Vascular calcification is dependent on plasma levels of pyrophosphate

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.

Figure 1

Aortic calcification in Enpp1−/− mice, alizarin red stain. A. Aorta from 4 month-old wild-type mouse fed a 0.4% phosphorus diet. B. Aorta from 4 month-old Enpp1−/− mouse fed a 0.4% phosphorus diet. C. Aorta from 4 month-old Enpp1−/− mouse fed a 1.5% phosphorus diet for 6 weeks.

Figure 2

Aortic calcium content in wild-type and Enpp1−/− mice. Mice were between 2 and 4 months old. Error bars, standard errors. Numbers in parentheses indicate the number of animals. *p<0.001 vs. wild-type.

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.

Figure 4

Histology of aortic allografts. Left-hand column: normal mouse aorta. Right-hand column: wild-type allograft in wild-type mouse 5 months after transplantation. Top row: hematoxylin and eosin stain. Middle row: elastin stain. Bottom row: trichrome stain.

Figure 5

Calcification of transplanted aortas, alizarin red stain. Aortas were harvested 2 months after transplantation. A. Wild-type aorta transplanted into a wild-type mouse. There is no staining of donor or recipient aorta. B. Enpp1−/− aorta transplanted into an Enpp1−/− mouse. There is extensive staining of both the donor and recipient aorta. C. Enpp1−/− aorta transplanted into a wild-type mouse. There is no staining of the recipient aorta with scattered staining of the donor aorta (inset). D. wild-type aorta transplanted into an Enpp1−/− mouse. There are a few foci of staining in the donor aorta (inset). Arrows indicate suture lines.

Figure 6

Calcium content and histology in aortas transplanted between wild-type and Enpp1−/− mice. Aortas were harvested 2 months after transplantation. A. Calcium content. Error bars, standard errors. *p <0.001 vs. recipient aorta and p = 0.039 vs. WT into WT; **p = 0.078 vs. recipient aorta and p = 0.002 vs. Enpp1−/− into Enpp1−/−. Numbers in parentheses indicate the number of transplantations. B. von Kossa stain of a representative section of an Enpp1−/− aorta. C. von Kossa stain of a representative section of an Enpp1−/− aorta transplanted into an Enpp1−/− mouse. Calcifications are in the media and along the internal elastic lamina (arrows). The latter is often adjacent to the lumen due to loss of the overlying intima during tissue sectioning.