Human subjects and study meal

Volunteers were recruited under informed written consent according to a protocol approved by the Institutional Review Board at the University of California, Davis. The study included both normal subjects and individuals with hypertriglyceridemia (fasting serum triglycerides > 150 mg/dL), and excluded those on lipid-lowering or anti-inflammatory medications or with a fasting blood glucose > 110 mg/dL. Venous blood was collected after a 12-hour overnight fast and again 3.5 hours after a standardized moderately high-fat meal (1230 calories, 47% from fat; 32% of total fat is saturated fat) [4]. Fasting and postprandial serum lipid and glucose levels were determined by the UC Davis Medical Center Clinical Laboratory using the Beckman Coulter UniCel DxC 800 Synchron Clinical System. LDL cholesterol was calculated by the Friedewald equation [26].

TGRL isolation and characterization

TGRL (ρ < 1.0063 g/ml) were isolated from postprandial blood samples by ultracentrifugation, and normalized for ApoB content quantified by ELISA (Alerchek), as previously described [8]. The isolates tested negative for endotoxin by chromogenic test kit (Associates of Cape Cod). They were aliquoted under nitrogen at 4°C for immediate use. Particle size distribution was measured using a Nanotrac Particle Size Analyzer equipped with FLEX software (Microtrac Inc.) as described previously [4]. More than 95% of the isolated TGRL particles were categorized as very-low-density lipoproteins (VLDL) based on particle diameter. For each subject’s sample, lipid and apolipoprotein concentrations (total cholesterol, triglycerides, ApoB, ApoCIII and ApoE) were measured by spectrophotometric assay using a clinical chemistry analyzer (Polymedco Inc.).

HAEC culture and TGRL treatment protocol

Low passage HAEC (passage 4-6) (Genlantis) were cultured on collagen-coated (50µg/ml, Clontech) culture dishes or gelatin-coated (1%) coverslips and maintained in Endothelial Cell Growth Medium-2 (EGM^®-2, Lonza) supplemented with 1x antibiotic-antimycotic solution (Invitrogen). Unless otherwise indicated, HAEC were treated with TNFα (0.3 ng/ml, R&D system) alone or simultaneously with individual freshly isolated TGRL (10 mg ApoB/dL) for 4 hours. To quantify inflammatory activation relative to TNFα alone, HAEC were detached using enzyme-free cell dissociation buffer (Gibco), labeled with fluorescein-conjugated antibodies to human intercellular adhesion molecule 1 (ICAM-1) or VCAM-1, and analyzed by a FACScan flow cytometer (Becton Dickinson) [4]. Median fluorescence intensity (MFI) was quantified using FlowJo software (TreeStar, Ashland, OR) and used for further analysis.

Antibodies

For flow cytometry, the following antibodies from R&D systems were used: hVCAM-1 FITC mAb (BBA22), hICAM-1 FITC mAb (BBA20), and IgG fluorescein isotype controls (IC002F and IC003F). For Western blotting, the following antibodies were used: BiP (ab21685), phospho-IRE1α (ab48187), and IRF-1 (ab55330) from Abcam; total IRE1α (3294), total-eIF2α (2103), phospho-eIF2α (3597), CHOP (5554) from Cell Signaling Technology; sXBP1 (sc-32138) and Tubulin (sc-53646) from Santa Cruz Biotechnology; and GAPDH (NB-300-328) from Novus Biologicals. For immunofluorescence staining of ER structure, goat anti-human calreticulin antibodies (SC-6468) from Santa Cruz Biotechnology and Alexa Fluor^® 546 rabbit anti-goat IgG (A-21085) from Life Technologies were used.

