Resveratrol attenuates ICAM-1 expression and monocyte adhesiveness to TNF-α-treated endothelial cells: evidence for an anti-inflammatory cascade mediated by the miR-221/222/AMPK/p38/NF-κB pathway

Abstract

Resveratrol, an edible polyphenolic phytoalexin, improves endothelial dysfunction and attenuates inflammation. However, the mechanisms have not been thoroughly elucidated. Therefore, we investigated the molecular basis of the effects of resveratrol on TNF-α-induced ICAM-1 expression in HUVECs. The resveratrol treatment significantly attenuated the TNF-α-induced ICAM-1 expression. The inhibition of p38 phosphorylation mediated the reduction in ICAM-1 expression caused by resveratrol. Resveratrol also decreased TNF-α-induced IκB phosphorylation and the phosphorylation, acetylation, and translocation of NF-κB p65. Moreover, resveratrol induced the AMPK phosphorylation and the SIRT1 expression in TNF-α-treated HUVECs. Furthermore, TNF-α significantly suppressed miR-221/-222 expression, which was reversed by resveratrol. miR-221/-222 overexpression decreased p38/NF-κB and ICAM-1 expression, which resulted in reduced monocyte adhesion to TNF-α-treated ECs. In a mouse model of acute TNF-α-induced inflammation, resveratrol effectively attenuated ICAM-1 expression in the aortic ECs of TNF-α-treated wild-type mice. These beneficial effects of resveratrol were lost in miR-221/222 knockout mice. Our data showed that resveratrol counteracted the TNF-α-mediated reduction in miR-221/222 expression and decreased the TNF-α-induced activation of p38 MAPK and NF-κB, thereby suppressing ICAM-1 expression and monocyte adhesion. Collectively, our results show that resveratrol attenuates endothelial inflammation by reducing ICAM-1 expression and that the protective effect was mediated partly through the miR-221/222/AMPK/p38/NF-κB pathway.

Figure 1. Resveratrol-mediated reduction in ICAM-1 mRNA and protein levels in TNF-α-treated HUVECs.

(A) Treatment of HUVECs with different concentrations of resveratrol (Res); cell viability was assessed using the MTT assay. *P < 0.05 compared to untreated cells. (B) HUVECs were incubated with the indicated concentrations of resveratrol for 24 h and then with 3 ng/mL TNF-α for 4 h in the continued presence of resveratrol; ICAM-1 protein in cell lysates was then measured by Western blot. GAPDH was used as the loading control. The data are expressed as a fold value compared to the control value and are shown as the mean ± SEM for five separate experiments. Blots are cropped for clarity; full blots are shown in the Supplementary file. (C) HUVECs were incubated for 24 h with 50 μM of resveratrol (R); then, the cells were incubated with 3 ng/mL of TNF-α (T) for 4 h. ICAM-1 expression was analyzed by immunofluorescence staining. Bar = 100 μm. (D) Analysis of ICAM-1 mRNA levels in untreated HUVECs or HUVECs preincubated with or without 50 μM resveratrol for 24 h and then incubated with 3 ng/mL TNF-α for 4 h. Total RNA was analyzed by RT-PCR after normalization to 18S levels. (E) Representative fluorescence images showing the effects of resveratrol on the TNF-α-induced adhesion of fluorescein-labeled U937 cells to HUVECs. Cells were left untreated or were pretreated for 24 h with 50 μM resveratrol or for 1 h with 1 or 2 μg/mL anti-ICAM antibodies; then, they were treated with 3 ng/mL TNF-α for 4 h. BCECF-AM-labeled U937 cells were added to HUVECs and incubated at 37 °C for 45 min. The adherent cells were imaged by fluorescence microscopy. Bar = 100 μm. (F) The number of U937 cells bound per high power field in six randomly selected images was counted. The data are expressed as the mean ± SEM of three separate experiments. *P < 0.05 compared with the untreated cells. ^†P < 0.05 compared with the TNF-α-treated cells.

Figure 2. The resveratrol-mediated reduction in TNF-α-induced ICAM-1 expression is partly dependent on the inhibition of p38 phosphorylation.

