Atherosclerosis and Lipoproteins

Apolipoprotein E3- and Nitric Oxide–Dependent Modulation of Endothelial Cell Inflammatory Responses

Adam E. Mullick, Andrew F. Powers, Rama S. Kota, Sarada D. Tetali, Jason P. Eiserich, John C. Rutledge
https://doi.org/10.1161/01.ATV.0000253947.70438.99
Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:339-345
Originally published January 17, 2007

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Abstract

Objective— Although apolipoprotein E3 (apoE3) is known to be atheroprotective, its mechanisms of protection in endothelial cells remain unclear.

Methods and Results— Cultured human aortic endothelial cells were stimulated with tumor necrosis factor (TNF)-α in the presence of human recombinant apoE3 solubilized in dimyristoyl phosphatidylcholine liposomes. Using flow cytometry and real-time polymerase chain reaction, a significant increase of inflammatory cell adhesion proteins (vascular cell adhesion molecule-1 and E-Selectin), and MCP-1, interleukin-8, and intercellular adhesion molecule-1 gene expression was observed within 5 hours of TNF-α exposure, which was markedly attenuated in cells coincubated with apoE3. Treatment with apoE4 resulted in increased inflammatory gene expression relative to either TNF treatment alone or TNF + apoE3 treatment. NO synthase inhibition experiments demonstrated NO to be an active participant in the actions of both TNF and apoE. To clarify the role of NO, dose-response experiments were performed with 0.03 to 300 μmol/L DEA-NONOate. Using flow cytometry and real-time polymerase chain reaction, a modulatory role of NO in TNF-induced endothelial cell activation was observed.

Conclusions— These data suggest a role of vascular wall apoE3 to balance the intracellular redox state in injured endothelial cells via NO-dependent pathways.

Complications arising from atherosclerosis represent a major cause of morbidity and mortality worldwide.1 Animal and human studies have revealed a clear role of apolipoprotein E3 (apoE3) and nitric oxide (NO) as atheroprotective.2,3 Illustrative of the significance of apoE3 in mitigating atherosclerotic disease, results from the Framingham Offspring Study led investigators to conclude that the incidence of coronary artery disease (CAD) associated with defective apoE isoforms ε2 and ε4 appeared greater than that for any known genetic lipid abnormality.4,5 Because this multifunctional protein has many actions, it is difficult to mechanistically separate the atheroprotective properties of apoE3 on plasma lipid levels and on vascular cell responses to inflammation.

The atheroprotective effect on plasma lipoprotein metabolism is well known. However, the direct role of apoE3 in extrahepatic tissue remains less clear. Our data and others demonstrate that apoE3 can increase vascular tissue lipoprotein accumulation.6–8 Apart from the role of apoE3 in lipoprotein metabolism, animal studies have demonstrated an atheroprotective effect of apoE distinct from an effect on plasma lipoprotein levels.9–12 Furthermore, studies have described multiple mechanisms, distinct from lipoprotein metabolism, which may facilitate the antiatherogenic role of apoE in the vasculature. Candidate antiatherogenic functions of apoE include inhibition of platelet activation,13 immunomodulation of cytokine release,14,15 antiproliferative activity,16–18, and antioxidative properties.19 It has been demonstrated that apoE can directly increase intracellular NO via activation of NO synthase in both platelets and endothelial cells.13,20,21 Conversely, epidemiological and experimental data have demonstrated an opposing role of apoE4 on various inflammatory processes. For example, studies have demonstrated that the ε4 isoform is proinflammatory,22 possibly through direct oxidant properties19,23 or through deficits in NO signaling.21 In light of these findings, it appears that apoE3, but not apoE4, may act directly on vascular cells to attenuate processes that contribute to atherosclerosis.

The aim of this study was to determine the endothelial cell–specific effects of apoE3 on modulation of endothelial cell activation and inflammation using cultured human aortic endothelial cells (HAECs). Endothelial cell activation was assessed with both inflammatory protein and gene expression. Herein, we demonstrate that apoE3 can markedly attenuate TNF-α–induced inflammatory responses in cultured human endothelial cells, and that the mechanism underlying this is related to its ability to modulate endothelial NO. Furthermore, we demonstrate that the antiinflammatory effects of apoE3 on gene expression were reversed with apoE4.

