Nitric oxide induces the progression of abdominal aortic aneurysms through the matrix metalloproteinase inducer EMMPRIN

Abstract

Nitric Oxide (NO) is involved in the development and progression of abdominal aortic aneurysms (AAA). We found that inhibition of inducible NO synthase (iNOS) protects mice in an elastase-induced AAA model, significantly inhibiting the production of matrix metalloproteinase-13 (MMP-13). The extracellular MMP inducer (EMMPRIN; CD147) was increased in human AAA biopsies and in wild-type murine AAA but not in AAA from iNOS null mice. In cells overexpressing ectopic EMMPRIN, MMP-13 secretion was stimulated, whereas silencing of EMMPRIN by RNA interference led to significant inhibition of MMP-13 expression. In addition, elastase infusion of MMP-13 null mouse aortas induced a significant increase of EMMPRIN but reduced aortic dilatation when compared with wild-type mice, suggesting that NO-mediated AAA may be mediated through EMMPRIN induction of MMP-13. These findings were further verified in elastase-infused iNOS null mice, in which daily administration of NO caused a significant aortic dilatation and the expression of EMMPRIN and MMP-13. By contrast, in iNOS wild-type mice, pharmacological inhibition of iNOS by administration of 1400 W induced a reduction of aortic diameter and inhibition of MMP-13 and EMMPRIN expression when compared with control mice. Our results suggest that NO may regulate the development of AAA in part by inducing the expression of EMMPRIN and modulating the activity of MMP-13 in murine and human aneurysms.

Figure 1

Lack of iNOS protects mice during induction of AAA. A: Thrichrome Masson staining of aortic sections from C57BL/6 wild-type mice (WT), iNOS null mice, apoE null mice and iNOS/apoE double null mice, 15 days after surgery. Scale bars, 100 μm (n = 6). B: Immunoblot detection of iNOS in 12- and 40-week-old mice aortic lysates, 15 days after elastase infusion. C: Effect of NO on aortic dilatation overtime. Ten animals per group were either infused with elastase or saline buffer, and the aortas were monitored over time by magnetic resonance. Left, Graphical representation of AD 15 days after elastase infusion. Right, Graphical representation of the increase in diameter 15 days after surgery respect to sham operated mice (n = 10mice/genotype/assay by triplicate mean ± SD; ^∗P < 0.05 vs wild-type). L, lumen.

Figure 2

Lack of iNOS inhibits the expression of MMP-13 in AAA. A: Immunofluorescence detection of MMP-13 in aortic ring sections from wild-type (WT) and iNOS null mice, collected after elastase infusion (left panels), or saline infusion (right panels) over time (n = 3 mice/time point by triplicate, mean ± SD; ^∗P < 0.05 vs wild-type) L: lumen. Scale bars, 50 μm. B: Immunoblot detection of MMP-13 in the same mice as in A. GAPDH was also detected for control loading purposes. Right, Densitometric analysis of the immunoreactive bands detected at time points 0 and 15 days after surgery (n = 3 mice/time point by triplicate, mean ± SD; ^∗P < 0.05 vs wild type).

Figure 3

EMMPRIN expression is inhibited in iNOS null mice. A: Detection of EMMPRIN (FITC, green) and the endothelial marker ICAM-1 (Cy3, red) by immunohistofluorescence in aortic rings isolated from elastase-infused mice 15 days after surgery. Scale bars, 50 μm. B: Immunoblot analysis of EMMPRIN expression in wild-type (WT) and iNOS null mice (knockout (KO)). Three major immunoreactive bands of EMMPRIN were detected, corresponding to different glycosylation levels. GAPDH was also detected for control loading purposes. Right, Densitometric analysis of signals detected at time points 0 and 15 after surgery (n = 3 mice/time point by triplicate, mean ± SD; P < 0.05 wild-type elastase versus iNOS null elastase). C: Diameter of iNOS null mouse and iNOS null mouse aortas in which 5 mmol/L Spermidine-nonoate was administered (iNOS^−/− + NO), 15 days after elastase infusion ((n = 3 mice by triplicate, mean ± SD; ^∗P < 0.05 versus iNOS null). D: Representative micrographs of aortic serial sections of the same mice as in C, in which HE, Masson Trichrome, Verhoeff-van Gieson staining, EMMPRIN, protein tyrosine nitration, and MMP-13 expression were detected. Arrows point endothelial positive cells (n = 3). Scale bars, 50 μm. E: Representative micrographs of aortic serial sections isolated from iNOS wild-type mice in which 500 μmol/L 1400W was daily administered, 15 days after elastase infusion, showing HE, Masson Trichrome, Verhoeff-van Gieson staining, and protein tyrosine nitration (n = 3 mice/group by tryplicate). Scale bars, 50 μm. F: Immunohistochemistry detection of MMP-13 and EMMPRIN in aortic sections of the same mice as in E. A detailed selection was highlighted in the open squares. Scale bars, 50 μm.

Figure 4

EMMPRIN regulates MMP-13 expression and activity in vascular endothelium. A: Immunoblot detection of ectopic FLAG-EMMPRIN, and endogenous MMP-13 in cell lysates and culture medium of FLAG-EMMPRIN expressing murine aortic endothelial cells. GAPDH was also detected for control loading purposes. B: Immunoblot detection of ectopic FLAG-EMMPRIN and endogenous MMP-13 in cell lysates and culture medium from murine aortic endothelial cells in which EMMPRIN was silenced. GAPDH was also detected for control loading purposes (n = 3 by triplicate). Right, Expression of MMP-13 in endothelial cells treated with 100 μmol/L DEA-NO in silenced-EMMPRIN cells. C: Detection of EMMPRIN in aortic rings of wild-type and MMP-13 null mice, 15 days after surgery. L: lumen. n = 3. Scale bars, 50 μm. D: 10 animals/group were either infused with elastase or saline buffer and the aneurysms (arrow) were monitored over time by magnetic resonance. Right, Graphical representation of aortic dilatation 15 days after surgery respect to sham operated mice (n = 10 mice/genotype/assay by triplicate mean ± SD; ^∗P < 0.05 versus wild-type).

Figure 5

EMMPRIN, MMP-13, iNOS, and nitro-tyrosine detection in human AAA. A: EMMPRIN, MMP-13, and iNOS, expression in AAA samples (n = 18) (upper) or in control aortas (n = 3) (lower). Serial sections showing colocalization of EMMPRIN and MMP-13 (arrows) are depicted. B: Aortic serial sections showing immunofluorescence detection of nitrotyrosine by confocal microscopy (left, Red-Cy3, DAPI-nuclei), and immunohistochemistry detection of iNOS by using bright field microscopy (right). Colocalization of both signals was also detected.