Published online before print January 18, 2007, doi: 10.1161/01.RES.0000258023.76515.a3
© 2007 American Heart Association, Inc.
Molecular Medicine |
Triglyceride-Rich Lipoproteins Prime Aortic Endothelium for an Enhanced Inflammatory Response to Tumor Necrosis Factor-
From the Departments of Biomedical Engineering (H.J.T., U.Y.S., A.G.P., S.I.S.) and Cardiovascular Medicine (J.P.S., J.C.R., A.A.K.), University of California, Davis; and Department of Pathology (D.Y.H.), University of Cincinnati, Ohio.
Correspondence to Dr Scott I. Simon, Department of Biomedical Engineering, University of California, Davis, 451 E Health Sciences Dr, Genome and Biomedical Sciences Facility, Davis, CA 95616-5294. E-mail sisimon{at}ucdavis.edu
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Abstract |
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High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-
. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-
B, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.
Key Words: triglyceride-rich lipoprotein • endothelium • monocyte • NFB • vascular adhesion molecule-1 • atherosclerosis
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Introduction |
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Atherosclerosis is initiated by an inflammatory response resulting in monocyte recruitment to arterial intima and the gradual development of lipid-rich plaques. Whether because of diet or genetic predisposition, elevated triglyceride-rich lipoproteins (TGRL) levels have been associated with an increased risk of atherogenesis.1–3 Although published data implicate lipid oxidation as a critical link between vascular inflammation and atherosclerosis, it has not been established that lipoprotein oxidation is the primary mediator early in disease progression.
Oxidized lipoproteins are taken up by scavenger receptors and oxidized low-density lipoprotein receptor-1 (LOX-1)4 and, on internalization, trigger endothelial inflammation. In the absence of oxidation, low-density lipoprotein (LDL), and TGRL remnant particles infiltrate the vascular endothelium and accumulate in the intima. Chiefly composed of cholesterol-rich very-low-density lipoproteins (VLDLs) and chylomicron particles, TGRL is taken up by LDL family receptors such as VLDL receptor and LDL receptor-related protein (LRP) that bind to apolipoproteins (ie, apoE and apoB-100).5,6 Uptake of TGRL through this pathway is vital for clearance as LDL-receptor-deficient mice accumulate postprandial TGRLs after a high fat meal.1,7,8 Although epidemiological evidence has shown a clear link between chronic hyperlipidemia and atherogenesis, there is a paucity of data on how repetitive exposure to elevated TGRL exacerbates vascular inflammation. We focus on the mechanisms by which unmodified TGRL alters the inflammatory response of human aortic endothelial cells (HAECs) to cytokine.
Atherosclerosis develops within discrete vascular lesions, associated with focal expression of acute inflammatory cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-1.9,10 This in turn triggers activation of mitogen-activated protein kinases (MAPKs) such as Akt, extracellular signal-regulated kinase 1/2 (Erk1/2), and p38.11,12 The transcription NF-B translocates from the cytosol to the nucleus where it promotes transcription of VAMs (ie, endothelial [E]-selectin, ICAM-1, and VCAM-1) and chemokines, which guide the recruitment of leukocytes.9,13,14 Costimulation of aortic endothelium by lipoproteins and cytokines results in the preferential recruitment of monocytes and T cells from the circulation by upregulation of E-selectin and VCAM-1.9,10 This is central to atherogenesis as apoE knockout mice have been shown to exhibit decreased vascular lesion formation when these adhesion molecules are genetically deleted as compared with wild-type animals.15,16
Here we explore the link between dietary hyperlipidemia and inflammatory atherogenesis. We mimicked a high-fat diet by repetitively exposing HAECs to unmodified postprandial TGRLs, freshly-isolated from human blood, for 2 hours over a period of 1 to 3 days. Inflammatory responses of HAECs were then assessed by acute stimulation (ie, 4 hours) with TNF-. Membrane binding of TGRL receptor was monitored along with its effect on intracellular signaling pathways, leading to VAM expression and monocyte recruitment on inflamed HAECs in a custom-designed microfluidic flow chamber. We report that although repetitive treatment with TGRL did not itself elicit an inflammatory response, it enhanced the acute response to cytokine stimulation in a manner dependent on LDL family receptor binding and activation of the p38 MAPK and NF-B pathways. These data reveal how chronic exposure to TGRL primes aortic endothelium for enhanced monocyte recruitment to incipient sites of atherosclerosis.
