In the present study, we have documented that external elastase application to the infrarenal aortic results in the ability to generate AAAs in two strains of mice, and equally in male and female mice. As opposed to previous work, the ME B6129 mice formed larger aneurysms than their C57BL/6 counterparts. In this model the AAA phenotype (as determined by aortic diameter and disrupted elastic laminae by van gieson stain) occurs in association with an increase in Mac-2 stained cells in both the male and female C57BL/6 animals. However, the B6129 females did not have a significant increase in Mac-2 cells after elastase application, and B6129 ME had a muted increase as compared with C57BL/6 ME. CD+ lymphocytes were greater in C57 ME than FE, while PMNs were not found to be different. MMP9 activity was significantly higher in C57BL/6s than B6129s, but still significantly greater in treated vs. control groups in males and females and in both strains. However, unlike Mac-2 counts, MMP9 activity was equivalent between genders within the strains. MMP2 activity was less elucidating, with no clear significant pattern except C57BL/6 ME with greater activity than B6129 ME. The TIMPs also did not show any differences based on gender. These data suggest that this model therefore is good for inducing AAAs in a wider range of mouse strains, but may not be useable for helping us to understand gender differences. Further studies are needed to see if there are other methods of distinguishing the response of males versus females when the phenotype and proteinase activity are equivalent, but macrophages and lymphocytes show gender differences.
Presently, there are multiple methods for producing experimental in vivo AAAs, including topical calcium chloride, subcutaneous angiotensin II infusion, and elastase intraluminal perfusion. Each of these have advantages and disadvantages, as summarized in . However, none of these models completely recapitulate human disease. In addition, there have been knockout mice strains developed to produce AAAs, such as the Lox −/−, MMP3 −/−, and TIMP-1 −/−. These animals are either not hardy or have aortic wall destruction that is too generalized, and have other unknown phenotypic changes making translation to humans potentially problematic. An ideal model for experimental AAA development should replicate the shear stresses, hemodynamic forces, and perianeurysmal environment present in humans. While all of these animal models attempt to reproduce human disease with fidelity, it is difficult as information regarding early stage AAA development is lacking. External elastase application represents a novel, reliable and easily reproducible model for producing infrarenal AAAs. It produces the desired effect of destruction of elastic tissue within the adventitia and media, while eliminating the physical component of dilatation of the intra-luminal aorta that is needed in the standard elastase perfusion AAA model.
Descriptive characteristics of different AAA animal models
There are many different interrelated processes implicated in the development of AAAs, including chronic inflammation in the aortic adventitia, neovascularization, production and early recruitment of proinflammatory cytokines, excessive local production and dysregulation of MMPs leading to destruction of collagen, medial elastic lamellae, and other structural matrix proteins and depletion of medial SMCs. This leads to progressive weakening and dilatation of the aortic wall, partially due to an impaired capacity for connective tissue repair. Genetically altered mice have helped establish temporal expression of cytokines and hierarchy of inflammatory responses in AAA development. Thompson et al. reviewed the studies that were done in genetically altered mice in order to elucidate the roles of different molecular mediators, chemokines, proinflammatory cytokines, and cellular signaling molecules1
. With all of these processes involved, there are many possibilities for why different strains have different results in all models. Our data suggest the development of AAAs in B6129 mice may be through a different mechanism as compared with the C57BL/6 mice since the MEs have fewer Mac-2 positive cells and MMP9 activity, yet equal or greater AAA phenotype with C57BL/6 ME. Thompson summarized the fact that different wild type strains have different susceptibilities toward forming aneurysms as “consistent with an inherited, genetically determined susceptibility to the development of elastase-induced AAAs”1
. The differences documented in this study between C57BL/6 and B6129 could reflect that susceptibility is at least somewhat associated with genetically determined inheritable traits.
The gender disparities seen in AAA formation may also be due to genetic differences. Our lab has done many studies elucidating the mechanisms behind the gender differences in AAA formation. Female rats after elastase perfusion were found to exhibit an early five-fold decrease in proinflammatory TNFα superfamily ligands. Also, females had fourfold lower expression of TGFβ and VEGF families, possible mediators of AAA development5
. Interleukins, CC chemokine receptors, and CC ligand families were too low to be detected as compared with males5
, again indicating possible mechanisms for less inflammation in female aortas. Another recent study from our lab correlatively documented decreased collagen types I and III along with increase in MMP-13 after elastase perfusion in male rats compared with females2
Many studies have looked at the estrogen receptor as having a role in protecting females from cardiovascular disease, including AAAs. It is now known that there are multiple estrogen receptors with possible overlapping and counter-acting functions. The effects are complex and involve eNOS, increased nitric oxide, G-protein pathways, and transcription regulated genomic changes. This complexity was witnessed when previously in our lab estrogen and testosterone were given to male and female rats before elastase perfusion. Males given estrogen experienced decreased aortic diameter, but oophorectomized females did not have increased aortic diameters4
. The exact mechanisms for the elastase perfusion model maintaining a gender difference, but this topical elastase model forming AAA in both genders is not clear; the female protection may be largely overwhelmed by the topical method. An AAA phenotype may therefore be manifested in females, despite the expected decrease in Mac-2 cells, secondary to an altered inflammatory response.
Future studies include using the model to analyze IL-6, IL-1β, TNFα, estrogen receptors, and EDPs (elastin-degradation peptides). These studies will hopefully serve to shed light on the pathogenesis behind the differences seen between strains and genders in AAA development.