High-phosphate Causes Endothelial Extracellular Matrix Calcification by Inducing Endothelial Cell Mesenchymal Transition and Osteoblastic Differentiation

Chronic kidney disease (CKD) and diabetes are prevalent conditions characterized by increased cardiovascular risk, also due to vascular calcification (VC). Vascular smooth muscle cells actively participate to VC, conversely, the role of endothelial cells (ECs) has been less studied. Therefore, we investigated whether high-phosphate (Pi) has a pro-calcifying potential on ECs both in vitro and ex vivo, analyzing calcium deposition, mesenchymal transition (EndMT) and osteoblastic differentiation. An ex vivo model of arterial ring was developed to study intima calcification. The effect of CKD serum and intima calcification in arteries of CKD patients were also investigated. We demonstrated that Pi induces EC calcification dependent on Pi influx into the cell. Between day 2 and 4 Pi induces EndMT with increase of both mesenchymal genes and markers together with the acquisition of migratory capabilities. From day 5 of Pi treatment ECs differentiated in osteoblastic-like cells with up-regulation of osteoblastic genes and proteins together with a modification of extracellular matrix that acquires osteo-chondrogenic characteristics. Interestingly, EndMT modulation decreased calcium deposition, suggesting a relationship between the two differentiation processes. Moreover, in an ex vivo model of arterial ring intimal calcification, Pi induced endothelial calcification and expression of osteogenic markers. Moreover, in vitro, CKD serum increased calcium deposition by exacerbating EndMT and simil-osteoblastic differentiation. Finally, in arteries of CKD patients were detectable intima calcification and EC osteoblastic transformation. In this study we demonstrated that both Pi and CKD induce intimal calcification and that endothelial calcification is an active process characterized by EndMT and osteoblastic differentiation.