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Methods and Materials |
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HAEC Culture and Treatment With TGRLs
HAECs were purchased at passage 4 (Cascade Biologics),
13 cell divisions following primary isolation, expanded in T75 flasks to 80% to 90% confluence, and then seeded onto 6-well tissue culture plates (BD) between passages 5 and 6 (16 to 19 total cell divisions). HAECs were conditioned in growth media for 2 h/d for up to 3 days with freshly isolated TGRLs (at 2.5 mg/mL unless otherwise indicated) or a control lipid emulsion, Intralipid (at 3 mg/mL). Following incubation, unbound lipoprotein was removed by washing with Hanks’ balanced salt solution (without Ca2+ or Mg2+). Acute inflammation was stimulated by incubating HAECs with TNF- for 4 hours and then washing. Where indicated, LDL receptor-associated protein-1 (RAP-1) (100 µg/mL) was added before each TGRL treatment; it remained throughout the incubation and was used to block VLDL receptor as previously described.1,17
Triglyceride-Rich Lipoprotein Purification
Blood from healthy human volunteers, as approved by the Human Subjects Institutional Review board of the University of California, Davis, was drawn and prepared at the Clinical Nutrition Research Unit. For the TGRL studies, volunteers consumed controlled meals typical of a Western diet containing 30% total fat (20% saturated fat), 15% protein, and 55% carbohydrate. Postprandial blood was drawn 3.5 hours following meal ingestion, a time point previously shown to correlate with elevated circulating TGRL.18 Blood was collected in 10-mL Vacutainer tubes containing Na+ EDTA from BD (San Jose, Calif). TGRL, isolated as previously described,18 contained 
0.5 endotoxin unit/mL according to a Pyrochrome kit from Associates of Cape Cod (Falmouth, Mass). Samples were stored over nitrogen gas in sealed tubes at 4°C. VLDL and chylomicrons were isolated at an average concentration of 75 and 32 mg/dL of whole blood, respectively.
Quantitative Real-Time RT-PCR
Gene transcripts for E-selectin, ICAM-1, VCAM-1, p50, and p65 were quantified using PCR as described in the online data supplement, available at http://circres.ahajournals.org. Briefly, HAECs were lysed, RNA isolated and quantified, and first-strand cDNA synthesized. The cDNA was then amplified for 40 cycles and results quantified by the 
CT method.
Protein Isolation and Western Immunoblot Analysis
Protein levels for total and phosphorylated Akt, Erk1/2, p38, IB, IB kinase (IKK), p50, and p65 were quantified using Western immunoblot and densitometry of the bands as described in the online data supplement. Briefly, HAECs were lysed, and protein was isolated, run on SDS-PAGE gel, and transferred to nitrocellulose.
Statistical Analysis
Analysis of data were performed using GraphPad Prism version 4.0 software (GraphPad Software Inc, San Diego, Calif). All data are reported as mean±SEM, as indicated. Data were analyzed by ANOVA and secondary analysis for significance with Newman-Keuls post tests. Group comparisons were deemed significant for 2-tailed P<0.05.
