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. Author manuscript; available in PMC: 2020 Jan 29.
Published in final edited form as: Circulation. 2019 Jan 29;139(5):679–693. doi: 10.1161/CIRCULATIONAHA.118.034615

Figure 3. Loss of LMO7 induced elevated expression of TGF-β1 and αvβ3 integrin, a latent TGF-β1 activator.

Figure 3

(A) qPCR analysis of Tgfb1 gene expression in medial layers of freshly isolated mouse aortas (n=4 independent experiments). (B) Mouse SMCs were treated with 0.5 ng/ml TGF-β1 for 24hrs and mRNA was harvested for qPCR analysis of Tgfb1 gene (n=4 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and TGF-β1 treatment on Tgfb1 mRNA expression but there was no significant interaction between Lmo7−/− and TGF-β1 treatment. (C) Lmo7+/+ or Lmo7−/− mouse SMCs with the same confluency were cultured in 0.5% FBS for 24 hrs. Conditioned media was harvested to quantitate total TGF-β secretion by ELISA. (n=4 independent experiments). (D) qPCR analysis of ECM genes from Lmo7+/+ or Lmo7−/− mouse SMCs treated with 40 μg/ml TGF-β neutralizing antibody (Clone #1D11) or Vehicle for 24 hrs (n=3 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and anti-TGF-β neutralizing antibody treatment on mRNA expression. LMO7 depletion resulted in a greater magnitude of reduction by neutralizing antibody treatment for Col1a1 mRNA (p=0.0022). (E) Immunostaining of ACTA2 (red), β3 Integrin (green) and αv Integrin (white) on tissue sections from control and Lmo7iΔSM mice 21 days after femoral artery injury. Scale bar=50 μm. Cells from each mouse were randomly picked and β3 and αv Integrin staining intensity was measured and normalized to cell area. Quantification is shown on right (n=4). (F) Mouse SMCs were treated with 0.5ng/ml TGF-β1 for 24hrs and mRNA was harvested for qPCR analysis of Itgav and Itgb3 genes (n=3 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and TGF-β1 treatment on mRNA expression, but there was no significant interaction between Lmo7−/− and TGF-β1 treatment. (G, H) Mouse SMCs were treated with 20 μM Cilengitide or Vehicle for 24hrs and cell lysates were harvested for (G) western analysis of SMAD3 phosphorylation (n=3 independent experiments) or (H) qPCR analysis of ECM and Tgfb1 genes (n=3 independent experiments). Hprt1 was used as a negative control in qPCR analysis. Two-way ANOVA revealed a significant effect of LMO7 depletion and Cilengitide treatment on SMAD3 phosphorylation and mRNA expression, and the magnitude of reduction was greater with Cilengitide treatment in Lmo7−/− SMCs for all the readouts except Tgfb1 mRNA (SMAD3 phosphorylation p=0.013, Col1a1 p=0.030, Col1a2 p=0.0098, Col3a1 p=0.0001, Fn1 p=0.015), indicating LMO7 regulates TGF-β activity as well as Tgfb1 and ECM gene expression by modulating αvβ3 Integrin activity. Data are expressed as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001