During the past few years several studies have addressed
During the past few years, several studies have addressed macrophage phenotype in experimental models, such as angiotensin II-induced AAA. A first study investigated macrophage phenotype using flow cytometry to identify M1 and M2 macrophages and qPCR analysis for expression of NOS2 and Ym1/2. The authors found an increase of M1/M2 ratio in the suprarenal TDZD-8 of ApoE mice from 7 days after angiotensin II infusion, and this effect was maintained during the 28 days of the experiment. A second study revealed that infusion of angiotensin II for an additional 56 days led to AAA expansion and rupture, which was associated with an increase of macrophage infiltration in the AAA wall. The authors used CD68 and MOMA2 immunostaining to identify macrophages and found that the expression of the M2 marker CD206 in the AAA wall was more commonly found than the M1 marker NOS2, suggesting that advanced aneurysm may be associated with a shift toward M2 polarization. Given the role of macrophages in tissue healing, it has been hypothesized that this shift toward M2 polarization might result from a compensatory mechanism to prevent AAA expansion. The mechanisms underlying macrophage polarization during AAA are not completely understood. A recent study pointed out the role of SIRT1, a member of the class III deacetylase family. In this study, global deletion and macrophage-specific deletion of SIRT1 exacerbated the severity of AAA induced by angiotensin II perfusion. Interestingly, this was associated with an increase of NOS2 and a decrease of ARG1 in the aorta, suggesting the role of SIRT1 into a shift toward M2 polarization. In our study, analysis of gene expression in the infrarenal abdominal aorta revealed an increase of M1 markers (CCL2, NOS2, CCL5), whereas M2 markers were increased (ARG1, Ym1/2) or decreased (FIZZ1, MGL2) during the different time points of aneurysm development in elastase and elastase + anti-TGF-β models. The expression of M1 and M2 markers over time is variable, underlining the interest in performing kinetic experiments when characterizing macrophage phenotype. Note that M1 and M2 markers are not cell specific, and for some of them, macrophages do not mainly account for their expression in the aneurysmal wall. In addition, clodronate depletes circulating monocytes and aortic monocyte-derived macrophages but does not affect vascular-associated resident macrophages. This could explain, at least partly, why the expression of the M1 markers CCL2, NOS2, and CCL5 and the M2 markers including FIZZ1 and MGL2 did not significantly vary after injection of clodronate liposomes. Our results indicate that TGF-β blockade in elastase-induced AAA does not strictly induce a shift toward M1 or M2 polarization but rather modulates macrophage phenotype into a complex spectrum of activation. The strict paradigm of M1/M2 activation has been revisited recently, highlighting a more complex and broader repertoire for macrophages.8, 22 Even if the angiotensin II perfusion model reproduces the main features of human aneurysm including ECM degradation, leukocyte extravasation, and concomitant presence of atherosclerosis, it is associated with medial dissection and intramural hematoma. In addition, the AAA usually develops in the suprarenal or the ascending aorta, whereas the most frequent localization in humans involves the infrarenal aortic region. The topical application of elastase associated with the systemic neutralization of TGF-β has the advantage of inducing AAA in the infrarenal region of the abdominal aorta and is associated with a high percentage of AAA rupture within 14 days. This model produces features of human-like AAA, including ECM degradation, immune cell accumulation in the aorta, loss of VSMCs, intraluminal thrombus formation, and adventitial neoangiogenesis. In this model, AAA development was precisely characterized by serial in vivo ultrasound imaging and synchrotron-based ultrahigh-resolution ex vivo imaging at different time points (day 0, 7, and 14 for ultrasound and day 0, 3, 5, 7, 10, and 14 for synchrotron imaging). Ultrasound analysis revealed that at day 7, the aortic dilation was increased in both the elastase and elastase + anti-TGF-β groups compared with controls. Between day 7 and day 14, the aortic dilation significantly progressed in the elastase + anti-TGF-β group but not in the elastase alone group. Hence, differences between these two groups were more marked between day 7 and day 14. In addition, synchrotron-based ultrahigh-resolution ex vivo imaging in the elastase + anti-TGF-β group revealed more marked changes in aortic perimeters and adventitial thickness after day 7. Based on these findings, we hypothesized that changes of macrophage phenotype in the aortic tissue would be preferentially observed from day 7 to day 14. Note, however, that whereas the model offers the opportunity to study aortic rupture, the survival rate of mice receiving elastase + anti-TGF-β is estimated at between 50% and 80% at day 14. Analysis performed at day 14 may thus potentially lead to select animals that better resist rupture, underlining the real interest in performing kinetic experiments and analysis at different time points in this model.