Dipeptidyl peptidase-4 inhibitor decreases abdominal aortic aneurysm formation through GLP-1-dependent monocytic activity in mice

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening situation affecting almost 10% of elders. There has been no effective medication for AAA other than surgical intervention. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown to have a protective effect on cardiovascular disease. Whether DPP-4 inhibitors may be beneficial in the treatment of AAA is unclear. We investigated the effects of DPP-4 inhibitor sitagliptin on the angiotensin II (Ang II)-infused AAA formation in apoE-deficient (apoE-/-) mice. Mice with induced AAA were treated with placebo or 2.5, 5 or 10 mg/kg/day sitagliptin. Ang II-infused apoE-/- mice exhibited a 55.6% incidence of AAA formation, but treatment with sitagliptin decreased AAA formation. Specifically, administered sitagliptin in Ang II-infused mice exhibited decreased expansion of the suprarenal aorta, reduced elastin lamina degradation of the aorta, and diminished vascular inflammation by macrophage infiltration. Treatment with sitagliptin decreased gelatinolytic activity and apoptotic cells in aorta tissues. Sitaglipitn, additionally, was associated with increased levels of plasma active glucagon-like peptide-1 (GLP-1). In vitro studies, GLP-1 decreased reactive oxygen species (ROS) production, cell migration, and MMP-2 as well as MMP-9 activity in Ang II-stimulated monocytic cells. The results conclude that oral administration of sitagliptin can prevent abdominal aortic aneurysm formation in Ang II-infused apoE-/-mice, at least in part, by increasing of GLP-1 activity, decreasing MMP-2 and MMP-9 production from macrophage infiltration. The results indicate that sitagliptin may have therapeutic potential in preventing the development of AAA.

Fig 1. Sitagliptin attenuated AAA formation in Ang II-infused apoE^-/- mice.

(A) Histogram representing the percent incidence of AAAs for each treatment group. (n = 10–25 per group) (B) Representative photographs showing the features of aneurysms induced by Ang II. The arrow indicates typical AAAs. (C) Quantification of the maximal outer diameters of the suprarenal aortas in apoE^-/- mice infused with NS or Ang II given as percentages. (*P < 0.05, n = 10 per group) (D) Representative staining with H&E of the suprarenal aortas of mice. The magnification of images shown is 40x. (n = 5 per group) (E and F) Quantification of the luminal diameters and wall thickness of suprarenal aortas in apoE^-/- mice infused with NS or Ang II given as percentages. (*P < 0.05, n = 5 per group).

Fig 2. Sitagliptin ameliorated Ang II-induced morphological and biochemical changes in the aortic wall.

Representative aortic sections of the suprarenal aorta stained with Verhoeff -Van Giessen (VvG) and Masson’s trichrome from apoE^-/- mice treated with NS or Ang II (n = 5 per group). Elastin staining in Ang II-infused mice revealed that the internal elastic lamina was discontinuous compared with NS-infused mice. Treatment with 2.5 mg/kg/day sitagliptin attenuated the rupture of the elastin laminas, but the elastic fibers still had less loose elastin elements. Administration of 5 and 10 mg/kg/day of sitagliptin significantly ameliorated the curve and density of the elastin laminas. Compared with NS-infused mice, trichrome staining of the aortas from Ang II-infused mice showed that medial smooth muscle and fibrous cells were markedly separated by stainable collagen. Treatment with 2.5 mg/kg/day of sitagliptin showed decreased adventitial matrix staining. Administration of 5 and 10 mg/kg/day of sitagliptin significantly prevented changes in the cellular components of the aortic wall. Masson's Trichrome stain, muscle is stained red, collagen—blue, fibrin—pink.

Fig 3. Sitagliptin suppressed macrophage infiltration and gelatinase activity in Ang II-infused apoE^-/- mice.

(A) Representative immunostaining of F4/80, a specific marker of mature macrophages, MMP-2 and MMP-9. In the Ang II-infused aorta, the macrophage staining appeared to be much more prominent in the adventitia than in the media. Treatment with sitaglitin significantly decreased macrophages infiltration as well as MMP-2 and MMP-9 expression. The black arrow indicated macrophage and MMP-2 and MMP-9. The magnification of immunostaining images is 200x. (n = 5, per group) (B) Gelatin gel zymography to detect MMPs activity in aortic tissue extracts from different groups of mice. Aortic samples were pooled into 1 set of 3 samples each. (C) Quantitative densitometric analysis of MMP expression.

Fig 4. Treatment with sitagliptin suppresses apoptosis in Ang-infused apoE^-/- mice.

Representative photographs of deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Apoptotic cells are shown in green and the cell nuclei in blue. The white arrowheads indicate the apoptotic cells. Ang II significantly increased apoptotic cells, particularly in the adventitia layer, but sitagliptin greatly suppressed cell apoptosis (n = 5 per group). The images of the upper panel represent the visible light phase. The magnification of the middle and lower panels was 100x and 200x, respectively.

Fig 5. Liraglutide reduced AAA formation in Ang II-infused apoE^-/- mice.

(A) Histogram representing the percent incidence of AAAs treated with or without liraglutide in Ang II-induced apoE^-/- mice. Liraglutide decreased the incidence of Ang II-induced AAA (n = 3–6 per group). (B) Representative aortic sections of the suprarenal aorta stained with H&E, VvG, F4/80, MMP-2 and MMP-9. Liragultide not only preserved elastic elements but also decreased macrophages infiltration and MMP-2 and MMP-9 expression (n = 3 per group). The magnification of immunostaining images is 200x.

Fig 6. GLP-1 decreased ROS generation, migration, and MMPs activity in monocytic cells by Ang II stimulation.

(A) Intracellular ROS was measured by DCF fluorescence. U937 cells were pre-treated with GLP-1 analog, liragultide, at dose of 10, 50 and 100 nM or vehicle for 1 hour, and then cells were incubated with or without Ang II (1μM) for 24 hours. (B) Chemotaxis assay was used to evaluate the effect of liragultide on U937 cell migration to fibroblast. 2x10⁵ U937 cells were treated with liragultide at a concentration of 10, 50 and 100 nM for 1 hour, and then added to the upper chamber of transwells. The lower chamber contained conditional media from adventitial fibroblast with Ang II (1μM) stimulation for 24 hours. (C) Zymography was used to determinate the gelatinolytic MMPs activity. The conditional media were obtained from the U937 cells which were pre-treated with liragultide (10, 50 and 100 nM) for 1 hour, then cells were incubated with or without Ang II (1μM) for 24 hours. (*P < 0.05, all experiment was performed in triplicate.)