Published online before print October 22, 2007, doi: 10.1161/CIRCULATIONAHA.107.693382
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(Circulation. 2007;116:2173-2181.)
© 2007 American Heart Association, Inc.
Vascular Medicine |
Apolipoprotein C-I Is Crucially Involved in Lipopolysaccharide-Induced Atherosclerosis Development in Apolipoprotein E–Knockout Mice
From The Netherlands Organization for Applied Scientific Research–Quality of Life (M.W., J.F.P.B., N.M.M.P., R.K., L.M.H., P.C.N.R.), Department of Biomedical Research, Gaubius Laboratory, Leiden, the Netherlands; and Departments of General Internal Medicine, Endocrinology, and Metabolic Diseases (M.W., J.F.P.B., J.A.R., L.M.H., P.C.N.R.), Cardiology (N.M.M.P., L.M.H.), Rheumatology (G.J.D.v.M.), and Vascular Surgery (R.K.), Leiden University Medical Center, Leiden, the Netherlands.
Correspondence to Marit Westerterp, Leiden University Medical Center, Department of Endocrinology and Metabolic Diseases, C4-R, PO Box 9600, 2300 RC Leiden, the Netherlands. E-mail M.Westerterp{at}lumc.nl
Received January 30, 2007; accepted August 31, 2007.
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Abstract |
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Background— Lipopolysaccharide (LPS), which is released from Gram-negative bacteria on multiplication or lysis, aggravates atherosclerosis in humans and rodents by inducing inflammation via toll-like receptors. Because apolipoprotein C-I (apoCI) enhances the LPS-induced inflammatory response in macrophages in vitro and in mice, we investigated the effect of endogenous apoCI expression on LPS-induced atherosclerosis in mice.
Methods and Results— Twelve-week-old apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice received weekly intraperitoneal injections of LPS (50 µg) or vehicle for a period of 10 weeks, and atherosclerosis development was assessed in the aortic root. LPS administration did not affect atherosclerotic lesion area in apoe–/–apoc1–/– mice but increased it in apoe–/–apoc1+/+ mice. In fact, apoCI expression increased the LPS-induced atherosclerotic lesion area by 60% (P<0.05), concomitant with an increase in LPS-induced plasma levels of fibrinogen and E-selectin. This indicated that apoCI increased the LPS-induced inflammatory state, both systemically (ie, fibrinogen) and at the level of the vessel wall (ie, E-selectin). In addition, both macrophage-derived apoCI and HDL-associated apoCI increased the LPS-induced tumor necrosis factor-
response by macrophages in vitro.
Conclusions— We conclude that apoCI is crucially involved in LPS-induced atherosclerosis in apoe–/– mice, which mainly relates to an increased inflammatory response toward LPS. We anticipate that apoCI plasma levels contribute to accelerated atherosclerosis development in individuals who have chronic infection.
