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Induction of ATF3 Gene Network by Triglyceride-Rich Lipoprotein Lipolysis Products Increases Vascular Apoptosis and Inflammation

Originally published, Thrombosis, and Vascular Biology. 2013;33:2088–2096



Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated.

Approach and Results—

We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)–mediated inhibition of ATF3 blocked lipolysis products–induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB–dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3−/− mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes.


This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.


Elevation of triglyceride-rich lipoprotein (TGRL) is a known atherosclerotic cardiovascular disease risk factor and can induce endothelial dysfunction and inflammation. Although epidemiological evidence has shown a clear link between hypertriglyceridemia and atherogenesis,1 the mechanism of vascular injury related to elevated TGRL remains incompletely understood. TGRL are hydrolyzed on the endothelial cell surface by lipoprotein lipase (LpL), thus potentially inducing very high concentrations of lipolysis products along the blood–endothelium interface. Increased concentrations of TGRL lipolysis products may contribute to the pathogenesis of atherosclerotic cardiovascular disease by affecting the expression of multiple proinflammatory, procoagulant, and proapoptotic genes.

In our studies, proinflammatory pathways are activated and predominate when endothelial cells are exposed to high physiological and pathophysiological concentrations of TGRL lipolysis products, but not TGRL alone.24 TGRL lipolysis releases neutral and oxidized free fatty acids that induce endothelial cell inflammation.5 Also, we have found that TGRL lipolysis products injured endothelial cells by increasing very low-density lipoprotein remnant deposition in the artery wall, augmented endothelial monolayer permeability, perturbed zonula occludens-1 and F-actin, and induced apoptosis.6,7 TGRL lipolysis products also significantly increased the production of reactive oxygen species in endothelial cells and altered lipid raft morphology.8 Thus, TGRL lipolysis products in high physiological and pathophysiological concentrations have multiple proinflammatory actions on endothelial cells.

The p38 and stress-activated protein kinase/c-Jun N-terminal kinase (JNK) subset of mitogen-activated protein kinase signaling is a key cellular stress response pathway leading to induction of apoptosis.9 Induction of this pathway has been documented using a variety of physiochemical stressors in several cell types including endothelial cells.1012 Signaling through small G proteins, such as Rac, Rho, Ras, and CDC42, activates mitogen-activated protein kinase kinase pathways leading to phosphoactivation of JNK and subsequent nuclear translocation and transcriptional activation of several genes.1315 Activated JNK also phosphorylates c-Jun, a component of the activator protein 1 (AP-1) transcription complex. Response elements induced by JNK signaling include the cAMP response element-binding protein family that contains additional AP-1–associated activating transcription factor isoforms.16

We hypothesized that TGRL lipolysis products activate stress response pathways that induce expression of multiple proinflammatory and proapoptotic genes leading to endothelial dysfunction. Our studies identified transcription of activating transcription factor 3 (ATF3), a member of the cAMP response element-binding protein family, as a key response gene after treatment with TGRL lipolysis products and demonstrated that its induction was essential for the expression of a subset of proinflammatory responses. Thus, ATF3 could represent a key regulatory protein in TGRL lipolysis product–mediated vascular inflammation and atherosclerosis.

Materials and Methods

Materials and Methods are available in the online-only Supplement.


TGRL Lipolysis Products Activate Genes Encoding Transcription Factors and Proinflammatory Activities

The response of the human aortic endothelial cell (HAEC) transcriptome to media, LpL, TGRL, or TGRL lipolysis products (TGRL+LpL) was determined with high-density oligonucleotide arrays containing 22 000 probe sets (Human Genome U133A2.0 array, Affymetrix) that represent a large fraction of the expressed human genome. Transcription of ≈14 500 genes was reliably detected (detection P≤0.05) in all treatment groups with no significant difference in gene numbers evident between groups. Table I in the online-only Data Supplement records the percentage of genes altered by LpL, TGRL, or TGRL + LpL, and Figure I in the online-only Data Supplement depicts the functional classifications for the affected genes. Verification of the gene chip results with qRT-PCR is depicted in Figure IIA and IIB in the online-only Data Supplement. Specific genes sensitive to TGRL lipolysis treatment are listed in Tables II and III in the online-only Data Supplement.