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Results |
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TGRL Binding and Internalization by HAECs Induces Expression of LDL Receptor
Endothelial response to postprandial lipidemia was modeled by repetitive exposure of cultured HAECs to TGRL, freshly isolated from postprandial blood. TGRL binding to endothelium was directly imaged by phase contrast and fluorescence microscopy (Figure 1). A pericellular distribution of fluorescent TGRL was observed on the majority of HAECs (Figure 1a). Membrane-bound TGRL was endocytosed within 30 minutes of incubation, and confocal sections at
1 µm beneath the plasma membrane clearly revealed fluorescent endosomes (Figure 1b). The specificity of binding was assessed by coincubating HAECs with TGRL and RAP-1, a high-affinity LRP antagonist previously reported to block VLDL receptor binding.17,19 RAP-1 virtually abrogated TGRL membrane fluorescence to the level of IgG2a control (Figure 1c and 1d).
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LDL family receptors are reported to be upregulated within atheromatous lesions of apoE knockout mice.17,20 To elucidate the mechanism of TGRL binding to HAECs, we assessed expression of 2 members, LRP and LR11, by immunofluorescence. Both were expressed on untreated HAECs at levels significantly greater than nonspecific IgG2a (Figure 1e and 1f). Repetitive exposure to TGRL was observed to upregulate LRP by 7-fold and LR11 nearly more than 10-fold more than baseline. Coincubation with RAP-1 significantly reduced LRP and LR11 upregulation as indicated by the shift in mean fluorescence intensity (MFI) toward that of untreated HAECs (MFI of TGRL versus untreated: LRP, 135 versus 59; LR11, 203 versus 84; P<0.05). Taken together, these data reveal a positive-feedback mechanism, by which TGRL binds LDL family receptors, is subsequently internalized, and then signals their upregulation on HAECs.
TGRL Primes HAECs for Enhanced Upregulation of Adhesion Molecules
We next determined whether binding of TGRL alone, or in combination with acute TNF- stimulation, alters transcription and membrane expression of adhesion molecules on HAECs (Figure 2). A 2-hour incubation with TGRL was chosen for priming studies, as elevated TGRL has been detected at this time point in postprandial plasma. Furthermore, 4-hour exposures did not further augment priming of inflammation (data not shown). Incubation with TGRL, whether as a single exposure or repetitively over 3 days, failed to significantly upregulate VAM mRNA or expression. In contrast, acute stimulation for 4 hours with TNF- (0.3 ng/mL) upregulated both VAM transcription and expression. A single 2-hour exposure to TGRL followed a day later by TNF- stimulation primed a 30% increase in E-selectin expression over TNF- alone. Expression was not further enhanced over 2 additional days of TGRL exposure, but correlated with a doubling of E-selectin mRNA. TNF- stimulation upregulated ICAM-1 expression by 6-fold more than unstimulated control, however, priming with TGRL even over 3 days did not enhance mRNA or protein expression over TNF- alone. In contrast, TGRL priming over 3 days increased VCAM-1 mRNA and expression by 70% more than that stimulated by TNF- alone. Taken together, these data suggest that TGRL alone is not inflammatory to HAECs but differentially amplifies VAM expression elicited by cytokine.
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3).
We then examined the sensitivity of HAECs to priming with TGRL by measuring its capacity to augment VAM upregulation elicited over a 1000-fold dose range in TNF- (Figure 3). VAM expression was compared between culture media and that supplemented with TGRL or Intralipid, a control lipid emulsion lacking apolipoproteins associated with LDL receptor binding. A low concentration of TNF- (0.003 ng/mL) did not elicit expression of E-selectin or VCAM-1 above unstimulated control. Significantly, priming HAECs with TGRL, but not Intralipid, lowered the threshold dose of TNF- that stimulated upregulation (0.003 versus 0.03 ng/mL). In this case, TNF- at 0.03 ng/mL effectively doubled membrane expression of E-selectin and VCAM-1. Though ICAM-1 was expressed on unstimulated HAECs, and was upregulated at 0.003 ng/mL, TGRL did not enhance its expression over the entire dose range of TNF-. In contrast, VCAM-1 exhibited the greatest sensitivity to TGRL priming, increasing expression relative to Intralipid by 100% over the entire dose range of TNF-. We conclude that TGRL primes HAECs for enhanced inflammation via lowering by 10-fold the threshold dose of TNF- necessary to elicit upregulation of VCAM-1 and E-selectin.
