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Vascular calcification is dependent on plasma levels of pyrophosphate.

Lomashvili KA, Narisawa S, Millán JL, O'Neill WC - Kidney Int. (2014)

Bottom Line: However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels.Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification.Histology revealed medial calcification with no signs of rejection.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

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Calcification factors in calcified Enpp1−/− aortas. A. Activity of TNAP in intact mouse aortas. WT: wild-type. B. Immunoblots of TNAP. Each lane was loaded with 100 μg protein. Samples from one male and one female of each genotype are shown. Recombinant human TNAP (approximately 8 ng) was used as positive control. The faster mobility is due to the lack of glycosylation. C and D. Immunohistochemistry of osterix in calcified Enpp1−/− aorta and neonatal mouse spine.
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Figure 3: Calcification factors in calcified Enpp1−/− aortas. A. Activity of TNAP in intact mouse aortas. WT: wild-type. B. Immunoblots of TNAP. Each lane was loaded with 100 μg protein. Samples from one male and one female of each genotype are shown. Recombinant human TNAP (approximately 8 ng) was used as positive control. The faster mobility is due to the lack of glycosylation. C and D. Immunohistochemistry of osterix in calcified Enpp1−/− aorta and neonatal mouse spine.

Mentions: As shown in Table 1, plasma PPi, measured on the high-phoshorus diet, was approximately 30-fold lower in the Enpp1−/− mice (p=0.0013), consistent with the absence of the synthetic enzyme. Other potential mechanisms for the calcification were also explored. Specifically, plasma phosphate and calcium, also shown in Table 1, were not elevated in Enpp1−/− mice. Plasma phosphate tended to be lower in Enpp1−/− mice, probably explained by the phosphaturia in these mice that may be due to elevated fibroblast growth factor-23 levels 12. Activity and content of tissue non-specific alkaline phosphatase (TNAP) did not differ between wild-type and Enpp1−/− aortas from mice aged 2–4 months, an age at which some calcification is present (Fig. 3A,B). To assess osteogenic trans-differentiation, immunohistochemistry for osterix, an osteogenic transcription factor, was performed in 6 sections from 6 different calcified Enpp1 aortas (age 4–6 months). There was no cellular staining (Fig. 3C) while nuclear staining was readily apparent in neonatal spine used as a positive control (Fig. 3D). Non-specific staining of the calcifications was present in some of the sections.


Vascular calcification is dependent on plasma levels of pyrophosphate.

Lomashvili KA, Narisawa S, Millán JL, O'Neill WC - Kidney Int. (2014)

Calcification factors in calcified Enpp1−/− aortas. A. Activity of TNAP in intact mouse aortas. WT: wild-type. B. Immunoblots of TNAP. Each lane was loaded with 100 μg protein. Samples from one male and one female of each genotype are shown. Recombinant human TNAP (approximately 8 ng) was used as positive control. The faster mobility is due to the lack of glycosylation. C and D. Immunohistochemistry of osterix in calcified Enpp1−/− aorta and neonatal mouse spine.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4308968&req=5

Figure 3: Calcification factors in calcified Enpp1−/− aortas. A. Activity of TNAP in intact mouse aortas. WT: wild-type. B. Immunoblots of TNAP. Each lane was loaded with 100 μg protein. Samples from one male and one female of each genotype are shown. Recombinant human TNAP (approximately 8 ng) was used as positive control. The faster mobility is due to the lack of glycosylation. C and D. Immunohistochemistry of osterix in calcified Enpp1−/− aorta and neonatal mouse spine.
Mentions: As shown in Table 1, plasma PPi, measured on the high-phoshorus diet, was approximately 30-fold lower in the Enpp1−/− mice (p=0.0013), consistent with the absence of the synthetic enzyme. Other potential mechanisms for the calcification were also explored. Specifically, plasma phosphate and calcium, also shown in Table 1, were not elevated in Enpp1−/− mice. Plasma phosphate tended to be lower in Enpp1−/− mice, probably explained by the phosphaturia in these mice that may be due to elevated fibroblast growth factor-23 levels 12. Activity and content of tissue non-specific alkaline phosphatase (TNAP) did not differ between wild-type and Enpp1−/− aortas from mice aged 2–4 months, an age at which some calcification is present (Fig. 3A,B). To assess osteogenic trans-differentiation, immunohistochemistry for osterix, an osteogenic transcription factor, was performed in 6 sections from 6 different calcified Enpp1 aortas (age 4–6 months). There was no cellular staining (Fig. 3C) while nuclear staining was readily apparent in neonatal spine used as a positive control (Fig. 3D). Non-specific staining of the calcifications was present in some of the sections.

Bottom Line: However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels.Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification.Histology revealed medial calcification with no signs of rejection.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Show MeSH
Related in: MedlinePlus