TGRL lipolysis products activated genes encoding transcription factors, including cAMP responsive element modulator (2-fold), v-rel reticuloendotheliosis viral oncogene homolog/nuclear factor of κ light polypeptide (2-fold), Jun (3.5-fold), JunB (8.6-fold), CCAAT/enhancer binding protein (C/EBP) β (4.9-fold), activating transcription factors 3 (ATF3; 36.8-fold) and ATF4 (2.1-fold; Table III in the online-only Data Supplement). Transcription of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA/nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor alpha [IκBA]), an inhibitor of nuclear factor-κB (NFκB) action, was increased 4.3-fold, whereas Kruppel-like factor 4 was increased 7-fold. Proinflammatory genes activated included E-selectin (7-fold), interleukin-1α (IL-1α; 4-fold), IL-8 (6.1-fold), and vascular endothelial growth factor (VEGF) (3.5-fold).

TGRL Lipolysis Products Induce ATF3 and Phosphorylated JNK Protein Expression and Activate the Stress-Activated Protein Kinase/JNK Pathway

Western blot analysis showed lipolysis product treatment–induced ATF3 and phosphorylated JNK (p-JNK) protein expression (Figure 1A and 1B). To determine the selectivity of p-JNK activation, we evaluated downstream proteins of this pathway. Compared with control treatments, only lipolysis products significantly increased expression of c-Jun and p-c-Jun (Figure 1C). Despite increased mRNA transcription of JunB (associated with NFκB signaling), protein expression was downregulated by lipolysis product treatment (Figure 1C).

Figure 1.

Figure 1. Triglyceride-rich lipoproteins (TGRLs) lipolysis products increase activating transcription factor 3 (ATF3), phosphorylated c-Jun N-terminal kinase (p-JNK), p-c-Jun, and c-Jun protein expression and translocation of ATF3 and p-JNK from the cytoplasm to the nucleus. Human aortic endothelial cells were exposed to TGRL lipolysis products (T+L), media (M), lipoprotein lipase (LpL; L), or TGRL (T) for 3 hours. Cell lysates were analyzed by Western blotting (a) and (b) densitometry. A, Protein expression of ATF3 after the 4 treatments described above. B, Protein expression of p-JNK. C, Protein expression of c-Jun, phosphorylated c-Jun (p-c-Jun), and JunB. A–C, n=3, #P≤0.05, T+L compared with M, L, or T. D, Immunofluorescence images of ATF3 and p-JNK showing transition of cytosolic to nuclear localization translocation of ATF3 and p-JNK from the cytoplasm to the nucleus after lipolysis product exposure (bar=40 µm). 4',6-diamidino-2-phenylindole (DAPI) for nucleus stain. E, Percentage of cells with nuclear staining for ATF3 and p-JNK after lipolysis product exposure, n=5 coverslips/treatment group, *P≤0.04 for comparisons between TGRL+LpL and TGRL alone.

Cellular location of ATF3 and p-JNK also changed in response to lipolysis product treatment. In cells treated with only TGRL or LpL, ATF3 and p-JNK were present at very low levels and not concentrated in the nucleus (Figure 1D). After 3 hours of TGRL+LpL treatment, ATF3 and p-JNK production were upregulated with rapid accumulation in nuclei. After TGRL+LpL treatment, 93% and 79% of cells had nuclei that stained positive for ATF3 and p-JNK, respectively (Figure 1E). Our observations are consistent with previous studies that show ATF3 is expressed at a low level in normal and quiescent cells but can be rapidly induced in response to extracellular signals and is involved in controlling a wide variety of stress-related cellular activities.1719

ATF3 siRNA Suppresses Induction of a Subset of Inflammatory Genes

To determine the relationship between ATF3 and induction of proinflammatory gene networks, we performed 18 hours of ATF3 siRNA (Ambion, Carlsbad, CA) knockdown experiments in HAECs treated with TGRL+LpL for 1, 2, and 3 hours. In scrambled siRNA-treated HAECs, ATF3 expression was upregulated significantly after treatment with lipolysis products for up to 3 hours (Figure 2A). Oligo-based siRNA targeting effectively decreased the amount of ATF3 mRNA (90%) in HAECs after 3 hours of lipolysis product treatment (Figure 2B). Immunofluorescence analysis also showed that siRNA knockdown in HAECs attenuated ATF3 protein expression (Figure 2C).

