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

Objective: The calcium chloride (CaCl ₂) model is a widely accepted rodent model for abdominal aortic aneurysms (AAAs). Calcium deposition, mainly consisting of calcium phosphate (CaPO ₄) crystals, has been reported to exist in human and experimental aneurysms. CaPO ₄ crystals have been used for in vitro DNA transfection by mixing CaCl ₂ and phosphate-buffered saline (PBS). Here, we describe accelerated aneurysm formation resulting from a modification of the CaCl ₂ model. Methods: A modified CaCl ₂ model, the CaPO ₄ model, was created by applying PBS onto the mouse infrarenal aorta after CaCl ₂ treatment. Morphologic, histologic, and immunohistochemical analyses were performed on arteries treated with the CaPO ₄ model and the conventional CaCl ₂ model as the control. In vitro methods were performed using a mixture of CaCl ₂ and PBS to create CaPO ₄ crystals. CaPO ₄- induced apoptosis of primary cultured mouse vascular smooth muscle cells (VSMCs) was measured by DNA fragmentation enzyme-linked immunosorbent assay. Results: The CaPO ₄ model produces AAA, defined as an increase of <50% in the diameter of the aorta, faster than in the CaCl ₂ model. The CaPO ₄ 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 CaCl ₂ 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 CaPO ₄ model compared with the CaCl ₂ model (2.7 ± 0.2 vs 1.5 ± 0.2; n = 6; P <.05). A significantly higher level of apoptosis occurred in the CaPO ₄ 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 CaCl ₂ model; n = 3; P <.05). An enhancement of macrophage infiltration and calcification was also observed at 3 and 7 days in the CaPO ₄ model. CaPO ₄ induced approximately 3.7 times more apoptosis in VSMCs than a mixture of CaCl ₂ (n = 4; P <.0001) in vitro. Conclusions: The CaPO ₄ 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.