Published online before print February 16, 2009, doi: 10.1161/CIRCULATIONAHA.108.810721
(Circulation. 2009;119:1135-1145.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
Inflammation Impairs Reverse Cholesterol Transport In Vivo
From the Cardiovascular Institute and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia (F.C.M., C.C.H., E.H.C., J.T.B., M.P.R.); and Gastrointestinal and Nutrition Division, Children’s Hospital of Philadelphia, Philadelphia, Pa (M.d.l.L.M., G.H.R.).
Correspondence to Muredach P. Reilly, Cardiovascular Institute, University of Pennsylvania Medical Center, 609 BRB 2/3, 421 Curie Blvd, Philadelphia, PA 19104-6160. E-mail muredach{at}spirit.gcrc.upenn.edu
Received July 29, 2008; accepted December 17, 2008.
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Abstract |
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Background— Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT.
Methods and Results— We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans. Endotoxemia (3 mg/kg SC) reduced 3H-cholesterol movement from macrophage to plasma and 3H-cholesterol associated with HDL fractions. At 48 hours, bile and fecal counts were markedly reduced consistent with downregulation of hepatic expression of ABCG5, ABCG8, and ABCB11 biliary transporters. Low-dose lipopolysaccharide (0.3 mg/kg SC) also reduced bile and fecal counts, as well as expression of biliary transporters, but in the absence of effects on plasma or liver counts. In vitro, lipopolysaccharide impaired 3H-cholesterol efflux from human macrophages to apolipoprotein A-I and serum coincident with reduced expression of the cholesterol transporter ABCA1. During human (3 ng/kg; n=20) and murine endotoxemia (3 mg/kg SC), ex vivo macrophage cholesterol efflux to acute phase HDL was attenuated.
Conclusions— We provide the first in vivo evidence that inflammation impairs RCT at multiple steps in the RCT pathway, particularly cholesterol flux through liver to bile and feces. Attenuation of RCT and HDL efflux function, independent of HDL cholesterol levels, may contribute to atherosclerosis in chronic inflammatory states including obesity, metabolic syndrome, and type 2 diabetes.
Key Words: atherosclerosis • cholesterol • inflammation • lipoproteins • macrophages
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Introduction |
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Reverse cholesterol transport (RCT) is the process by which cholesterol in peripheral cells (eg, lipid-laden foam cells) is effluxed onto circulating high-density lipoprotein (HDL) and transported back to the liver for secretion into bile and feces.1,2 Promotion of RCT is considered a major antiatherogenic function of HDL.3 Inflammation is proposed to impair HDL function and RCT. This may be of pathophysiological significance because attenuation of RCT might contribute to atherosclerosis in chronic inflammatory states, including metabolic syndrome and type 2 diabetes. In the present study, we provide the first in vivo and ex vivo proof of concept that inflammation impairs RCT and does so at multiple steps in the RCT pathway.
Clinical Perspective p 1145
Previous studies in vitro and in vivo suggest that acute inflammation induces changes in HDL composition and metabolism that may impair RCT.4–6 These include induction of acute phase lipases that catabolize HDL phospholipids,6–8 increased HDL content of acute phase serum amyloid A (SAA) with displacement of apolipoprotein (apo) A-I,9 downregulation of the hepatic HDL cholesterol receptor scavenger receptor class B type I (SR-BI),10 and reduced expression of hepatic transporters involved in excretion of cholesterol11 and bile acids.12 However, the integrated effect of in vivo inflammation on RCT and the relative impact on individual RCT steps have not been examined.
We employed subacute endotoxemia in the rodent macrophage-to-feces RCT model, previously described by our group,13–15 to assess the effects of inflammation on RCT in vivo. Briefly, this model tracks 3H-cholesterol from intraperitoneally injected J774 macrophages onto HDL in plasma and subsequent uptake by liver and clearance into bile and feces. Furthermore, ex vivo cholesterol efflux studies to HDL isolated from control and inflamed mice were performed to examine the capacity of acute phase HDL to accept cholesterol from macrophages. Finally, we performed translational studies in humans that extend the proof of concept by demonstrating that inflammation may also retard RCT in humans.