Figure 2.

Figure 2. Effect of activating transcription factor 3 (ATF3) small interfering RNA (siRNA). The expression of each gene was normalized to that of GAPDH, and the fold change was calculated as the difference in expression with triglyceride-rich lipoprotein (TGRL) lipolysis products in the presence of scrambled siRNA and ATF3 siRNA (n=3; *P≤0.05). Samples pretreated with siRNA 18 hours before lipolysis product exposure. A, Effect of ATF3 siRNA relative to time on ATF3 levels. B, Alterations in the transcription of ATF3, E-selectin, interleukin (IL)-8, IL-1α, Kruppel-like factor 4 (KLF4), and vascular endothelial growth factor (VEGF). C, ATF3 protein expression, as monitored by immunofluorescence, was suppressed by ATF3 siRNA (bar=40 µm). D, Alterations of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), NFKB1, and IL-6 gene expression. LpL indicates lipoprotein lipase.

The transcription of mRNAs encoding E-selectin, IL-8, IL-6, NFKBIA/IκBA, and NFKB1/ NFκB(p50) was quantified in ATF3 siRNA–transfected cells. ATF3 blockade resulted in a large and significant decrease in lipolysis product–induced E-selectin (89%) and IL-8 (60%) mRNA levels (Figure 2B). Similar results were retained with a different construct of ATF3 siRNA (IDT, Coralville, IA; Figure IIIA and IIIB in the online-only Data Supplement). IL-1α, Kruppel-like factor 4, and VEGF transcription also were moderately decreased (Figure 2B). siRNA inhibition of ATF3 did not alter lipolysis product induction of NFKB1 or IL-6 (Figure 2D); the latter is known to be regulated by NFκB. Also, knockdown of ATF3 after lipolysis product treatment significantly increased NFKBIA. These data suggest a specific and possibly a novel activating effect of endothelial cell ATF3, a transcription factor that was initially identified as a repressive transcription factor for E-selectin and IL-8 expression.

JNK siRNA Decreased ATF3 Expression

To examine the association between JNK activation and ATF3 expression, we transfected HAECs with scrambled siRNA or JNK siRNA for 48 hours followed by treating cells with TGRL+LpL for 3 hours. JNK siRNA significantly repressed transcription of JNK (80%; Figure IVA in the online-only Data Supplement) compared with scrambled siRNA. The JNK siRNA treatment also produced a decrease in JNK protein (Figure IVB in the online-only Data Supplement). siJNK resulted in significantly decreased ATF3, E-selectin, JunB, and IL-8 mRNA expression (Figure 3A). In contrast, mRNA expressions of IL-6, NFKBIA/IκB, and NFKB1/NFκB were not altered by JNK siRNA (Figure 3A). JNK siRNA transfection significantly decreased lipolysis product–induced protein expression of ATF3 and p-c-Jun (Figure 3B and 3C). In contrast, lipolysis product–induced increase of c-Jun protein expression was not significantly altered by JNK knockdown. A different construct of JNK siRNA produced similar results (Figure VA and VB in the online-only Data Supplement).

Figure 3.

Figure 3. Effect of c-Jun NH2-terminal kinase (JNK) small interfering RNA (siRNA) on gene transcription and translation. A, Alterations in gene transcription are normalized to that of GAPDH, with fold changes calculated as the difference in expression of factors in the presence of triglyceride-rich lipoprotein lipolysis products and the exposure to either scrambled siRNA or JNK siRNA. Cells were exposed to siRNA 48 hours before lipolysis product treatment (n=3; *P≤0.05). B, Alterations in activating transcription factor 3 (ATF3) protein. C, Alterations in p-c-Jun and c-Jun. For both B and C, (a) Western blot, (b) densitometry quantification; n=3, *P≤0.05 for comparisons between scrambled siRNA exposed to media (M) and TGRL lipolysis (T+L) and JNK siRNA exposed to M and T+L; #P≤0.05 difference between T+L in scrambled siRNA and JNK siRNA. IL indicates interleukin; and NFKBIA, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α.