Key Words: apolipoproteins • atherosclerosis • inflammation • lipoproteins • hypercholesterolemia • leukocytes
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Introduction |
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Cardiovascular disease (CVD) is the principal cause of death in Europe, the United States, and much of Asia. The main cause of CVD is atherosclerosis.1–3
Clinical Perspective p 2181
Although hypertension and hyperlipidemia are common risk factors for atherosclerosis, it is also evident that bacterial infections worsen the outcome of atherosclerosis by maintaining a heightened state of inflammatory response, thus propagating this inflammatory disease.1–4 Lipopolysaccharide (LPS) is a highly inflammatory constituent of the outer membrane of Gram-negative bacteria. When these bacteria multiply or lyse, LPS is released and then activates the MD-2/toll-like receptor 4 (TLR4) receptor complex on endothelial cells, neutrophils, and macrophages, which results in a proinflammatory response through activation of the nuclear factor–
B pathway.5 TLR4 is expressed in macrophages and endothelial cells within atherosclerotic lesions,6,7 and indeed, impaired LPS signaling in human carriers of the TLR4 polymorphisms Asp299Gly or Thr399Ile is associated with LPS hyporesponsiveness,8 and the Asp299Gly mutation is associated with decreased CVD.4 Conversely, infection with the Gram-negative bacterium Chlamydia pneumoniae has been associated with increased CVD in humans.9–11
In rodents, bacterial infection and LPS also promote atherogenesis. For example, local delivery of C pneumoniae in the vessel wall of carotid arteries increases the development of atherosclerosis in low-density lipoprotein (LDL) receptor gene–deficient (ldlr–/–) mice,12 and infection with C pneumoniae accelerates the development of atherosclerosis in apolipoprotein (apo) E gene–deficient (apoe–/–), ldlr–/–, and APOE*3-Leiden transgenic mice.13–15 In addition, LPS accelerates intimal lesion development in a periadventitial cuff model in wild-type mice, which is largely decreased when TLR4 expression is absent.16 Furthermore, repeated intravenous and intraperitoneal administration of LPS accelerates atherosclerosis in rabbits and apoe–/– mice, respectively.17,18
Until recently, apolipoprotein C-I (apoCI) was known primarily for its role in lipoprotein metabolism. ApoCI circulates in plasma with a concentration of 6 mg/dL and is mainly bound to the lipoproteins very-low-density lipoprotein (VLDL), chylomicrons, and high-density lipoprotein (HDL).19 ApoCI inhibits lipoprotein lipase (LPL),20,21 and endogenous apoCI expression was associated with modest hyperlipidemia in apoe–/– mice.21 Recently, we have discovered that apoCI strongly binds to LPS, thereby augmenting the inflammatory response to LPS and the Gram-negative bacterium Klebsiella pneumoniae in mice in vivo and to LPS in macrophages in vitro.22
In the present study, we investigated the effect of endogenous apoCI on the development of LPS-induced atherosclerosis in apoe–/–apoc1–/– versus apoe–/–apoc1+/+ mice. We found that LPS-induced atherosclerosis is enhanced in apoCI-expressing mice, in association with a higher inflammatory state achieved with LPS.
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Methods |
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For an extended version of the Methods section, see the online-only Data Supplement.
Animals
Apoe–/–apoc1–/–23 and apoe–/–24 mice were back-crossed at least 8 times to the C57Bl/6 background. Apoe–/–apoc1–/– and apoe–/–apoc1+/+ littermates were generated, and female mice were used for experiments, housed under standard conditions with a 12-hour light cycle (7 AM to 7 PM), and fed ad libitum with regular chow.
Analysis of LPS Response in Time
Twelve-week-old apoe–/–apoc1–/– and apoe–/–apoc1+/+ littermates (n=8 per genotype) received an intraperitoneal injection of LPS (50 µg; Escherichia coli serotype 055:B5, Sigma, St Louis, Mo) in 200 µL of PBS. Twenty-four hours before LPS injection (t=0) and every 24 hours thereafter for a period of 1 week, 35 µL of blood was collected after a 4-hour fast for measurement of total cholesterol (TC), fibrinogen, and E-selectin. At t=0, 24 hours after LPS injection, and 1 week after the injection, triglyceride (TG) levels were also measured.
Atherosclerosis Study
Twelve-week-old apoe–/–apoc1–/– and apoe–/–apoc1+/+ littermates received weekly intraperitoneal injections of LPS (50 µg) in 200 µL of PBS or PBS alone (vehicle; n=8 to 11 per genotype per group), exactly as described previously.18,25 Plasma lipid and inflammatory parameters of these mice were analyzed 1 week before the start of the injections and 24 hours after the first, fifth, and tenth injections. Twenty-four hours after the first injection, lipid distribution over lipoproteins was assessed.
Plasma Lipid and Lipoprotein Analysis
Blood was collected, and plasma TC and TG, as well as TC and TG distribution over lipoproteins, were determined.26 For analysis of the apolipoprotein distribution over lipoproteins, 2 adjacent fractions of 50 µL were pooled, and apolipoproteins were separated.27
HDL Analysis
To determine the apoCI level on HDL relative to HDL lipid concentrations, HDL from apoe–/–apoc1+/+ mice that received either vehicle or LPS was isolated via precipitation of apoB-containing lipoproteins,27 and HDL phospholipids, HDL cholesterol, and apoCI21 were determined.