TGRL labeling and uptake assay

TGRL were conjugated to Alexa-Fluor 488 (AF488) using a protein labeling kit (Invitrogen) as described previously [4], and stored at 4°C, protected from light. For the uptake assay, HAEC were incubated with AF488-TGRL (10 mg ApoB/dL in phenol red-free culture media) at 37°C for the designated period, washed twice with Hank's Balanced Salt Solution (HBSS, w/Ca²⁺, Life Technologies), detached using TrypLE Express (Life Technologies), pelleted at 1,000 g at 4°C, and resuspended in HBSS (w/Ca²⁺ + 0.2% human serum albumin) on ice. The samples were immediately analyzed by a FACScan flow cytometer (Becton Dickinson) with CellQuest software. Data represent MFI from a single Gaussian population of 10,000 cells for each sample.

Lipid droplet staining and imaging

HAEC were incubated with TGRL (10 mg ApoB/dL) for the indicated period, washed with 37°C HBSS w/ Ca²⁺, and incubated with BODIPY 493/503 (Life Technologies, 2 µM in HBSS) at 37°C for 1 min. HAEC were washed with ice cold HBSS, collected using TrypLE Express, and analyzed by a FACScan flow cytometer. Samples were protected from light throughout these procedures. Data represent MFI from a single Gaussian population of 10,000 cells for each sample. For visualization of LD by confocal microscopy, HAEC were cultured on 1% gelatin-coated coverslips, incubated with TGRL for 4 hours, and stained with BODIPY as described above. Cells were maintained in phenol red-free Leibovitz-15 medium (Life Technologies) at 37°C, and imaged under 488nm laser excitation by a Zeiss LSM 5 Pascal confocal microscope.

Immunofluorescence

HAEC were grown on 1% gelatin-coated coverslips to 80% confluence. After treatment, cells were rinsed with phosphate-buffered saline (PBS), fixed and permeabilized with 100% methanol at -20°C for 6 min. The coverslips were then rinsed with PBS and blocked in 5% bovine serum albumin (BSA) + 0.2% Triton X-100 in PBS. HAEC were then incubated with goat anti-human calreticulin antibody (1 µg/ml) in PBS w/ 1% BSA for 1 hour at room temperature, washed with PBS, and incubated with Alexa Fluor 546 rabbit anti-goat IgG (2 µg/ml) in PBS at room temperature for 1 hour. Nuclei were counterstained with Hoechst (Sigma). The samples were imaged using a Zeiss LSM 5 Pascal confocal microscope. Images were analyzed using ImageJ. The coefficient of variation (CV) in the pixel fluorescence intensity of labeled calreticulin in the cytoplasm of each cell, a global index of spatial heterogeneity, was applied to assess ER morphological changes. Cells with expanded ER yielded higher CV values as calreticulin became less evenly distributed [27]. 30-40 cells were analyzed for each condition.

RNA isolation and quantitative RT-PCR

Total RNA was isolated using the High Pure Total RNA isolation kit (Roche), which includes a DNase digestion step to remove contaminating genomic DNA. RNA (~500 ng) was converted to 1st strand cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche). Quantitative PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems, VCAM-1: Hs00365485_m1; ICAM-1: Hs00164932_m1; RPLP0: Hs99999902_m1; IRF-1: Hs00971960_m1) and TaqMan Gene Expression Master Mix (Applied Biosystems) with a RealPlex Mastercycler (Eppendorf). Quantification relative to the housekeeping gene acidic ribosomal protein P0 (RPLP0) was determined by the ΔΔC_t method, where C_t is threshold cycle number.

Protein extraction and Western immunoblot analysis

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer containing 1 mM NaF, 1 mM sodium orthovanadate and protease inhibitor cocktail (Sigma) with freshly added 0.1 mM phenylmethanesulfonylfluoride (PMSF). The lysate was sonicated and protein concentration determined by the bicinchoninic acid (BCA) assay. Samples were normalized to 1 mg/ml of protein content, and resolved by SDS-PAGE, transferred to nitrocellulose, and blocked. The blots were incubated overnight at 4°C with primary antibody, then at room temperature for 1.5 hours with horseradish peroxidase-conjugated secondary antibody. They were developed with Chemiluminescent Substrate. Signals were captured by ChemiDoc MP gel imaging system (Bio-Rad). Tubulin, GAPDH or the total protein forms of phosphorylated proteins of interest served as the loading control for quantification using ImageJ [5].

