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Journal of Lipid Research, Vol. 47, 1406-1415, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

The role of lipolysis in mediating the proinflammatory effects of very low density lipoproteins in mouse peritoneal macrophages

Viswanathan Saraswathi and Alyssa H. Hasty¹

Vanderbilt University Medical Center, Department of Molecular Physiology and Biophysics, Nashville, TN 37232

Published, JLR Papers in Press, April 25, 2006.

¹ To whom correspondence should be addressed. e-mail: alyssa.hasty{at}vanderbilt.edu

	ABSTRACT

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Hypertriglyceridemia is an important risk factor for atherosclerosis, especially in obesity. Macrophages are one of the primary cell types involved in atherogenesis and are thought to contribute to lesion formation through both lipid accumulation and proinflammatory gene expression. In this study, we sought to determine the direct impact of triglyceride (TG)-rich VLDL-induced lipid accumulation on macrophage proinflammatory processes. Incubation of mouse peritoneal macrophages with 100 µg/ml VLDL for 6 h led to 2.8- and 3.7-fold increases in intracellular TGs and FFAs, respectively (P < 0.05). The inflammatory proteins tumor necrosis factor-, interleukin-1ß, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase 3 (MMP3), and macrophage inflammatory protein-1 (MIP-1) were all upregulated by at least 2-fold (P < 0.05) in a dose-dependent manner in VLDL-treated macrophages. The increase in inflammatory gene expression coincided with the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway members extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38 MAPK and was ameliorated by U0126, an inhibitor of ERK1/2. Inhibition of extracellular TG hydrolysis with tetrahydrolipstatin (Orlistat) resulted in the absence of intracellular TG and FFA accumulation and was accompanied by the amelioration of ERK1/2 phosphorylation and MIP-1 gene expression. These data indicate that VLDL hydrolysis, and the subsequent accumulation of intracellular FFAs and TGs, plays a substantive role in mediating the proinflammatory effects of VLDL. These data have important implications for the direct proatherogenic effects of VLDL on macrophage-driven atherosclerosis.

Abbreviations: CE, cholesteryl ester; ERK, extracellular signal-regulated kinase; IL, interleukin; LRP, low density lipoprotein receptor-related protein; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; mmLDL, minimally modified low density lipoprotein; PBMC, peripheral blood mononuclear cell; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; TG, triglyceride; TNF, tumor necrosis factor

Supplementary key words inflammation • gene expression • mitogen-activated protein kinases

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Traditionally, LDLs have been thought to be the key plasma lipoproteins involved in atherosclerotic lesion development. However, with the dramatic increase in the prevalence of obesity around the world, associated lipoprotein abnormalities such as hypertriglyceridemia have been brought into focus. In fact, hypertriglyceridemia is now regarded as one of the main contributing factors to the development of atherosclerotic disease (1, 2), although the exact mechanisms by which triglycerides (TGs) function in atherogenesis are unknown. TGs in fasting plasma are carried in VLDLs, and these VLDLs have been found within human and experimental atherosclerotic lesions (3, 4), providing a rationale to study their direct effects on macrophage functions such as foam cell formation and inflammation. It is well established that VLDL causes the accumulation of TGs and FFAs in macrophages (5–9). This can occur through three different mechanisms: 1) uptake of intact VLDL particles; 2) uptake of VLDL remnants resulting from lipoprotein lipase-mediated lipolysis; and 3) uptake of FFAs produced by VLDL lipolysis, which can be taken into cells through both passive diffusion and receptor-mediated mechanisms. Despite these known pathways of macrophage-mediated VLDL catabolism, the consequences of VLDL-induced lipid accumulation on macrophage inflammatory processes are not known.

Macrophages not only transform into foam cells via the uptake of modified lipoproteins, they also express a battery of inflammatory genes such as cytokines, chemokines, and adhesion molecules that play pivotal roles in atherosclerotic lesion formation. It has been reported that cholesterol-loaded macrophages are an abundant source of tumor necrosis factor (TNF)- and interleukin (IL)-6 (10). In addition, it was recently reported that VLDL potentiates LPS-induced TNF- expression in human macrophages (11) and that VLDL can directly induce IL-1ß and monocyte chemoattractant protein-1 (MCP-1) (12, 13).

