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.
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.
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