Cell Transfection

To deplete CHOP or sXBP1 expression, cells (5×10⁵) were transfected with 150 pmol siRNA to CHOP or XBP1 (Santa Cruz Biotech), using a 4D-Nucleofector system (Lonza). After 48-96 hr, cells were treated as described followed by Western blot or flow cytometry analysis.

NF-κB and activator protein-1 (AP-1) activation

NF-κB and AP-1 activities were measured by ELISA targeting active phospho-p65 and phospho-c-Jun in the nuclear extract as previously described [5].

Statistical analysis

Data were analyzed using GraphPad Prism v5.0 software. All data are presented as mean ± standard error (SE) unless otherwise indicated. Differences among multiple groups were assessed by one-way ANOVA with a post hoc Newman-Keuls test. Two experimental groups were compared using Student’s t test, with pairing where appropriate. Comparisons were considered significant for 2-tailed p values < 0.05. Correlations between groups were assessed using Pearson's correlation coefficient (r).

TGRL uptake induces extensive lipid droplet formation independent of its atherogenicity

To investigate the origin of the heterogeneity in the atherogenicity of TGRL isolated from blood collected following consumption of a high-fat meal, we recruited thirty-four subjects with normal fasting glucose, but exhibiting a broad range in anthropometric characteristics and fasting serum triglycerides from normal to hypertriglyceridemic (Tables S1 and S2). We categorized each subject’s postprandial TGRL based on its capacity to alter the inflammatory response of HAEC stimulated with TNFα, a pivotal inflammatory cytokine expressed locally at sites of atherosclerotic plaques [28] and a biomarker for CVD that is found at elevated levels in the serum of individuals at increased risk [29]. The dose of TNFα in use (0.3 ng/ml) was previously reported to correspond to the EC₅₀ for upregulation of membrane adhesion receptors on HAEC (VCAM-1, ICAM-1, or E-selectin) [8]. TGRL that enhanced VCAM-1 expression relative to TNFα stimulation alone was designated as pro-atherogenic and that which suppressed VCAM-1 expression as anti-atherogenic [5,16]. Pro-atherogenic TGRL were enriched in triglyceride, cholesterol, ApoE, and ApoCIII content per particle as compared to anti-atherogenic that uniformly expressed lower levels (Figure S1).

We next investigated whether the differential atherogenic response of HAEC to inflammatory stimulation was a function of the extent of TGRL uptake and subsequent lipid droplet (LD) formation. The internalization of fluorophore-conjugated TGRL particles increased rapidly upon addition to the EC monolayer and rose steadily up to 6 hr (Figure 1A), while the uptake rate reached a steady state after 2 hr (Figure 1A, inset). Intracellular LD formation was visualized by confocal fluorescence microscopy of BODIPY stained EC (Figure 1B). Discrete punctate BODIPY-positive droplets measuring ~0.1-0.5 micrometer in diameter were resolved in HAEC cytoplasm. To determine whether the rate and extent of LD formation correlated with TGRL atherogenicity, we quantified their cytoplasmic abundance using flow cytometry. LD formation increased with a time course similar to that of TGRL uptake at a rate that plateaued at 2 hr and maintained a steady state up to 5 hr (Figure 1C, inset). LD abundance in TGRL treated HAEC was increased by ~6-fold at 4 hr with no significant difference observed in the extent of LD formation between anti- and pro-atherogenic TGRL (Figure 1D), which decreased VCAM-1 expression by 10 ± 2 %, and increased it by 29 ± 7 %, respectively, compared to TNFα alone. Noteworthy was the observation that the extent of LD formation as measured by BODIPY fluorescence correlated closely with the side scatter (SSC) signal obtained by flow cytometry (r = 0.94, P < 0.0001, Figure S2), suggesting that SSC provides a simple label-free approach to assess LD abundance in EC. Together, these data demonstrate a rapid and sustained TGRL uptake that results in the extensive formation of LD in the cytoplasm of HAEC. Heterogeneity of particle size and composition that discriminated anti- from pro-atherogenic TGRL did not result in a discernible difference in the extent of LD formation. We conclude that the relative atherogenicity of TGRL on HAEC is not simply a function of its relative abundance or extent of cytoplasmic internalization.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. TGRL induces extensive lipid uptake and lipid droplet formation.