Plasma Analysis of Fibrinogen and E-Selectin
Plasma fibrinogen levels were determined with a homemade ELISA,28 and plasma E-selectin levels were ascertained via a commercially available ELISA (R&D Systems Europe, Abingdon, UK).
Incubation of Macrophages With LPS In Vitro
Apoe–/–apoc1–/– and apoe–/–apoc1+/+ peritoneal macrophages were isolated and cultured27 and then incubated with LPS (0.1 to 100 ng/mL) in Dulbecco’s Modified Eagle Medium supplemented with 0.01% human serum albumin (4 hours at 37°C). Tumor necrosis factor (TNF)- in the medium was determined.22 Murine RAW 264.7 macrophages were cultured22 and incubated with LPS (100 ng/mL) with or without HDL isolated from apoe–/–apoc1–/– and apoe–/–apoc1+/+ littermates (24 hours at 37°C), and TNF- was determined in the medium.22
Atherosclerotic Lesion Analysis
For determination of atherosclerosis development, mice were euthanized at 24 hours after the tenth injection. Hearts were fixed and embedded in paraffin, and sections were made.27 Lesion area and composition with regard to adherent monocytes, macrophages, collagen, smooth muscle cells (SMCs), and T cells were determined.29
Statistical Analysis
The Mann-Whitney nonparametric test for 2 independent samples was used to define differences between data sets from experimental groups. The criterion for significance was set at P<0.05. Linear regression analysis was used to evaluate correlations between HDL apoCI levels versus HDL lipid parameters. Statistical analyses were performed with SPSS version 11.5 (SPSS Inc, Chicago, Ill).
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Effect of ApoCI on Plasma Lipid Levels After LPS Injection
To examine the effect of LPS on plasma lipid levels, we measured plasma levels of TC and TG before and during a period of 7 days after a single LPS injection (Figure 1). LPS injection increased TC levels in both apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice at 24 hours after LPS injection by
2.5 mmol/L. The level of TC decreased thereafter to return to its starting value after 7 days (Figure 1). LPS only modestly increased levels of TG, by 0.1 mmol/L, and this also returned to starting values within 7 days (results not shown). ApoCI expression apparently did not affect the total LPS-induced increase in TC (Figure 1) and TG levels (results not shown).
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) mice received an injection of LPS (50 µg). Twenty-four hours before LPS injection (t=0) and every 24 hours thereafter for a period of 7 days, plasma samples were taken after a 4-hour fast. TC was measured, and the values shown indicate the increase compared with t=0. Basal levels of TC were 7.0±0.5 and 9.3±1.8 mmol/L in apoe–/–apoc1–/– and apoe–/– apoc1+/+ mice, respectively. Values are mean±SD; n=8.
We next investigated the effect of repeated injections of LPS or vehicle on plasma TC and TG levels in apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice. LPS or vehicle was injected weekly for a period of 10 weeks, and plasma lipid levels were determined 24 hours before the first injection and 24 hours after the first, fifth, and tenth injections. The lipid response to the first injection was similar to that observed in the previous experiment. The response declined somewhat after the subsequent injections up to 50% after the tenth injection, which indicates that repeated LPS injections led to tolerance to some extent (data not shown). Over the entire period of these injections, LPS increased TC exposure for both genotypes by only 5%, which indicates that the transient increases in TC levels as a consequence of LPS injections only contributed marginally to total TC exposure. We also examined the effect of LPS and vehicle on cholesterol distribution over various lipoproteins in this experiment at 24 hours after the first injection. It appeared that the LPS-induced increased levels of TC in mice of both genotypes were mainly confined to intermediate-density lipoprotein (IDL)/LDL and HDL (Figure 2A and 2B). At later time points after the first injection, cholesterol levels decreased to a similar extent in IDL/LDL and HDL in both genotypes, and values similar to those of the vehicle group were reached after 7 days (results not shown). TG levels in the various lipoprotein fractions were only marginally affected (results not shown). Analysis of the apolipoprotein composition of the lipoproteins revealed that LPS did not affect apolipoprotein composition of VLDL and IDL/LDL (results not shown). However, in both genotypes, LPS similarly increased apoAI and led to the appearance of serum amyloid A in the HDL fractions. Representative fractions of HDL are shown in Figure 2C.