Atherosclerosis is considered to be a chronic inflammatory disease of the vessel wall (14). Several chemokines and their receptors are important in the pathogenesis of atherosclerosis. Chemokines such as MCP-1, macrophage inflammatory protein-1 (MIP-1), and IL-8 are reported to be expressed within atherosclerotic plaques (15). MCP-1 is one of the CC family chemokines, whose role in atherosclerosis is well known (15, 16). VLDL has been shown to induce MCP-1 in macrophages (13) and mesangial cells (17). In addition to MCP-1, there are several other CC chemokines associated with advanced atherosclerotic lesions. MIP-1, MIP-1ß, and, to a lesser extent, RANTES are expressed by activated T-cells in human plaques (18). Because the role of MIP-1 is increasingly recognized in cardiovascular diseases (19, 20), we investigated the role of VLDL-induced TG and FFA accumulation in macrophage inflammatory responses, with particular emphasis on MIP-1.

In this study, we show that VLDL induces a dose- and time-dependent increase in the expression of several inflammatory genes, including MIP-1. The induction of inflammation is associated with the phosphorylation of mitogen-activated protein kinase (MAPK) pathway members and is blocked by the inhibition of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Finally, inhibition of VLDL lipolysis with tetrahydrolipstatin (Orlistat) results in a significant inhibition of VLDL-induced ERK1/2 phosphorylation and subsequent MIP-1 gene expression. Thus, our data are consistent with the hypothesis that hydrolysis of VLDL induces inflammation in macrophages via the activation of the ERK1/2 pathway.

	METHODS

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Materials
Antibodies against phosphorylated and unphosphorylated forms of MAPKs, ß-actin, and U0126 were purchased from Cell Signaling Technology (Danvers, MA). JNK inhibitor peptide was purchased from Calbiochem (La Jolla, CA), and Orlistat was purchased at the pharmacy. SB203580 and all other chemicals were from Sigma (St. Louis, MO).

Macrophage collection
Mouse primary peritoneal macrophages Peritoneal macrophages were collected from female C57BL/6 mice 3 days after injection of thioglycollate medium. Cells were washed and plated at 1.5 x 10⁶ cells per well of 12-well dishes in DMEM containing 10% FBS. After incubation at 37°C for 4 h, nonadherent cells were removed, and adherent cells were incubated for an additional 48 h at 37°C before treatment. VLDL was used at the indicated concentrations in DMEM containing 5% FBS.

Human peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were obtained from human blood by Ficoll gradient centrifugation. Cells were plated in RPMI medium containing 5% FBS at a density of 1.5 x 10⁶cells per well in 12-well culture dishes.

Lipoproteins
Plasma VLDL (d < 1.019 g/ml) for all studies was isolated from fasted human donors by ultracentrifugation as described previously (21). After centrifugation, the lipoprotein fraction was filtered and endotoxin content in the lipoprotein preparations was tested using the LAL assay (Cambrex, Walkersville, MD). All samples were found to contain <10 pg endotoxin/mg protein. The VLDL was characterized by SDS-PAGE, lipoprotein gel electrophoresis, and fast performance liquid chromatography. It contained primarily apolipoprotein B-100 and apolipoprotein E, displayed preß mobility, and eluted in the size range of VLDL with small amounts of intermediate density lipoprotein-sized particles. A modified Lowry assay was used to determine protein concentration of the VLDL preparations, and cells were treated with 0, 50, 100, or 150 µg/ml VLDL based upon protein concentration.

Analysis of macrophage and VLDL lipid content
Oil Red O staining Cells were plated on eight-well chamber slides and treated with VLDL for 6 and 24 h. The cells were washed and fixed with 4% paraformaldehyde, followed by staining with Oil Red O for 4 h. In separate experiments, cells were pretreated with Orlistat followed by cotreatment with VLDL for 6 h, and lipid accumulation was monitored by Oil Red O staining.

