OC 56.5 - Detection and inhibition of nascent vascular calcification with by protein S Gla-domain

Background: Early-stage vascular calcification (microcalcification) is strongly associated with progression of vascular disease and subsequent myocardial infarction, stroke and death. Although vascular calcification is accepted as independent risk factor for vascular disease, conventional imaging still falls short of early detection of calcification and treatment is currently unavailable. The Gla-domain of vitamin K-dependent coagulation factors could offer a solution to both these challenges. The Gla-domain has calcium ion-binding properties via γ-carboxyglutamic acids (Gla) and was recently shown to interact with ectopic calcium-crystals. For this study we chose the protein S Gla-domain.

Aims: Assess the efficacy of protein S Gla domain as an imaging agent and inhibitor of nascent vascular calcification.

Methods: Protein S Gla-domain (11 times fully carboxylated) and negative control protein S Glu-domain (uncarboxylated) were chemically synthesized and assessed for their ability to detect calcification through fluorescence microscopy. We used an in vitro setting, using primary vascular smooth muscle cell (hVSMC) assays, and ex vivo, using aortic tissue from high-fat diet fed ApoE-/- mice. Further, RNA sequencing was performed on calcifying hVSMC, both treated and untreated with protein S Gla-domain.

Results: In vitro, fluorescent protein S Gla showed binding before any clear calcification deposits could be detected with the o-Cresolphtalein assay. Protein S Gla also interfered with progression of calcification and allowed detection to follow the development of calcification. Ex vivo, protein S Gla detects microcalcification in atherosclerotic lesions, that were undetectable by Alizarin Red S staining. RNA sequencing revealed that treatment of calcifying hVSMC with protein S Gla induced differential gene expression to untreated calcifying hVSMC, which helps maintain a more contractile phenotype.

Conclusion(s): Protein S Gla holds potential as an early detector and in vivo acting inhibitor for early vascular calcification.