Angiotensin II Receptor Antagonism Reduces Transforming Growth Factor Beta and Smad Signaling in Thoracic Aortic Aneurysm

Abstract

Background: The expression of transforming growth factor beta (TGF-β) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-β receptor-dependent and -independent pathways.

Methods: To determine the expression of AngII type 1 (AT1R) and type 2 receptors (AT2R), TGF-β, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors.

Results: MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-β and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-β and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-β expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-β was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT1R and AT2R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT1R antagonist losartan caused both reduced TGF-β vesicle localization and nuclear expression of Smad3.

Conclusions: Increased TGF-β and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-β expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-β expression and the cytoplasmic localization of Smad3 support a role for AT1R antagonism in the inhibition of aneurysm progression.

Keywords: Aneurysm; Smad3 protein; aorta; immunohistochemistry; transforming growth factor beta; vascular smooth muscle.

Figure 1.

Increased accumulation of Smad3 (A-C) and transforming growth factor beta (TGF-β) (D-F) observed in the area of pathological remodeling/focal degeneration in the aortic wall of Marfan syndrome (MFS) (B, E) and bicuspid aortic valve (BAV) (C, F) aneurysms. Positive expression of Smad3 and TGF-β is identified as areas of dark brown color (arrows). The control aortic wall (A, D) showed weak to negligible expression (hematoxylin;magnification 250×).

Figure 2.

Increased expression (identified as dark brown color) of angiotensin II type 1 (AT₁R) and type 2 receptors (AT₂R) was observed in vascular smooth muscle cells of Marfan syndrome (MFS) (B, E) and bicuspid aortic valve (BAV) (C, F) aortic media compared to control aortic media (A, D) (hematoxylin;magnification 250×).

Figure 3.

Smad3 expression is identified as dark brown areas of color in cultured aortic vascular smooth muscle cells (VSMCs). Nuclear accumulation is shown in Marfan syndrome (MFS) and bicuspid aortic valve (BAV) VSMCs (open arrows; B, C). Lack of Smad3 nuclear expression in the control (A) is indicated with arrows. Losartan treatment of MFS and BAV VSMCs reduced nuclear accumulation of nuclear Smad3 (E, F) (hematoxylin;magnification 250×).

Figure 4.

Transforming growth factor beta (TGF-β) expression (brown color) in cultured aortic vascular smooth muscle cells (VSMCs). Increased expression of TGF-β is shown in VSMCs from Marfan syndrome (MFS) and bicuspid aortic valve (BAV) samples where vesicular localization is also increased (B, C). Losartan treatment resulted in decreased expression and diffuse localization of intracellular TGF-β (E, F) similar to control VSMCs (A, D) (hematoxylin;magnification 500×).

Figure 5.

Angiotensin II type 1 receptor (AT₁R) expression (brown color) in cultured aortic vascular smooth muscle cells (VSMCs). Increased expression of AT₁R in cultured Marfan syndrome (MFS) and bicuspid aortic valve (BAV) VSMCs (B, C) was found. Losartan treatment reduced AT₁R in cultured MFS and BAV VSMCs (E, F) to the same level as losartan-treated control VSMCs (D) (hematoxylin;magnification 500×).