IRF-1 and miRNA126 modulate VCAM-1 expression in response to a high-fat meal

Abstract

Rationale: A high-fat diet accompanied by hypertriglyceridemia increases an individual's risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect.

Objective: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal.

Methods and results: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-α alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1.

Conclusions: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

Figure 1. Atherogenicity of TGRL varies with postprandial particle composition

TGRLs were analyzed for their composition using a clinical chemistry analyzer and each component was normalized to ApoB concentration in the sample to obtain a per particle density. TGRLs (10 mg/dl ApoB) were characterized for their atherogenicity, defined by the ability to positively (pro) or negatively (anti) modulate TNFα (0.3 ng/ml)-induced VCAM-1 expression in HAEC at 4 hr. Correlation of particle density for (A) TG, (B) ApoCIII, (C) ApoE, and (D) Cholesterol with the % change in VCAM-1 expression from TNFα. (E-H) Particle compositions for the same samples categorized by their ability to positively or negatively modulate VCAM-1 expression. (*P<0.05, **P<0.01, ***P<0.001 from paired TNFα; n=22–28).

Figure 2. PUFAs suppress VCAM-1 expression and reverse the effects of pro-atherogenic TGRL

VCAM-1 and ICAM-1 expression as % change from TNFα-stimulated (0.3 ng/ml) HAEC treated simultaneously with increasing concentrations of (A) DHA or (B) αLA for 4 hr. (*P < 0.05 from TNFα; n=5–9). Change in (C) VCAM-1 or (D) ICAM-1 expression from TNFα-stimulated HAEC treated with TGRL (10 mg/dl ApoB) with or without DHA (10 µM). (*P<0.05, **P<0.01 from paired TNFα) or by Student’s t-test (#P<0.05 and ##P<0.001 from TGRL. NS=not significant from TGRL; n=4–10).

Figure 3. TGRL and DHA do not affect basal or TNFα-induced NF-κB and AP-1 activity

(A) Active NF-κB (phospho-p65) measured by ELISA in nuclear extract of HAEC treated for 30 min with DHA (10µM) or TGRL (10 mg/dl ApoB) alone or simultaneously with TNFα (0.3ng/ml). n=3–8. (B) Phosphorylated c-Jun expression measured by ELISA in nuclear extract of HAEC treated as in (A). n=3–9. *p<0.05; **p<0.01; ***p<0.001 vs. non-stimulated.

Figure 4. IRF-1 expression varies with particle TG density and correlates directly with TGRL atherogenic potential

HAEC were treated for 4 hr with TNFα (0.3ng/ml) alone or simultaneously with DHA (10 µM) or TGRL (10 mg/dl ApoB), characterized for its effect on VCAM-1 expression. (A) Correlation between TGRL modulation of TNFα-induced VCAM-1 expression and IRF-1 expression. Linear regression to data for n=25. (B) Correlation between TGRL modulation of TNFα-induced IRF-1 expression and particle TG density. n=13. (C) HAEC were treated for 4 hr with 10 µM DHA or pro- or anti-atherogenic TGRL in the absence or presence of TNFα. IRF-1 protein expression was detected using Western blot and expressed relative to non-stimulated HAEC. (*P< 0.05; ***P<0.001 from non-stimulated; # P<0.05; ### P<0.001; n = 5–14.) (D) HAEC were treated as described above except TNFα was applied at 3 ng/ml. Chromatin was immunoprecipitated at 2 hr. IRF-1 binding to VCAM-1 promoter was quantified by qPCR and normalized to input DNA (means ± SE from 2 independent experiments).

Figure 5. TGRL-modulated VCAM-1 upregulation is IRF-1 dependent

HAEC were transfected with human IRF-1 siRNA (siIRF-1) (A-D) or pCMV6-XL5/human Irf-1 cDNA (E-H). Scrambled siRNA (siCtrl) and empty plasmid vector (sham) were used as controls. After 24 hr (knockdown) or 72 hr (overexpression), cells were stimulated with different doses of TNFα for 4 hrs prior to Western blot detection of IRF-1 (A, E), and flow cytometric analysis of VCAM-1 (B, F) and ICAM-1 (C, G) surface expression. Significance was determined by paired t-student test. (*P<0.05 vs. control. n=4). Flow cytometric analysis of VCAM-1 surface expression (D, H) Post-transfection, cells were treated with TNFα (0.3ng/mL) alone or plus TGRL (10mg/dl ApoB) for 4 hr (*P<0.05, **P<0.01 vs. siCtrl or sham; # P<0.05, ##P<0.01 pro- vs. anti-atherogenic TGRL group with the same transfection. NS, P>0.05. n=4–6).

Figure 6. miR-126 expression correlates inversely with TGRL atherogenic potential

(A) Correlation between TGRL modulation of TNFα-induced VCAM-1 expression and miR-126 expression at 1 hr. Linear regression for n=15. (B) miR-126 expression in HAEC treated for 1hr with TNFα (0.3 ng/ml) alone or simultaneously with DHA (10µM) or TGRL (10 mg/dl ApoB). (C) Change in VCAM-1 and miR-126 expression relative to TNFα-stimulated HAEC treated simultaneously with increasing concentrations of DHA for 1 hr (miR-126) or 4 hr (VCAM-1). (D) miR-17-3p expression in HAEC treated as in (B). (*P < 0.001 from TNFα; n=3–5).

Figure 7. Monocyte adhesion to activated HAEC correlates with changes in VCAM-1 expression

HAEC were treated with pro- or anti-atherogenic TGRL plus 0.3ng/mL TNFα, or transfected with IRF-1 siRNA or cDNA construct, or miR-126 inhibitor prior to TNFα stimulation. Monocyte adhesion was performed as described in Methods and plotted relative to the number arrested in response to 0.3ng/mL TNFα plus control transfection. Black dots represented 0.1ng/ml (−27%), 0.3ng/ml (0%), or 1.0ng/ml (25%) ng/ml TNFα, respectively. Ellipse centroid represents the mean value, the width and height correspond to the SEM of VCAM-1 expression or monocyte adhesion, respectively. (n=3–5 individual experiments for each treatment.)