Potential Roles of Necroptosis in the Development and Progression of Vascular Calcification, in Vitro

Abstract

Vascular calcification (VC) is a major complication in individuals with chronic kidney disease (CKD). A constant inflammatory state remains a key characteristic in the development of VC. Necroptosis is a programmed form of cell death that results in an inflammatory phenotype. Early descriptions of necroptosis involve phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting serine/threonine-protein kinase (RIPK) 1/3 signaling. Many studies have shown that necroptosis is a key contributor in various inflammatory diseases, but none have explored its place in VC. In this study, we aim to examine the roles of necroptosis in a model of VC in vitro. We establish VC by utilizing Human Aortic Smooth Muscle Cells (HA-SMCs) treated with 5mM CaCl2 and β-glycerolphosphate for 7, 14, 21 days. VC was confirmed by Arsenazo III and Alizarin Red Staining and by expression of Klotho and runt-related transcription factor 2 (RUNX2). Necroptosis is assessed through the expression of MLKL, phosphorylated-MLKL, RIPK 1 and 3, using Western Blot. Pan-caspase, apoptotic inhibitor ZVAD.fmk (20 µM), RIPK inhibitor necrostatin-1 (20 µM, 40 µM), and MLKL inhibitor necrosulfonamide (0.5 µM, 1 µM), were used to assess the effects of necroptosis inhibition. VC was confirmed by down regulation of Klotho and up regulation of RUNX2. RIPK1 and RIPK3 expression were down regulated in a time-dependent manner in our VC model. The opposite was seen in MLKL and phosphorylated-MLKL, indicating the presence of necroptosis in VC. Furthermore, treatment with necrosulfonamide, alone displayed a dose-dependent reduction of calcification. Neither ZVAD.fmk nor necrostatin-1 resulted in significant changes in calcification. Calcification of HA-SMCs highlight increased activity of MLKL, but not RIPK1/3. Our results indicate that RIPK-independent activation of MLKL may play a significant role in the development of VC. These findings suggest a novel pathway of necroptosis whose inhibition may be a target in the treatment of VC.