GC-MS Macrophage monolayers were washed extensively, and cells were harvested in PBS. Lipids were extracted from the lipoprotein fractions using the method of Folch, Lees, and Sloane Stanley (22). Individual lipid classes were separated by TLC using Silica Gel 60 A plates developed in petroleum ether-ethyl ether-acetic acid (80:20:1) and visualized by rhodamine 6G. Phospholipids, TG, and cholesteryl ester (CE) were scraped from the plates and methylated using BF₃/methanol as described by Morrison and Smith (23). The methylated fatty acids were extracted and analyzed by GC using an HP 5890 gas chromatograph equipped with a Supelcowax 10 capillary column (0.25 mm x 30 m, 0.25 µm film), a flame ionization detector, and an HP 3365 Chemstation. Fatty acid methyl esters were identified by the computer comparing the retention times with those of known standards. Odd chain fatty acids were used as internal standards to quantify the amount of lipid in the sample. Unesterified cholesterol was analyzed by the method of Rudel et al. (24). Aliquots of the total lipid extract were dried under nitrogen, dissolved in hexane, and analyzed by GC using the Hewlett Packard 5890 gas chromatograph equipped with a DB-17 column (0.53 mm inner diameter x 15 m, 1 µm film; J&W Scientific). 5-Cholestane was added to the aliquots before drying and used as an internal standard.

RT-PCR
Total RNA was isolated from cells using TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RT-PCR was performed in a two-step process. First, RNA was reverse-transcribed to cDNA and amplified by PCR using random hexamers with the Reverse Transcription System kit from Promega. cDNA synthesis reactions were performed simultaneously under the same conditions for all RNA samples for each experiment. From this cDNA, PCR analyses for different genes were performed according to their optimal cycling conditions based upon the Tm of the primers. ß-actin PCR was performed with each new cDNA synthesis to confirm similar starting cDNA concentrations for all PCR procedures. The sequences of the primer pairs were as described previously (25–28).

Western blotting for MAPK pathway proteins
Macrophages were collected in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 0.5 mM PMSF. A modified Lowry protein estimation was performed, and equal concentrations of protein were electrophoresed through 4–12% SDS gels (Invitrogen), transferred to nitrocellulose membranes, and immunoblotted for proteins with appropriate antibodies. Antibodies against unphosphorylated and phosphorylated forms of ERK1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), p38 MAPK, and ß-actin were used at a 1:1,000 dilution. Detection of immunoreactive proteins was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ), and signal was quantified using Quantity One software from Bio-Rad.

MAPK inhibition studies
Macrophages were preincubated in 10 µM U0126, JNK inhibitor peptide, or SB203580 for 30 min, followed by incubation with VLDL (100 µg/ml) for an additional 2 h. Cells were used for isolation of either RNA for RT-PCR or protein for Western blotting.

Inhibition of VLDL hydrolysis and intracellular TG reesterification
Macrophages were preincubated with 10 µM Orlistat for 1 h, followed by incubation with VLDL (100 µg/ml) for an additional 6 h. Cells were used for isolation of either RNA for RT-PCR or protein for Western blotting.

Statistical analysis
One-way ANOVA with the Bonferroni post hoc test was used to compare mean responses among the treatment groups. Statistical probability of P < 0.05 was considered significant.

	RESULTS

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Effect of VLDL on macrophage intracellular lipid accumulation
Primary peritoneal macrophages were collected from C57BL/6 mice and treated with 100 µg/ml VLDL for 6 or 24 h. Lipid accumulation was increased dramatically after 6 h, and an even further increase was detected after 24 h of treatment, as assessed morphologically by Oil Red O staining (Fig. 1A ).

View larger version (68K): [in this window] [in a new window]   Fig. 1. Foam cell formation in VLDL-treated mouse peritoneal macrophages. Mouse peritoneal macrophages were treated with 100 µg/ml VLDL for 6 and 24 h. A: Cells were fixed, dried, and stained with Oil Red O. Magnification, 10x. B: Fatty acid composition of triglyceride (TG) from VLDL (white bars) and 6 h VLDL-treated macrophages (black bars) was analyzed from three individual samples by GC-MS as described in Methods. Data are presented as the percentage of total fatty acids (means ± SEM).