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Methods |
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Cell Culture
J774 Macrophage Preparation for RCT and Ex Vivo Cholesterol Efflux Studies
RCT studies were performed as described previously.13–16 J774 macrophages were grown in suspension and incubated for 48 hours in labeling media containing acetylated low-density lipoprotein (LDL) (25 µg/mL) and 3H-cholesterol (5 µCi/mL). Cells were washed, equilibrated, centrifuged, and resuspended in minimal essential media before intraperitoneal injection. For murine ex vivo efflux studies, J774 macrophages were labeled and loaded in a manner identical to that in in vivo studies (see Methods in the online-only Data Supplement). For human ex vivo efflux studies, we used a simple, established model of ABCA1 efflux17,18-unloaded J774 macrophages plus ACAT inhibitor (2 µg/mL) plus or minus cAMP (0.3 mmol/L).
In ex vivo cellular cholesterol efflux studies, plasma from mice was collected at 0, 6, 24, and 48 hours after lipopolysaccharide treatment (3 mg/kg SC). Serum from humans was collected at 4, 8, 12, and 24 hours after lipopolysaccharide treatment (3 ng/kg IV). ApoB-containing lipoproteins were removed from plasma/serum by polyethylene glycol precipitation.19 Ex vivo efflux from labeled macrophages to 2.8% HDL supernatant or minimal essential media control was measured over 4 hours. Efflux of 3H-cholesterol was normalized to cholesterol content in supernatants.
Human Macrophage Culture and In Vitro Cholesterol Efflux Studies
Human monocytes, isolated from healthy volunteers, were purified by countercurrent elutriation and incubated in macrophage differentiation media for 7 days (RPMI, 20% fetal bovine serum, 100 ng/mL macrophage colony-stimulating factor). Macrophages were loaded and labeled for 24 hours (RPMI, 10% fetal bovine serum, 50 µg/mL acetylated LDL, 6 µCi/mL 3H-cholesterol), then washed, equilibrated, and treated with or without lipopolysaccharide (100 ng/mL) for 4 hours. Cholesterol efflux to human apoA-I (20 µg/mL), HDL3 (50 µg/mL), serum (2.5%), and minimal essential media control was assessed over a 4-hour period. Protein and mRNA expressions of cholesterol transporters were also assessed after 4-hour lipopolysaccharide treatment.
Rodent In Vivo RCT Studies
C57BL/6 mice, in metabolic cages (Tecniplast, USA, Inc) were fed chow diet ad libitum. Our rationale for choice of route and dose of lipopolysaccharide and pilot study data are presented in the online-only Data Supplement. Anesthetized mice were injected subcutaneously with lipopolysaccharide (0.3 or 3 mg/kg) or PBS 4 hours before and 24 hours after intraperitoneal injection of labeled macrophages (
4.5 million cpm/2.3 million cells per mouse). Blood was collected, and liver and gall bladder were isolated and prepared for lipid, protein, and mRNA studies as described previously13,14 and in the online-only Data Supplement. 3H-label counts in plasma, liver, and feces are expressed as a percentage of total 3H-cholesterol injected. The University of Pennsylvania Institutional Animal Care and Use Committee approved rodent studies, and mice were handled according to Institutional Animal Care and Use Committee guidelines.
Human Endotoxemia Studies
Healthy volunteers (n=20, 50% female, aged 18 to 40 years), without medical history, tobacco or prescription medication use, or laboratory abnormality were recruited from the Delaware Valley region.20,21 Whole blood samples were collected at indicated time points before and after intravenous infusion of 3 ng/kg US standard reference endotoxin (lot No. CC-RE-LOT-1+2, Clinical Center, National Institutes of Health), a dose that we have shown produces a robust inflammatory response.20 The University of Pennsylvania institutional review board approved the study, and written informed consent was provided.
Laboratory Methods
A description of general laboratory methods including lipoprotein analysis, quantitative real-time polymerase chain reaction, and immunoblot analysis are available in Methods in the online-only Data Supplement.
