Visualization of Mineral‐Targeted Alkaline Phosphatase Binding to Sites of Calcification In Vivo

• Published in: Journal of bone and mineral research Vol. 35; no. 9; pp. 1765 - 1771
• Main Authors: Amadeu de Oliveira, Flavia, Narisawa, Sonoko, Bottini, Massimo, Millán, José Luis
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01-09-2020; Wiley Subscription Services, Inc
• Subjects: Alkaline phosphatase; Animal models; Aorta; ASFOTASE ALFA; Calcification; Calcification (ectopic); Calvaria; CHONDROCYTE AND CARTILAGE BIOLOGY; DENTAL BIOLOGY; ECTOPIC CALCIFICATION; Fluorescence; Fluorescent indicators; HYPOPHOSPHATASIA; Immunohistochemistry; Jaw; Mineralization; Phosphatase; Renal artery; Spine; Stroma; Vertebrae; ASFOTASE ALFA; DENTAL BIOLOGY; HYPOPHOSPHATASIA; CHONDROCYTE AND CARTILAGE BIOLOGY; ECTOPIC CALCIFICATION
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1002/jbmr.4038

ABSTRACT
A mineral‐targeted form of recombinant tissue‐nonspecific alkaline phosphatase (TNAP), asfotase alfa, was approved multinationally as an enzyme replacement therapy for hypophosphatasia in 2015. Two reports to date have shown evidence of binding of this drug to mineralizing tissues using histochemistry and immunohistochemistry. Here, we sought to expand on those earlier studies by directly visualizing the in vivo binding of asfotase alfa conjugated with AnaTag HiLyte Fluor 750 or Alexa Fluor 647 fluorescent dye to sites of skeletal/dental mineralization and ectopic calcification. We utilized 40‐day‐old Tagln‐Cre; HprtALPL/Y mice, a model of severe medial vascular calcification; Tie2‐Cre; HprtALPL/Y mice, a model of severe intimal calcification; and sibling WT HprtALPL/Y mice, devoid of soft‐tissue calcification. A single dose of 8 mg/kg labeled asfotase alfa was injected via the retro‐orbital route. Skeletal tissues and soft organs were imaged ex vivo 2 days after the injection. Strong fluorescence signal was observed in all skeletal tissues (calvaria, vertebra, long bones, jaw, and mandibles) from mutant and WT mice. Fluorescence analysis of histological sections from bones revealed strong binding of asfotase alfa. Asfotase alfa binding to sites of ectopic calcification in the heart, aorta, and renal artery were found in both the Tagln‐Cre; HprtALPL/Y and Tie2‐Cre; HprtALPL/Y mice but not in WT mice. In addition, asfotase alfa binding was also found in the kidney stroma and brain of the Tie2‐Cre; HprtALPL/Y mice. Our results show that fluorescence‐labeled asfotase alfa administered in vivo binds not only to sites of skeletal and dental mineralization but also to sites of ectopic calcification in these animal models. © 2020 American Society for Bone and Mineral Research.