Both ATF3 and JNK Contribute to TGRL Lipolysis Product–Induced Endothelial Cell Apoptosis

TGRL lipolysis products increased caspase-3/7 activity significantly 3 hours after treatment (Figure 4A). Two lipids previously characterized as injurious to endothelium, 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC; 40 µg/mL) and 13-hydroxyoctadecadienoic acid (50 µmol/L) also significantly increased caspase-3/7, but to a much lesser extent than TGRL lipolysis products. Additionally, both ATF3 siRNA (Figure 4B) and JNK siRNA (Figure 4C) significantly suppressed lipolysis products–induced caspase-3/7 activity. Figure 4D shows the results of a different measure of apoptosis, a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, which again depicts the involvement of ATF3 and JNK in lipolysis-induced apoptosis. A set of representative images relative to the terminal deoxynucleotidyl transferase dUTP nick end labeling assay are recorded in Figure VI in the online-only Data Supplement.

Figure 4.

Figure 4. Triglyceride-rich lipoprotein (TGRL) lipolysis increased apoptosis as measured by caspase-3/7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. A, Caspase-3/7 activity was significantly increased with TGRL lipolysis (TGRL + lipoprotein lipase [LpL]) after 3 hours of incubation. *TGRL+LpL compared with media (M), LpL, or TGRL alone and oxPAPC or 13-hydroxyoctadecadienoic acid (13-HODE) compared with media; #oxPAPC (40 µg/mL) or 13-HODE (50 µmol/L) compared with TGRL+LpL treatment. B, The caspase-3/7 activity significantly decreased in human aortic endothelial cells (HAECs) pretreated with activating transcription factor 3 (ATF3) small interfering RNA (siRNA) transfected for 18 hours. C, HAECs transfected with c-Jun N-terminal kinase (JNK) siRNA for 48 hours and treated with TGRL lipolysis products for 3 hours. *TGRL+LpL compared with media; #Scrambled compared with either ATF3 or JNK siRNA treatment for T+L. For A, B, and C, n=5 treatments/group, P≤0.05. D, TGRL lipolysis product–induced apoptosis is significantly reduced by ATF3 siRNA and JNK siRNA. n=4 treatments/group. *P≤0.05 compared with media control, #P≤0.05 scrambled compared either ATF3 or JNK siRNA treatment for T+L assayed by TUNEL.

Infusion of Lipolysis Products Activates ATF3 in Intact Carotid Arteries

To determine the applicability of cell culture results to intact tissues, we treated mouse carotid arteries in situ by cardiac puncture and perfuse with media, TGRL, or TGRL lipolysis products. As shown in Figure 5A, ATF3 was present at very low levels in arterial cell cytoplasm in media-treated C57BL/6 mice but was extensively expressed and accumulated in the nucleus of carotid endothelial and medial cells from TGRL lipolysis products–infused mice. Counts of ATF3 positive nuclei by a treatment-blinded observer confirmed that the nuclear accumulation of ATF3 was significantly higher in carotid arteries perfused with TGRL lipolysis products relative to media or TGRL alone (Figure 5B). These results demonstrate that activation of ATF3 in response to TGRL lipolysis occurs in the endothelium of intact vessels.

Figure 5.

Figure 5. Triglyceride-rich lipoprotein (TGRL) lipolysis products activate activating transcription factor 3 (ATF3) expression in mouse carotid arteries. A, Immunostaining for ATF3 protein expression. B, Quantitative evaluation of ATF3 expression in the mice carotid arteries after perfusion with media alone, TGRL alone, or TGRL lipolysis (TGRL + lipoprotein lipase [LpL]) for 15 minutes. n=4 mice/group, *P≤0.02, **P≤0.05. Original magnification ×60. White arrow denotes ATF3 accumulation in nucleus, and yellow arrow, nuclear staining absent. DAPI indicates 4',6-diamidino-2-phenylindole.

Mice Genetically Deficient in ATF3 (ATF3−/−) Fail to Activate Carotid Artery Endothelial Cell Apoptosis in Response to Treatment With Lipolysis Products

We asked whether lipolysis products induce vascular apoptosis in vivo and whether this is ATF3 dependent. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining of carotid arteries 3 hours after femoral vein infusion showed apoptotic endothelial cells in carotid arteries of male C57BL/6 mice treated with either TGRL or TGRL lipolysis products. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining (Figure 6A) was not present in carotid artery endothelium from mice injected with either PBS or LpL alone. Counts of endothelial cells showed carotid arteries perfused with TGRL alone, and TGRL lipolysis products had significantly increased numbers of apoptotic cells (47% and 30%) compared with PBS control (Figure 6B). There was no statistical difference in apoptosis induced by TGRL alone compared with TGRL lipolysis products. We reasoned that TGRL underwent lipolysis as a result of interacting with endogenous LpL, thus increasing endothelial cell apoptosis. ATF3−/− mice had no increase in the incidence of apoptosis in response to either TGRL or TGRL lipolysis product infusion.