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TGRL Primes HAECs for Enhanced Monocyte Recruitment Under Shear Flow
An early event in atherogenesis is monocyte recruitment to sites of arterial inflammation, whereas neutrophils are typically not observed in developing plaques. We examined the role of TGRL in priming TNF-–inflamed endothelium for enhanced monocyte recruitment using a custom microfluidic shear flow chamber under a stress of 2 dynes/cm2 (Figure 4). Recruitment of monocytes and neutrophils onto HAECs were imaged by phase contrast microscopy and categorized as rolling, arrested, and transmigrated across the endothelial monolayer. Over a dose range of TNF- stimulation, neutrophils transitioned from rolling to arrest almost twice as efficiently as monocytes (Figure 4a). Priming HAECs with TGRL doubled monocyte recruitment efficiency at each concentration of TNF-, whereas neutrophil recruitment did not increase. Consistent with VAM upregulation, TGRL priming did not directly stimulate monocyte recruitment even following repetitive conditioning over 3 days at concentrations up to 5.0 mg/mL (data not shown). However, increasing the concentration of TGRL from 2.5 to 5.0 mg/mL doubled the number of monocytes recruited and transmigrating across TNF-–inflamed HAECs (Figure I in the online data supplement).
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We next examined the VAMs supporting increased monocyte recruitment. In the presence of TNF- stimulation alone, monocyte arrest and transmigration was supported by 4ß1 and ß2 integrins, as blocking each with antibody added up to the control level of 6 monocytes per field (ie, 4ß1, 4/field; ß2, 2/field) (Figure 4b). ICAM-1 and VCAM-1 supported firm adhesion; however, capture and rolling required expression of E-selectin, as determined by antibody inhibition (data not shown). Priming HAECs with TGRL doubled monocyte recruitment efficiency, which remained dependent on both ß1 and ß2 integrins (Figure 4c). However, inhibition of ß2 integrins blocked proportionally less monocyte arrest on TGRL-primed HAECs versus TNF- alone, suggesting that ß1 integrin is predominant. Interestingly, greater cooperativity between ß1 and ß2 integrins was apparent for the TGRL-primed HAECs, as the total number of monocytes arrested was 30% greater than the sum of monocytes recruited when blocking integrins individually (ie, 12/field versus 9/field, P<0.05). These data reveal that priming with TGRL increases monocyte recruitment to inflamed HAECs in a ß1- and ß2-integrin-dependent manner.
VLDL Primes for Enhanced Inflammation Through LDL Family Receptors
We further analyzed TGRL to determine which components were associated with the priming effect by isolating VLDL and chylomicrons separately and incubating HAECs with each before assaying TNF-–stimulated monocyte arrest (Figure 5a). Neither VLDL nor chylomicron fractions at concentrations commensurate with that applied during TGRL incubation (2.5 mg/mL) stimulated monocyte arrest in the absence of TNF-. Repetitive exposure with VLDL elicited 70% of the increase in monocyte arrest observed for priming with TGRL. In contrast, chylomicron did not significantly increase monocyte arrest above that stimulated by TNF- alone. To confirm that TGRL priming was not attributable to oxidized lipoprotein, we treated HAECs with native and oxidized VLDL in the absence of TNF- and assayed VCAM-1 expression (Figure 5b). Oxidized VLDL significantly upregulated VCAM-1 to levels observed with TGRL and TNF-. This effect was inhibited by treatment of oxidized VLDL with the antioxidant ascorbic acid, whereas VLDL alone or treated with antioxidant did not significantly alter VCAM-1 expression. Together, these data reveal that the inflammatory priming effect of TGRL is largely mediated by VLDL and not associated with oxidized lipoprotein.
