Introduction

Arterial calcification, which is defined by pathological mineral deposition in the arterial wall, is a risk factor for cardiovascular mortality. The calcification process is driven by the osteogenic transition of vascular smooth muscle cells (SMCs), that release extracellular vesicles (EVs) serving as mineralization nucleation sites. Although mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been linked to arterial calcification, the involvement of their contact sites remains unknown. Mitochondria-associated membranes (MAMs) are inter-organelle contacts that connect the outer mitochondrial membrane to the ER membrane via protein-protein interactions. Glucose-regulated protein 75 (GRP75) is a linker protein, tethering the mitochondrial Voltage-dependent anion channel 1 (VDAC1) to the ER-bound Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3). We hypothesized that GRP75 is involved in SMC calcification and EV formation.

Methods and Results

Calcifying SMCs cultured in osteogenic media expressed higher GRP75 mRNA (2.2-fold ± 0.7, p=0.043) and protein (1.3-fold ± 0.2, p= 0.009) levels compared to control SMCs. ITPR3 protein expression was enhanced in calcifying SMCs (2.9-fold ± 0.7, p=0.045), whereas VDAC1 was not altered on mRNA and protein level. Subcellular fractionation of SMCs was performed to isolate MAMs from crude mitochondria by percoll gradient centrifugation. Long-chain-fatty-acid-CoA ligase 4 served as a MAM marker and the absence of Cytochrome c validated MAM purity. MAMs isolated from calcifying SMCs had 9.4-fold higher GRP75 levels than control MAMs, while MAMs from pre-calcifying SMCs showed no difference. Electron microscopy images demonstrated a closer proximity of ER and mitochondria in calcifying SMCs. Silencing of GRP75 using siRNA reduced the activity of tissue-nonspecific alkaline phosphatase (TNAP) (-36% ± 8%, p=0.017) and matrix mineralization (-35% ± 13%, p<0.001) in calcifying SMCs. Moreover, GRP75 inhibition by MKT-077 reduced the expression of the osteogenic marker bone morphogenetic protein 2 (BMP2) and decreased matrix mineralization in a dose-dependent manner. Furthermore, GRP75 (1.3-fold ± 0.1, p=0.040) and ITPR3 (5.1-fold ± 0.5, p<0.001) were enriched in EVs released from calcifying SMCs, while VDAC1 (-67% ± 13%, p=0.002) was less abundant compared to control EVs.

Conclusion
Our data indicate that ER-mitochondria contacts tethered by GRP75 are altered in calcifying SMCs. MAMs may modulate the osteogenic transition of SMCs and might be involved in the formation of calcifying EVs. Therefore, MAMs might serve as potential targets to prevent arterial calcification.