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  • Bioengineered baculovirus-derived extracellular vesicles loaded with of γ-carboxylated Gla-rich protein : dual modulation of inflammation and vascular calcification
    Publication . Viegas, Carla; Pichard, Simon; Carreira, Joana; Ova, Adélia; Troffer-Charlier, Nathalie; Maia, Teresa M.; Edelweiss, Evelina; Macedo, Anjos L.; Matos, António; Faria, Tiago Q.; Calado, Sofia M.; Monico, Carina; Devos, Simon; Impens, Francis; Schaeffer-Reiss, Christine; Cianférani, Sarah; Peixoto, Cristina; Poterszman, Arnaud; Simes, Dina
    Chronic inflammation and ectopic calcification are interrelated processes driving major chronic inflammatory diseases such as cardiovascular and chronic kidney diseases. Gla-rich protein (GRP), a vitamin K–dependent protein (VKDP) with dual anti-inflammatory and anti-calcific properties, has emerged as a promising therapeutic molecule. However, its biomedical development has been limited by difficulties in producing the γ-carboxylated (cGRP) form and by its poor solubility at physiological pH, constraining formulation and delivery. To address these challenges, we established a baculovirus expression vector system (BEVS) designed to couple GRP post-translational maturation with its secretion in extracellular vesicles (EVs). Co-expression of GRP with γ-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR), and the convertase Furin enabled efficient γ-carboxylation, propeptide removal, and secretion of mature cGRP. GGCX and VKOR were essential for γ-carboxylation, while Furin mediated propeptide processing. EVs were isolated by differential ultracentrifugation into 30 K and 100 K fractions and characterized by NTA, TEM, Western blot, ELISA, and proteomics. All vesicles displayed physical and molecular features resembling mammalian EVs, including canonical EV markers and distinct proteomic profiles, with GRP, GGCX, VKOR, and Furin preferentially enriched in the 30 K population. Functional assays demonstrated that the resulting EVs associated with human THP-1 macrophages and vascular smooth muscle cells (VSMCs) without inducing cytotoxicity, and both cGRP-EVs and uncarboxylated GRP-EVs reduced pro-inflammatory cytokine release while exerting dual anti-inflammatory and anti-mineralizing effects. This study establishes the first bioengineered platform capable of generating functional γ-carboxylated GRP and its vesicular formulation, providing a dual innovation for VKDP research and therapeutic biomaterial development.
    2026-07Contributo em revista Acesso aberto Ver mais