Figure 6.

Figure 6. Triglyceride-rich lipoprotein (TGRL) lipolysis product–induced apoptosis is reduced in ATF3−/− mouse carotid arteries. A, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of mouse carotid artery. B, Percentage of apoptosis of endothelial cells based on FITC and nuclear staining. Both TGRL (47%) and TGRL lipolysis (TGRL + lipoprotein lipase [LpL]; 39%) significantly induced apoptosis compared with PBS control only in wild type (WT) carotid arteries, not in ATF3−/− mice. Positive control: DNAse I treated; negative control: without rTdT enzyme. n=4 mice/group, *P≤0.05 compared with PBS control of WT mice, #P≤0.05 ATF3−/− compared with WT treatment group. Bar, 20 µm. ATF3 indicates activating transcription factor 3; and DAPI, 4',6-diamidino-2-phenylindole.

ATF3 Expression in Human Peripheral Blood Mononuclear Cells

We previously have shown that ingestion of a moderately high-fat meal or treatment with TGRL lipolysis products activates monocytes and causes lipid droplet formation.20 Results of the present study suggest augmented ATF3–mediated inflammatory responses in the postprandial state. We compared ATF3 and cytokine (IL-6 and chemokine [C-C motif] ligand-2) gene expression in fasting and postprandial peripheral blood mononuclear cells isolated from healthy individuals. mRNA expressions of ATF3, IL-6, and chemokine (C-C motif) ligand-2 (monocyte chemoattractant protein-1) were increased 1.6-, 1.9-, and 3.4-fold (Figure VII in the online-only Data Supplement) in postprandial peripheral blood mononuclear cells compared with expression in peripheral blood mononuclear cells from fasting individuals. This work suggests that even a single moderately high-fat meal with increased generation of TGRL lipolysis products can regulate monocyte gene expression to enhance proinflammatory pathways.


Our experiments demonstrated a robust proinflammatory and proapoptotic response when endothelial cells were treated with TGRL lipolysis products in high physiological to pathophysiological concentrations. Among the 266 genes induced by lipolysis products, the 2 largest categories were transcription factors (23%) and inflammation (11%). The next largest categories were related to protein binding, signaling pathways, and cell cycle alterations. Four genes involved in cell stress response pathways, c-Jun, JunB, CCAAT/enhancer binding protein (C/EBP) β, and ATF3, were highly expressed in TGRL lipolysis–treated cells.

The 4 major mitogen-activated protein kinase pathways, extracellular signal-regulated kinase, JNK, P38, and BMK/extracellular signal-regulated kinase 5, perform activating phosphorylations of nuclear transcription factors. Of these, ERK has been shown to inhibit ATF3 expression, whereas JNK activates ATF3 through transcriptional regulation.12 JNK activation also results in phosphorylation of c-Jun, a component with ATF3 of a complex that binds AP-1 responsive promoter regions. Although both ATF3 and c-Jun promote apoptosis,21 this is opposed by JunB, another transcription factor activated through the NFκB pathway. CCAAT/enhancer binding protein (C/EBP) β acts to amplify NFκB-mediated IL-6 transcription through epigenetic modulation at the interface between ATF3 and NFκB signaling.22