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Relation Between ApoCI and HDL Lipid Composition
Because HDL cholesterol correlates with binding and neutralizing LPS,30,31 and apoCI mainly circulates on HDL,19 we examined the relation between HDL lipids and HDL apoCI in apoe–/–apoc1+/+ mice at 24 hours after vehicle or LPS injection. In both vehicle- and LPS-injected mice, HDL apoCI did not correlate with HDL cholesterol (Pearson correlation coefficient r=–0.16 and 0.18, respectively; P=NS; Figure 3A and 3B). In contrast, HDL apoCI correlated positively with HDL phospholipids in vehicle-treated mice (r=0.68, P<0.05) and LPS-treated mice (r=0.58, P=0.08; Figures 3C and 3D).
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Effect of ApoCI on Plasma Inflammatory Markers After LPS Injection
Concomitant with the investigation of the effect of apoCI on the lipid response induced by a single LPS injection in time, as described above, we investigated the extent and duration of the LPS-induced inflammation and the effect of apoCI on it. Hereto, we measured plasma levels of fibrinogen and E-selectin before and during a period of 4 days after a single LPS injection into apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice. Basal plasma levels of fibrinogen and E-selectin before LPS injection (t=0) were not different between apoe–/– apoc1–/– and apoe–/–apoc1+/+ mice (Table). LPS injection resulted in a marked increase in fibrinogen and E-selectin in both apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice. Twenty-four hours after injection, plasma levels of these inflammation markers were maximal. Both markers decreased thereafter gradually and reached their starting value after a period of 4 days (Figure 4A and 4B). ApoCI expression augmented the LPS-induced inflammatory response with respect to both plasma markers, which was significant at 2 days after injection, as evident by a comparison of the response between apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice (P<0.05; Figures 4A and 4B). Fibrinogen and E-selectin were also measured after repeated injections of LPS and vehicle. The response to the first injection was similar to that described above and declined 15% after repeated LPS injections (data not shown), which indicates the development of some tolerance to LPS injections in both genotypes.
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In all, these data show that LPS transiently increases both plasma lipid levels and the inflammatory response in apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice. Whereas the lipid response after LPS injection was relatively small, LPS injection led to a marked increase in fibrinogen and E-selectin levels. The presence of apoCI had no effect on LPS-induced changes in plasma lipid concentrations but increased the magnitude of the LPS-induced inflammatory response.
Effect of ApoCI on LPS-Induced TNF- Response of Macrophages
Because we found previously that exogenous apoCI increased the LPS-induced TNF- response in macrophages,22 and apoCI mainly circulates on HDL,19 we next examined the effect of HDL (0.1 and 1 µg protein/mL) from apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice on the LPS-induced TNF- response by macrophages. ApoCI presence on HDL increased the TNF- response by 77% (P<0.05; HDL=0.1 µg protein/mL) and 66% (P<0.05; HDL=1 µg protein/mL) as evident from a comparison of the response between apoe–/–apoc1+/+ and apoe–/–apoc1–/– HDL (Figure 5A). In addition, we investigated the effect of apoCI expression by macrophages on the LPS-induced TNF- response using apoe–/–apoc1–/– and apoe–/–apoc1+/+ peritoneal macrophages. Endogenous apoCI expression by macrophages increased the TNF- response elicited by 1, 10, and 100 ng/mL LPS, which reached significance with 100 ng/mL LPS (45%; P<0.05; Figure 5B). Collectively, both apoCI on HDL and macrophage-derived apoCI contributed to enhancement of the LPS-induced TNF- response by macrophages.
