IL-4-mediated induction of VCAM-1 expression in the intact endothelium. Mice were pretreated with saline (control) or blocking antibody against IL-4Rα and then injected i.v. with control saline, 0.5 mg/kg IL-4, or 0.1 mg/kg TNF-α. Tissues were harvested 6 h later, and cryosections of the aorta (A and B) and heart (C) were collected and stained with anti-VCAM-1 (A and C) or anti-ICAM-1 (B) antibody (left panels, red), ICAM-2 (right panels, green), and DAPI (right panels, blue). Merged images are shown in the right panels. Bar, 50 μm. L, lumen. (D) Total RNA was harvested from hearts and assayed for VCAM-1 or ICAM-1 mRNA levels by quantitative real-time PCR. Ab, antibody. The results show the means and standard deviations of expression levels relative to cyclophilin A from at least four independent sets of mice. *, P < 0.001 compared with VCAM-1 or ICAM-1 expression levels with saline treatment. N.S., nonsignificant.

Genome-wide analysis of IL-4-mediated up- or downregulated genes in primary cultured endothelial cells. (A) Heat map representation of IL-4-regulated genes. The color intensity indicates the expression level (upregulated in red and downregulated in blue relative to the median in white). A total of 983 probes demonstrating >5-fold upregulation or downregulation in the presence of IL-4 for 16 h were clustered at indicated time points. (B) Kinetics of IL-4-induced genes (526 probes) using Gene Spring software (Agilent). The bold line indicates the median of all probes in each cluster. (C) Kinetics of IL-4-repressed genes (457 probes) using Gene Spring software. The bold line indicates the median of all probes in each cluster. (D) Venn diagram showing overlap of IL-4-induced genes in HUVECs and CD4⁺ T cells. Whole probes were selected based on >2-fold induction after IL-4 treatment at 1, 4, and 16 h (HUVECs) or 1, 4, and 24 h (T cells).

Genome-wide analysis of STAT6-regulated genes in primary cultured endothelial cells. (A) Combined representation of microarrays and ChIP-seq results. Array genes were selected by the criteria with more than 2-fold up-or downregulated via IL-4 and were aligned with the results from ChIP-seq (middle column). (B) Representative ChIP-seq data (genome browser view) from SOCS1, P-selectin, PMCH, and BCL6. STAT6 binding signals are shown in red. H3K4me1 signals are shown in blue. Chr.16, chromosome 16. (C) Venn diagram showing the STAT6-dependent IL-4-inducible genes in HUVECs and CD4⁺T cells. Each number indicates the gene volume, which was induced via IL-4 and bound by STAT6 within the region from kb −20 upstream promoter, TSS, 5′ UTR, or 1st intron.

IL-4-mediated induction of VCAM-1 expression in primary cultured endothelial cells. (A) Confluent HUVECs were serum starved for 16 h and treated with 20 ng/ml IL-4 for the indicated times. Total RNA was harvested and assayed by quantitative real-time PCR for VCAM-1 and ICAM-1 mRNA levels. Data are expressed relative to untreated cells as means ± standard deviations (n = 4). *, P < 0.001 compared with untreated control. (B) HUVECs were treated with IL-4 and/or TNF-α for the indicated times and then assayed for VCAM-1 mRNA levels using quantitative real-time PCR. Data are expressed relative to untreated cells as means ± standard deviation (n = 4). *, P < 0.01 compared with untreated cells; #, P < 0.01, and ##, P < 0.05, compared with IL-4-treated cells at each time point. (C) HUVECs were treated as described above for 24 h. Western blotting was performed using anti-VCAM-1 antibody. The membrane was stripped and reprobed with anti-β-actin antibody as a loading control. Shown are representative data from three independent experiments.

Role of STAT6 in IL-4-mediated induction of VCAM-1 and monocyte adhesion in primary cultured endothelial cells. (A) HUVECs were treated in the presence or absence of 20 ng/ml IL-4 and then processed for cytosolic and nuclear fractions. Western blot analysis was carried out using antibodies against total STAT6, phospho-STAT6, nuclear lamin A, cytosolic α-tubulin, and p65 NF-κB. (B) HUVECs were treated with Ad-control or Ad-CA-STAT6. STAT6 (left) and VCAM-1 (right) mRNA levels were measured by quantitative real-time PCR. The results show the means and standard deviations of expression levels relative to Ad-control, derived from three independent experiments. *, P < 0.001 compared with Ad-control. (C) HUVECs were transfected with si-control or two independent siRNAs against STAT6 (oligo1 or oligo2), serum starved, and then treated with 20 ng/ml IL-4 for 24 h. STAT6 (left) and VCAM-1 (right) mRNA levels were measured by quantitative real-time PCR. The results show the means and standard deviations of expression levels relative to si-control in the absence of IL-4 treatment, derived from at least four independent experiments. *, P < 0.01 compared with si-control-treated cells in the presence of IL-4. N.S., nonsignificant. (D and E) U937 monocytic cell adhesion assays were carried out as described in Materials and Methods. HUVECs were infected with Ad-control or Ad-CA-STAT6 or transfected with si-control or si-STAT6, preincubated with control IgG or neutralizing antibody against VCAM-1, treated with 20 ng/ml IL-4 for 24 h, and then washed and incubated with U937 monocytes. The results are representative of four independent optical images from three independent experiments. Bar, 50 μm. Adhesion levels were quantitated (E). *, P < 0.01, compared with Ad-control minus IL-4; **, P = 0.045 compared with Ad-CA-STAT6 minus IL-4; #, P < 0.01 compared with si-control plus IL-4; and $, P < 0.01 compared with control IgG.

