Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1 mice

Abstract

Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency

“…From this data, we concluded that Enpp1 deficiency resulted in no changes in osteoclast formation or resorptive activity. We noted that these observations concur with similar findings in Enpp1 null mice …”

“…Murine models of Enpp1 deficiency exhibit the essential characteristics of human GACI, including arterial calcifications, neointimal hyperplasia, cardiac dysfunction, and myocardial infarctions . However, in contrast to the rickets present in human ARHR2, the skeletal phenotype present in Enpp1‐deficient mice is reported to be osteopenic/osteoporotic . Osteomalacia, the defining histology of rickets in ARHR2 children, has thus far not been observed in murine models.…”

Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms

“…This PP i can then act locally to regulate the level of calcification. Consistent with this, NPP1 knockout mice ( Enpp1 −/− ) display extensive ectopic calcification in a variety of soft tissues including the aorta, kidney, cartilage, ear pinna, and whisker vibrissae (Hajjawi et al, ; Johnson et al, ; Mackenzie et al, ). Furthermore, mutations in the gene encoding NPP1 lead to the recessive condition Generalised Arterial Calcification of Infancy (GACI) which is characterized by extensive vascular calcification (Rutsch et al, ).…”

Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

“…A number of factors could contribute towards the differences between the in vivo and in vitro data. Firstly, Enpp1 −/− mice have a complex phenotype and thus it is possible that alterations in other tissues could indirectly influence bone mass (Mackenzie et al, ); for example, Enpp1 −/− animals have increased levels of serum sclerostin, a key inhibitor of bone formation (Hajjawi et al, ). Secondly, Enpp1 −/− mice display an unusual walking gate (Sali et al, ), mineralisation of the joints and muscle damage (Mackenzie et al, ), which combined are likely to affect the mechanical loading of bones.…”