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Effect of ApoCI on LPS-Induced Atherosclerosis Development
We next examined the effect of repeated LPS injections on atherosclerosis development and the effect of apoCI on LPS-induced atherosclerosis. For this purpose, mice were euthanized 24 hours after the tenth LPS injection, and the development of atherosclerosis was studied at the level of the aortic root. Representative lesions are shown in Figure 6A through 6D, and the data of all mice are summarized in Figure 6E. LPS increased atherosclerotic lesion area by 53% (P<0.05) in apoe–/–apoc1+/+ mice, as derived from the comparison between vehicle- and LPS-treated apoe–/–apoc1+/+ mice (Figure 6C versus 6D). In contrast, LPS did not affect the atherosclerotic lesion area in apoe–/–apoc1–/– mice (Figure 6A versus 6B). Furthermore, apoCI expression increased LPS-induced atherosclerosis by 60% (P<0.05), as evident from a comparison of LPS-injected apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice (Figure 6B versus 6D). In contrast, apoCI expression did not affect atherosclerosis in mice injected with vehicle (Figure 6A versus 6C). These data thus indicate that endogenous apoCI expression is a strong determinant of the LPS-induced increase in atherosclerotic lesion size (Figure 6E).
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Effect of ApoCI on LPS-Induced Atherosclerotic Lesion Composition
To study whether LPS treatment affected lesion composition, we characterized the atherosclerotic lesions with respect to monocyte adhesion, the content of CD3+ T cells, macrophages, SMCs, and collagen. In mice of both genotypes, LPS treatment tended to stimulate monocyte adhesion (Figure 7A) and T-cell recruitment (Figure 7B), although the effects did not reach statistical significance. These effects are consistent with previous observations that LPS stimulates monocyte adhesion and T-cell recruitment.5,18 In mice deficient in apoCI, LPS treatment did not affect macrophage area (Figure 7C), SMC area (Figure 7D), or collagen area (Figure 7E), as evident from a comparison of vehicle-treated and LPS-treated apoe–/–apoc1–/– mice. In contrast, in mice expressing apoCI, LPS increased the macrophage area significantly (P<0.05; Figure 7C), tended to increase SMC area (Figure 7D), and significantly increased collagen area (P<0.05; Figure 7E), as evident from a comparison of vehicle-treated apoe–/–apoc1+/+ mice and LPS-treated apoe–/–apoc1+/+ mice.
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In both the vehicle- and LPS-treated groups, apoCI expression did not affect monocyte adhesion (Figure 7A), yet it showed a tendency to increase T-cell recruitment (Figure 7B) and macrophage area (Figure 7C), as evident from a comparison of the vehicle- or LPS-treated apoe–/–apoc1–/– mice and the vehicle- or LPS-treated apoe–/–apoc1+/+ mice. Furthermore, in the LPS-treated groups, apoCI expression significantly increased SMC (Figure 7D) and collagen (Figure 7E) area, as evident from a comparison of LPS-treated apoe–/–apoc1–/– mice and LPS-treated apoe–/–apoc1+/+ mice.
Taken together, apoCI expression accelerated atherosclerosis after treatment with LPS. As a result, the atherosclerotic lesions increased in size and concomitantly contained more SMCs and collagen, which reflected the progression of atherosclerosis.
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Discussion |
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Gram-negative bacteria such as C pneumoniae release LPS on multiplication or lysis during infections, which leads to a chronic inflammatory state that accelerates atherosclerosis in humans and rodents.9–18 We have shown previously that apoCI binds to LPS, thereby augmenting the inflammatory response to LPS and K pneumoniae in mice and in macrophages in vitro.22 In the present study, we investigated the significance of these observations for a chronic inflammatory disease, atherosclerosis, by assessing the effect of apoCI expression on LPS-induced atherosclerosis in apoe–/– mice in vivo. We found that endogenous apoCI increased atherosclerosis development in apoe–/– mice induced by chronic treatment with LPS.
