New ex vivo calcification model for intact murine aortic valves

Introduction Calcific aortic valve disease (CAVD) is a common, progressive disease of the aortic valves for which no medical treatment exists and surgery is the only therapeutic solution. The discovery of novel pharmacological treatment for CAVD has been hampered due to the lack of suitable test-systems. Purpose We aimed to establish an ex vivo calcification model for intact mouse aortic valves. Methods We used the ex vivo flow model for mouse hearts (Miniature Tissue Culture System) to induce calcification with culture media supplemented with 1) β-glycerophosphate, ascorbic acid, and dexamethasone (OSM) or 2) inorganic phosphates (PI) and compared this with in vitro calcification of mouse valvular interstitial cells (mVICs). Results Ex vivo cultured aortic valve leaflets calcified in the presence of PI, but not OSM. RUNX was upregulated in both OSM and PI conditions, whereas alkaline phosphatase (ALP) and cyclooxygenase-2 (COX2) were differentially expressed. Apoptosis was not observed, together indicating that osteogenic calcification took place. Interestingly, both OSM and PI were able to induce calcification in vitro, revealing in vitro-ex vivo differences. In addition to the calcification in the aortic valves, endochondral calcification was present in the sinus near the hinge of the aortic valve in both PI and OSM conditions as evidenced by the expression of collagen II, aggrecan and ALP. Conclusions Together these data show that we have established an ex vivo calcification model for mouse aortic valves. Osteogenic calcification is induced in the aortic leaflets, whereas endochondral calcification is induced in the aortic sinus, reflecting the calcification found in human CAVD. Using this model, we can now study the initiation and progression of aortic valve calcification. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Dutch Heart Foundation, grant number 2013T093. Grants received from GE Healthcare, Lantheus medical imaging, St Jude Medical, Medtronic, Boston Scientific, Biotronik, and Edwards Lifesciences

Initiation of endochondral calcification is related to changes in the redox state of hypertrophic chondrocytes

The level of pyridine nucleotides (NADH and NAD+) in the mineralizing growth plate of the chick was ascertained by high-resolution scanning microfluorimetry and biochemical analysis. Scanning electron microscopy and light microscopy were used to relate the concentrations of NADH and NAD+ to stages of chondrocyte maturation. A dramatic increase was found in the relative concentration of reduced pyridine nucleotides in the hypertrophic zone. On either side of this zone, in proliferating and calcifying cartilage, there was a decrease in NADH fluorescence, and the NADH/NAD+ ratio was depressed. The finding that NADH accumulated in the tissue zone associated with the earliest deposition of bone mineral supports the hypothesis that a change in the redox state initiates tissue mineralization.