Materials and Methods

Cell Culture

HAECs were purchased from Cascade Biologics and grown to confluence in Medium 200 supplemented with low serum growth supplement and penicillin, streptomycin, and amphotericin B. Experiments were conducted with cells at passage 5 to 6 and removed from culture dishes using a trypsin/EDTA solution followed by a trypsin neutralizer (Cascade Biologics). A typical experiment involved cells grown to confluence in a T-25 flask (≈800 000 cells), with 120K cells used for flow cytometry, 280K cells used for mRNA assays, and 400K cells used for protein assays. An acute inflammatory stimulus was produced by coincubation of human recombinant TNF-α (Roche Applied Science).

Generation of ApoE3 and ApoE4 Liposomes

Small unilamellar vesicles of the saturated phospholipid 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC; Avanti Polar Lipids) were produced by sonication for lipidation of apoE3 or apoE4. Briefly, a solution of 10 mg/mL DMPC was sonicated with a tip sonicator set to 20 watts for 10 minutes in a solution of 0.15 mol/L NaCl, 10 mmol/L Tris-HCl, and 1 mmol/L EDTA (pH 7.6). Generation of 30 to 50 nmol/L vesicles was detected by visual inspection of the DMPC solution, which turns from cloudy to clear on vesicle generation. Additionally, laser light scattering was performed on sonicated liposomes to characterize particle sizes and distribution. ApoE3 or apoE4 was added to liposomes at a lipid to protein ratio of 4:1 (w/w).24 This solution was vortexed for 10 seconds and incubated for 1 to 2 hours at 25°C, above the Tm of DMPC (23°C), to facilitate apoE protein lipidation. The expression and purification of apoE4 is described in the supplemental Methods (available online at http://atvb.ahajournals.org).

Inflammatory Gene Chip Analysis

Inflammatory gene profiling was performed using a NF-κB signaling-specific gene chip array designed to profile 96 NF-κB–dependent inflammatory genes (GEArray Q Series HS-016, SuperArray). Candidate genes were considered if their expression levels were at least 5% of the average of housekeeping gene expression levels (GAPDH, cyclophilin A and β-actin). Please refer to the supplemental Methods addendum for a detailed description of the methodology of gene chip hybridization and detection.

Quantitative Real-Time Polymerase Chain Reaction Analysis

Detection of gene transcript levels was performed using the GeneAmp 5700 sequence detection system with SYBR Green (Applied Biosystems). Real-time quantitation of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), intracellular adhesion molecule-1 (ICAM-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene levels were performed from cDNA prepared from total RNA extracts isolated from ≈300 000 HAECs grown to confluence. Each treatment group was performed at least in duplicate. Optimal sample cDNA and primer concentration was performed to determine efficiency and specificity of all primers. Verification of appropriate polymerase chain reaction (PCR) product formation was determined by analysis of product heat-dissociation curves and amplification plots, with product quantification values taken from the linear region of the PCR curve. Inflammatory gene expression levels were normalized to GAPDH levels from each sample. Please refer to supplemental Methods for MCP-1, IL-8, ICAM-1, and GAPDH sequence information and PCR protocol.

Measurement of Vascular Cell Adhesion Molecule-1 and E-Selectin by Flow Cytometry

Cell surface expression of vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin was performed with a Beckman Coulter Epics XL FACScan with Expo 32 XL 4 Color software. Positive and negative controls for each fluorophore were performed to determine optimal levels of PMV gain and sensitivity and set correct parameters for compensation. Between 5000 and 10 000 events were collected for each sample, with samples performed in duplicate. Please refer to supplemental Methods for complete description of sample preparation for flow cytometry.