We showed that injection of LPS similarly increased the TC content of IDL/LDL and HDL in both apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice and marginally increased TG levels. This response was transient, which indicates that the effects were induced after LPS injection, returning to their starting value before the next LPS injection. It is tempting to speculate about the mechanism underlying the lipid changes in the present study after LPS injection. Serum amyloid A and apoAI, which were both elevated after LPS injection, can contribute to an increased HDL plasma level by stimulating HDL generation.32,33 In addition, serum amyloid A can inhibit HDL clearance.34 In contrast to the present findings, HDL cholesterol decreases in humans upon challenge with LPS.35 We speculate that the increased HDL cholesterol in the present mouse model is mainly related to reduced clearance of HDL as a consequence of the absence of apoE that mediates the clearance of HDL, as reported previously.36 The increased cholesterol content in IDL/LDL might be secondary to reduced LPL activity, because LPS injection decreases LPL activity.37 The effects of LPS on lipid levels were transient and similar in both mouse models, and because the increased levels of plasma cholesterol contributed only 5% to the total cholesterol exposure over time until atherosclerosis assessment (not shown), it is unlikely that the transient increase in plasma lipid levels after LPS injection was a primary contributor to development of atherosclerosis.
The augmenting effect of apoCI on LPS-induced atherosclerosis is related to a greater inflammatory status in LPS-treated apoe–/–apoc1+/+ mice than in apoe–/–apoc1–/– mice. We showed that apoCI expression enhanced the effect of LPS on fibrinogen and E-selectin. Fibrinogen is an acute-phase protein that is secreted from the liver and that reflects the general inflammatory status of mice and putatively participates in atherosclerotic lesion development.38 Fibrinogen is primarily regulated by interleukin-6.39 E-selectin is a target gene of the nuclear factor–B pathway in macrophages and reflects the inflammatory state of the vessel wall.40 ApoCI thus increased LPS-induced inflammatory responses both systemically and at the vascular level. Although the difference in magnitude of the LPS-induced inflammatory response was relatively small, it has recently been stated in humans that small effects on inflammatory responses contribute substantially to CVD.41 ApoCI expression per se did not affect these inflammatory markers in vehicle-treated mice, an observation that is in agreement with the observed comparable atherosclerotic lesion area in vehicle-treated apoe–/–apoc1–/– and apoe–/–apoc1+/+ mice. In addition, apoCI expression by apoe–/– macrophages per se increased the LPS-induced inflammatory response, and apoCI on HDL from apoe–/– mice was crucial for increasing the inflammatory response of macrophages toward LPS.
A recent human study showed that HDL cholesterol negatively correlates with the response to a single administration of LPS.30 Because apoCI is mainly localized on HDL, it may have been expected that apoCI would be associated with a decreased LPS response in the present mouse study. However, although HDL apoCI correlated with HDL phospholipids, owing to the distribution of both components in the particle shell, HDL apoCI did not correlate at all with HDL cholesterol. Therefore, the present study does not exclude the possibility that apoCI also increases the LPS response in humans.
We thus conclude that apoCI accelerates LPS-induced atherosclerosis progression in apoe–/– mice mainly as a consequence of increasing inflammation. This is in line with the finding that LPS-treated apoe–/– mice expressing human apoA-IV show reduced production of proinflammatory cytokines and reduced atherosclerosis compared with their apoe–/– littermates.25
LPS treatment did not affect lesion composition of mice deficient in apoCI, yet it increased macrophage and collagen area in mice expressing apoCI. The increased macrophage content might have been the consequence of the increased inflammation in macrophages after apoCI expression, as we showed in vitro. However, comparison of lesions from both vehicle groups and LPS-injected groups with the same atherosclerotic lesion area revealed no differences regarding atherosclerotic lesion composition (not shown). Also, in advanced lesions, we observed adventitial infiltrates of activated lymphocytes, which have been reported to be caused by LPS injections in apoe–/– mice.18 Because we only observed these infiltrates in advanced atherosclerotic lesions, we suggest that these infiltrates were the consequence of the severity of the lesion rather than being caused by LPS specifically. In addition, it has been reported that apoCI increases apoptosis in human aortic SMCs by recruiting neutral sphingomyelinase.42 It is unlikely that this effect contributed to atherosclerosis in the aortic root in the present study, because apoCI did not affect SMCs in vehicle-treated mice.