(A) The kinetics of TGRL uptake. HAEC were incubated in the presence (“●”) or absence (“○”) of Alexa fluor488-labeled TGRL (10 mg ApoB/dL) for 0-6 hr, and examined for internalized TGRL by flow cytometry. Data presented as median fluorescence intensity (MFI). Inset: uptake rate defined as ∆MFI/∆hr. (B) Representative confocal images (i. and zoomed-in version ii.) of lipid droplets formed in HAEC. Cells were treated for 4 hr with native TGRL (10 mg ApoB/dL) or culture media (“Ctrl”), and stained for lipid droplets with BODIPY 493/503. (C) The kinetics of lipid droplet formation. HAEC were incubated with native TGRL for designated periods, stained with BODIPY and analyzed by flow cytometry. n=3. Inset: the rate of lipid droplet formation = ∆MFI of BODIPY/∆hr. (D) Quantification of lipid droplets formed in HAEC after 4 hr incubation with anti- or pro-atherogenic TGRL. n=3-6. ns, not significant.

https://doi.org/10.1371/journal.pone.0078322.g001

TGRL atherogenicity is reflected in ER morphological changes associated with ER stress

Excess fatty acids and sterols released from lipoproteins are mainly converted to neutral lipids and stored in LD by a series of ER-resident enzymes [9]. We next examined whether the atherogenic phenotype induced by uptake and metabolism of individual TGRL was reflected in alteration of ER [9]. One readout of ER dysfunction is distortion in the composition and structure of its membrane, as previously observed in smooth muscle cells [30] and livers of obese mice fed diets high in fat [31]. To investigate if the metabolic challenge induced by TGRL uptake and subsequent LD formation effectively disrupts ER function, we examined ER morphology after 4 hr incubation with pro- or anti-atherogenic TGRL or control media in the presence of TNFα stimulation. HAEC ER morphology was imaged by confocal microscopy and immunofluorescence staining of calreticulin, an ER luminal resident protein [32]. Image analysis revealed a significant extent of ER membrane expansion in HAEC treated with pro-atherogenic TGRL (Figure 2A), similar to that previously reported following skeletal muscle uptake of saturated fatty acid [30]. ER membrane expansion was characterized by an increase in tubular and web structures with a significant 33% increase in the spatial heterogeneity of labeled calreticulin in HAEC treated with pro-atherogenic TGRL and stimulated with TNFα (Figure 2B). In contrast, anti-atherogenic TGRL suppressed ER expansion by 14%. To test whether ER stress was associated with the differential effects of pro- versus anti-atherogenic TGRL, we examined ER membrane alteration in the presence of the ER stress inhibitor, 4-phenyl butyric acid (4-PBA). Treatment with 4-PBA abrogated the effect of pro-atherogenic TGRL on ER expansion (Figure 2C-D). Further, we confirmed that TNFα itself did not cause obvious morphological changes compared to control cells; however, treatment of HAEC with pro-atherogenic TGRL alone induced a significant increase in ER expansion that was augmented upon TNFα stimulation (Figure S3). These data demonstrate that ER membrane morphology provides a reliable readout of the relative effects of TGRL and suggest that ER stress may directly influence downstream inflammatory responses of HAEC.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. TGRL-induced ER morphological changes are alleviated by an ER stress inhibitor.

