Figure 2. TGF-β signaling and ECM gene expression are augmented in LMO7 deficient SMCs.

(A, B) Immunostaining of (A) p-SMAD3 (green) or ACTA2 (red) or (B) TGF- β or CTGF in tissue sections from control and Lmo7^iΔSM mice after carotid artery ligation. (C) CTGF Immunohistochemistry of arterial sections of control and Lmo7^iΔSM mice after carotid artery ligation for durations as indicated. (D) Western analysis of Lmo7^+/+ or Lmo7^−/− mouse SMCs treated with TGF-β1 at indicated doses for 24 hrs. Quantification of SMAD3 phosphorylation is shown on right (n=6 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and TGF-β1 dose effect on p-SMAD3 phosphorylation, but there was no significant interaction between Lmo7^−/− and TGF-β1 treatment. (E) Lmo7^+/+ or Lmo7^−/− mice were subjected to carotid ligation and injected intraperitoneally with SB4341542 (10mg/kg/d) or Vehicle (1% DMSO/30% polyethylene/ 1% Tween 80) daily from days 7–28 post-ligation. Trichrome staining of cross-sections of injured arteries is shown. (F) qPCR analysis of ECM genes in Lmo7^+/+ or Lmo7^−/− mouse SMCs treated with 10 μM SB431542 or Vehicle for 24 hrs (n=5 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and TGF-β receptor inhibition on mRNA expression. For Col1a1, Col1a2 and Col3a1 mRNA, the magnitude of reduction was greater in Lmo7^−/− SMCs (Col1a1 p=0.0057, Col1a2 p=0.019, Col3a1 p=0.0022), indicating LMO7 regulates many ECM genes via TGF-β signaling. Scale bar=50 μm. Data are expressed as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001