Next, we asked whether the anti-inflammatory effect of TA is KLF2-dependent. Because systemic KLF2 knockout was embryonically lethal2, Klf2 +/− mice were used to determine KLF2-dependency of TA. Lung ECs from Klf2 +/− and Klf2 +/+ mice were isolated and then stimulated with TNFα in the presence or absence of TA. As shown in Fig. 6, KLF2 expression in lung ECs from Klf2 +/− mice was significantly decreased compared that from Klf2 +/+ mice (Fig. 6A). VCAM1 mRNA and protein expression was greatly induced by TNFα in Klf2 +/− and Klf2 +/+ mouse lung ECs (Fig. 6B,C). TA significantly decreased VCAM1 mRNA and protein expression in KLF2+/+ mouse lung ECs. However, in lung ECs from Klf2 +/− mice, the inhibitory effects of TA on TNFα-induced VCAM1 expression was partially reversed (Fig. 6B,C). These data demonstrate that the anti-inflammatory effect of TA was KLF2-dependent.Figure 6TA inhibited vascular inflammation via KLF2. Mouse lung ECs were isolated from Klf2 +/+ or Klf2 +/− mice as described in methods. Pooled lung ECs from 3 mice with identical genotype were included in each group. (A) The levels of KLF2 mRNA expression in isolated lung endothelial cells from Klf2 +/+ and Klf2 +/− mice were analyzed by qPCR. (B) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 3 h. qPCR was performed to detect VCAM1 mRNA expression. Statistical comparisons and analyses between 2 groups were performed by 2-tailed, paired Student’s t test. *P < 0.05, **P < 0.01, n = 5. (C) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 6 h. Western blot assays were performed to examine VCAM1 and GAPDH protein expression.