Results

ApoE3 Attenuates Surface Adhesion Molecule Expression

Treatment of HAECs with TNF-α (50 ng/mL) and the apoE3 lipid carrier DMPC (200 μg/mL) for 5 hours resulted in increased VCAM-1 and E-Selectin expression (P<0.001; Figure 1B and 1D). Before TNF administration, less than 1% expression of VCAM-1 or E-Selection could be recorded, whereas, after TNF administration, VCAM-1 and E-Selectin expression were 65.0±2.4% and 28.7±2.5%, respectively (Figure 1D). Cells coincubated with 50 μg protein/mL of apoE3-DMPC had significantly decreased VCAM-1 and E-Selectin expression (Figures 1C, 1D, and 2⇓A). This apoE3-dependent reduction of inflammatory adhesion molecule expression was similar to that observed in cells coincubated with the strong antioxidant pyrrolidine dithiocarbamate (PDTC; Figure 2B).

Figure1

Figure 1. ApoE3 mitigated TNF-induced protein expression of VCAM-1 and E-Selectin. HAECs were stimulated with TNF-α (50 ng/mL) for 5 hours and cells were analyzed by flow cytometry for surface expression of VCAM-1 and E-Selectin using flow cytometry. A, forward and side scatter properties of HAECs. Cells analyzed for antibody staining were derived from gate A. B, HAECs exposed to only TNF. C, Cells coincubated with TNF and apoE (50 μg protein/mL). D, Quantitation of VCAM-1 and E-Selectin expression. A significant reduction in both VCAM-1 and E-Selectin expression was seen in cells with apoE3 coincubation. *P<0.05.

Figure2

Figure 2. The antiinflammatory effect of apoE3 was reduced in the presence of L-NMMA. Representative histogram plots illustrate fluorescence intensity shift of VCAM-1 positive staining. A, gray=TNF; black=TNF + apoE3 (50 μg protein/mL); B, gray=TNF; black=TNF + PDTC (100 μmol/L); C, gray=TNF + L-NMMA; black=TNF + L-NMMA (1 mmol/L)+apoE3 (50 μg protein/mL). All cell populations were exposed to 50 ng/mL TNF-α for 5 hours. DMPC was added to the media in all treatments lacking apoE3.

Becaues the actions of apoE3 have been shown to be related to NO, experiments were performed to address the role of NO in TNF-induced inflammatory responses in HAECs. TNF-treated cells were coincubated with apoE3-DMPC and NG-Monomethyl-l-Arginine Monoacetate (L-NMMA), a general NO synthase (NOS) inhibitor. Relative to cells exposed to TNF-α alone, L-NMMA resulted in a decrease in VCAM-1 expression (Figure 2C versus 2A or 2B, gray histograms), suggesting NO to be partly involved in TNF-induced VCAM-1 expression. With the addition of apoE3 to similarly treated cells, we observed reduced VCAM-1 expression (Figure 2C, black histogram). Comparing the effects of apoE3 in the presence or absence of NO inhibition (Figure 2C and 2A, black histograms), we see that apoE3 did not decrease VCAM-1 staining to the same extent with L-NMMA coincubation.

These data suggested that part of the ability of apoE3 to blunt acute responses induced by TNF-α was partly mediated through NO. Experiments with D-NMMA, an inactive isomer of L-NMMA, resulted in no appreciable change of TNF-induced expression of VCAM-1. Further, an analysis of cell viability using propidium iodide (PI) uptake after exposure of cells to TNF-α and/or apoE3 demonstrated no changes in viability following treatments.