Statistical Analysis
Data are reported as mean±SEM. For mouse experiments with lipopolysaccharide and saline treatments over multiple time points, we performed 2-way repeated-measures ANOVA to test for differences in means. When the ANOVA test was significant, post hoc Bonferroni corrected t tests were applied. For comparison of data between lipopolysaccharide- and saline-treated groups at a single time point (liver, bile, feces, cellular efflux, and mRNA data), unpaired t tests were performed. Endotoxemia effects over time in humans were tested by 1-way repeated-measures ANOVA, and, when significant, post hoc Bonferroni corrected t tests were performed comparing time points after lipopolysaccharide treatment to baseline. GraphPad Prism 5 (GraphPad Software Inc, San Diego, Calif) and Stata 9.0 software (Stata Corp, College Station, Tex) were used for statistical analyses. Statistical significance is presented as *P<0.05, **P<0.01, and ***P<0.001 in all figures.
The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Inflammatory and Lipoprotein Responses in Rodents After Endotoxin Challenge
Subcutaneous administration of lipopolysaccharide (3 mg/kg) induced systemic and hepatic inflammation (Figure I in the online-only Data Supplement). Plasma levels of tumor necrosis factor-
increased transiently, whereas interleukin-6 had more sustained elevation (Figure IA and IB in the online-only Data Supplement). Hepatic mRNA levels of both cytokines were elevated at 6 hours (Figure IC in the online-only Data Supplement). No evidence of hepatotoxicity, as reflected in plasma alanine aminotransferase levels, was observed during endotoxemia (Figure ID in the online-only Data Supplement).
As reported previously,4,22–24 lipopolysaccharide increased total plasma cholesterol levels (Figure IE in the online-only Data Supplement). Fast protein liquid chromatography (FPLC) profiles (48 hour) confirmed increased LDL cholesterol mass with lipopolysaccharide treatment (2-fold) but minimal effect on HDL cholesterol mass (Figure IF in the online-only Data Supplement). Increased apoB protein in FPLC LDL fractions was observed (Figure 1F and 1G) and coincided with reduced hepatic expression of the LDL receptor (Table I in the online-only Data Supplement). An increase in cholesterol mass levels in the shoulder region between LDL and HDL peaks was also observed with lipopolysaccharide treatment (1.7-fold increase); these particles were apoA-I/apoB poor and apoE/SAA enriched (Figure 1F and 1G and Figure 2D). Increased HDL-associated apoE was observed despite reduced hepatic apoE mRNA levels during endotoxemia (Table I in the online-only Data Supplement).
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Initial RCT experiments were performed with 3 mg/kg lipopolysaccharide (Figures 1 to 3
), a dose that induced significant lipoprotein changes as well as systemic and hepatic inflammation. Subsequently, we used a lower dose of lipopolysaccharide (0.3 mg/kg; Figure 4) to determine the impact of low-grade inflammation on RCT.
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Endotoxemia Reduced Macrophage 3H-Cholesterol Efflux to Plasma HDL In Vivo
The effect of lipopolysaccharide (3 mg/kg SC) on 3H-cholesterol movement from intraperitoneal macrophages into the plasma compartment was monitored over time. Analysis of pooled data across multiple studies (9 studies; n=6 mice per treatment group per study) showed a moderate but consistent reduction in plasma counts at 4 and 24 hours with no difference observed at 48 hours (Table, Figure 1A).
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FPLC profiles of plasma from 3 separate experiments were analyzed to assess the distribution of cholesterol mass and label across lipoproteins during endotoxemia. In control mice, the majority of counts (65%) were found in HDL fractions. At 4 hours, counts in HDL fractions were reduced by 61% with lipopolysaccharide and remained attenuated at 24 hours (–44%) and 48 hours (–16.5%) (Figure 1D and 1E). In contrast, little change in HDL cholesterol mass was observed with lipopolysaccharide at 4 hours (1.17-fold control), 24 hours (1.07-fold control), and 48 hours (1.04-fold control) (Figure 1B and 1C). There was a reduction in the ratio of cholesterol counts/mass, ie, the rate of enrichment of HDL with new 3H-cholesterol from macrophages after lipopolysaccharide treatment (Table II in the online-only Data Supplement). Analysis of FPLC fractions revealed decreased apoA-I and increased apoE and SAA content in HDL fractions after lipopolysaccharide treatment (Figure 1F and 1G and Figure 2C and 2D). A marked induction in hepatic SAA mRNA (Figure 2A) coincided with increased plasma protein levels (Figure 2B). In summary, during endotoxemia we found that (1) HDL cholesterol mass was unchanged but HDL apoA-I content was reduced; (2) HDL particles were enriched with SAA and apoE; and (3) there was reduced 3H-cholesterol movement from macrophages to HDL.
