ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP

Abstract

Generalized arterial calcification of infancy (GACI) is associated with widespread arterial calcification and stenoses and is caused by mutations in ENPP1. ENPP1 encodes for ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which cleaves ATP to generate inorganic pyrophosphate (PP_i) and adenosine monophosphate (AMP) extracellularly. The current study was designed to define the prevalence of arterial stenoses in GACI individuals and to identify the mechanism through which ENPP1 deficiency causes intimal proliferation. Furthermore, we aimed to effectively prevent and treat neointima formation in an animal model of GACI through the systemic administration of recombinant human (rh)ENPP1-Fc protein. Based on a literature review, we report that arterial stenoses are present in at least 72.4% of GACI cases. We evaluated the effect of rhENPP1-Fc on ENPP1-silenced human vascular smooth muscle cells (VSMCs) and on induced intimal proliferation in Enpp1-deficient ttw/ttw mice treated with carotid ligation. We demonstrate that silencing ENPP1 in VSMCs resulted in a tenfold increase in proliferation relative to that of cells transfected with negative control siRNA. The addition of rhENPP1-Fc, AMP or adenosine restored the silenced ENPP1-associated proliferation. In contrast, neither PP_i nor etidronate, a current off-label treatment for GACI, had an effect on VSMC proliferation. Furthermore, subcutaneous rhENPP1-Fc protein replacement was effective in preventing and treating intimal hyperplasia induced by carotid ligation in an animal model of GACI. We conclude that ENPP1 inhibits neointima formation by generating AMP. RhENPP1-Fc may serve as an approach for the effective prevention and treatment of arterial stenoses in GACI.

Fig. 1. ENPP1 deficiency leads to decreased levels of PP_i and elevated levels of ATP in the plasma of GACI individuals and ttw/ttw mice.

Plasma levels of PP_i (a, b) and ATP (c, d) were determined from healthy controls (n = 10) and GACI patients (n = 4) (a, c) and from 7-week-old WT (n = 6) and ttw/ttw mice (n = 5) (b, d). Plasma levels of ATP from healthy controls (c) correspond to normal human EDTA plasma concentrations from previous publications. Values are presented as the mean ± SD. ^**p < 0.005, ^***p < 0.001 (Student’s t test, unpaired two-sample testing for means)

Fig. 2. Silencing of ENPP1 by siRNA in hiPSC-derived VSMCs results in a reduction of ENPP1 RNA expression and an increase in hiPSC-derived VSMCs proliferation.

SMC markers calponin (first panel) and SM-MHC11 (second panel) confirmed the SMC phenotype of hiPSC-derived VSMCs (a). hiPSC-derived VSMCs were transfected overnight with ENPP1 siRNA or control siRNA, followed by 24-h starvation in 0.25% FBS. Then, the cells were cultured in basal media. ENPP1 RNA expression (b) and hiPSC-derived VSMCs cell number (c), and proliferation (d) were investigated. b, c Values are presented as the mean ± SEM, n = 4; ^***p < 0.001 vs. control nontargeting siRNA transfected cells. d Cell proliferation was evaluated by [³H] thymidine uptake. [³H]thymidine was added in the last 18 h of culture. The results are expressed as the CPM ± SEM, n = 4. ^***p < 0.001 vs. control nontargeting siRNA transfected cells (Student’s t test, unpaired two-group testing for means)

Fig. 3

Effects of bisphosphonates (a), AMP (b), adenosine (c), and rhENPP1-Fc (d) on accelerated proliferation in ENPP1-silenced hiPSC-derived VSMCs. hiPSC-derived VSMCs were transfected overnight with ENPP1 siRNA or control siRNA, followed by 24-h starvation in 0.25% FBS. Then, cells were cultured in basal media, supplemented with etidronate (0.1–100 µM, a), AMP (0.3–300 µM, b), and adenosine (0.3–300 µM, c) for 3 days before determining proliferation. Cell proliferation was evaluated by [³H] thymidine uptake. [³H]thymidine was added in the last 18 h of culture. Extracellular PP_i levels in the cell culture medium of cells supplemented with 300 µM ATP and rhENPP1-Fc (0.01–5 µg/ml, d) were determined (e). Values are presented as the mean ± SEM, n = 4. ^***p < 0.001 (one-way ANOVA multiple group comparison followed by the Bonferroni’s post hoc test)

Fig. 4. Administration of rhENPP1-Fc prevents intimal proliferation after carotid ligation in ttw/ttw mice.

RhENPP1-Fc treatment was started 7 days prior to carotid ligation, and serial sections of the left carotid arteries were taken 14 days after carotid ligation. Histological analysis (Von Gieson’s stain) was performed on sections taken either 100 (upper panel) or 200 (lower panel) µm from the point of ligation from WT, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw- mice, shown from left to right (a). The internal elastic lamina (IEL), external elastic lamina (EEL), and lumen (L) are indicated by arrows. The scale bar represents 100 µm. Morphometric quantitation was performed on intimal (b) and medial (c) areas, and the I/M ratio was calculated (d). Values are presented as the mean ± SEM, n = 7 each group, ^***p < 0.001 (one-way ANOVA multiple group comparison followed by the Bonferroni’s post hoc test)

Fig. 5. Therapeutic administration of rhENPP1-Fc inhibits intimal proliferation after carotid ligation in ttw/ttw mice

RhENPP1-Fc treatment was started 7 days after carotid ligation, and serial sections of the left carotid arteries were taken 14 days after carotid ligation. Histological analysis (Von Gieson’s stain) was performed on sections taken either 100 (upper panel) or 200 (lower panel) µm from the point of ligation from WT, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw− mice, shown from left to right (a). The scale bar represents 100 µm. Morphometric quantitation was performed on intimal (b) and medial (c) areas, and the I/M ratio was calculated (d). Values are presented as the mean ± SEM, n = 7 for WT, n = 10 for vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw- mice, ^***p < 0.001, ^*p < 0.05 (one-way ANOVA multiple group comparison followed by the Bonferroni’s post hoc test)

Fig. 6. The two roles of ENPP1: inhibiting arterial calcification and myointimal proliferation by modulating extracellular PP_i and ATP metabolism.

The transmembrane ectoenzyme ENPP1 converts extracellular ATP to AMP and thereby generates PP_i. AMP is further hydrolyzed by CD73 (5-exonucleotidase) to adenosine and P_i. PP_i is a physiologic inhibitor of hydroxyapatite formation, regulates chondrogenesis and is therefore important in the prevention of soft tissue calcification. A decreased PP_i/P_i ratio leads to calcification. Elevated extracellular ATP levels lead to increased VSMC proliferation, while AMP and adenosine are known to inhibit VSMC proliferation. In ENPP1 deficiency, independent of low PP_i levels, proproliferating extracellular ATP levels increase, whereas antiproliferating AMP and adenosine levels decrease, leading to accelerated VSMC proliferation and arterial stenosis. We conclude that a decreased PP_i/P_i ratio leads to ectopic calcification, whereas an increased extracellular ATP/AMP ratio leads to increased VSMC proliferation and arterial stenosis in ENPP1 deficiency