Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Robert Dzhanaev - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Christian Hasberg - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Andrea Gorgels - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Carlo Schmitz - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Camilla Franziska Winkler - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Hanna Malyaran - , Universitätsklinikum Aachen (Autor:in)
  • Steffen Gräber - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)
  • Anouk Gentier - , Maastricht University (Autor:in)
  • Armand Jaminon - , Maastricht University (Autor:in)
  • Stijn Agten - , Universitätsklinikum Aachen (Autor:in)
  • Tilman Hackeng - , Maastricht University (Autor:in)
  • Asim Cengiz Akbulut - , Maastricht University (Autor:in)
  • Leon Schurgers - , Maastricht University (Autor:in)
  • Felix Manuel Mottaghy - , Akademisches Krankenhaus Maastricht (UMC+) (Autor:in)
  • Claudia Goettsch - , Universitätsklinikum Aachen (Autor:in)
  • Willi Jahnen-Dechent - , Rheinisch-Westfälische Technische Hochschule Aachen (Autor:in)

Abstract

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Details

OriginalspracheEnglisch
Seiten (von - bis)659-672
Seitenumfang14
FachzeitschriftTheranostics
Jahrgang13
Ausgabenummer2
PublikationsstatusVeröffentlicht - 2023
Peer-Review-StatusJa
Extern publiziertJa

Externe IDs

PubMedCentral PMC9830449
Scopus 85145288135
ORCID /0000-0002-7973-1329/work/184443309

Schlagworte

Schlagwörter

  • Animals, Calcinosis/diagnostic imaging, Calcium Phosphates/metabolism, Calcium/metabolism, Mice, Protein Binding, alpha-2-HS-Glycoprotein/metabolism, fluorescent proteins, ectopic calcification, fetuin-A, calcification imaging