Genome-wide ChIP-seq analysis of IL-4-mediated STAT6 binding in primary cultured endothelial cells. (A) ChIP-seq analysis was used to survey genome-wide STAT6 binding in HUVECs treated with IL-4 for 0, 1, 4 and 16 h. (B, upper) De novo search for STAT6 binding recognition sequence based on sustained or transient binding in HUVEC. (Lower) Second and third enriched motifs calculated by the MEME method. The E-value indicates the probability of de novo enriched sequences obtained from ChIP-seq. (C) Distribution of STAT6 binding sites in the proximal promoter (within 5 kb upstream of the 5′ untranscribed region [UTR] from the transcriptional start site [TSS]), exon, intron, and intergenic regions (defined by regions 25 kb upstream and 10 kb downstream from the TSS). (D) IL-4-responsive genes (based on 17,444 probes [representing ∼8,500 genes] in DNA microarrays) at 1, 4, and 16 h, sorted according to the induction ratio at 16 h (left). Genes are aligned with results of ChIP-seq (blue bars indicate STAT6 binding). The vertical black bar indicates a group of highly induced IL-4-responsive genes at 16 h that are enriched in STAT6 binding. A graphic representation of the GSEA enrichment score is shown on the right.

Identification of the STAT6-bound VCAM-1 enhancer. (A) ChIP-seq data in the VCAM-1 locus with the genome browser view. STAT6 binding signals are shown in red. H3K4me1 signals are shown in blue. The box (dashed red line) indicates the H3K4me1-positive region associated with stable binding of STAT6. Chr.1, chromosome 1. (B, upper) Schematic representation of the luciferase constructs. (Lower) HUVECs were transiently transfected with Enhancer-VCAM-1-luc, serum starved for 16 h, incubated with Ad-control or Ad-CA-STAT6 for 24 h, and then assayed for luciferase activity. The results show the mean ± standard deviations of luciferase light units (relative to VCAM-1-luc- and Ad-control-transfected cells) obtained in triplicate from at least 3 independent experiments. *, P < 0.01 compared with Ad-control-transfected cells. N.S., nonsignificant. (C) HUVECs were transiently transfected with VCAM-1-luc or VCAM-1-luc containing the enhancer, serum starved for 16 h, and incubated with IL-4 for 24 h. The results show the mean ± standard deviation of luciferase light units (relative to VCAM-1-luc in the absence of IL-4 treatment) obtained in triplicate from at least 3 independent experiments. *, P < 0.01 compared without IL-4 treatment. (D) HUVECs were transiently transfected with VCAM-1-luc, wild-type Enhancer (−16-kb)-VCAM-1-luc, or (Enhancer −16-kb)-VCAM-1-luc containing a point mutation of one or both STAT6 binding elements. Cells were assayed as described above. The results show the mean ± standard deviation of luciferase light units (relative to VCAM-1-luc- and Ad-control-transfected cells) obtained in triplicate from at least 3 independent experiments. *, P < 0.01, compared with Ad-control-transfected cells. (E) HUVECs were transiently transfected with plasmids, serum starved, and incubated with IL-4 for 24 h. The results show the mean ± standard deviation of luciferase light units (relative to VCAM-1-luc-transfected cells in the absence of IL-4 treatment) obtained in triplicate from at least 3 independent experiments. *, P < 0.05 compared with IL-4-nontreated cells.

IL-4-mediated epigenetic modification of the kb −16 VCAM-1 enhancer. HUVECs were treated in the absence (0 h) or presence (1, 4, and 16 h) of IL-4, formalin fixed, and processed for immunoprecipitation of H4Ac, H3K4me3, p300, and H3K4me1. Precipitated genomic DNA was subjected to quantitative real-time PCR using primers specific to the VCAM-1 TSS, the kb −16 VCAM-1 enhancer, and the MyoD1 promoter. Shown is the ChIP enrichment level (mean ± standard deviation) relative to the ChIP-PCR value from the MyoD1 promoter without IL-4 (negative control). *, P < 0.05 compared to the same condition without IL-4 treatment. N.S., nonsignificant.