Bone morphogenetic protein-2 and -4 and its receptors mRNA expression in an in-vitro model of vascular calcification.

Vascular calcification (VC) is a complex ectopic calcification process considered the most important hallmark of atherosclerosis. The onset and progression of VC are similar to bone formation, thus based on the bone-vascular axis theory, and many studies have highlighted the role of bone morphogenetic proteins (BMP) in VC. They are a group of at least 30 proteins belonging to the TGF-? superfamily that act by binding to a heterodimeric complex of transmembrane receptors (BMPR). The aim was to study the involvement of BMP system (BMP-2/4, BMPR1a/1b, and BMPR2) expression in an advanced in-vitro model able to simulate the biological environment of the vascular wall assessing the ability of phosphates mixture to induce the osteoblastic switch of Human Coronary Artery Smooth Muscle cells (HCASMCs), a key event in VC. The HCASMCs were cultured in a double-flow bioreactor (LiveBox2, IVTech Srl, IT), allowing static and/or dynamic conditions. The induction of HCASMCs calcification was obtained by supplementing DMEM HG with 1.9 mM phosphates solution (NaH2PO4/Na2HPO4) for 7 days. After the incubation period, HCASMCs viability and calcium quantification were tested. Real Time-PCR of the BMP system was performed at the end of each experiment. The analysis of cell viability demonstrated that calcifying media significantly decreased HCASMCs viability in static conditions. Real Time-PCR of the BMP system in HCASMCs revealed a marked increase in the phosphate-induced calcification of both BMP-2 and BMP-4. In particular, we observed a higher amount of BMP2 transcript (p=0.0096), as expected, in parallel with the mRNA expression increase of the receptors BMPR1a (p=0.0023) and BMPR1b transcripts with respect to controls. BMPR2 remains in a steady state in the experimental setting. Despite the roles of the BMPs in osteogenesis being well documented, their involvement in VC is more complex and less defined. Our studies provide new pieces of evidence of how osteotropic factors, such as BMP-2 in synergy with its receptors, are modulated in VC caused by increased phosphate uptake and are involved in the osteogenic phenotype of HCASMCs.