Previous studies have shown upregulation of ATF3 to act as either proinflammatory23 or anti-inflammatory.2426 The dimeric state of ATF3 gives us clues as to the ultimate effects of ATF3 on inflammation. As a homodimer, ATF3 acts as a transcriptional regulator inhibiting expression of proapoptotic molecules. Alternatively, ATF3 can form a heterodimer with activated c-Jun that enhances transcription of stress response genes.27 Cellular responses to increases in ATF3,28 c-Jun,16 and JunB27,29 reflect the increased probability of these factors interacting with promoter sites independently or in combination.27,29 Our results suggest lipolysis of TGRL initiates JNK-mediated signaling that drives ATF3 complexes toward proapoptotic and proinflammatory responses through formation of a phospho-c-Jun/ATF3 AP-1–binding complex. Furthermore, although JunB mRNA levels increase, protein levels are decreased in lipolysis product–exposed cells. JNK regulates the turnover of both c-Jun and JunB by phosphorylation-dependent activation of the E3 ligase Itch.30,31 Because JunB is associated with NFκB signaling, increased transcription of IκBA and downregulation of JunB in response to lipolysis products emphasize inhibition of NFκB signaling. Correlatively, siRNA knockdown of ATF3 was without effect on the NFκB-associated expression of IL-6. In general, JNK activation in endothelial cells is considered a proapoptotic event, whereas NFκB promotes cell survival. This observation also implies a predominance of proapoptotic responses that correlate well with our previous demonstration that lipolysis products induce apoptosis in HAECs.7

siRNA knockdown experiments suggest the apoptotic response depends on both JNK and ATF3. This is correlated with prevention of carotid apoptosis in ATF3−/− mice treated with lipolysis products. JNK siRNA decreased ATF3, E-selectin, JunB, and IL-8 transcription. Similarly, ATF3 siRNA decreased E-selectin and IL-8 transcription but was without effect on NFκB-associated genes. These results suggest that JNK is an upstream regulator of AP-1 signaling through both phosphorylation of c-Jun and transcription of ATF3. We performed JNK siRNA experiments with 2 sequences with both treatments demonstrating significant but partial reduction in ATF3 expression (Figure 3; Figure V in the online-only Data Supplement). This suggests that JNK signaling augments constitutive ATF3 expression through uncertain mechanisms that could include transcriptional activation by an unidentified transcription factor or mRNA stabilization. As a consequence, expression of both JNK and ATF3 is important in stimulating vascular inflammation through ATF3/c-Jun–mediated upregulation of IL-8 and E-selectin.

Our microarray data suggest a 30-fold induction of the superoxide dismutase 3 gene (SOD3), 10-fold induction of prostaglandin-endoperoxide synthase 2, also known as COX2 (Table III in the online-only Data Supplement), which are oxidative stress response genes. A related gene, phospholipase A2 (PLA2G6), had a 2-fold induction. A recent study suggests cross-talk between PLA2 and the mitogen-activated protein kinase pathway.32 These systems interact principally to increase the duration of JNK signaling with prolonged signaling necessary for induction of apoptosis. In contrast, decreased JNK signaling is regulated through NFκB-mediated upregulation of a variety of inhibitors of JNK activation, including tumor necrosis factor receptor type 1-associated DEATH domain protein inhibitors, iron, and reactive oxygen species scavengers, and specific inhibitors of activating enzymes involved in JNK signaling.33 The constellation of findings in our study implies that lipolysis products create an imbalance between MAPK-JNK and NFκB pathways that favor ATF3/AP-1–mediated inflammation and apoptosis.

A number of other biological molecules and pathways could contribute to the proinflammatory and proapoptotic effects of TGRL lipolysis products. For example, key inflammatory and anti-inflammatory genes were upregulated by lipolysis products, including IL-1α, IL-6, IL-8, VEGF, and peroxisome proliferator–activated receptor γ. Most of these genes activate monocytes and endothelial cells. IL-1α is likely pivotal in inflammatory activation because it has been shown to act as an autocrine stimulus upregulating IL-6, IL-8, and VEGF in complement-treated endothelium.34,35 In addition, 3 leukocyte adhesion molecules, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, were upregulated by lipolysis products. Furthermore, induction of IL-8 and chemokine (C-C motif) ligand-2 (monocyte chemoattractant protein-1) has been shown to be increased by oxPAPC.36 oxPAPC is a prototypic biologically active oxidized phospholipid first isolated from minimally modified low-density lipoprotein, and oxPAPC is a known stimulator of ATF3.37 Also, 13-hydroxyoctadecadienoic acid is the most prevalent oxylipid in atherosclerotic plaque, a TGRL lipolysis product,5 and is a ligand for PPARs.38,39 More studies are needed to further define proinflammatory and anti-inflammatory factors in lipolysis products.

