Fig 1.
Myeloid-specific ablation of Tgfbr2 ameliorated aortic aneurysm in Fbn1C1039G/+ mice.
(A, B) Representative macroscopic appearance (A) and ultrasound images (B) of the thoracic aorta at 30 weeks of age in Tgfbr2 fl/fl, Tgfbr2MyeKO, Fbn1C1039G/+;Tgfbr2 fl/fl, and Fbn1 C1039G/+;Tgfbr2MyeKO mice. Scale bar, 1 mm. Arrows indicate aortic root (AoR); AAo, Ascending aorta. (C) Aortic root diameter measured by ultrasonography at 8, 12, and 30 weeks of age. n = 3–12 per group. Data are shown as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
Myeloid-specific ablation of Tgfbr2 reduced aortic medial thickening and elastic fiber fragmentation in Fbn1C1039G/+ mice.
(A) Representative images of Elastica van Gieson staining of the proximal ascending aorta at 30 weeks of age in Tgfbr2 fl/fl, Tgfbr2MyeKO, Fbn1C1039G/+;Tgfbr2 fl/fl, and Fbn1 C1039G/+;Tgfbr2MyeKO mice. Scale bar, 100 μm. (B) Average number of elastic fiber breaks per section. All mice were male. n = 4–7 per group. Data are shown as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3.
Myeloid-specific ablation of Tgfbr2 reduced macrophage infiltration into the aortic wall in Fbn1C1039G/+ mice.
(A) Representative images of F4/80 (marker of macrophage, red arrowheads) of the proximal ascending aorta at 30 weeks of age in Tgfbr2 fl/fl, Tgfbr2MyeKO, Fbn1C1039G/+;Tgfbr2 fl/fl, and Fbn1 C1039G/+;Tgfbr2MyeKO mice. Cell nuclei were counterstained with hematoxylin. All mice were male. Scale bar, 50 μm (20 μm in inset). (B) Average number of F4/80-positive macrophages per area of aortic adventitia. Four random 40× magnification fields were investigated. All mice were male. n = 4–7 per group. Data are shown as mean ± standard deviation. *p < 0.05, ***p < 0.001.
Fig 4.
Myeloid-specific ablation of Tgfbr2 attenuated phospho-Smad2/3 and phospho-ERK1/2 signals in Fbn1C1039G/+ mice.
(A, B) Representative phospho-Smad2/3 (A) and phospho-ERK1/2 (B) staining of the proximal ascending aorta at 30 weeks of age in Tgfbr2 fl/fl, Tgfbr2MyeKO, Fbn1C1039G/+;Tgfbr2 fl/fl, and Fbn1 C1039G/+;Tgfbr2MyeKO mice. Scale bar, 50 μm. Arrowheads show the representative positive staining. (C) Average percentage of positive staining of phospho-Smad2/3 per nuclei. Three random 20× magnification fields were investigated. All mice were male. n = 4–7 per group. Data are shown as mean ± standard deviation. *p < 0.05, **p < 0.01. (D) Average percentage of positive stained area of phospho-ERK1/2 per aortic area. Three random 40× magnification fields were investigated. All mice were male. n = 4–7 per group. Data are shown as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5.
Effects of TGF-β treatment on the proliferation and migration capacity of RAW264.7 macrophages.
(A) MTS cell proliferation assay. n = 6 per group. Data are shown as mean ± standard deviation. n.s, not significant. (B, C) In vitro cell migration assay. A monolayer of RAW264.7 cells was scratched with a P200 pipette tip, which induced migration of cells into the denuded area to close the wound in the presence or absence of TGF-β (5 ng/mL) for 24 hours. (B) Representative images obtained immediately after the scratch and after 24 hours. (C) The migration capacity was quantified from the area between the edges of cells in the scratch zone. n = 10 per group. Data are shown as mean ± standard deviation. ***p < 0.001.