Spectroscopic and echocardiographic assessment of AV calcification in a rabbit Vitamin D2 model

Abstract

Aortic valve (AV) calcification is the major characteristic of AV sclerosis. Several forms of calcium salts accumulate including hydroxyapatite in late phases. We sought to examine whether a modified cholesterol – Vitamin D2 diet can produce AV calcification and stenosis in a rabbit model. AV calcification was introduced in New Zealand male rabbits using the modified diet: normal chaw+ 0.5%w/w cholesterol+ 3.500IU Vitamin D2/kg/d (ergocalciferol). Thirty six rabbits were equally randomized into control (normal chaw) and experimental group (modified diet) for 12 weeks. Echocardiography was performed at baseline and the day before the sacrifice. AV Vmax, Transaortic pressure gradient and AV area (AVA) were measured (Vivid 7, GE). AV cusps (collected every 2 weeks, 3 per group) were processed for Fourier-Transformed Infrared Spectroscopy (FT-IR) to detect and characterize calcium salt deposition longitudinally (Nicolet 6700 spectrometer, OMNIC 7.3 software). AVs from surgical patients with severe AV stenosis were used for qualitative comparisons. None of the echocardiographic parameters was significantly altered with modified diet; baseline AVA: 0.32 ± 0.06 cm 2 vs. 12 weeks: 0.37 ± 0.07, P = 0.18. FT-IR vibrations in the region of 1800-800 cm-1 demonstrated the peroxidation of lipids, advanced glycation end products (AGEs), deposition of CaCO3 and non-hydroxyapatite Ca3 (PO4)2. Further, the 840 cm-1 (HCO3-) band is in accordance to those obtained from human AVs. Overall, our rabbit Vitamin D2 model did not produce any AV hemodynamic changes. Interestingly, FT-IR spectroscopy showed that Vitamin D2 stimulated formation of AGEs, deposition of carbonic and phosphate calcium salts in the rabbit AV, both present in human AVs. This study demonstrates that acidosis is an important pathway of AV calcification. Further analysis is needed to elucidate molecular mechanisms in this particular animal model.