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To confirm that TGRL binding and internalization are required for the priming response, we assayed VCAM-1 upregulation and monocyte recruitment in the presence of the LRP antagonist RAP-1 (Figure 6). VCAM-1 expression nearly doubled in response to TNF-, and priming with TGRL augmented expression by 100% (Figure 6a). RAP-1 blocked this augmented VCAM-1 expression down to the level induced by TNF- alone and likewise reduced monocyte arrest elicited on TGRL-primed HAECs to the level stimulated by TNF- alone (Figure 6b). These data confirmed that blocking membrane binding and internalization of TGRL by coincubation with RAP-1 abrogates its capacity to augment inflammation, but does not inhibit the response to TNF-.
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TGRL Primes for Inflammation Through Enhanced Activation of p38 MAPK and NFB
TNF-–induced inflammatory responses are partially mediated via activation of MAPKs, which in turn trigger the transcription factor NF-B, known to play an important role in atherogenesis. We focused on this pathway to reveal how repetitive exposure to TGRL itself elicits, or amplifies signaling through TNF-. Consistent with its lack of direct inflammatory activity, TGRL alone failed to increase MAPK activation above that of untreated HAECs. However, TNF- significantly activated MAPKs, detected by an increase in phosphorylated Akt, Erk1/2, and p38 (Figure 7a through 7c). Priming with TGRL had the greatest influence in amplifying activation of p38, with an increase of 3-fold more than TNF- alone. We assessed the role of p38 activation by application of the pharmacological p38 inhibitor SB202190 during TGRL priming. SB202190, in a dose-dependent manner, reduced expression of E-selectin and VCAM-1 in both TNF-–treated and TGRL-primed HAECs (Figure 7d). Significantly, blocking p38 activity effectively reduced VCAM-1 upregulation in TGRL-primed HAECs to the level observed with TNF- alone, whereas upregulation of ICAM-1 was unaffected (data not shown). This result is consistent with previous reports on the role of p38 in modulating expression of VAM in response to inflammatory stimulation.21,22
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TGRL alone did not elicit phosphorylation of IKK but on stimulation with TNF- increased activation by 4-fold, whereas IKK synthesis did not increase (Figure 8a). A concomitant downregulation in the inhibitory protein IB was observed in HAECs stimulated with TNF-, which was reduced to undetectable levels in TGRL-primed HAECs (Figure 8b). Notably, priming with TGRL alone resulted in a 2.5-fold increase in mRNA for NF-B subunit p65 (Figure 8c). Despite the increase in p65 at the transcript level, an increase in p65 and p50 protein was not detectable by Western blot (data not shown). Likewise, TGRL alone did not increase the amount of activated p65 detected in nuclear fractions over untreated HAECs (Figure 8d). In contrast, within 30 minutes of TNF- stimulation, there was a dramatic increase in nuclear translocation of active phospho-p65. Moreover, TGRL-primed HAECs exhibited a 30% increase in NF-B activation over TNF- alone. This correlated with amplification in expression of inflammatory gene products and augmented monocyte recruitment. We confirmed that TGRL primed for enhanced inflammatory response through NF-B by pretreating HAECs with consensus decoy oligonucleotides that prevented its binding to DNA promoter regions, as previously reported for cytokine mediated inflammation.23 Transfection of HAECs with decoy oligonucleotides, but not a nonspecific scrambled DNA sequence, abrogated upregulation of all 3 adhesion molecules (supplemental Figure II). Taken together, these data reveal that TGRL primes upregulation of p65 along with its inhibitory partner IB, thereby providing a readily accessible reservoir of cytoplasmic NF-B for translocation to the nucleus in response to acute inflammatory stimulus.
