Proinflammatory Role of Angiotensin II in the Aorta of Normotensive Mice

Abstract

Angiotensin II plays important functions in cardiovascular system mediating actions leading to inflammatory responses such as activation of VSMC in order to produce ROS, inflammatory cytokines, chemokines, and adhesion molecules. Changes in angiotensin II production could stimulate the recruitment and activation of myeloid cells initiating local inflammatory response without effect on BP. We aimed to verify if angiotensin II induces an inflammatory response in the aorta and if it correlates with variations in BP. C57Bl/6 mice treated with saline solution (0.9%, control group) or angiotensin II (30ng/kg, Ang II group) were used. BP and HR levels were measured. Immunohistochemistry for IL1-β, TGF-β, iNOS, CD45, and α-actin was performed in the aorta. BP and HR do not change. A biphasic response was observed both for IL1-β and TGF-β expression and also for the presence of CD45 positive cells, with an acute increase (between 30 and 60 minutes) and a second increase, between 24 and 48 hours. Positive staining for iNOS increased in the earlier period (30 minutes) in perivascular adipose tissue and in a longer period (48 hours) in tunica adventitia. Immunoblotting to α-actin showed no alterations, suggesting that the applied dose of angiotensin II does not alter the aortic VSMCs phenotype. The results suggest that angiotensin II, even at doses that do not alter BP, induces the expression of inflammatory markers and migration of inflammatory cells into the aorta of normotensive mice. Thus, angiotensin II may increase the propensity to develop a cardiovascular injury, even in normotensive individuals.

Figure 1

Hemodynamic evaluation showing mean values for dyastolic, systolic, and mean arterial pressure, as well as heart rate. The subpressor dose of angiotensin II did not change any of the evaluated parameters. N=7/group.

Figure 2

Immunostaining for inflammatory markers in aorta of mice. Positive staining for IL1-β increased in earlier period (30 minutes) in tunica adventitia and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media, tunica adventitia, and PVAT after angiotensin II injection. Angiotensin II increased immunostaining for TGF-β in acute and late periods in tunica media and later in tunica adventitia. iNOS immunostaining increased in adventitia 48 hours after Ang II injection, while in PVAT increased earlier, after 30 minutes. N=7/group. ∗≤0.05; ∗∗≤0.01; ∗∗∗≤0.001 compared to control.

Figure 3

Photomicrography showing immunostaining for IL1-β in aorta of mice. Positive staining for IL1-β (arrows) increased in earlier period (30 minutes) in tunica adventitia (TA) and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media (TM), tunica adventitia and PVAT after angiotensin II injection (Magnification: 40x).

Figure 4

Photomicrography showing immunostaining for TGF-β in aorta of mice. Angiotensin II increased immunostaining for TGF-β (arrows) in acute and late periods in tunica media and later in tunica adventitia (Magnification: 40x).

Figure 5

Photomicrography showing immunostaining for iNOS in aorta of mice. iNOS immunostaining (arrows) increased in adventitia 48 hours after Ang II injection, while in PVAT increased earlier, after 30 minutes (Magnification: 40x).

Figure 6

Macrophage counting in aorta of mice. The subpressor dose of angiotensin II (30ng/kg) was able to induce acute macrophage migration, after 30min in perivascular adipose tissue. The injection of angiotensin II was also able to increase macrophage density in adventitia in two different moments after 1 hour and 24 hours. N=7/group. ∗≤0.05 compared to control.

Figure 7

Photomicrography showing immunostaining for CD45 in aorta of mice. The subpressor dose of angiotensin II (30ng/kg) was able to induce acute macrophage migration (arrow), after 30min in perivascular adipose tissue (PVAT). The injection of angiotensin II was also able to increase macrophage density in tunica adventitia (TA) in two different moments after 1 hour and 24 hours (Magnification: 40x).

Figure 8

Immunostaining for α-actin to study contractile phenotype of vascular smooth muscle cells in tunica media. The analysis showed a slight reduction (not significant) of positive staining in mice injected with angiotensin II, after 48 hours. N=7/group.

Figure 9

Photomicrography showing immunostaining for α-actin to study contractile phenotype of vascular smooth muscle cells in tunica media. There was a slight reduction (not significant) of positive staining in mice injected with angiotensin II, after 48 hours (Magnification: 40x).

Figure 10

Proinflammatory responses of angiotensin II in aorta of mice. In the presented scheme, Ang II induces a fast macrophage migration to PVAT, possibly increasing iNOS and IL1-β expression. A secondary macrophage migration to tunica media may be related to a late increase of iNOS and IL1-β expression. On the other hand, TGF-β quickly increases in tunica media. The reason why the late expression of TGF-β in adventitia and PVAT could be influenced by earlier TGF-β expression or by macrophages infiltration needs to be investigated.