A-B: Pro-atherogenic TGRL induces ER expansion, defined as increased spatial heterogeneity (Standard Deviation/Mean of pixel fluorescence intensity within cell cytoplasm), while anti-atherogenic TGRL suppresses it. HAEC grown on coverslips were treated for 4 hr with TNFα alone or simultaneously with pro- or anti-atherogenic TGRL (10 mg ApoB/dL). Cells were then visualized by indirect immunofluorescence and confocal microscopy. Representative confocal images shown in (A) red, calreticulin; blue, Hoechst nuclear stain. Bottom panels show zoomed images of calreticulin signal from the boxed 20µm × 5µm region. Statistical analysis of ER expansion shown in (B). 30-40 cells were analyzed for each condition. ***P<0.001, *P<0.05 from TNFα-stimulated. C-D: ER stress inhibition normalizes TGRL’s effect on ER morphology. 4-phenyl butyric acid (4-PBA) was added to the above treatments with additional 1 hr pretreatment. Confocal images shown in (C) and statistical analysis in (D). 30-40 cells were analyzed for each condition. ns, not significant.

https://doi.org/10.1371/journal.pone.0078322.g002

ER stress plays a key role in TNFα and TGRL modulation of VCAM-1 expression

The distinct alterations of ER morphology by pro- and anti-atherogenic TGRL led us to further investigate whether inhibition of ER stress with 4-PBA would alter TNFα-stimulated upregulation of adhesion receptor expression. Treatment of HAEC with 4-PBA significantly inhibited VCAM-1 upregulation in a dose-dependent manner, yielding an IC₅₀ of 2mM and ~70% inhibition at 3mM 4-PBA following 4 hrs of stimulation (Figure 3A and Figure S4 A-B). In contrast, 4-PBA did not affect ICAM-1 upregulation within this dose range (Figure 3A and Figure S4 C-D). Similar results were observed for regulation of mRNA production at the 2 hr time point (Figure 3B). This suggested that ER stress was modulating a VCAM-1-specific regulatory pathway. To determine whether inhibition of ER stress also modulated the atherogenic effect of TGRL on VCAM-1 expression, HAEC were treated with pro- or anti-atherogenic TGRL and stimulated with TNFα in the presence or absence of 4-PBA. Suppression of ER stress by 4-PBA abrogated the pro-atherogenic effect of TGRL in regulating TNFα-stimulated VCAM-1 expression (Figure 3C). These results clearly implicate ER stress in TNFα and TGRL-modulated adhesion molecule upregulation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. ER stress plays a key role in TGRL modulation of TNFα-stimulated VCAM-1 expression.

A-B: ER stress inhibition dose-dependently reduced TNFα-induced VCAM-1 expression but did not affect ICAM-1 expression. HAEC were conditioned for 4 hr (A) or 2 hr (B) with TNFα (0.3 ng/ml) and 4-phenyl butyric acid (4-PBA) at different doses (0, 0.3, 1, or 3 mM, with 1 hr pretreatment). VCAM-1 and ICAM-1 protein or mRNA expression analyzed by flow cytometry (A) or quantitative RT-PCR (B). n=3-5. *P<0.05, **P<0.01 from TNFα stimulated alone. (C) ER stress inhibition abolished TGRL modulation of TNFα-stimulated VCAM-1 expression. HAEC were treated for 4 hr with TNFα (0.3 ng/ml) alone or simultaneously with pro- or anti-atherogenic TGRL (10 mg ApoB/dL) in the presence (1 mM, 1 hr pretreatment and 4 hr co-treatment) or absence of 4-PBA. n=4-7. ***P<0.001, **P<0.01 from TNFα-stimulated alone. ns, not significant.

