Development of a novel murine model of aortic aneurysms using peri-adventitial elastase

Abstract

Background: Our aim was to establish a novel model of abdominal aortic aneurysms (AAA) in mice using application of peri-adventitial elastase.

Methods: C57BL/6J male mice underwent infrarenal peri-adventitial application of either (1) sodium chloride (control; n = 7), (2) porcine pancreatic elastase (PPE; n = 14), or (3) PPE and doxycycline (PPE + doxycycline 200 mg/kg; n = 11) for 14 days. Aortas were analyzed by video micrometry, immunohistochemistry, qualitative polymerase chain reaction, and zymography. Groups underwent Mann-Whitney U comparisons.

Results: At day 14 compared with baseline, control animals had minimal aortic dilation, whereas fusiform aneurysms were seen in PPE (control, 20 ± 3%; PPE, 82 ± 15%; P ≤ .003). Doxycycline abrogated aneurysm formation (PPE, 82 ± 15%; PPE + doxycycline, 37 ± 10%; P ≤ .03). Compared with control and PPE + doxycycline, immunohistochemistry demonstrated greater elastin fiber degradation, macrophage infiltration, and matrix metalloproteinase-9 expression in PPE. Ki-67 and cleaved caspase-3 were lower in control versus PPE. The loss of smooth muscle marker expression seen with PPE was preserved in PPE + doxycycline. Zymography confirmed that both MMP-2 and -9 were more active in PPE than PPE + doxycycline.

Conclusion: Peri-adventitial application of elastase is a simple, reproducible in vivo model of aneurysm formation leading to consistent infrarenal aortic aneurysm development by day 14, with inflammatory cell infiltration and MMP upregulation. Doxycycline inhibits AAA progression in this model via limiting matrix degradation and preserving differentiated smooth muscle cells.

Fig 1

(A) The peri-adventitial elastase application model. The infra-renal murine aorta is first circumferentially dissected, and 10 μL of 100% PPE is topically applied to the adventitial aorta for 10 minutes. The aorta is monitored closely for phenotypic changes after which the animal is closed and recovered for 14 days. The AAA at day 14 is explanted for analysis. The in situ, naïve aorta at the beginning of the procedure has a fibrous matrix (solid white arrows). (B) Effects of elastase and time-dependent exposure on aortic dilation. Nonlinear regression curves for elastase volume and time of exposure where intersected with targeted aortic dilation between 30% and 40%. When considered with visual aortic changes during the operation, the most reproducible results were anticipated when 10 μL of elastase was applied for 10 minutes.

Fig 2

(A) Progression of aneurysm formation after peri-adventitial elastase application. The aorta at 5 minutes after PPE application (A) develops wall thinning and early qualitative changes. At 10 minutes after PPE application (B), the aorta develops an enriched red coloration, as the matrix seems to be visually degraded. The infrarenal murine abdominal aorta at 14 days after PPE application, has a sheen that is noted with fusiform changes (C). (B) Relative change in aortic diameter above baseline. The relative change in the aortic diameter over baseline is highest in the PPE group. An aneurysm was defined as an increase in the aortic diameter by ≥50% above baseline. *P = .03; **P = .003.

Fig 3

(A) Modified Russell-Movat Pentachrome (Movat). Movat staining (10×) revealed degraded elastin and smooth muscle layer(s) with PPE application (solid white arrows on anterior surface of aorta) that were preserved among doxy-cycline treated animals. (B) Activated macrophages (Mac2). Mac2 stain (10×) show increased presence with elastase administration (solid white arrows). Activated macrophages are reduced with doxycycline treatment. (C) Matrix metalloproteinase-9 (MMP-9). Total MMP-9 stain (10×) shows increased presence with elastase administration. MMP-9 expression is attenuated with doxycycline treatment at day 14. (D) Quantified immunostaining for activated macrophages (Mac2) and matrix metalloproteinase-9 (MMP-9). Threshold gated quantification of immunohistochemistry in aortic sections for Mac2 and MMP-9 show increased presence of macrophages, and MMP-9 at day 14 in the aortic wall after elastase administration. *P < .05; **P < .01.

Fig 4

(A) Smooth muscle 22-α (SM22α). SM22α immunostain (10×) shows that the smooth muscle layer are preserved among doxycycline-treated animals. The solid white arrows are on the anterior surface of the aorta. (B) Smooth muscle α-Actin (SMαA). SMαA immunostain (10×) shows increased smooth muscle degradation after topical elastase exposure (solid white arrow), which is decreased after treatment with doxycycline. (C) myosin heavy chain (MHC-SM1). MHC-SM1 immunostain (10×) shows increased smooth muscle degradation after topical elastase exposure, which is reduced after treatment with doxycycline. (D) Ki-67. Ki-67 immunostaining (10×) for cell proliferation, was greater in PPE group after peri-adventitial elastase application as shown by the arrows and decreased with doxycycline treatment. (E) Cleaved caspase-3 (Caspase). Caspase staining (10×) showed increased apoptosis from baseline after peri-adventitial elastase application as highlighted by the solid black arrows. (F) Quantified immunostaining for smooth muscle 22-α (SM22α), Smooth muscle α-Actin (SMαA), myosin heavy chain (MHC-SM1), and antigen Ki-67. Threshold-gated quantification of immunohistochemistry in aortic sections for smooth muscle cell markers were reduced among elastase administered animals. Cellular proliferation with marked increase in Ki-67 protein was noted with peri-adventitial elastase application. ** P < .01; ***P < .001.

Fig 5

(A) Gelatin zymography for MMP-2 and 9. Gelatin zymography was performed as described with Coomassie brilliant blue for visualization of lytic bands. Faint bands are seen in the control and doxycycline-treated animals, whereas PPE-treated animals had intense bands. Relative densitometry was completed on all groups. B, Relative densitometry of gelatin zymograms showed differences in protein expression between groups. Relative densitometry adjusting for background influence shows that PPE exposure increases pro-MPP9 expression, which is attenuated with doxycycline treatment (A). Similarly, cleaving of the MMP-9 protein to the active form was greater after PPE (B). Doxycycline treatment decreased active MMP-9 expression. (C, D) Although pro–MMP-2 expression is not different between groups, the active form of the protein is expressed in greater amounts after PPE, whereas doxycycline limited active MMP-2 expression. *P < .05; **P < .01.