GRP75 inhibition attenuates arterial calcification

Abstract

Background and aims: Arterial calcification is a risk factor for cardiovascular mortality. The calcification process is driven by the osteogenic transition of vascular smooth muscle cells (SMCs), which release extracellular vesicles (EVs) that act as mineralization nucleation sites. While mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in arterial calcification, the role of their contact sites remains unknown. Mitochondria-associated membranes (MAMs) are inter-organelle contacts connecting the outer mitochondrial membrane to the ER membrane through protein-protein interactions. This study investigated the role of Glucose-regulated protein 75 (GRP75), a MAM linker protein, in SMC calcification and EV cargo.

Methods: Human coronary artery SMCs were cultured in osteogenic medium to induce calcification. MAMs were isolated from SMCs and human carotid artery by subcellular fractionation and visualized using transmission electron microscopy. SMC-derived EVs were isolated from the conditioned culture medium by ultracentrifugation. GRP75 inhibition was achieved using silencing RNA or the inhibitor MKT-077. Mitochondrial respiration and ER stress were analyzed using Seahorse analysis and Western blotting.

Results: Calcifying SMCs expressed higher GRP75 mRNA (2.2-fold ± 0.7, p = 0.043) and protein (1.3-fold ± 0.2, p = 0.008) levels compared to control SMCs. GRP75 was enriched at MAMs, and electron microscopy imaging demonstrated closer mitochondria-ER contacts in both calcifying SMCs in vitro and human calcified carotid artery specimens. GRP75 inhibition by silencing RNA (-35 % ± 13 %, p < 0.001) or MKT-077 (-57 % ± 3 %, p < 0.001) attenuated matrix mineralization and reduced close mitochondria-ER contacts along with attenuating mitochondrial respiration capacity. Additionally, GRP75 was enriched in EVs released by calcifying SMCs (1.3-fold ± 0.1, p = 0.040).

Conclusions: Our findings demonstrate that MAMs are altered in calcifying SMCs. GRP75 inhibition disrupted close mitochondria-ER contact formation, decreased mitochondrial respiration, modulated the osteogenic transition of SMCs, and reduced vascular calcification. Therefore, GRP75 could serve as a potential target for preventing arterial calcification.

Keywords: Arterial calcification; Extracellular vesicles; Mitochondria-ER contacts; Mitochondria-associated membranes; Vascular smooth muscle cells.