Endotoxemia Impairs Murine HDL Efflux Capacity Ex Vivo
These findings suggest that lipopolysaccharide reduced 3H-cholesterol transfer from macrophages to remodeled HDL particles in vivo. Therefore, we determined, ex vivo, the capacity of the inflammatory HDL fraction (2.8% supernatant after polyethylene glycol precipitation of apoB) to promote 3H-cholesterol efflux from loaded J774 macrophages. Efflux from macrophages to HDL supernatant collected at 6, 24, and 48 hours after lipopolysaccharide treatment (from a 3-mg/kg SC non-RCT study) was significantly reduced compared with saline-treated control (Figure 2E). These studies support the concept that during inflammation, alteration in HDL structure/composition impairs HDL acceptor function in vivo.
Endotoxemia Markedly Impairs Hepatic to Bile and Fecal 3H-Cholesterol Secretion
We assessed the effects of inflammation on later steps in the RCT pathway involving hepatic 3H-cholesterol uptake and flux through liver to bile and feces. Analysis of hepatic counts across multiple studies revealed a nonsignificant reduction (–13.5±6.2%) in counts at 48 hours with lipopolysaccharide (Figure 3A and Table). Despite a significant reduction in hepatic SR-BI mRNA during endotoxemia (Figure 3B), little change in SR-BI protein was observed (Figure 3F). Little difference in hepatic ABCG1 and ABCA1 protein levels was observed at any time point, consistent with minimal effects on hepatic mRNA of these transporters (Figure 3B and 3F).
The most pronounced effect of endotoxemia on the RCT pathway was on 3H-cholesterol secretion from liver to bile and feces (Table, Figure 3). At 48 hours, lipopolysaccharide markedly reduced bile counts (–56.3±4.5%) coincident with reduced mRNA levels for ABCG5, ABCG8, and ABCB11 transporters and the bile-acid synthesis enzyme CYP7A1 (Figure 3C and 3D). Effects on transporter mRNAs were observed as early as 6 hours after lipopolysaccharide treatment: ABCG5 (–84.1±1.5%), ABCG8 (–91.1±1.7%), and ABCB11 (–88.9±2.3%) (n=5; P<0.001 for all). A reduction in ABCG5/8 heterodimer (150 kDa)25 protein levels was observed at all time points, consistent with reduced mRNA levels (Figure 3F, 24-hour blots presented).
A consistent reduction in fecal counts was also observed after lipopolysaccharide treatment (–62.1±5.7%) (Table, Figure 3E). Lipopolysaccharide had similar effects on fecal free cholesterol (–71.9±4.8%) and bile-acid (–65.2±5.9%) levels (pooled data representative of 3 studies; n=6 per treatment group per study; P<0.001).
Low-Dose Endotoxin Selectively Blocks Hepatic Cholesterol and Bile-Acid Secretion
Because the effects of a moderate subcutaneous lipopolysaccharide dose on bile counts were dramatic, we hypothesized that mild "subclinical" inflammation with low-dose lipopolysaccharide might attenuate liver to bile 3H-cholesterol even in the absence of significant effects on lipoprotein levels or HDL functionality. Low-dose lipopolysaccharide (0.3 mg/kg SC) had minimal effects on plasma tumor necrosis factor- levels (P=0.56) (Figure 4A), induced modest increase in plasma SAA levels (76.3±21.1 to 167.4±11.0 mg/dL at 48 hours; n=6; P<0.01), but had no effect on serum lipoprotein FPLC profiles (Figure 4C). Low-dose lipopolysaccharide did not reduce 3H-cholesterol plasma counts (Figure 4B), had no impact on HDL cholesterol counts/mass ratio (1.1) (Figure 4C and 4D), and did not reduce liver counts (Figure 4E). Nonetheless, low-dose lipopolysaccharide significantly reduced bile (–36%) and fecal counts (–69%) (Figure 4F and 4G), which correlated with reduced ABCG5 (–23%), ABCG8 (–37%), and ABCB11 (–44%) mRNAs (Figure 4H).
