Abstract 245: Activation of Receptor for Advanced-Glycation End Products (Rage) Induces Notch-msx2-Dependent Osteoblastic Differentiation of Vascular Smooth Muscle Cells
Activation of receptor for advanced-glycation end products (RAGE) plays an important role in the development of atherosclerotic lesion in the patients with diabetes and chronic renal failure (CRF). However, little is known about effects of RAGE on calcification of vascular smooth muscle cells (VSMC). We first examined effects of RAGE on osteogenic conversion of VSMC by monitoring the expression and enzymatic activity of alkaline phosphatase (ALP). RT-PCR analysis showed that expression of ALP was up-regulated in VSMC when RAGE was transduced by adenovirus. The ALP up-regulation by RAGE overexpression was obviously decreased when fetal bovine serum (FBS) was pre-treated with soluble-RAGE, a decoy receptor, or when 1% FBS was used instead of 15%, suggesting that the FBS contains RAGE ligands. The induction of ALP mRNA was accompanied by ALP activity, calcium deposition detected by Kossa staining, and an inducible expression of osteogenic transcription factors, Msx2 and Runx2, and components of Notch signaling, Jagged1 and Notch1. The induction of Notch components is in line with our previous report that Notch signaling induces Msx2-dependent calcification of VSMC. Consistent with this, the induction of Msx2 and ALP mRNA by RAGE overexpression was blocked by DAPT, a Notch signal inhibitor. Immunohistochemistry revealed that RAGE antigen overlapped with Jagged1, Notch1, Msx2, and calcium deposition in calcified plaque of human carotid artery. We further studied whether human serum from patients with both diabetes and CRF promotes ALP activity in cultured VSCM. The serum from healthy volunteers did not up-regulate ALP activity while those from the patients significantly up-regulated it under overexpression of RAGE. Interestingly, multiple regression analyses revealed that induction of ALP activity by the patients serum was positively co-related with serum creatinine level, but not hemoglobin-A1c, suggesting a more important role of renal dysfunction than plasma glucose level in vascular calcification. These findings suggest that RAGE induction in VSMC contributes to the development of calcifying atherosclerotic plaque characterizing diabetes- and CRF-associated vascular lesion by inducing Notch-Msx2-dependent osteogenic differentiation.