Effect of NO Oxide on Surface Adhesion Molecule Expression

To clarify the role of NO in acute inflammatory responses induced by TNF-α in HAECs, dose–response experiments were performed using DEA-NONOate (DEANO; A.G. Scientific), an NO donor. In cells exposed to TNF-α, we coincubated the cells with 0.03 to 300 μmol/L DEANO and analyzed VCAM-1 and E-Selectin surface protein expression from 1.5 to 5 hours after treatment. Similar doses of DEANO have previously been shown to result in modulation of cell inflammation in murine macrophages.25 Preliminary studies examining the effects of DEANO in modulating TNF-induced VCAM-1 or E-Selectin expression suggested a dose–response effect with similar dose effects of DEANO at 0.03 and 0.3 or 30 and 300 μmol/L. Therefore, we used 0.03, 3.0, and 300 μmol/L DEANO for all subsequent experiments. Peak values of [NO] were measured with an NO electrode and were as follows: 0.03 μmol/L DEANO=70.5±4.6 nmol/L NO, 3 μmol/L DEANO=1347.7±110.0 nmol/L NO, 300 μmol/L DEANO=22009±1885 nmol/L NO. Please refer to supplemental Methods for complete description of NO measurement.

After 5 hours of incubation with TNF-α (50 ng/mL), significant induction of VCAM-1 or E-Selectin was recorded in cultured HAECs (24.4±5.2% and 14.5±2.5% cellular positive VCAM-1 and E-Selectin expression, respectively). Coincubation of TNF with 3.0 and 300 μmol/L DEANO resulted in a significant decrease in VCAM-1 and E-Selectin expression (Figure 3A). Early time points of TNF-α treatment in HAECs did not result in a significant induction of VCAM-1, but did cause an increase in E-Selectin expression. E-Selectin positive cells could be measured as early as 1.5 hours after TNF-α incubation. At 2 hours, 44±2.0% cells were positive for E-Selectin, which was greater than that seen at the 5 hour time point described above. With 2 hours coincubation of DEANO with TNF-α, a dose-dependent biphasic response in E-Selectin expression was observed. E-Selectin expression peaked with 3.0 μmol/L DEANO and was reduced with 300 μmol/L DEANO (Figure 3C).

Figure3

Figure 3. Modulation of TNF-induced protein expression of VCAM-1 and E-Selectin by NO. HAECs were exposed to TNF-α (50 ng/mL) and DEANO and then analyzed with flow cytometry for surface VCAM-1 or E-Selectin expression. A, Flow cytometric analysis of cell surface VCAM-1 and E-Selectin demonstrated attenuation of these inflammatory proteins in the presence of increasing levels of DEANO after 5 hours coincubation of DEANO and TNF-α. B, Representative histogram plots illustrate fluorescence intensity shift of VCAM-1 positive staining in the presence of TNF and/or DEANO for data summarized in A. C, HAECs exposed to TNF-α (50 ng/mL) and DEANO for 2 hours demonstrated biphasic E-Selectin expression, with maximal expression at 3.0 μmol/L DEANO and minimal expression at 300 μmol/L DEANO. Data in graphs are presented as % cells positive for antibody staining relative to positive control (TNF). *P<0.05 vs TNF treatment, †P<0.05 vs 3.0 μmol/L DEANO.

Experiments were performed to evaluate the effect of DEANO on cellular activation in the absence of TNF-α. At all time points up to 5 hours, we could not detect a change in E-Selectin or VCAM-1 expression in cells exposed to 0.03 to 300 μmol/L DEANO. Furthermore, at these doses of DEANO, we did not detect a change in cell viability (PI staining) after up to 6 hours of exposure.

Effects of ApoE3, ApoE4, or NO on Inflammatory Gene Expression

HAECs were exposed to TNF-α (50 ng/mL) and DMPC (200 μg/mL) or apoE3-DMPC (50 μg protein/mL) for 5 hours. Real-time quantitative PCR analysis was performed for MCP-1, IL-8, ICAM-1, and GAPDH (used as a housekeeping gene). All treatments with TNF-α had significant increases in MCP-1, ICAM-1, and IL-8 relative to untreated cells. HAECs stimulated with TNF-α and coincubated with apoE3-DMPC expressed significantly less MCP-1, IL-8, and ICAM-1 transcript levels relative to apoE3-untreated cells (Figure 4). However, coincubation of cells with apoE4 demonstrated a proinflammatory role of this apoE isoform, with increased in MCP-1, IL-8, and ICAM-1 gene expression observed relative to either TNF treatment alone or TNF + apo3-treated cells.