(A–C) The effects of resveratrol treatment on the phosphorylation of ERK1/2, p38, or JNK in TNF-α-treated HUVECs. HUVECs were incubated for 24 h with or without 50 μM resveratrol; then, the cells were incubated with 3 ng/mL of TNF-α for the indicated time, and aliquots of cell lysates containing equal amounts of protein subjected to immunoblotting with antibodies against (A) p-ERK1/2 and t-ERK1/2, (B) p-p38 and t-p38, (C) or p-JNK and t-JNK. (D–F) HUVECs were incubated for 23 h with or without 50 μM resveratrol, with or without the subsequent addition of the indicated concentrations of (D) PD98059 (an ERK1/2 inhibitor), (E) SB203580 (a p38 inhibitor), or (F) SP600125 (a JNK inhibitor) for 1 h in the continued presence of resveratrol. This was followed by incubation with or without TNF-α for 4 h, and then the cell lysates were analyzed for ICAM-1 expression by Western blot. The data are expressed as a fold of the control value and are shown as the mean ± SEM of three separate experiments. GAPDH was used as the loading control. Blots are cropped for clarity; full blots are shown in the Supplementary file. (G) Representative fluorescence images showing the effects of MAPK inhibitors on the TNF-α-induced adhesion of fluorescein-labeled U937 cells to HUVECs. Cells were left untreated or were pretreated for 1 h with PD98059 (30 μM), SB203580 (30 μM), or SP600125 (30 μM). Then, they were treated with 3 ng/mL TNF-α for 4 h in the continued presence of the inhibitor. BCECF-AM-labeled U937 cells were added to HUVECs and incubated at 37 °C for 45 min. The adherent cells were imaged by fluorescence microscopy. Bar = 100 μm. The number of U937 cells bound per high power field in six randomly selected images was counted. The data are expressed as the mean ± SEM of three separate experiments. *P < 0.05 compared with the untreated cells. ^†P < 0.05 compared with the TNF-α-treated cells.

Figure 3. Resveratrol decreases TNF-α-induced IκB phosphorylation and the phosphorylation, acetylation, and translocation of NF-κB p65 in HUVECs.

(A,B) Western blot analysis for the phosphorylation of IκB (A) and NF-κB p65 (B). HUVECs were preincubated for 24 h with 50 μM resveratrol and then were treated with 3 ng/mL TNF-α for the indicated time. (C) Cells were co-incubated for 24 h with 0–10 μM Bay117082 (an NF-κB inhibitor) and then with 3 ng/mL TNF-α. Cell lysates were prepared and assayed for ICAM-1 by Western blot. (D) Nuclear extracts were prepared from untreated cells or from cells with or without the 24 h pretreatment with 50 μM resveratrol and incubation with 3 ng/mL TNF-α for 1 h; these extracts were then tested for NF-κB DNA binding activity by EMSA. (E) Immunofluorescence staining for NF-κB p65. HUVECs were preincubated for 24 h with 50 μM resveratrol and were then treated with 3 ng/ml TNF-α for 4 h. Representative results from three separate experiments are shown. (F) Western blot for NF-κB p65 expression in nuclear extracts. (G) Western blot for the acetylation of NF-κB p65. HUVECs were preincubated for 24 h with 50 μM resveratrol and were then treated with 3 ng/mL TNF-α for the indicated time. The data are expressed as a fold of the control value and are shown as the mean ± SEM of three separate experiments. GAPDH and t-p65 were used as loading control. (H) The effect of Bay 117082 on U937 cells adhered to TNF-α-treated HUVECs. (I–L) HUVECs were incubated for 23 h with or without 50 μM resveratrol, and then with or without the indicated concentrations of PD98059 (I), SB203580 (J), or SP600125 (K) for 1 h in the continued presence of resveratrol. This was followed by incubation with or without TNF-α for 5 min, and then the cell lysates were analyzed for the phosphorylation of IκB using Western blot. The data are expressed as the means ± SEM for three separate experiments. *P < 0.05 compared with the untreated cells. ^†P < 0.05 compared with the TNF-α-treated cells. Blots are cropped for clarity; full blots are shown in the Supplementary file.

Figure 4. Resveratrol-mediated decreases in TNF-α-induced ICAM-1 expression and partly dependent on AMPK phosphorylation.