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Discussion |
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We addressed the hypothesis that repetitive exposure of human aortic endothelium to TGRL exacerbates the inflammatory response to cytokine activation. TGRL levels found to prime for enhanced inflammation in this study were commensurate with circulating levels in normolipidemic individuals (189±20 mg/dL) hours after consumption of a typical Western diet (
30% fat, 20% saturated fat). By comparison, TGRL in metabolic syndrome patients can reach 800 mg/dL and remain elevated for 4 hours following a high-fat meal.2 Conventional wisdom is that progression of atherosclerosis involves oxidized lipoprotein (OxLDL) binding to scavenger receptors, thereby triggering inflammation. In contrast, we show here that although native TGRL does not directly signal inflammation without first being oxidized, it lowers the threshold dose at which a cytokine elicits VAM expression and increases the efficiency of monocyte recruitment in shear flow. We provide the first evidence that TGRL binds aortic endothelium and elicits upregulation of LRP and LR11, known to recognize apoE-laden lipoproteins.17,19 LRP has been reported to function in VLDL and chylomicron remnant clearance by endocytosis and can also bind tissue factor, resulting in transmembrane signaling involving tyrosine phosphorylation.5,24,25 LR11 is expressed on smooth muscle cells and its upregulation shown to correlate with cell proliferation and intimal thickening of atherosclerotic arteries.19,20 Upregulation of lipoprotein receptors suggests that TGRL can elicit a positive-feedback mechanism for increased uptake of apolipoproteins. The finding that RAP-1 blocked TGRL from binding to HAECs may be explained by its capacity to directly antagonize ligand binding to LRP alone, or in a complex with LR11 and other VLDL receptors.5,17 Experiments to determine whether LR11 and LRP directly colocalize with TGRL and the dependence on caveolae-rich membrane domains during endocytosis of TGRL are underway.26
Atherosclerosis exhibits many elements common to chronic inflammatory diseases including upregulation of VAMs and extensive monocyte recruitment.9,13,14,27 Targeted deletion of E-selectin, ICAM-1, or VCAM-1 in apoE knockout mice revealed that these adhesion molecules are critical for monocyte extravasation into arterial intima.15,16 A novel vascular mimetic flow chamber was used here to image the multistep process of monocyte recruitment to inflamed HAECs. Monocyte rolling transitioned to arrest and transmigration within a few cell diameters of capture, as also observed by intravital microscopy of mouse models of atherosclerosis.28 Priming HAECs with TGRL followed by acute inflammation resulted in a doubling of VCAM-1 expression and a 2-fold boost in monocyte arrest and transmigration. Under these conditions, the fraction of monocytes transitioning to arrest and transmigration exceeded that of neutrophils. This may be attributed to an increase in production of the chemokine monocyte chemoattractant protein-1 (data not shown) and activation of ß1 and ß2 integrins on monocytes to bind VCAM-1 and ICAM-1 with high affinity.29 We hypothesize that repetitive exposure of aortic endothelium to TGRL may provide cues that amplify the efficiency of monocyte recruitment by sensitizing HAECs to inflammatory stimuli and increasing the efficiency of integrin mediated adhesion in shear flow.
