Figure 1.
Inflammatory cells in the aortic vessel wall.
Immunohistochemical staining (positive area/total aortic wall area) for leukocytes (A; CD45) and macrophages (B; Mac3) revealed that placebo-treated Marfan mice contained significantly more leukocytes and macrophages in the aortic wall as compared to wildtype mice. Losartan significantly reduced both leukocyte and macrophage influx. While methylprednisolone revealed a trend in decreased leukocytes (* p = 0.050), abatacept did not. Yet, all drugs significantly decreased the macrophage influx. Each group of mice comprises 11 mice, except Marfan placebo with n = 12, with equal male/female distribution.
Figure 2.
Aortic wall thickness, elastin breaks and GAG accumulation.
A) The area of the aortic media of placebo-treated Marfan mice was significantly thickened compared to wall thickness in wildtype mice. Methylprednisolone showed a trend towards enhanced thickening of the aortic media in Marfan mice (* p = 0.066). B) There were significantly more elastic lamina breaks in the aortic wall of Marfan mice compared to wildtype mice. Methylprednisolone revealed a trend towards enhanced elastic lamina breaks in the aortic media in Marfan mice (* p = 0.076). C) There was enhanced alcian blue positive area in the aortic media of methylprednisolone-treated mice, as compared to Marfan placebo mice, as a marker for medial necrosis. Abatacept showed a trend towards increased GAG accumulation as visualized by alcian blue (* p = 0.066). D) Alcian blue staining (blue) is present in the media (black line) in placebo-treated Marfan mice, yet it is more pronounced in the Methylprednisolone-treated aortic root. Pink stain = cytoplasm, red dots = nuclei, A = adventitia, L = lumen.
Figure 3.
Aortic dilatation in Marfan mice reduced by losartan.
The aortic root dilatation rate was determined. Placebo-treated Marfan mice had a significantly higher dilatation rate compared to wildtype mice. Losartan attenuated the aortic root dilatation rate in Marfan mice significantly, whereas the other treatment strategies did not change the aortic root dilatation rate compared to placebo-treated Marfan mice.
Figure 4.
A) Phosphorylation of Smad2 (pSmad2) and localization in the nucleus of vascular cells in the aortic wall (positive area/total aortic wall area) is expressed in arbitrary units (AU). pSmad2 was significantly reduced by losartan treatment, as compared to placebo-treated Marfan mice. The other anti-inflammatory drugs did not affect the number of pSmad2-positive nuclei. B) An example of pSmad2 staining in placebo-treated Marfan mice and reduced pSmad2 in losartan-treated Marfan mice. A = adventitia, L = lumen, line indicates media.
Figure 5.
Losartan is currently the only drug that effectively inhibits aortic root dilatation in mice and men, and specifically targets the angiotensin-II receptor type 1. Losartan clearly decreases TGF-β/pSmad2 signaling, decreases total leukocyte and macrophage influx into the vessel wall, and diminishes aortic root dilatation. TGF-β is known to polarize macrophages into a repair phenotype and at the same time induces collagen synthesis and matrix metalloproteinase activity to degrade extracellular matrix proteins (ECM). Methylprednisolone and abatacept decreased macrophage influx significantly, which resulted in increased GAG accumulation in the aortic vessel wall, thus disturbing ECM homeostasis, which may be potentially harmful.