Abstract 233: Novel Roles of Notch2 and Notch3 in Differential Regulation of Cardiovascular Calcification
Background: We previously demonstrated that Notch receptors contribute to aortic valve calcification, however the underlying mechanisms, particularly relative contributions of each receptor remain obscure. Here we tested the hypothesis that Notch homologues differentially regulate calcification.
Methods and Results: The activation of Notch1– 4 (N1-N4) was examined during osteogenic differentiation of mouse mesenchymal stem cells (mMSC), potential contributors to calcification. Cleavage of Notch intracellular domain (ICD), which acts as a transcriptional regulator, indicates activation of this pathway. Western blot analysis of Notch expression and cleavage during osteogenic treatment showed increasing N3 (2-fold), transient N2 and decreasing N1 (0.21-fold) and N4 (0.72-fold) activation. We therefore, investigated further the roles of N2 and N3 in calcification by gain-of-function (transfection of constitutively active N2-ICD or N3-ICD) or loss-of-function (transfection of N2 or N3 RNAi) approaches in mMSC treated with osteogenic medium. Calcification evaluated by alkaline phosphatase (ALP) activity and hydroxyapatite (HA) crystal formation (von Kossa, Figure⇓), suggested that N3 promotes and N2 inhibits osteogenic activity. Histological analysis showed N3 but not N2 expression in calcified aortic sinuses of apoE−/− mice (n=7). Co-immunoprecipitation of N2/N3 and their ligands suggested that a glycosylation-regulated switch from N2- to N3-Jagged1 complex precedes calcification.
Conclusion: Competing N2/N3 signals regulate osteogenic differentiation and cardiovascular calcification.
This research has received full or partial funding support from the American Heart Association, National Center.