https://doi.org/10.1371/journal.pone.0078322.g003

TGRL modulates VCAM-1 expression through activation of ER stress

We next investigated the ER stress signaling pathways that may contribute to the atherogenic effect of TGRL specifically on VCAM-1 expression. We first measured changes in BiP protein expression and the phosphorylation of IRE1α (Ser⁷²⁴) and eIF2α (Ser⁵¹), adjusted for their relative levels of protein expression. Western immunoblotting revealed that TNFα at 0.3 ng/ml elicited 80% upregulation of BiP expression and 50% of IRE1α phosphorylation over baseline, which equals 42% and 58% of the elevation by TNFα at 10 ng/ml (Figure S5). Pro-atherogenic TGRL on average upregulated BiP expression by ~80% and increased the phosphorylation of both IRE1α (35%) and eIF2α (15%) from the modest effect of TNFα alone, whereas anti-atherogenic TGRL had comparatively less of an effect on BiP and IRE1α (Figure 4A-C). Further, a significant direct correlation was observed in activity of all three ER stress markers versus regulation of VCAM-1 expression elicited by treatment with each subject’s TGRL relative to TNFα alone. To better understand the effect of TGRL on downstream mediators of ER stress we assessed activation of sXBP1 and CHOP. Baseline expression of sXBP1 protein was detected at low levels and not significantly elevated by treatment with TGRL alone. TNFα stimulation significantly increased the level of sXBP1, which was further elevated by pro-atherogenic TGRL (15%) and decreased by anti-atherogenic TGRL (63%) relative to TNFα stimulation alone (Figure 4D). In contrast, baseline expression of CHOP was increased by 80% following treatment with pro-atherogenic TGRL, but remained unchanged in response to anti-atherogenic TGRL. TNFα stimulation alone increased CHOP expression by 3-fold and this rise was suppressed by anti-atherogenic TGRL (Figure 4E). Both sXBP1 and CHOP levels significantly correlated with regulation of VCAM-1 expression elicited by treatment with each subject’s TGRL compared to TNFα alone (Figure S6). As expected, inhibition with 4-PBA down regulated cytokine activation of ER stress as indicated by an attenuated phosphorylation of IRE1α and reduced expression of CHOP and sXBP1 (Figure 4F). These results indicate that ER stress and activation of the UPR is directly influenced by stimulation with TNFα and its activity further modulates VCAM-1 expression as a function of the relative atherogenicity of TGRL.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. TGRL modulates ER stress markers as a function of its atherogenicity.

HAEC were treated for 4 hr with TNFα alone or simultaneously with TGRL (10 mg ApoB/dL). Protein extracts from whole cell lysates were analyzed by Western immunoblot. Representative blots are shown. A-C: Protein expression level or phosphorylation ratio presented as % change from TNFα-stimulated alone. Pearson correlations between TGRL modulation of TNFα-induced VCAM-1 expression and (A) BiP expression, (B) IRE1α or (C) eIF2α phosphorylation. n = 15-18. Insets: the same data binned into two categories based on TGRL’s atherogenicity. *P<0.05, **P<0.01 from TNFα. D-E: Pro-atherogenic TGRL enhanced (D) sXBP1 expression, while anti-atherogenic TGRL suppressed (D) sXBP1 and (E) CHOP expression. ns, not significant. *P<0.05, ***P<0.001 from TNFα-stimulated. #P<0.05 from non-stimulated control. n=3-9. (F) HAEC were treated for 4 hr with TNFα (0.3 ng/ml) with or without pretreatment with 1 mM 4-PBA, followed by Western blot analysis, representative of 3 separate experiments.

https://doi.org/10.1371/journal.pone.0078322.g004

IRF-1-regulation of VCAM-1 expression correlates with TGRL-induced ER stress response

