergic signaling, the expression of genes involved in purine and phosphate transport/metabolism was dramatically modified in Abcc6^-/- mouse aorta, with much less impact on the liver. ABCC6 deficiency causes impaired vascular homeostasis and tissue perfusion. Our findings suggest that these alterations are linked to changes in extracellular nucleotide metabolism that are remote from the liver. This opens new perspectives for the understanding of PXE pathophysiology.

Figure 1.. Quantification of plasma adenine nucleotide, ADO, and PPi levels during ABCC6 deficiency.

Concentrations of (a) PPi, (b) ATP, and (c) ADP were determined in blood plasma from healthy control (CTRL) and PXE individuals. (d) PlasmaPPi, (e) ATP, (f) AMP, and (g) adenosine were measured in WT and Abcc6^−/− mice. Data represent the mean ± standard error of the mean of the indicated number of plasma samples. #P < 0.05, ^###P < 0.005. ADO, adenosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; CTRL, control; PPi, pyrophosphate; PXE, pseudoxanthoma elasticum; WT, wild type.

Figure 2.. Changes in soluble nucleotide-converting activities in PXE patients and Abcc6^−/− mice.

The activities of key soluble enzymes, (a, f) NTPDase1/CD39 (ADPase), (b, g) ecto-5′-nucleotidase/CD73, (c, h) alkaline phosphatase (AP), (d, i) adenylate kinase (AK), and (e, j) nucleotide pyrophosphatase phosphodiesterase (NPP) were determined in (a–e) the serum of healthy control individuals and PXE patients and in (f–j) Abcc6^−/− and WT mice. Data represent the mean ± standard error of the mean of the indicated number of serum samples. ^#P < 0.05; ^##P < 0.01. CTRL, control; IU, international unit; nM/mL/hr, nano molar/milliliter/hour; PXE, pseudoxanthoma elasticum.

Figure 3.. Gene expression pattern in liver and aorta of Abcc6^−/− mice.

Heat map of gene expression in the (a) liver and (b) aorta of young (5 months, n = 7) and old (24 months, n = 6, except for Abcc6^−/−aorta, for which n = 5) Abcc6^+/+ and Abcc6^−/− mice. Results represent color-coded expression relative to the control group (WT young). On the right are noted genes that are differently expressed (P < 0.05, two-way analysis of variance) according to genotype (red box) and to age (blue box) and the significance of the interaction between the two factors (green box). WT, wild type.

Figure 4.. Gene expression pattern in liver and aorta of Abcc6^−/− mice.

Bar graphs illustrating representative gene expression pattern in young andold WT and Abcc6^−/− mice in the (a) liver and (b) aorta. Genes represented were selected according to (i) the significant effect of Abcc6 genotype and(ii) the significance of aging/genotype interaction. Black bars indicate WT mice, and gray bars indicate Abcc6^−/− mice; solid fill indicatesadult mice, and hatched fill indicates old mice. Significant differences according to age (^#) or genotype (*) (P < 0.05). Volcano plots showing the distribution of relative gene expression (log of fold changes) and P-values (log₁₀) in (c) Abcc6^−/− mice liver (n = 13) and (d) aorta (n = 13 WT and n = 12 Abcc6^−/−). Data are the mean of five to seven values obtained from different animals. Values of log P above 1.3 (P < 0.05) are statistically significant. ^#P < 0.05; ^###P < 0.001; *P < 0.05; **P < 0.01; ***P < 0.001. WT, wild type.

Figure 5.. Proposal of a summary diagram for the pathophysiology of PXE.

In the context of liver (central) ABCC6 deficiency, plasma levels of PPi and adenine nucleotides (ATP, ADP) are decreased and 5′-nucleotidase activity is increased each of these, representing potential disease biomarkers. ABCC6 deficiency is also associated with modifications of purine and phosphate gene expression in (remote) affected tissues like arteries. Both systemic and local alterations in purine and phosphate metabolism contribute to PXE-associated peripheral arterial disease. ADP, adenosine diphosphate; ATP, adenosine triphosphate; MGP, matrix gla protein; Pi, inorganic phosphate; PPi, pyrophosphate; PXE, pseudoxanthoma elasticum; Vit, vitamin.