Cysteinyl leukotriene receptor 1 antagonism prevents experimental abdominal aortic aneurysm

Abstract

Cysteinyl-leukotrienes (cys-LTs) are 5-lipoxygenase-derived lipid mediators involved in the pathogenesis and progression of inflammatory disorders, in particular asthma. We have previously found evidence linking these mediators to increased levels of proteolytic enzymes in tissue specimens of human abdominal aortic aneurysm (AAA). Here we show that antagonism of the CysLT1 receptor by montelukast, an established antiasthma drug, protects against a strong aorta dilatation (>50% increase = aneurysm) in a mouse model of CaCl₂-induced AAA at a dose comparable to human medical practice. Analysis of tissue extracts revealed that montelukast reduces the levels of matrix metalloproteinase-9 (MMP-9) and macrophage inflammatory protein-1α (MIP-1α) in the aortic wall. Furthermore, aneurysm progression was specifically mediated through CysLT1 signaling since a selective CysLT2 antagonist was without effect. A significantly reduced vessel dilatation is also observed when treatment with montelukast is started days after aneurysm induction, suggesting that the drug not only prevents but also stops and possibly reverts an already ongoing degenerative process. Moreover, montelukast reduced the incidence of aortic rupture and attenuated the AAA development in two additional independent models, i.e., angiotensin II- and porcine pancreatic elastase-induced AAA, respectively. Our results indicate that cys-LTs are involved in the pathogenesis of AAA and that antagonism of the CysLT1 receptor is a promising strategy for preventive and therapeutic treatment of this clinically silent and highly lethal disease.

Keywords: abdominal aortic aneurysm; inflammation; leukotriene; montelukast.

Fig. 1.

The effect of montelukast on aorta dilatation in CaCl₂-induced AAA. (A) 5-LO, FLAP, LTA₄H, and LTC₄S mRNA was determined by qPCR in aortic wall of C57Bl6/J mice 21 d after the treatment with CaCl₂ (n = 4) and compared with control mice (n = 4) treated with NaCl. (B) Bright field and representative immunofluorescence staining for 5-LO, FLAP, LTA₄H, and LTC₄S (Cy3, red), CD68 (FITC, green), and DNA (DAPI, blue) and merged picture. Dashed yellow lines indicate external elastic lamina. Dashed line squares represent selected area for high magnification. (Scale bars: 100 μm, inner square 10 μm.) (C) AAA was induced on mice by periaortic application of CaCl₂ (n = 25) or NaCl (n = 6). CaCl₂-induced AAA mice were divided into four groups: group 1, no treatment (n = 10); group 2, mice treated with montelukast 0.1 mg/kg/d (n = 7); group 3, mice treated with montelukast 1 mg/kg/d (n = 8); and group 4, mice treated with CysLT2 antagonist 3 mg/kg/d (HAMI3379; n = 5); AAA was also induced on Alox5^−/− mice by periaortic application of CaCl₂ (n = 7) or NaCl (n = 4) as control. After 21 d, aortas were harvested, stained, and circumferences were calculated. (D) AAA was induced by CaCl₂ application and mice were divided into three groups receiving montelukast 1 mg/kg/d for 21 (n = 8), 14 (n = 6), and 7 (n = 6) d after AAA induction. CaCl₂-treated mice without montelukast (n = 10) and NaCl-treated mice (n = 6) were used as positive and negative control, respectively. At the end of the experiment, aortas were harvested, stained, and circumferences were calculated. (E) Homogenated aortas from C57Bl6/J mice treated with NaCl (n = 8), CaCl₂ (n = 8), CaCl₂ + montelukast 0.1 mg/kg/d (n = 4), CaCl₂ + montelukast 1 mg/kg/d (n = 4), and from Alox5^−/− mice treated with NaCl (n = 4) and CaCl₂ (n = 5) were analyzed by zymography and bands were quantified. (F) Homogenated aortas from separate C57Bl6/J mice treated with NaCl (n = 3), CaCl₂ (n = 3), CaCl₂ + montelukast 0.1 mg/kg/d (n = 3), and CaCl₂ + montelukast 1 mg/kg/d (n = 3), and from Alox5^−/− mice treated with NaCl (n = 3) and CaCl₂ (n = 3) were analyzed for the presence of MIP-1α by Bio-Plex mouse cytokine assay. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, L, lumen; n.s., nonsignificant.

Fig. 2.

Montelukast inhibits LTD₄-induced expression of MIP-1α and TNFα in human MonoMac6 (MM6) cells. MM6 cells were pretreated with montelukast or vehicle before challenge with LTD₄ (100 nM, 1 h). (A) qPCR analysis of MIP-1α and TNFα transcript levels in MM6 cells treated with vehicle only (control), challenged with LTD₄ only (LTD₄), or challenged with LTD₄ following pretreatment with montelukast (montelukast+LTD₄). (B) Analysis of MIP-1α and TNFα protein levels in supernatants of MM6 cells treated as in A. The graphs depict results from analysis of duplicate and triplicate samples obtained in three independent experiments. Data represent mean of experiments ±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Effect of montelukast in AngII-infused ApoE^−/− mouse model. (A) 5-LO, FLAP, LTA₄H, and LTC₄S mRNA was determined by qPCR in aortic wall of AngII-infused ApoE^−/− (n = 4) and compared with ApoE^−/− mouse (n = 4). (B) Bright field and representative immunofluorescence staining for 5-LO, FLAP, LTA₄H, and LTC₄S (Cy3, red), CD68 (FITC, green), and DNA (DAPI, blue) and merged picture of suprarenal aorta in AngII-infused ApoE^−/−. Dashed yellow lines indicate external elastic lamina. Dashed line squares represent selected area for high magnification. (Scale bars: 100 μm, inner square 10 μm.) (C) Aortic rupture rate was determined in AngII-infused ApoE^−/− treated with placebo (45%, 9 of 20) vs. montelukast group (15%, 3 of 20). (D) Homogenated aortas from AngII-infused ApoE^−/− treated with placebo (n = 4) and montelukast (n = 5) were analyzed by zymography and bands were quantified. Data represent mean of experiments ±SEM. The aortic rupture was analyzed by χ² test. *P < 0.05, L, lumen; n.s., nonsignificant.

Fig. 4.

Effect of montelukast in PPE infusion model. (A) 5-LO, FLAP, LTA₄H, and LTC₄S mRNA was determined by qPCR in aortic wall of PPE (n = 3) compared with sham unoperated animals (n = 3). (B) Bright field and representative immunofluorescence staining for 5-LO, FLAP, LTA₄H, and LTC₄S (Cy3, red), CD68 (FITC, green), and DNA (DAPI, blue) and merged picture of infrarenal aorta in PPE. Dashed yellow lines indicate external elastic lamina. Dashed line squares represent selected area for high magnification. (Scale bars: 100 μm, inner square 10 μm.) (C) Representative ultrasound imaging of infrarenal aorta from mice treated with placebo and montelukast after 28 d of PPE infrarenal infusion. (D) Luminal diameter (relative dilatation from baseline) after 28 d of PPE infrarenal infusion in mice treated with placebo (n = 7) and montelukast (n = 5). (E) Homogenated infrarenal aortas from mice treated with placebo (n = 6) and montelukast (n = 5) were analyzed by zymography and bands were quantified. Data represent mean of experiments ±SEM. *P < 0.05, ***P < 0.001, L, lumen; n.s., nonsignificant.