Vascular calcification (VC) is an indicator of atherosclerotic plaque burden and is a strong, independent predictor for future cardiovascular complications, significantly increasing the risk for cardiovascular morbidity and mortality. Type II diabetes (T2D), the 7th leading cause of death in the US, is associated with increased incidence of VC clinically, as well as in our recent animal study. Although VC was considered a degenerative process involving passive accumulation of calcium-phosphate salts associated with tissue necrosis, numerous studies have indicated that VC is an active, cell-mediated process that resembles embryonic bone formation and remodeling. Smooth muscle cells (SMCs) have been implicated in giving rise to bone- and cartilage-like cells in calcifying blood vessels by undergoing lineage reprogramming in response to local environmental cues. We investigated the association of S100A11, a calcium binding protein, with a multi-ligand receptor, the receptor for advanced glycation end products (RAGE), as a possible signaling mechanism involved in the lineage reprogramming of SMCs under T2D settings. We used a genetic fate mapping strategy to trace SM-derived cells in the model T2D mice that developed vascular calcification. Immunofluorescent dual staining for S100A11 and RAGE, along with immunohistochemical staining for Runx2/Cbfa1, an early osteochondrogenic marker, was performed on aortic sections of the T2D mice. The results revealed that S100A11-RAGE signaling is most likely to be involved in cartilage lesion development. This is supported by co-localization of S100A11-RAGE markers within cells of chondrocyte morphology in cartilaginous and calcific areas of blood vessels. Interestingly, almost all S100A11-RAGE positive cells were derived from SMCs that have been labeled by specific transgenes. These results highlight the significance of S100A11-RAGE signaling in VC and possibly provide for the development of preventative or therapeutic targets against T2D-related VC.

Diabetes is a serious and prevalent disease affecting the lives of many people. Studies have shown that cardiovascular calcification, or calcium-phosphate mineral deposition in blood vessels, cardiac valves, and myocardium, is prominent in patients with Type II diabetes (T2D), accounting for the heightened cardiovascular morbidity and mortality in this population. Using a T2D mouse model, our laboratory has shown a positive correlation of hyperglycemia with cardiovascular calcification. A multi-ligand receptor, namely the receptor for advanced glycation-end products (RAGE), was high in cartilaginous and calcific areas of blood vessels and aortic valves and translocated to the cell membrane of the cells with osteochodrogenic (bone and cartilage precursor) and chondrocytic (cartilage) properties. To study whether RAGE is involved in the early signaling of osteochodrogenic differentiation and calcification of cardiac valves, we cultured bovine valve interstitial cells (BVICs) in procalcific medium containing normal and high glucose in a range of hyperglycemia. We found that high glucose increased calcification of BVICs by up to 3 folds. Expression levels of RAGE and its ligands are determined by reverse-transcription PCR and Western blot analysis. The biological activity of RAGE are blocked by a commercially available neutralizing antibody to see whether RAGE is required for the high glucose-associated BVIC calcification. Our results show a decrease in BVIC calcification when RAGE activity is blocked by a neutralizing antibody. For future studies, we will create a cell line with lower expression levels of RAGE via small RNA interference.