Diseases of medial wall vascular and connective tissue calcification associated with low plasma pyrophosphate (PPi) range from orphan diseases such as Pseudoxanthoma Elasticum (PXE) and Generalized Calcification of Infancy (GACI) to ubiquitous diseases of medial wall vascular calcification affecting every aging adult such as hardening of the arteries associated with aging. Although recognized as a potent mineralization inhibitor for over a generation, PPi has never been established as a causative agent in the broad class of connective tissue diseases and effective therapeutics capable of treating the calcifications driving the morbidity and mortality of these conditions is lacking. We have recently demonstrated that elevation of plasma PPi using a novel biologic containing the pyrophosphate generating enzyme ENPP1 is curative in rodent models of the rare orphan disease GACI - a severe lethal neonatal disease of arterial calcification. Here, we demonstrate the efficacy of this approach in a more common disorder of vascular and connective tissue calcification and low plasma PPi called PXE.

PXE is a monogenic, hereditable connective tissue disease characterized by aberrant mineralization of the skin, eye, and vasculature. PXE is associated with loss of function mutations in the ABCC6 gene - a multi-pass ATP dependent membrane transport protein. Two opposing theories regarding the genetic etiology of PXE are held - a metabolic hypothesis postulating that ABCC6 mutations deprive the body of a global mineralization inhibitor, and a cellular hypothesis postulating that ABCC6 mutations compromise a stem cell progenitor locally present within specific tissues. Both etiologies are proposed to account for the regional mineral and elastic fiber alterations present in the disease.

Although the substrate of ABCC6 is unknown, patients with PXE (and ABCC6 knockout mice) have plasma [PPi] at about 20-30% of unaffected (or wild-type) individuals. Furthermore, transfections of ABCC6 into HEK cells increase extracellular [ATP] in vitro. ATP is metabolized into PPi by the extracellular enzyme ENPP1, and the combined findings support the notion that ABCC6 mutations decrease extracellular [ATP] thereby limiting ENPP1 production of PPi by limiting the enzymatic substrate of the enzyme.

Here, we demonstrate that subcutaneous administration of ENPP1-Fc, a fusion protein containing the enzyme ENPP1, normalizes serum PPi concentrations in ABCC6 knockout mice. Furthermore, we show that normalization of serum PPi by this means potently inhibits the regional connective tissue calcifications present in ABCC6 knockout mice. Our findings provide in vivo experimental evidence for the metabolic basis of PXE. Furthermore, because ENPP1 is downstream of ABCC6, the efficacy of ENPP1-Fc is surprising and indicates that reduced extracellular [ATP] may not be the etiology for the decreased plasma PPi concentrations reported in PXE patients as previously proposed. Finally, the efficacy of ENPP1-Fc on the connective tissue calcifications in PXE suggests that elevation of plasma PPi by this means may be efficacious in spectrum of severe and poorly treated connective tissue calcification disorders associated with low plasma PPi.


Braddock:Yale University: Patents & Royalties: Yale University owns a patent on the technology, in which Demetrios Braddock is listed as an inventor.

Author notes


Asterisk with author names denotes non-ASH members.