Induction of inflammatory gene expression in macrophages by VLDL
Macrophages treated for 6 h with physiologic concentrations of VLDL ranging from 50 to 150 µg/ml demonstrated a dose-dependent increase in the expression of inflammatory genes, such as TNF-, IL-1ß, MCP-1, intercellular adhesion molecule-1, and matrix metalloproteinase 3 (MMP-3), reaching 10.4-, 2.1-, 4.8-, 6.3-, and 2.0-fold increases in expression, respectively, in VLDL treated cells (150 µg/ml) compared with untreated cells (normalized to ß-actin expression; P < 0.05 for all genes) (Fig. 2 ). We also observed a dose-dependent increase in MIP-1 in macrophages upon treatment with 0–150 µg/ml VLDL (Fig. 3A , B). Treatment of macrophages with 100 µg/ml VLDL resulted in a time-dependent increase in MIP-1 expression, which was seen as early as 2 h and was maintained for up to 24 h (Fig. 3C, D).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Dose-dependent effects of VLDL on inflammatory genes. Mouse primary peritoneal macrophages were incubated with 0, 50, 100, or 150 µg/ml VLDL for 6 h. RNA was isolated, and the expression of the inflammatory cytokines tumor necrosis factor- (TNF-), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and matrix metalloproteinase 3 (MMP-3) was measured by RT-PCR. ß-Actin was used as a control to confirm equal RNA concentrations. Representative amplifications of duplicate samples for each gene are shown.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Dose- and time-dependent effects of VLDL on macrophage inflammatory protein (MIP)-1 expression in mouse peritoneal macrophages. A, B: Mouse primary peritoneal macrophages were incubated with 0, 50, 100, or 150 µg/ml VLDL for 6 h. RNA was isolated, and MIP-1 gene expression was measured by RT-PCR. ß-Actin was used as a control to confirm equal RNA concentrations. A: Representative amplification of duplicate samples. B: Band intensities from six samples were quantified using Quantity One software and plotted as means ± SEM. C, D: Mouse primary peritoneal macrophages were incubated with 100 µg/ml VLDL for 0, 2, 4, 6, or 24 h. C: RNA was isolated from cells and analyzed for MIP-1 expression by RT-PCR. D: Band intensities from six samples were quantified using Quantity One software and plotted as means ± SEM. *P < 0.01 compared with 0 µg/ml VLDL; **P < 0.001 compared with 0 and 50 µg/ml VLDL.

The role of VLDL-induced phosphorylation of MAPKs in MIP-1 production
Because MAPK proteins are associated with the induction of inflammatory gene expression (29), activation of MAPKs was considered one mechanism whereby VLDL could mediate its effects. All three members of the MAPK family, ERK1/2, SAPK/JNK, and p38 MAPK, were activated upon treatment with VLDL, as shown by increases in their phosphorylation at 50, 100, and 150 µg/ml VLDL (2.2-, 2.8-, and 2.8-fold at 150 µg/ml dose; P < 0.05 for all) after a 6 h treatment (Fig. 4 ).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Analysis of VLDL-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Mouse primary peritoneal macrophages were treated for 6 h with increasing concentrations of VLDL (0–150 µg/ml), and cellular lysates were collected. Western blotting was performed for the phosphorylated (P) and unphosphorylated (U) forms of extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), and p38 MAPK. A: Representative duplicates of samples are as indicated. B–D: Band intensities of phosphorylated and unphosphorylated forms from six individual samples were quantified and plotted as fold increase of the ratio P/U over untreated samples (means ± SEM). *P < 0.05, **P < 0.005 compared with 0 µg/ml VLDL.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Impact of ERK1/2 inhibition on MIP-1 production in VLDL-stimulated macrophages. Mouse primary peritoneal macrophages were pretreated with U0126 (10 µM), an inhibitor of ERK1/2 phosphorylation, for 1 h and then stimulated with VLDL (100 µg/ml) for 2 h. A: Lysates from cells treated with U0126 were immunoblotted with antibodies against phosphorylated (P) and unphosphorylated (U) forms of ERK1/2. B: Quantification of phosphorylated and unphosphorylated forms of the proteins was performed using Quantity One software. Data are expressed as means ± SEM of the phosphorylated/unphosphorylated bands relative to vehicle-treated cells. C, D: RNA was isolated from cells and analyzed for MIP-1 mRNA expression level by RT-PCR. Data are expressed as means ± SEM of MIP-1/ß-actin compared with vehicle-treated cells from six separate samples. *P < 0.01 compared with controls and cells treated with VLDL + U0126; **P < 0.001 compared with cells treated with VLDL.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Impact of extracellular lipolysis on macrophage lipid accumulation. Primary peritoneal macrophages were pretreated for 1 h with Orlistat (OR), an inhibitor of lipoprotein lipase, before incubation with 100 µg/ml VLDL for an additional 6 h. A: Cells were stained with Oil Red O to detect neutral lipids. B: Cell lysates were analyzed for lipid content by GC-MS as described in Methods. Data are presented as means ± SEM of three individual samples. ND, not detected. *P < 0.001, **P < 0.01 compared with control and VLDL + OR.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of OR treatment on ERK1/2 phosphorylation and MIP-1 expression in VLDL-treated cells. Primary peritoneal macrophages were pretreated for 1 h with 10 µM Orlistat (OR) before incubation with 100 µg/ml VLDL for an additional 6 h. A: Protein was prepared from cell lysates, electrophoresed through 4–12% gradient gels, and blotted for phosphorylated (P) and unphosphorylated (U) forms of ERK1/2. Representative samples from two experiments performed in duplicate are shown. B: Quantification of phosphorylated and unphosphorylated forms of the proteins was performed using Quantity One software. Data are expressed as means ± SEM of the phosphorylated/unphosphorylated bands relative to control cells from four separate samples. C: RNA was isolated and used for RT-PCR analysis of MIP-1 and ß-actin. Representative samples from two experiments performed in triplicate are shown. D: Band intensities from six samples were quantified using Quantity One software and plotted as means ± SEM. *P < 0.01 compared with control and VLDL + OR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Macrophage foam cells are a prominent cellular component of the atherosclerotic plaque in all stages of its development (31). Our findings show that VLDL induces pronounced increases in the TG and FFA contents of macrophages (Figs. 1, 6). These findings are in agreement with previous reports suggesting that incubation of macrophages with TG-rich chylomicron remnants and VLDL promotes TG and FFA accumulation to a greater extent (32) than CEs, which are known to accumulate upon acetylated and oxidized LDL treatment (9, 32).

