Tannic acid as a plant-derived polyphenol exerts vasoprotection via enhancing KLF2 expression in endothelial cells

Abstract

The transcription factor Kruppel-like factor 2 (KLF2) is a critical anti-inflammatory and anti-atherogenic molecule in vascular endothelium. Enhancing KLF2 expression and activity improves endothelial function and prevents atherosclerosis. However, the pharmacological and molecular regulators for KLF2 are scarce. Using high-throughput luciferase reporter assay to screen for KLF2 activators, we have identified tannic acid (TA), a polyphenolic compound, as a potent KLF2 activator that attenuates endothelial inflammation. Mechanistic studies suggested that TA induced KLF2 expression in part through the ERK5/MEF2 pathway. Functionally, TA markedly decreased monocyte adhesion to ECs by reducing expression of adhesion molecule VCAM1. Using lung ECs isolated from Klf2 ^+/+ and Klf2 ^+/- mice, we showed that the anti-inflammatory effect of TA is dependent on KLF2. Collectively, our results demonstrate that TA is a potent KLF2 activator and TA attenuated endothelial inflammation through upregulation of KLF2. Our findings provide a novel mechanism for the well-established beneficial cardiovascular effects of TA and suggest that KLF2 could be a novel therapeutic target for atherosclerotic vascular disease.

Figure 1

Identification of TA as a KLF2 activator. (A) Chemical structure of the TA. (B) KLF2 luciferase activity was analyzed in COS-7 cells transfected with KLF2-luciferase plasmids treatment with DMSO (vehicle), TA (0.1, 1.0, 10 and 20 μM) or simvastatin (1.0 μM). **P < 0.01, Student’s t test. Values represent mean ± SEM; n = 4.

Figure 2

TA induced KLF2 expression in HCAECs. (A,B) HCAEC cells were treated with DMSO, TA (0.1, 1, 10 and 20 μM), or simvastatin (1.0 μM) for 24 h, then KLF2 (A) and ET-1 (B) mRNA expression was detected by qPCR. **P < 0.01, *P < 0.05 (TA or vs vehicle), Student’s t test. Values represent mean ± SEM; n = 4.

Figure 3

The ERK5/MEF2 pathway is involved in TA-induces KLF2 expression. (A) COS-7 cells were transfected with KLF2 -221 WT or KLF2 -221 mutant plasmids, treated with TA (10.0 µM), DMSO (vehicle) or simvastatin (1.0 µM, positive control) for 24 h, and luciferase activities were then detected. *P < 0.05. n = 3. (B–D) HCAECs were pretreated with TA (10.0 µM) for 0, 5, 10, 15, 30 and 60 min, respectively, then protein expression of phospho-ERK5, ERK5 and GAPDH were determined by Western blotting. *P < 0.05. n = 3.

Figure 4

TA attenuated monocyte adhesion to ECs. (A,B) HCAECs were pretreated with vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (A) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (B) THP-1 cells in panel A were counted and statistically analyzed. *p < 0.05, **p < 0.01, n = 4. (C–E) HUVECs were transfected with siRNA (control siRNA) or siKLF2 (KLF2 siRNA) for 48 h. Then the cells were pretreated vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (C) The levels of KLF2 mRNA in ECs treated with control siRNA and KLF2 siRNA were analyzed by q-PCR. (D) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (E) THP-1 cells in panel C were counted and statistically analyzed. One way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, n = 3.

Figure 5

TA decreased TNFα-stimulated inflammatory response in HCAECs. (A) HCAECs were treated as described in Fig. 3 except for the exposure of TNFα for 3 h, then mRNA expression of VCAM1 and ICAM1 were determined by qPCR. Values are mean ± SEM, *p < 0.05, **p < 0.01, n = 5.B.HCAECs were treated as described in Fig. 4, then protein expression of VCAM1 and ICAM1 were determined by Western blot. (C) Quantification of panel B, values are mean ± SEM, *p < 0.05, **p < 0.01, n = 3.

Figure 6

TA 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.

Figure 7

A working model depicting TA-mediated vasoprotective effects via a KLF2-dependent mechanism. Kruppel-like factor 2 (KLF2), tannic acid (TA), myocyte enhancing factor 2 (MEF2), extracellular-signal related kinase 5 (ERK5), phosphorylation (p-ERK5), vascular cell adhesion molecule 1 (VCAM1), endothelial cells (ECs), tumor necrosis factor alpha (TNFα).