ATF3 has been reported to be the transcriptional inhibitor responsible for PPAR activator modulation of E-selectin upregulation in response to tumor necrosis factor-α.40 In contrast to previous reports suggesting an inhibitory role for ATF3 in inflammation, we found ATF3 expression was necessary for induction of some, but not all, proinflammatory responses to lipolysis product treatment. In particular, transcription of IL-1α, E-selectin, IL-8, and VEGF was inhibited by ATF3 siRNA. In contrast, ATF3 inhibition had no effect on IL-6 or NFκB transcription, suggesting a complex inter-relationship between these 2 pathways.

Lipolysis-activated cellular apoptosis was suppressed by pretreatment with ATF3 siRNA. After knockdown of ATF3, cell viability was significantly increased compared with scrambled siRNA-treated cells. This suggests that ATF3 inhibits cell growth and could reflect its proapoptotic properties. It also suggests that there is basal expression of the ATF3 gene that may contribute to the phenotype of endothelial cells. Furthermore, JNK siRNA also suppressed lipolysis products–induced mRNA expression of ATF3, E-selectin, JunB, and IL-8 and decreased protein expression of ATF3 and p-c-Jun. Knockdown of JNK also suppressed apoptosis. These results suggest that JNK signaling is upstream of ATF3.

Experiments in cell culture are inherently limited by the altered extracellular context and absence of multicell interactions. We performed 2 experiments with mice asking whether our findings in vitro were applicable to tissues from living animals. In this study, infusion of lipolysis products into carotid arteries confirmed upregulation and nuclear accumulation of ATF3 in endothelium and cells of the media in intact vessels. The second experiment demonstrated activation of apoptosis in endothelial cells of carotid arteries in mice infused with either TGRL or TGRL lipolysis products intravenously. The activation of apoptosis in these in vivo studies seemed to be dependent on ATF3 activity because mice genetically deficient in ATF3 did not have a vascular apoptosis response to lipolysis product infusions. Activation of apoptosis in vivo by TGRL alone suggests that endogenous lipolysis is sufficient to generate products that injure endothelium. We did not test the ability of in vivo infusion of lipolysis products to induce carotid inflammation in this study. Our findings correlate with previous demonstration of ATF3 expression in association with apoptosis in atherosclerotic plaques.41

Our study with human peripheral blood monocytes suggests a dynamic upregulation of ATF3 correlated with monocyte cytokines expression in the postprandial state of a moderately high-fat meal. Because expression of LpL is thought to be a local process regulated by specific tissues, the concept that hypertriglyceridemia could lead to increased concentrations of lipolysis products at the blood–endothelial cell interface is an intriguing phenomenon that deserves further study.

In conclusion, our study showed a robust and rapid induction of transcription factor ATF3–related genes in the JNK pathway and cytokines associated with AP-1 signaling in endothelial cells treated with TGRL lipolysis products. Furthermore, our mouse carotid artery and monocyte experiments documented the pathophysiological relevance of our observations. These studies indicate that high physiological to pathophysiological concentrations of TGRL lipolysis products can cause vascular inflammation and apoptosis that potentially render the artery more susceptible to the development of atherosclerosis.


We thank Dr Tsonwin Hai, Department of Molecular and Cellular Biochemistry and Center for Molecular Neurobiology, The Ohio State University, Columbus, OH for providing ATF3-deficient mice.


The online-only Data Supplement is available with this article at

Correspondence to Hnin H. Aung, PhD, Division of Cardiovascular Medicine, 5404 Genome and Biomedical Sciences Facility, 451 E Health Sciences Dr, University of California, Davis, CA 95616. E-mail


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Elevation of triglyceride-rich lipoproteins contributes to the risk of atherosclerotic cardiovascular disease and can induce endothelial dysfunction and inflammation. Our studies identified transcription factor–activating transcription factor 3, a member of the cAMP response element-binding protein (CREB) family, as a key response gene after treatment with triglyceride-rich lipoprotein lipolysis products and demonstrated that its induction was essential for the expression of a subset of proinflammatory responses. These findings correlate with previous demonstration of activating transcription factor 3 expression in association with apoptosis in atherosclerotic plaques. This study demonstrates that triglyceride-rich lipoprotein lipolysis products activate activating transcription factor 3–c-Jun NH2-terminal kinase transcription factor networks and induce endothelial cells inflammatory response. Thus, activating transcription factor 3 could represent a key regulatory protein in triglyceride-rich lipoprotein lipolysis product–mediated vascular inflammation and atherosclerosis.