There are at least three mechanisms that can contribute to the accumulation of TGs and FFAs in VLDL-treated macrophages. First, holo-VLDL particle uptake can occur via receptor-mediated mechanisms. Although modified lipoprotein uptake occurs via scavenger receptors, the most likely receptor for VLDL uptake is the low density lipoprotein receptor-related protein (LRP). Second, lipolysis of VLDL can lead to the uptake of VLDL remnants by receptor-mediated mechanisms (33). Third, FFA generated during the lipolysis of VLDL can also be brought into the cells via receptors or by passive diffusion (34). Macrophages are known to secrete active lipoprotein lipase (35), and lipoprotein lipase activity has been shown to be upregulated by exposure to VLDL (8, 30). Our data showing the absence of TG and FFA accumulation in macrophages treated with the lipoprotein lipase inhibitor Orlistat are in agreement with other reports (30, 33, 36) and implicate VLDL remnant and/or FFA uptake as the primary mechanisms by which VLDL induces macrophage foam cell formation.

Macrophages not only convert to foam cells within atherosclerotic lesions, they also contribute to lesion formation by secreting inflammatory cytokines and chemokines that direct and amplify local immune response in the lesions. Yet, few studies have focused on the potential of VLDL to stimulate inflammation in macrophages. One study demonstrated that VLDL in hypertriglyceridemic patients augments TNF- production in whole blood samples ex vivo (37). Stollenwerk et al. (11, 12) have reported that VLDL potentiates LPS-induced TNF- in THP-1 cells. They have also shown that VLDL directly induces IL-1ß expression in human monocyte-derived macrophages. In addition, Wang et al. (13) have shown that VLDL induces macrophage MCP-1 expression. Our current data suggest that VLDL independently induces a strong and direct proinflammatory response in macrophages by stimulating the expression of MIP-1 and other inflammatory cytokines. In addition, our study showing the upregulation of only TNF- in undifferentiated PBMCs provides evidence that inflammatory responses in undifferentiated monocytes and differentiated macrophages are different. Further studies are required to determine the bases of these differences.

MIP-1 is a CC chemokine that is increasingly recognized as an inflammatory mediator that has a critical role in the development of atherosclerotic lesions (15, 38). Increased plasma levels of MIP-1 have been reported in hypertriglyceridemic mice (39). In addition, an increase in MIP-1 mRNA has been reported in aortas of apolipoprotein E-deficient mice fed a high-cholesterol diet, providing further evidence for the involvement of MIP-1 in atherosclerosis (40). T-cells have been reported to be the major source of MIP-1 in arterial lesions (18); however, in the present study, we show increased expression of MIP-1 in TG-loaded macrophages, suggesting that the foam cells may also act as a source of this chemokine within the vessel wall.