Figure4

Figure 4. The induction of inflammatory gene expression by TNF was sharply attenuated by apoE3 and increased by apoE4. HAECs were exposed to TNF-α (50 ng/mL), apoE3 (50 μg protein/mL), or apoE4 (50 μg protein/mL) for 5 hours. Quantitative real-time PCR analysis was performed for MCP-1, IL-8, ICAM-1, and GAPDH. Treatment with TNF resulted in significant increases in inflammatory gene expression relative to control, which was significantly reduced with apoE3. Contrary to the antiinflammatory effects of apoE3, apoE4 increased the proinflammatory effects of TNF. DMPC was added to the media in all treatments lacking apoE3 or apoE4. Gene expression levels are reported relative to GAPDH ±SEM. Addition of TNF in all treatment groups caused a significant increase in MCP-1, IL-8, and ICAM-1 expression. Significant differences among treatment groups for all three genes are denoted by * (TNF vs Media), † (TNF + apoE3 vs TNF or TNF + apoE4), or ‡ (TNF + apoE4 vs TNF); P<0.05.

To confirm that our real-time PCR gene targets provided sensitive markers of TNF-induced endothelial inflammation, a 96 NF-κB–specific inflammatory gene array was performed. HAECs were exposed to TNF-α (50 ng/mL) for 5 hours. Of the 96 inflammatory genes profiled, 26 were expressed at levels of at least 5% relative to the average expression levels of the 3 housekeeping genes, GAPDH, cyclophilin A, and β-actin. Of these 26 genes, 15 demonstrated at least a 100% change in expression after TNF-α incubation. From these 15 TNF-induced genes, our real-time quantitative PCR targets represented 3 of the top 10 expressed genes (Table).

View this table:

Top 10 TNF-Induced Inflammatory Genes Measured From Cultured HAECs

Using the inflammatory gene array, an analysis of the effects of 3.0 or 300 μmol/L of DEANO on TNF-induced inflammation were performed. With a cutoff threshold of at least a 100% change, 16 genes were reduced in expression and 5 were increased in expression (supplemental Table I). Additionally, 4 genes demonstrated a biphasic response, with increased expression levels in 3.0 μmol/L DEANO (at least a 100% increase from TNF treatment alone) and decreased expression levels in 300 μmol/L DEANO (at least a 100% decrease from 3.0 μmol/L DEANO; supplemental Table I).

Cellular Uptake of ApoE3-DMPC

Studies were performed in HAECs to determine whether DMPC-solubilized apoE3 could be endocytosed in this model vascular endothelial cell. After a 5-hour coincubation with TNF-α (50 ng/mL), Western blot analysis of HAEC cytoplasmic extracts demonstrated an intracellular localization of apoE, which was absent in cells not incubated with apoE3 (Figure 5).

Figure5

Figure 5. ApoE3 was increased in cell extracts after apoE3 incubation. Cytosolic extracts from HAECs incubated for 5 hours in the absence (lane 1) or presence (lane 2) of 50 μg/mL apoE3-DMPC.

Discussion

The association between atherosclerosis and apoE3 has been known for some time.26 Studies have demonstrated the beneficial role of apoE3 in preventing or attenuating cardiovascular diseases.2,27 What remains unclear are the mechanisms by which apoE3 exerts its beneficial effects. Although many studies have illustrated the effects of apoE3 on blood cholesterol levels and preventing cardiovascular diseases, such as atherosclerosis, less is known about the direct effects of apoE3 in mitigating endothelial inflammatory responses.

Studies have provided clues on the immunomodulatory role of apoE3. For example, it has been demonstrated that the absence of apoE alone was key in the increased mortality following lipopolysaccharide (LPS) treatment in apoE knockout mice.28 The protection conferred by apoE could be attributable to a number of factors, such as direct detoxification of LPS or immunomodulation of immune and/or endothelial cells. In vitro studies have demonstrated the ability of apoE3 to attenuate endothelial cell activation29 and regulate NO production.21 Herein, we extend the results of a protective effect of apoE3 in endothelial cells. To our knowledge, this is the first report that demonstrates a protective effect of solubilized recombinant apoE3 against endothelial cell activation. Our data demonstrate that apoE3, but not apoE4, can effectively blunt the inflammatory effects of TNF-α exposure in aortic endothelial cells. Our criteria of endothelial cell activation includes both functional (cell surface expression of inflammatory adhesion molecules) and genomic (inflammatory gene expression) assays.