(A) Representative immunoblot analysis of the time-dependence of the resveratrol-mediated phosphorylation of AMPK (p-AMPK) in HUVECs. The density of the p-AMPK bands was quantified and normalized to the protein loading control GAPDH, and the mean ± SEM are shown (n = 3). (B) Immunoblot analysis of the time-dependence of AICAR (an AMPK activator)-mediated AMPK phosphorylation. (C) Western blot analysis of the p-AMPK expression. HUVECs were preincubated for 24 h with 50 μM resveratrol and were then treated with 3 ng/mL TNF-α for 4 h. (D) Western blot analysis of ICAM-1 expression. HUVECs were incubated with the indicated concentrations of AICAR for 24 h and then with 3 ng/mL TNF-α for 4 h in the continued presence of AICAR; then, the ICAM-1 protein expression in cell lysates was measured by Western blot. (E) Western blot analysis of ICAM-1 expression. HUVECs were incubated with the indicated concentrations of Dorsomorphin (an AMPK inhibitor) and 50 μM resveratrol for 24 h and then with 3 ng/mL TNF-α for 4 h; then, the ICAM-1 protein expression in the cell lysates was measured by Western blot. (F) Western blot analysis of SIRT-1 expression. HUVECs were preincubated for 24 h with 50 μM resveratrol and were then treated with 3 ng/mL TNF-α for 4 h. (G) Western blot analysis of ICAM-1 expression. HUVECs were incubated with the indicated concentrations of Sirtinol (a SIRT-1 inhibitor) and 50 μM resveratrol for 24 h and then with 3 ng/mL TNF-α for 4 h; then, the ICAM-1 protein expression in the cell lysates was measured by Western blot. (H-K) The effects of AICAR treatment on p-ERK1/2, p-JNK, p-p38, and p-IκB in TNF-α-treated HUVECs. HUVECs were incubated for 24 h with or without 0.5 mM AICAR, and then the cells were incubated with 3 ng/mL of TNF-α for the indicated time. Cell lysates were subjected to immunoblotting with the antibodies against (H) p-ERK1/2 and t-ERK1/2, (I) p-p38 and t-p38, (J) p-JNK and t-JNK, or (K) p-IκB and t-IκB. *P < 0.05 compared with the untreated cells. ^†P < 0.05 compared with the TNF-α-treated cells at the same time point. Blots cropped for clarity; full blots are shown in the Supplementary file.

Figure 5. Resveratrol-mediated reduction in ICAM-1 expression in TNF-α-treated HUVECs involves miR-221/-222 upregulation.

(A) Effect of resveratrol on miR-221/222 expression by RT-PCR in HUVECs after 4 h of exposure to TNF-α. RUN6B (U6) was used as the control. (B) The bioinformatics analysis of the potential target sites in the ICAM-1 3′ UTR for miR-221 or miR-222. (C) miR-221 and miR-222 significantly inhibit the TNF-α-induced ICAM-1 luciferase activity. HUVECs were co-transfected with the ICAM-1promoter plasmid and with miR-221 or miR-222 precursors for 48 h. The luciferase activity was measured in these cells after further incubated for 4 h with TNF-α. (D) Functional miR-221/222 overexpression decreased ICAM-1, p-p38, and p-p65 expression levels. HUVECs were transfected with miR-221 or miR-222 precursors for 48 h and then exposed to TNF-α for 4 h, followed by Western blot analysis for ICAM-1, p-p38, and p-p65 expression. (E) The effects of miR-221/222 inhibitors on resveratrol-reduced ICAM-1 expression in TNF-α-treated HUVECs. HUVECs were transfected with miR-221/222 inhibitors for 48 h and then treated with 50 μM resveratrol for 24 h and 3 ng/mL TNF-α for 4 h in the continued presence of miR-221/-222 inhibitors and resveratrol. ICAM-1 expression in the cell lysates was measured by Western blot. GAPDH was used as the loading control. Blots are cropped for clarity; full blots are shown in the Supplementary file. (F) Representative fluorescence images showing the effects of miR-221/222 overexpression on the adhesion of fluorescein-labeled U937 cells to TNF-α-treated HUVECs. The data are expressed as the mean ± SEM of three separate experiments. *P < 0.05 compared with the untreated cells. ^†P < 0.05 compared with the TNF-α-treated cells.

Figure 6. Resveratrol reduces ICAM-1 expression in the thoracic aorta ECs of TNF-α-treated WT mice but not miR-221/-222 KO mice.

(A) Immunohistochemical staining for CD31 (an EC marker) and ICAM-1 expression in serial sections from the thoracic aortas of WT and miR-221/-222 KO mice. Mice were treated with DMSO, TNF-α, resveratrol + TNF-α, or resveratrol alone. The lumen is the uppermost portion in all sections. The reaction product is indicated by arrowheads. Bar = 40 μm. (B) Western blot analysis of ICAM-1 expression in aortic tissues of C57BL6J and miRNA-221/222 KO mice. Blots are cropped for clarity; full blots are shown in the Supplementary file.