At first glance, the finding that apoCI expression did not significantly affect atherosclerosis in the vehicle-treated mice appears to be in contrast to our previous data showing that apoCI accelerates atherosclerosis as related to increased VLDL lipid levels in mice of 26 weeks of age.26 However, in the present study, VLDL lipid levels were somewhat increased on apoCI expression, yet the absolute values in both mouse genotypes were 20% lower than in our previous study. In fact, the relatively low lipid levels in the present study led to a relatively slow progression of atherosclerosis that was not different between mice of both genotypes, and this enabled us to investigate the effect of apoCI on LPS-induced atherosclerosis specifically. Indeed, mice need to express a certain level of hyperlipidemia for LPS to have an effect on atherosclerosis development, as was demonstrated in former studies using C pneumoniae, which mainly exerts its effects via LPS.15,43,44 We speculate that the effects of apoCI on LPS-induced atherosclerosis are mediated via TLR4 in macrophages, because TLR4 is primarily activated by LPS45 and has been demonstrated to be involved in LPS-induced atherosclerosis development in mice.16 The mechanism behind the augmenting effect of apoCI on the response toward LPS is currently under investigation.
We conclude that apoCI is crucially involved in LPS-induced atherosclerosis in apoe–/– mice, mainly as a consequence of enhancing the inflammatory response. We anticipate that in humans who suffer from chronic inflammation, plasma apoCI may enhance atherosclerosis development and CVD.
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Acknowledgments |
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We thank Erik Offerman and Karin Toet for excellent technical assistance.
Sources of Funding
This work was performed within the framework of the "Leiden Center for Cardiovascular Research LUMC-TNO" and was supported by the Netherlands Organization for Scientific Research (NWO-VIDI grant 917.36.351 to Dr Rensen, program grant 903.39.291 to Dr Havekes, and NWO-VENI grant 016.036.061 to Dr Kleemann), the Netherlands Heart Foundation (NHS grant 2005B226 to Dr Rensen), and the LUMC (Gisela Thier Fellowship to Dr Rensen).
Disclosures
None.
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CLINICAL PERSPECTIVE
Accumulating evidence indicates that Gram-negative bacteria such as Chlamydia pneumoniae and Porphyromonas gingivalis are involved in cardiovascular disease. Infection with these pathogens may cause a chronic inflammatory state as a result of the release of endotoxins such as lipopolysaccharide (LPS), which accelerates the development of atherosclerosis in humans and rodents. It has been generally assumed that high-density lipoprotein (HDL) binds LPS, thereby attenuating the inflammatory response, which may contribute to the antiatherosclerotic potential of HDL. However, we have shown previously that apolipoprotein C-I (apoCI), a small and highly positively charged surface apolipoprotein that is mainly HDL-associated, binds LPS and augments the inflammatory response toward LPS and Gram-negative bacteria in mice and macrophages. In the present study, we investigated the significance of these observations for atherogenesis. We assessed the effect of endogenous apoCI expression on atherosclerosis in apoE-knockout mice that were treated chronically with LPS. Although the present study confirmed that LPS itself aggravates atherosclerosis in apoE-knockout mice, apoCI appeared to be a crucial determinant of LPS-induced aggravation of atherosclerosis. Endogenous apoCI expression enhanced inflammation both systemically and at the level of the vessel wall. In addition, HDL-associated apoCI increased the inflammatory response toward LPS in macrophages in vitro. These data thus indicate that plasma apoCI may increase the cardiovascular disease risk associated with Gram-negative bacterial infections. Furthermore, the present study underscores the importance of the apolipoprotein composition of HDL for the antiatherosclerotic potential of HDL, which should be considered in the design and analysis of HDL-raising strategies aimed at reducing cardiovascular disease risk.
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*Drs Westerterp and Berbée contributed equally to this article.
The online-only Data Supplement, consisting of an expanded Methods section, is available with this article at http://circ.ahajournals.org/cgi/content/ full/CIRCULATIONAHA.107.693382/DC1.
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