Inhibition of enzymes involved in collagen cross-linking reduces vascular smooth muscle cell calcification

Vascular smooth muscle cells (VSMCs) transdifferentiate into osteoblast-like cells during vascular calcification, inducing active remodeling and calcification of the extracellular matrix (ECM). Intracellular and extracellular enzymes, such as lysyl hydroxylase 1 (PLOD1) and lysyl oxidase (LOX), contribute to ECM maturation and stabilization. We assessed the contribution of these enzymes to hyperphosphatemia-induced calcification. Human and murine VSMCs were differentiated into functional osteoblast-like cells by high-phosphate medium (HPM) conditioning. HPM promoted ECM calcification and up-regulated osteoblast markers associated with induction of LOX and PLOD1 expression and with an increase in ECM-insoluble collagen deposition. Murine VSMCs from transgenic mice overexpressing LOX (TgLOX) exhibited an increase in HPM-dependent calcification and osteoblast commitment compared with wild-type cells. Similarly, enhanced HPM-induced calcification was detected in aorta from TgLOX. Conversely, b-aminopropionitrile (a LOX inhibitor) and LOX knockdown abrogated VSMC calcification and transdifferentiation. We found a significant positive association between LOX expression and vascular calcification in human atherosclerotic lesions. Likewise, 2,29-dipyridil (a PLOD inhibitor) and PLOD1 knockdown impaired HPM-induced ECM mineralization and osteoblast commitment. Our findings identify LOX and PLOD as critical players in vascular calcification and highlight the importance of ECM remodeling in this processThis work was supported by Spanish Society of Cardiology-Daiichi-Sankyo Project 2012, Abbott Vascular research grant and Boston Scientific research grant, Instituto de Salud Carlos III (ISCIII) [PI15/01016, Red de Investigacion Cardiovascular (RD12/ ´ 0042/0049 and RD12/0042/0053)], Ministerio de Econom´ıa, Industria y Competitividad (MINECO; SAF2015-64767R), and Fondo Europeo de Desarrollo Regional (FEDER), a way to build Europe.Peer reviewe