High-phosphate causes endothelial extracellular matrix calcification by inducing endothelial cell mesenchymal transition and osteoblastic differentiation

Abstract

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 in VC; conversely, the role of endothelial cells (ECs) has been less studied. Therefore, we investigated whether high-inorganic phosphate (Pi) has a procalcifying potential on ECs both in vitro and ex vivo, analyzing calcium deposition, mesenchymal transition [endothelial-to-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 patients with CKD was also investigated. We demonstrated that Pi induces EC calcification dependent on Pi influx into the cell. Between days 2 and 4, Pi induces EndMT with an increase of both mesenchymal genes and markers together with the acquisition of migratory capabilities. From day 5 of Pi treatment, ECs differentiated into osteoblastic-like cells with the upregulation of osteoblastic genes and proteins together with a modification of extracellular matrix that acquires osteochondrogenic 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, intima calcification and EC osteoblastic transformation were detected in the arteries of patients with CKD. 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.NEW & NOTEWORTHY The significance of this study is the demonstration that, in chronic kidney disease (CKD), intima is not a passive actor but undergoes deep changes up to osteoblastic-like differentiation and calcification. Considering these new findings, the vessel probably needs to be considered as a unique organ in the pathogenesis of vascular calcification (VC). This new point of view may help in finding strategies and implementing targeted therapies to delay or block the development of VC in CKD.

Keywords: chronic kidney disease; endothelial cell; endothelium; phosphate; vascular calcification.