Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases

Abstract

Background: Elastin calcification is a widespread feature of vascular pathology, and circumstantial evidence exists for a correlation between elastin degradation and calcification. We hypothesized that matrix metalloproteinase (MMP)-mediated vascular remodeling plays a significant role in elastin calcification.

Methods and results: In the present studies, we determined that short-term periadventitial treatment of the rat abdominal aorta with low concentrations of calcium chloride (CaCl2) induced chronic degeneration and calcification of vascular elastic fibers in the absence of aneurysm formation and inflammatory reactions. Furthermore, the rate of progression of calcification depended on the application method and concentration of CaCl2 applied periarterially. Initial calcium deposits, associated mainly with elastic fibers, were persistently accompanied by elastin degradation, disorganization of aortic extracellular matrix, and moderate levels of vascular cell apoptosis. Application of aluminum ions (known inhibitors of elastin degradation) before the CaCl2-mediated injury significantly reduced elastin calcification and abolished both extracellular matrix degradation and apoptosis. We also found that MMP-knockout mice were resistant to CaCl2-mediated aortic injury and did not develop elastin degeneration and calcification.

Conclusions: Collectively, these data strongly indicate a correlation between MMP-mediated elastin degradation and vascular calcification.

Figures

Figure 1

Morphology and kinetics of aortic calcification. H&E stain for general morphology (A, B), Alizarin red for calcium deposits (C, D), and immunohistochemistry for macrophages (E) of abdominal aorta swabbed with 0.2 mol/L NaCl (A, C) or with 0.2 mol/L CaCl2 (B, D, E). Rat spleen cryosections (F) were used as positive control for macrophage staining (brown, round deposits, arrow). Original magnifications were ×400; aortic lumen is on top and brackets designate aorta thickness. Kinetics of aortic calcification (n=6 rats per time point per group) induced by 2 methods of periarterial application of 0.15 mol/L CaCl2 solution is shown in Figure 1G. Abbreviations are as defined in text.

Figure 2

Elastin-associated calcification occurs in rat abdominal aorta injury model. Abdominal aorta collected 7 days after CaCl2 injury was stained with Alizarin red/light green counter-staining (A, C, E, G) and DAPI nuclear stain (bright blue spots) superimposed with Alizarin red without light green counterstaining (B, D, F, H). Arrowheads and * designate calcium deposits; arrows depict elastic lamellae (wavy horizontal fibers). Original magnifications are as follows: A–F, ×400 and G and H, 3200. Brackets designate aorta; sections are oriented with aortic lumen on top. Abbreviations are as defined in text.

Figure 3

Extracellular matrix degradation accompanies aortic calcification in periadventitial injury model. Abdominal aorta collected after NaCl treatment (A, D), CaCl2 (B, E), and AlCl3 (C, F) stained with VVG (A–C), Masson’s trichrome (D, E), and Alizarin red (F). Arrowheads designate “transition” areas from wavy to flattened elastic fibers. Original magnifications were ×400; brackets designate aorta thickness; and sections are oriented with aortic lumen on top. Desmosine and calcium contents of abdominal aortas for control (no surgery) and for NaCl, CaCl2, and AlCl3 treatments (n=4 per group) are shown in G. Abbreviations are as defined in text.

Figure 4

Apoptosis of vascular cells in abdominal aorta injury model. Abdominal aorta after treatment with NaCl (A, D, G), CaCl2 (B, E, H), and AlCl3 (C, F, I) were stained with DeadEnd TUNEL for detection of apoptotic cells (A–F) and with DAPI nuclear stain to show cellular distribution (G–I). TUNEL-positive cells are brown (arrows). Negative control for TUNEL assay is shown in E and positive controls (DNase-treated sections) in D and F. Original magnifications were ×400x; brackets designate aorta thickness. L indicates lumen. All other abbreviations are as defined in text.

Figure 5

Periadventitial injury induces permeabilization of aortic endothelium to Evans blue. Cryosections collected from controls (A, E), NaCl-treated (B, F), CaCl2-treated (C, G), and AlCl3-treated (D, H) abdominal aortas were analyzed unstained under UV illumination with FITC filter (A–D). Under UV light and FITC filter, elastin fluoresces green, Evans blue appears red, and elastin to which Evans blue was bound appears as yellow–bright orange. Images obtained from DAPI nuclear staining (bright blue spots) were digitally superimposed with those obtained from sections by FITC filter (E–H). Arrowheads designate “transition” areas in calcified aortas (C, G). Original magnifications were ×200; brackets designate aorta thickness. L indicates lumen. All other abbreviations are as defined in text.

Figure 6

MMPs contribute to elastin-associated calcification in mouse abdominal aorta injury model. MMP2-knockout mice (MMP2 ko), MMP9 ko, and control wild-type (WT) mice were subjected to NaCl (A, D, G) or CaCl2 (B, C, E, F, H, I) abdominal aorta injury. Aortic segments were stained with Alizarin red/light green counterstaining (A, B, D, E, G, H), VVG (C, F), and Alizarin superimposed with DAPI nuclear staining (I). Original magnifications were ×200 (A, B, D, E, G, H) and ×400 (C, F, I); brackets designate aorta thickness. L indicates lumen. All other abbreviations are as defined in text.