The mechanism for this TGRL-mediated effect on endothelium is currently unknown. A recent report by Norata et al has shown that TGRL can modulate the activity of the p38 MAPK pathway, degradation of IB and expression of VCAM-1, E-selectin, and monocyte chemoattractant protein-1.30 Similarly, Dichtl et al has reported that VLDL from fasting, hypertriglyceridemic patients can activate NFB in vitro and when injected into rats can signal aortic expression of VCAM-1.31 Although these findings are consistent with our work, there are key points of divergence. Foremost, they found that TGRL alone activated acute inflammation and upregulated ICAM-1, whereas we found no evidence for direct activation in the absence of TNF-. Moreover, Norata et al reported transient activation of MAPKs including p38 and ERK1/2 within 10 minutes, fading to baseline by 30 minutes, whereas we found a more sustained priming activity of TGRL in that repetitive exposure of HAECs enhanced an acute inflammatory response to cytokine over several hours. Another key difference was that Norata et al and Dichtl et al studied TGRL isolated from hyperlipidemic fasting subjects, as compared with postprandial normolipidemic TGRL applied in our study. TGRL obtained from fasting subjects are metabolically different from postprandial TGRL in that they typically contain smaller particles, which may more readily infiltrate vascular endothelium.1
Priming HAECs with TGRL elicited an enhanced inflammatory response over a 1000-fold dose range in TNF- stimulation. The signaling pathway involved activation of p38 MAPK, as confirmed by pharmacological inhibition of p38 activity with SB202190. In fact, the highest dose of SB202190 effectively abrogated the TGRL priming response, although to a lesser extent blocking TNF-–stimulated upregulation of E-selectin and VCAM-1. This result might be expected because TNF- alone potently increased the activity of the Akt and Erk1/2 signaling pathways, whereas enhanced activity of p38 MAPK required costimulation with TGRL. We also assessed the importance of NFB in the signaling pathway by transfecting HAECs with decoy oligonucleotides that inhibited NF-B transcriptional activity. The decoy, but not a scrambled peptide, abrogated VAM upregulation in TGRL-primed as well as TNF-–stimulated HAECs. This confirmed a central role for NF-B in both cytokine and TGRL priming of enhanced inflammation. The increase in phosphorylated IKK (associated with activation of the kinase complex) and p65 mRNA, and concomitant decrease in IB inhibitory molecule on costimulation with cytokine and TGRL are consistent with the observed increase in transactivation of NF-B. We hypothesize that TGRL signals for enhanced p38 MAPK activity that, on cytokine stimulation, leads to more rapid and robust nuclear translocation and transcriptional activity of NF-B.
Priming of the NF-B pathway has been observed in vivo in relation to focal susceptibility to atherosclerosis. Hajra et al demonstrated nuclear translocation of NF-B and induction of proinflammatory genes in susceptible regions of the aortas of hypercholesterolemic LDLR–/– mice.32 In normal mice, although p65 and IBs were elevated in such regions, NF-B activation remained low, suggesting that the pathway was primed but not activated. In this regard, Passerini et al examined the effect of hemodynamics on the steady-state gene expression profiles in the arteries of swine fed a normal diet. In atherosusceptible regions of the vessel, they detected enhanced expression of several proinflammatory cytokines, chemokines, and receptors, as well as elevated transcript levels for components of NF-B and IB.33 They concluded that these regions are primed for inflammation, but activation is kept in check through a dynamic balance of antiinflammatory gene expression. Thus, metabolic risk factors such as elevated TGRL levels may tip the balance toward inflammatory-mediated atherogenesis in susceptible regions of the artery. Modeling repetitive injury of HAECs with native TGRL in our vascular mimetic flow system provides a "laboratory-on-a-chip" approach for gauging its inflammatory potential under defined hemodynamics and dissecting its mechanism(s) of action.
In summary, we demonstrate that repetitive exposure to native TGRL upregulated lipoprotein receptors and primed HAECs for an amplified inflammatory response to TNF-. Primed HAECs were 10-fold more sensitive to TNF-–stimulated upregulation of VCAM-1 and E-selectin. Monocytes were preferentially recruited over neutrophils on inflamed HAECs in shear flow as a function of the extent of TGRL priming. This vascular mimetic model of repetitive injury reveals how unmodified lipoproteins contribute to the inflammatory axis of atherogenesis.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants AI47294 (to S.I.S.); HL55667 (to J.C.R.); HL077281 and AG19327 (to A.A.K.); and HL61332 (to D.Y.H.). A.A.K. is also supported by the Department of Veteran Affairs. S.I.S. is an Established Investigator of the American Heart Association and supported by the Treadwell Innovative Grant Program. H.J.T. and S.I.S. are also supported by the National Science Foundation Center for Biophotonics Science and Technology.
Disclosures
None.
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Footnotes |
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Original received June 1, 2006; resubmission received October 5, 2006; revised resubmission received December 20, 2006; accepted January 5, 2007.
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