Background and aims Vascular smooth muscle cells (VSMC) transdifferentiate into osteoblast-like cells during vascular calcification inducing an active remodelling and calcification of the extracellular matrix (ECM). Intracellular and extracellular enzymes, such as lysyl hydroxylase 1 (PLOD1) or lysyl oxidase (LOX), contribute to ECM maturation and stabilisation towards collagen cross-linking. We aimed to assess the contribution of these enzymes to hyperphosphatemia (HPM)-induced calcification.

Methods and results Human and murine VSMC (hVSMC and mVSMC) were differentiated into functional osteoblast-like cells by HPM conditioning. ECM mineralization was demonstrated by von Kossa and calcium colorimetry or Alizarin Red staining, indicating the presence of calcium phosphates. Osteoblast markers were up-regulated at the transcript and protein levels and that was associated with the early induction of collagen-related genes such as LOX, PLOD1 and collagen type I. In addition, deposition of ECM insoluble collagen was enhanced by HPM. HPM-induced calcification and osteoblast transdifferentiation in mVSMC was limited by chemical inhibition of PLOD (2,2’-dipyridil). Moreover, mVSMC harvested from transgenic mice over-expressing LOX (TgLOX) exhibited an increase in HPM-dependent calcification and osteoblast commitment compared with wild-type cells. HPM-induced calcification was additionally studied ex vivo by using aortic rings harvested from wild-type or TgLOX mice, thus confirming our in vitro results. Therefore, we explored if regulation of PLOD1 and LOX could inhibit hVSMC calcification. Conversely, both β-aminopropionitrile (BAPN; LOX inhibitor), and LOX knockdown abrogated hVSMC calcification and transdifferentiation. Interestingly, deposition of mature collagen was significantly diminished by BAPN as shown by confocal imaging and in agreement with a consistent reduction of cross-linked collagen. The clinical impact of these results was supported by the positive association between the levels of both LOX expression and vascular calcification in human atherosclerotic lesions. Likewise, 2,2’-dipyridil (PLOD inhibitor) and PLOD1 knockdown impaired HPM-induced ECM mineralization and osteoblast commitment.

Conclusions Our findings identify LOX and PLOD as critical players in vascular calcification and highlight the importance of ECM remodelling in this process.