Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model

ObjectiveThe calcium chloride (CaCl2) model is a widely accepted rodent model for abdominal aortic aneurysms (AAAs). Calcium deposition, mainly consisting of calcium phosphate (CaPO4) crystals, has been reported to exist in human and experimental aneurysms. CaPO4 crystals have been used for in vitro DNA transfection by mixing CaCl2 and phosphate-buffered saline (PBS). Here, we describe accelerated aneurysm formation resulting from a modification of the CaCl2 model.MethodsA modified CaCl2 model, the CaPO4 model, was created by applying PBS onto the mouse infrarenal aorta after CaCl2 treatment. Morphologic, histologic, and immunohistochemical analyses were performed on arteries treated with the CaPO4 model and the conventional CaCl2 model as the control. In vitro methods were performed using a mixture of CaCl2 and PBS to create CaPO4 crystals. CaPO4- induced apoptosis of primary cultured mouse vascular smooth muscle cells (VSMCs) was measured by DNA fragmentation enzyme-linked immunosorbent assay.ResultsThe CaPO4 model produces AAA, defined as an increase of ≥50% in the diameter of the aorta, faster than in the CaCl2 model. The CaPO4 model showed significantly larger aneurysmal dilation at 7, 28, and 42 days, as reflected by a maximum diameter (measured in mm) fold-change of 1.69 ± 0.07, 1.99 ± 0.14, and 2.13 ± 0.09 vs 1.22 ± 0.04, 1.48 ± 0.07, and 1.68 ± 0.06 in a CaCl2 model, respectively (n = 6; P < .05). A semiquantitative grading analysis of elastin fiber integrity at 7 days revealed a significant increase in elastin degradation in the CaPO4 model compared with the CaCl2 model (2.7 ± 0.2 vs 1.5 ± 0.2; n = 6; P < .05). A significantly higher level of apoptosis occurred in the CaPO4 model (apoptosis index at 1, 2, and 3 days postsurgery: 0.26 ± 0.14, 0.37 ± 0.14, and 0.33 ± 0.08 vs 0.012 ± 0.10, 0.15 ± 0.02, and 0.12 ± 0.05 in the conventional CaCl2 model; n = 3; P < .05). An enhancement of macrophage infiltration and calcification was also observed at 3 and 7 days in the CaPO4 model. CaPO4 induced approximately 3.7 times more apoptosis in VSMCs than a mixture of CaCl2 (n = 4; P < .0001) in vitro.ConclusionsThe CaPO4 model accelerates aneurysm formation with the enhancement of apoptosis, macrophage infiltration, and calcium deposition. This modified model, with its rapid and robust dilation, can be used as a new model for AAAs.

Clinical RelevanceAnimal models of abdominal aortic aneurysms (AAAs) have been used in a range of experiments to explore various aspects of pathogenesis and potential methods of treatment. The conventional calcium chloride (CaCl2) model has been widely accepted as an experimental animal model with many pathologic similarities with human AAAs. This conventional model typically shows aneurysmal dilation at 4 to 6 weeks after injury. We have created a mouse experimental AAA model with rapid and robust aneurysmal dilation through the modification of the conventional CaCl2 model that uses calcium phosphate. This modified model, with its rapid and robust dilation, can be used as a new model for AAAs.