Recent work is beginning to illuminate the intracellular signaling pathways activated by apoE3.30 These include activation of G proteins to produce cyclic cAMP and activation of protein kinase A.31 Additionally, it has been demonstrated that apoE3 can activate eNOS via a tyrosine phosphorylation event.21 In support of this concept, we observed a decrease in the ability of apoE3 to protect against TNF-induced cell activation in the presence of the NOS inhibitor L-NMMA, suggesting apoE-induced NO release occurs in HAECs and is protective against cell activation. Our data also suggest that this effect does not completely explain the antiinflammatory effect of apoE3 observed in our cells. The ability of apoE3 to exert its antiinflammatory effects in endothelial cells may be related to cellular uptake of apoE3. In cells incubated with apoE3, we observed intracellular inclusion bodies possibly containing lipid and apoE3. Western blot analysis of cytoplasmic extracts of apoE3-treated cells revealed apoE3 uptake. The presence of intracellular apoE3 could be protective because of the ability of apoE3 to act as an antioxidant thereby scavenging TNF-induced production of superoxide anion.19

To further delineate the precise role of NO on endothelial cell activation, we performed dose–response experiments with the NO donor, DEANO. The effect of NO on TNF-induced E-Selectin protein expression was time- and concentration-dependent with augmentation of TNF-induced endothelial cell activation see early (<5 hours), which was reversed with higher doses of DEANO and/or longer (5 hours) incubation periods. Additionally, an inflammatory gene chip analysis illustrated biphasic regulation of several genes, which was dependent on the concentration of the NO donor. A similar response was observed in murine macrophages using identical doses of DEANO.25 At higher doses there was an attenuation of TNF-induced cell activation without a change in cell viability. The biphasic effect of NO on cell activation and inflammation underscores the observations that NO has been implicated in both pro- and antiinflammatory pathways.25,32 Our data also demonstrate that TNF-induced endothelial activation is, in part, dependent on NO. It is conceivable that the dual nature of NO is an important homeostatic means through which the cell uses NO to perform the myriad of actions ascribed to NO.

These results have important mechanistic implications for the roles of apoE and NO in modulation of endothelial cell inflammation and redox states. Importantly, our data demonstrate both a pro- and antiinflammatory role of NO in endothelial cells, suggesting that the ultimate effects of NO are dependent on a balance of NO and other factors that modulate the redox state of the cell. The role of apoE3 in the vascular wall may be to balance the intracellular redox state in injured endothelial cells via NO-dependent and independent pathways. The deleterious apoE isoform ε4 may lack these protective features and therefore contribute to injurious processes. Similar mechanisms have been suggested in apoE3- and apoE4-dependent modulation of cell inflammation after injury in the brain.22,33,34 Because macrophage-derived foam cells secrete apoE during lesion development, this work adds to the concept that apoE functions as an important vascular wall factor secreted in response to cell injury.

Acknowledgments

Sources of Funding

This work was supported in part by grants from the NIH (RO1 HL55667 to J.C.R.), the American Heart Association Western States Affiliate (A.E.M.), the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research (J.C.R.), and the Paul F. Gulyassy Endowed Professorship (J.P.E.).

Disclosures

None.

Footnotes

  • Original received January 8, 2006; final version accepted November 15, 2006.

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  • Apolipoprotein E3- and Nitric Oxide–Dependent Modulation of Endothelial Cell Inflammatory Responses
    Adam E. Mullick, Andrew F. Powers, Rama S. Kota, Sarada D. Tetali, Jason P. Eiserich and John C. Rutledge
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:339-345, originally published January 17, 2007
    https://doi.org/10.1161/01.ATV.0000253947.70438.99
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