Longitudinal visualization of calcification genesis and growth in vivo: novel implications for plaque vulnerability

Background: Clinical evidence links arterial calcification and cardiovascular risk. Fibrous cap microcalcifications promote atherosclerotic plaque failure, and large calcifications can stabilize the plaque. Therefore,... [ view full abstract ]

Background: Clinical evidence links arterial calcification and cardiovascular risk. Fibrous cap microcalcifications promote atherosclerotic plaque failure, and large calcifications can stabilize the plaque. Therefore, calcification morphology can determine cardiovascular morbidity, but temporal patterns of calcific mineral growth remain unknown.
Results: Apolipoprotein E–deficient (Apoe-/-) mice on an atherogenic diet develop plaque calcification. Longitudinal studies were performed using two fluorescent calcium tracers injected intravenously into Apoe-/- mice: calcein injection following 18 weeks of atherogenic diet (n=7) and alizarin red S injection into the same mice 1 (n=4) or 3 (n=3) weeks later. Imaging green (calcein) and red (alizarin red S) fluorescence provided snapshots of aortic calcification at 18, 19, and 21 weeks. Observations within histological sections revealed green microcalcifications at 18 weeks embedded within red larger calcifications that were formed by 19 weeks (a), demonstrating that microcalcifications present at the start of calcification become the core of larger calcifications over time. Serial histological sections from aortic root to arch (b) were digitally reconstructed into 3D volumes (c) to reveal total calcific burden and localization within the aorta (d). Total calcification volume increased at a significant rate of 6.0x10^6 µm^3 per week (R^2=0.99, p=0.007) and progressed from aortic arch to aortic root over time (p<0.001). Observations closely match calcification morphologies found by micro-computed tomography of human arteries.
Conclusion: Temporal and spatial understanding of calcification growth is crucial given the link between mineral morphology and cardiovascular risk. These techniques provide a method for testing therapeutic approaches to control calcification morphology over time in situ.