Abstract: SA-PO876

RIPK-Independent Necroptosis Plays Significant Roles in the Progression of Vascular Calcification In Vitro

Session Information

  • Vascular Calcification
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Mineral Disease

  • 1205 Vascular Calcification


  • Chang, Yu-Chun, Brigham and Women''s Hospital, Boston, Massachusetts, United States
  • Ding, Yan, None, Newton, Massachusetts, United States
  • Zhu, Langjing, The Eighth Affiliated Hospital, Sun Yat-sen University, Roxbury Xing, Massachusetts, United States
  • Liu, Qinghua, Brigham and Women''s Hospital, Boston, Massachusetts, United States
  • Hsiao, Li-Li, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States

Vascular calcification (VC) is a major complication in individuals with chronic kidney disease. 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 Mlkl by RIPK1/3 signaling. Many studies have shown that necroptosis is a key contributor in various inflammatory diseases, but none 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 Runx2. Necroptosis is assessed through the expression of Mlkl, phospho-Mlkl, RIPK1, RIPK3 using Western Blot. Pan-caspase inhibitor Z-VAD.fmk (10mM), RIPK inhibitor necrostatin-1 (20mM, 40mM), and Mlkl inhibitor necrosulfonamide (0.5mM, 1mM), were used to assess the effects of necroptosis inhibition.


The 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 phospho-Mlkl, indicating the presence of necroptosis in VC. Furthermore, treatment with Mlkl inhibitor, necrosulfonamide, alone displayed a dose-dependent reduction of calcification. Neither Z-VAD.fmk (Pan-caspase inhibitor) nor necrostatin-1 (RIPK inhibitor) resulted in significant changes in calcification.


Calcification of HA-SMCs highlight increased activity of Mlkl, but not RIPK1/3. Inhibition of Mlkl resulted in significant decrease of total degree of calcification, an effect not seen with treatment with necrostatin-1 or Z-VAD.fmk. 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 vascular calcification.


  • Private Foundation Support