Once thought to result from passive precipitation of calcium and phosphate, it now appears that vascular calcification is a consequence of tightly regulated processes that culminate in organized extracellular matrix deposition by osteoblast-like cells. These cells may be derived from stem cells (circulating or within the vessel wall) or differentiation of existing cells, such as smooth muscle cells (SMCs) or pericytes. Several factors induce this transition, including bone morphogenetic proteins, oxidant stress, high phosphate levels, parathyroid hormone fragments, and vitamin D. Once the osteogenic phenotype is induced, cells gain a distinctive molecular fingerprint, marked by the transcription factor core binding factor alpha1. Alternatively, loss of inhibitors of mineralization, such as matrix gamma-carboxyglutamic acid Gla protein, fetuin, and osteopontin, also contribute to vascular calcification. The normal balance between promotion and inhibition of calcification becomes dysregulated in chronic kidney disease, diabetes mellitus, atherosclerosis, and as a consequence of aging. Once the physiological determinants of calcification are perturbed, calcification may occur at several sites in the cardiovascular system, including the intima and media of vessels and cardiac valves. Here, calcification may occur through overlapping yet distinct molecular mechanisms, each with different clinical ramifications. A variety of imaging techniques are available to visualize vascular calcification, including fluoroscopy, echocardiography, intravascular ultrasound, and electron beam computed tomography. These imaging modalities vary in sensitivity and specificity, as well as clinical application. Through greater understanding of both the mechanism and clinical consequences of vascular calcification, future therapeutic strategies may be more effectively designed and applied.

Vascular Calcification Pathobiological Mechanisms and Clinical Implications

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP(i)). PP(i) suppresses the formation and growth of hydroxyapatite crystals. PP(i) is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP(i) content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP(i) concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP(i) insufficiency.

https://www.pnas.org/content/pnas/99/14/9445.full.pdf

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization

THIS year marks the 70th anniversary of the discovery of alkaline phosphatase (ALP) (1). For the greater part of the past seven decades there has been universal appreciation by physicians of the important clinical insight that comes from measuring ALP activity in serum to detect and follow the course of hepatobiliary and skeletal disease (2). Since the 1930s, quantification of ALP activity has been a routine test in the hospital laboratory and it is likely the most frequently performed enzyme assay (1). Nevertheless, the physiological function of this protein that is ubiquitous in nature remains unclear (1, 3–5). As reviewed here, however, recent molecular studies of hypophosphatasia, a rare heritable form of rickets, have confirmed the long-held notion that ALP has a significant role in skeletal mineralization in humans. How ALP acts was clarified by the discoveries that several phosphocompound substrates for tissue-nonspecific ALP (TNSALP) accumulate endogenously in this inborn error of metabolism.

Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization.

Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Deletion of the TNAP gene (Akp2) in mice results in hypophosphatasia characterized by elevated levels of PPi and poorly mineralized bones, which are rescued by deletion of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) that generates PPi. Mice deficient in NPP1 (Enpp1−/−), or defective in the PPi channeling function of ANK (ank/ank), have decreased levels of extracellular PPi and are hypermineralized. Given the similarity in function between ANK and NPP1 we crossbred Akp2−/− mice to ank/ank mice and found a partial normalization of the mineralization phenotypes and PPi levels. Examination of Enpp1−/− and ank/ank mice revealed that Enpp1−/− mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, suggesting that failure of ANK deficiency to correct hypomineralization in Akp2−/− mice reflects the lack of ANK activity in the matrix vesicle compartment. We also found that the mineralization inhibitor osteopontin (OPN) was increased in Akp2−/−, and decreased in ank/ank mice. PPi and OPN levels were normalized in [Akp2−/−; Enpp1−/−] and [Akp2−/−; ank/ank] mice, at both the mRNA level and in serum. Wild-type osteoblasts treated with PPi showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK coordinately regulate PPi and OPN levels. The hypomineralization observed in Akp2−/− mice arises from the combined inhibitory effects of PPi and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PPi and OPN pools that leads to hypermineralization.

/pdf/concerted-regulation-of-inorganic-pyrophosphate-and-59re4h506v.pdf

Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders.

Our knowledge of the structure and function of alkaline phosphatases has increased greatly in recent years. The crystal structure of the human placental isozyme has enabled us to probe salient features of the mammalian enzymes that differ from those of the bacterial enzymes. The availability of knockout mice deficient in each of the murine alkaline phosphatase isozymes has also given deep insights into their in vivo role. This has been particularly true for probing the biological role of bone alkaline phosphatase during skeletal mineralization. Due to space constraints this mini-review focuses exclusively on structural and functional features of mammalian alkaline phosphatases as identified by crystallography and probed by site-directed mutagenesis and kinetic analysis. An emphasis is also placed on the substrate specificity of alkaline phosphatases, their catalytic properties as phosphohydrolases as well as phosphodiesterases and their structural and functional relatedness to a large superfamily of enzymes that includes nucleotide pyrophosphatase/phosphodiesterase.

/pdf/alkaline-phosphatases-structure-substrate-specificity-and-55rwdsqc4l.pdf

Alkaline Phosphatases : Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes

1. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2. Both liver and intestinal preparations possessed pyrophosphatase activity at all stages of purification, and the two types of activity were not separated by gel filtration or by anion-exchange or cation-exchange chromatography. 3. After starch-gel electrophoresis of the tissue extracts, the zones of pyrophosphatase activity coincided exactly with alkaline-phosphatase zones. 4. Hydrolysis of each type of substrate was inhibited by the presence of the other, and a constant ratio of alkaline-phosphatase activity to pyrophosphatase activity was maintained during inactivation of the enzymes by incubation at 55°. 5. These results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases.

/pdf/association-of-inorganic-pyrophosphatase-activity-with-human-2e1y9d2px3.pdf

Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations.

/pdf/alteration-in-the-electrophoretic-mobility-of-alkaline-4njfxsx04h.pdf

R H Eaton

Papers

Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations.

Alteration in the Electrophoretic Mobility of Alkaline Phosphatases after Treatment with Neuraminidase