Appendix
Murine models of Vascular Calcifications
Genetic mutations in humans and murine models have identified a variety of genes that inhibit
VC processes that can be classified into three categories: modulation/transport of inorganic
phosphate (Pi) metabolism; inorganic pyrophosphate (PPi) availability; and bone formation and
homeostasis. Solute carrier family 20, member 1 and 2 are genes that encode the type III sodiumdependent phosphate transporters PiT1 and PiT2, which are predominantly expressed on vascular
smooth muscle cells (VSMCs) and transport extracellular Pi into the cytoplasm to regulate
phosphate homeostasis (1a). In vitro studies show VC is dependent on PiT1 (2a)2, and in vivo
murine studies have identified that these transporters serve redundant roles in regulating Pi
homeostasis (3a). Recently mutations in PiT2 have been linked to the brain-specific VC in the
basal ganglia of patients with idiopathic basal ganglia calcification (IBGC) (4a).
Inorganic pyrophosphate (PPi) is well-known potent endogenous inhibitor of calcium phosphate
precipitation in tissues (5a). Tissue non-specific alkaline phosphatase (ALPL in humans, Akp2 in
mice) hydrolyzes endogenous PPi and promotes ectopic mineralization. Akp2-/- mice exhibit high
levels of serum PPi and are hypophosphatemic. Conversely, humans with generalized arterial
calcification
of
infancy
(GACI)
mutations
in
the
gene
ectonucleotide
pyrophosphatase/phosphodiesterase 1 (ENPP1) cannot metabolize extracellular ATP to AMP
and PPi. The reduction in PPi results in drastic VC of the large arteries of GACI patients and
ENPP1-/- murine models (6a). Breeding of the Akp2-/- and the ENPP1-/- mice rescues the
phenotype (7). The tiptoe walking (ttw/ttw) mouse is a naturally occurring mutant line that
develops dystrophic cardiac calcinosis and the genetic defected was found to occur in the ABCC6
gene (8a), the genetic cause of pseudoxanthoma elasticum (PXE) in humans (9a-11a), which can
present clinically similar to GACI (12a). Though the substrate of ABCC6 remains to be
identified, a recent study identified that ABCC6-/- mice have very low levels of plasma PPi,
which suggests that like GACI, PXE is the result of insufficient circulating PPi (13). Similar to
the models above, mice with mutations in the progressive ankylosis (Ank) gene develop ectopic
calcification in the joint capsules and soft tissues that ultimately causes paralysis and death,
which stems from a drastic decrease in extracellular PPi (14a).
In addition to Pi/PPi homeostasis, murine models have identified protein factor can negatively
modulate VC and perhaps contribute to VC in MMS. Klotho (KL) is a gene that regulates insulin
sensitivity and vascular homeostasis, and KL-/- mice exhibit VC similar to that seen in the elderly
(15a) and humans with mutations in KL develop tumoral calcinosis due to diminished FGF23
signaling (16a). Mothers against decapentaplegic homolog 6 (Smad6) is a transcription factor
downstream of TGF- and BMP signaling expressed in the heart and blood vessels. Smad6-/mice develop medial VC and elevated blood pressure (17a), and families with congenital
cardiovascular malformations (CVM) were found to harbor nonsynonymous variants in the
Smad6 gene (18a). Matrix GLA protein (MGP) is produced by vascular smooth muscle cells and
chrondrocytes and binds to minerals in the extracellular matrix. MGP-/- mice develop extensive
calcification in the arteries and die due to vessel rupture, illustrating the potent inhibitory role
MGP plays in preventing ectopic mineralization (19a). Keutel syndrome (KS) is an autosomal
recessive human disease stemming from mutation in MGP, and these patients develop aberrant
calcification in soft tissues (20a). Similar to PPi, osteopontin (OPN) is a circulating protein that
inhibits the formation of hydroxyapatite (21a) and OPN-/- mice crossed to MGP-/- mice exhibit
exacerbated medial calcification on the arteries (22a).
Stemming from the above human and animal data, a few animal and clinical trials have been
undertaken to identify the role of pyrophosphate and alkaline phosphatase inhibitors for the
treatment of VCs. In vivo animal studies have showed that pyrophosphate injected into uremic
rats prevents VC in these animals (23a). Bisphosphonates are structurally similar to PPi and
inhibit both hydroxyapatite crystal formation and ALPL/Akp2 activity (24a). Proof-of-concept
experiments used VSMCs from ENPP1-/- and ank/ank mice to show that inhibition of tissue nonspecific alkaline phosphatase is a potential method to impede and/or prevent VC (25). A clinical
trail tested the effectiveness of bisphosphonates as treatment for GACI, and found that early
administration of bisphosphonates to GACI patients diagnosed shortly after birth increased
survival by slowing, and one case reversing, the progression of VC (26a, 27a).
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