To connect TGRL-modulated ER stress pathways to altered VCAM-1 expression, we analyzed changes in the expression of interferon regulatory factor 1 (IRF-1) with concomitant changes in ER stress markers over a range in activity from anti- to pro-atherogenic. The motivation for this set of experiments was our recent report that IRF-1 parallels inflammatory changes in VCAM-1 expression as a function of the atherogenicity of a subject’s TGRL [5]. Moreover, studies with siRNA have revealed that IRF-1 activity directly correlated with the up or down modulation of TGRL on VCAM-1 expression by regulating Vcam-1 transcription [5]. In the current cohort, we confirmed that IRF-1 expression strongly correlated with regulation of VCAM-1 expression in response to TGRL (Figure S7 A). Like VCAM-1 expression, IRF-1 protein levels in HAEC were directly and significantly correlated with IRE1α and eIF2α phosphorylation as modulated by TGRL from our subject pool (Figure 5A-B). TGRL that promoted sXBP1 upregulation by 20% and CHOP by 24% in response to TNFα, increased IRF-1 expression on average by 23%. Conversely, TGRL that suppressed sXBP1 upregulation by 63% and CHOP by 43%, reduced IRF-1 expression by 28%. The specificity in regulation of IRF-1 was assessed by pretreatment of HAEC over a dose range of 4-PBA, which revealed a decrease in TNFα-stimulated IRF-1 protein expression stimulated with TNFα for 4 hr (Figure 5C). Similar to inhibition of VCAM-1, we detected an IC₅₀ of ~1 mM and maximal suppression of IRF-1 protein production of 60% above 3mM (Figure 5C). Consistently, pretreatment of cells with 4-PBA at 1 mM significantly inhibited Irf-1 transcription at 1 hr post stimulation with TNFα (Figure S8). Depletion of IRF-1 with siRNA decreased TNFα-induced VCAM-1 expression at both mRNA and protein levels (Figure S7 B-C). In contrast, activation of NF-κB and AP-1, two other transcription factors of Vcam-1 that are activated by TNFα stimulation, was not affected by 4-PBA up to doses of 1 mM (Figure S9). This is consistent with our observation that ICAM-1 expression, which is primarily regulated by NF-κB and AP-1, is not significantly altered by 4-PBA treatment (Figure 3A-B). We conclude that IRF-1 activity is coupled to an ER stress response pathway that links TGRL metabolism with inflammatory regulation of VCAM-1 in HAEC.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. IRF-1 plays a central role in ER stress-mediated modulation of VCAM-1 expression by TGRL.

A-B: TGRL modulation of TNFα-induced VCAM-1 expression is positively correlated with IRF-1 expression. TGRL modulation of TNFα-induced IRF-1 expression positively correlated with the phosphorylation levels of (A) IRE1α and (B) eIF2α. n=11-12. (C) ER stress inhibition reduced TNFα-induced IRF-1 expression in a dose-dependent manner. HAEC were conditioned for 4 hr with TNFα (0.3 ng/ml) and 4-PBA over a dose range (0-10 mM, with 1 hr pretreatment). IRF-1 protein level characterized by Western blot. Representative blots are shown. n=3-5. *P<0.05 from TNFα stimulated. D-E: CHOP knockdown (E) and not sXBP1 knockdown (D) decreased TNFα-induced VCAM-1 expression. n=4. **P<0.01 vs. siCtrl+TNFα. (F) CHOP knockdown decreases VCAM-1 expression concomitantly with an inhibition on IRF-1 expression. Shown are representative blots from 3 independent experiments with similar results.

https://doi.org/10.1371/journal.pone.0078322.g005

CHOP is involved in the regulation of VCAM-1 expression via modulation of IRF-1

To specify the contribution of ER stress to VCAM-1 expression, the UPR downstream effectors sXBP1 and CHOP were depleted via transfection of siRNA. Western blotting confirmed successful specific knockdown of either sXBP1 or CHOP without affecting phosphorylation of IRE1α in response to TNFα stimulation (Figure 5D-E). We observed that siRNA suppression of CHOP but not sXBP1 knockdown decreased VCAM-1 expression (Figure 5D-E), concomitant with a reduction in IRF-1 expression (Figure 5F). These data are consistent with the higher propensity of TGRL to enhance CHOP expression and implicate it in activation of a pathway leading from ER stress to regulation of VCAM-1 expression.