We next examined the signaling pathways that led to the induction of MIP-1 in VLDL-stimulated macrophages. It has been reported that MAPKs play a key role in the induction of cytokine production in macrophages and also in the proliferative effect of oxidized LDL on macrophages (41–44). Our data suggest that ERK1/2 but not SAPK/JNK or p38 MAPK is involved in the induction of the inflammatory response by VLDL in macrophages. This is in accordance with another report showing that ERK1/2 activation is involved in upregulating inflammatory pathways in minimally oxidized LDL-loaded macrophages (45). The mechanism by which the incubation of macrophages with VLDL leads to MAPK activation remains uncertain. It is possible that the uptake of FFAs released as a result of the LPL-mediated hydrolysis of VLDL initiates downstream signaling events leading to the phosphorylation of MAPK family members. This is supported by our data that inhibition of VLDL lipolysis, resulting in the absence of FFA and TG accumulation, blunts ERK1/2 activation. An alternative mechanism relates to receptor-mediated endocytosis of VLDL remnants. One of the receptors involved in VLDL uptake in macrophages is LRP. Not only does LRP mediate lipoprotein endocytosis, it has also been shown to modulate the MAPK signaling cascade in other cells, such as osteoblasts and neurons (46–48). In our study, inhibition of VLDL lipolysis by Orlistat resulted in the absence of FFA and TG accumulation and blunted ERK1/2 activation and MIP-1 expression. These data provide evidence that the intracellular accumulation of FFAs and TGs activates these events. However, the role of receptor-mediated signal transduction cannot be ruled out, because Orlistat inhibition of VLDL hydrolysis resulted in only a partial inhibition of MIP-1. Further investigation is required to understand the involvement of receptor-mediated signaling in these events. However, we can conclude that hydrolysis of VLDL particles plays a crucial role in inducing macrophage inflammatory pathways. Our data on the role of VLDL hydrolysis in macrophage foam cell formation and inflammation support other studies showing that macrophage lipoprotein lipase activity potentiates atherosclerotic lesion formation in mice (49, 50).

The results we obtained regarding MAPK activation and inflammatory gene expression in VLDL-treated macrophages are similar to what has been reported regarding minimally modified low density lipoprotein (mmLDL) activation of macrophages. As mentioned above, Miller et al. (45) have reported that mmLDL induces an inflammatory response in macrophages via the activation of ERK1/2. In contrast to our results demonstrating that MIP-1 expression is increased for as long as 24 h of VLDL treatment, mmLDL treatment induced MIP-2 for <3 h. These authors also report that Toll-like receptor 4 was required for MIP-2 expression, whereas cytokines such as MCP-1, TNF-, and IL-6 were induced by mmLDL in a Toll-like receptor 4-independent manner. Further studies are necessary to determine whether similar mechanisms are involved in mediating the proinflammatory response stimulated by VLDL.

In summary, this study provides strong evidence that VLDL potently induces TG and FFA accumulation as well as inflammatory cytokine and chemokine production in macrophages. The stimulatory effect of VLDL on macrophage inflammatory responses is mediated by the activation of the ERK1/2 pathway and, in part, requires the lipolysis of VLDL particles. Thus, our studies have important implications regarding mechanisms by which TG-rich VLDL is a direct proatherogenic stimulus in human atherosclerotic disease.

	ACKNOWLEDGMENTS

 
The authors thank Michelle Plummer and Carla Harris for their technical assistance and the laboratory of Dr. Larry Swift for preparing VLDL. This work was supported by a Pilot and Feasibility Award to A.H.H. from the Diabetes Research and Training Center at Vanderbilt University (Grant DK-20593) and the Clinical Nutrition Research Unit (Grant DK-26657). A.H.H. is also supported by the American Heart Association (Grant 0330011N) and the American Diabetes Association (Grant 1-04-JF-20). The lipid profiles were performed at the Lipid Core Laboratory of the Mouse Metabolic Phenotyping Center at Vanderbilt University (Grant DK-59637).

Manuscript received July 15, 2005 and in revised form April 5, 2006.

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