Pharmacological inhibition of BLT1 diminishes early abdominal aneurysm formation

Abstract

Leukotriene B(4) (LTB(4)) is a pro-inflammatory lipid mediator generated by the enzymes 5-lipoxygenase (5-LO) and LTA(4)-hydrolase. LTB(4) signals primarily through its G protein-coupled receptor BLT1, which is highly expressed on specific leukocyte subsets. Recent genetic studies in humans as well as knockout studies in mice have implicated the leukotriene synthesis pathway in several vascular pathologies. Here we tested the hypothesis that pharmacological inhibition of BLT1 diminishes abdominal aortic aneurysm (AAA) formation, a major complication associated with atherosclerotic vascular disease. Chow-fed Apoe(-/-) mice were treated with a 4-week infusion of Angiotensin II (AngII, 1000 ng/(kg min)) beginning at 10 weeks of age, in a well-established murine AAA model. Administration of the selective BLT1 antagonist CP-105,696 beginning simultaneously with AngII infusion reduced the incidence of AAA formation from 82% to 40% (p<0.05). There was a concordant reduction in maximal aortic diameter from 2.35 mm to 1.56 mm (p<0.05). While administration of the antagonist on day 14 after the onset of AngII infusion diminished lesional macrophage accumulation, it did not significantly alter the size of AAA by day 42. Thus, pharmacological inhibition of BLT1 may ultimately hold clinical promise, but early intervention may be critical.

Figure 1. Inhibition of BLT1-mediated chemotaxis

(A, B) Chemotactic response curves of mouse splenocytes to SDF-1 and LTB₄. (C, D, E, F) Chemotaxis assays of isolated splenocytes from mice treated with CP-105,696 for 7 days at the indicated dose. Isolated splenocytes were placed in modified Boyden chambers and chemotaxis was assessed in response to SDF-1 (10 nM) and LTB₄ (100 nM). Data shown are from two experiments and are presented as cell number or mean chemotactic index (cells per four-high power fields in duplicate wells moving in response to SDF-1 or LTB₄ relative to media alone). *p<0.05 versus media alone.

Figure 2. AAA formation in AngII-infused mice treated concomitantly with vehicle or CP-105,696

Apoe^-/- mice were treated with AngII concomitant with vehicle or CP-105,696 for 4 weeks. (A) AAA incidence (*p<0.05), (B) maximal aortic diameter (*p<0.05), (C) Representative images of aortas from mice treated with vehicle or CP-105,696 from the prevention study. Circles represent individual mice, diamonds represent means and bars represent SEMs. The experiment included n=11 mice in the vehicle group, and n=10 mice in the CP-105,696 group. Two mice from the vehicle-control and two mice from the inhibitor-treated group died of AAA rupture.

Figure 3. Experimental design of AAA reversal study

Mice were infused with AngII for 2 weeks and then evaluated by ultrasound. Mice that had developed AAA at 2 weeks were randomly assigned to CP-105,696 (n=10) or vehicle control (n=12). All mice were evaluated again by ultrasound at weeks 4 and 8 after the onset of AngII infusion, and sacrificed at week 8 and evaluated for CBC, total cholesterol and aortic quantitation. The dashed line represents the AngII pumps that remained in place throughout the treatment with CP-105,696 for a total of 8 weeks.

Figure 4. Diminished macrophage accumulation and MMP-2 expression in aortas of mice treated with CP-105,696

(A) Representative images of macrophage (Mac-3) and (B) MMP-2 stain show intense macrophage infiltration and MMP-2 expression in the vehicle control and less macrophage accumulation and MMP-2 expression in the inhibitor-treated aorta (middle panels). No positive stain is detected in the isotype-matched control IgG for Mac-3 and MMP-2 stains (right panels). Magnification 20×. (C, D) Quantification of Mac-3 and MMP-2 immunostaining from vehicle- and inhibitor-treated AAA (*p<0.001).

Figure 5. Ultrasound measurements of aortas from mice treated with vehicle or CP-105,696 beginning 2 weeks after AngII infusion

N=12 mice were treated with vehicle and n=10 mice were treated with CP-105,696 at 2 weeks after AngII pumps were placed. (A) Maximal aortic diameters were measured periodically in the suprarenal aorta throughout treatment with vehicle or CP-105,696. Circles represent means and bars represent SEMs. (B) Correlation of maximal aortic diameter measurements by ultrasound versus histological measurements after dissection at 8 weeks (R=0.918).