Endotoxemia Modulates the RCT Pathway in Humans
We performed proof of concept studies in humans to establish whether experimental endotoxemia modulates components of human RCT in vivo. As reported previously, endotoxin (3 ng/kg IV) induced a short flu-like illness in humans with acute and transient increases in inflammatory cytokines that resolved within 6 to 8 hours.20 A significant and sustained elevation of C-reactive protein was evident after lipopolysaccharide treatment (Figure 5D). Minimal reductions in plasma HDL cholesterol and apoA-I were observed (Figure 5A and 5B). There were, however, substantial decreases in plasma (Figure 5C) and HDL (–27.8±8.1% at 24 hours; n=20; P<0.005) phospholipids, whereas plasma (Figure 5D) and HDL-associated (38.3±3.7-fold increase at 24 hours; n=20; P<0.001) SAA increased dramatically. Furthermore, compared with baseline there was a marked and progressive reduction in the ex vivo capacity of human HDL (baseline, 6, 24, and 48 hours) to promote 3H-cholesterol efflux from J774 macrophages after lipopolysaccharide treatment (Figure 5E). These findings are consistent with inflammatory-mediated remodeling of HDL and impaired RCT in humans during inflammation.
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Endotoxin Impairs Human Macrophage Efflux Function In Vitro
Inflammation may also directly impair human macrophage cholesterol efflux, the first step of the RCT pathway.9,26,27 Lipopolysaccharide (100 ng/mL for 4 hours) reduced in vitro 3H-cholesterol efflux from human monocyte-derived macrophages to apoA-I (–19.6%) and serum (–18.3%) with no effect on efflux to HDL3 (–3.1%) (Figure 5F). Significant reductions in cholesterol transporters ABCA1 and ABCG1 and SR-BI mRNA levels were apparent (Figure 5G). Immunoblot analysis revealed a marked reduction in ABCA1, a moderate reduction in SR-BI, and little change in ABCG1 protein levels (Figure 5H).
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Discussion |
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We demonstrate that acute inflammation retards RCT in vivo and provide evidence that this atherogenic response may occur in humans during inflammatory syndromes. Endotoxemia has a broad and integrated impact on RCT, attenuating several steps including macrophage cholesterol efflux, HDL acceptor function, and hepatic to bile/fecal cholesterol elimination. The profound impact on cholesterol secretion into bile, even with low doses of lipopolysaccharide, provides a novel insight into a major inflammatory regulation of the final steps of RCT. Overall, our studies suggest a substantial attenuation of atheroprotective RCT, with apparent conservation of body cholesterol stores, during inflammation.
Our findings showed reduced plasma 3H-cholesterol counts at early time points after lipopolysaccharide treatment with less pronounced effects at later time points. Because plasma counts are a single integrative measure of RCT, it is difficult to draw conclusions about effects on individual RCT processes solely from changes in plasma counts. Different components of RCT can influence plasma counts in opposite directions; impaired efflux from macrophages and reduced acceptor capacity of HDL reduce plasma counts, whereas reduced flux through liver to bile and feces may increase counts. Notably, in the setting of reduced RCT, plasma counts of SR-BI–deficient mice are increased because of reduced hepatic clearance of HDL cholesterol.13 Indeed, our data suggest that inflammation may reduce the flux of 3H-cholesterol from plasma through liver because of a marked decrease in clearance from liver to bile while also impairing macrophage efflux and HDL acceptor function. These opposing influences may account for the modest effect on 48-hour plasma counts.
We performed in vitro studies examining the direct impact of lipopolysaccharide on macrophage cholesterol efflux using a foam cell model of cholesterol-loaded, primary human monocyte-derived macrophages. Lipopolysaccharide impaired cholesterol efflux to human apoA-I and serum coincident with reduced expression of ABCA1. There was little change in ABCG1 protein, perhaps accounting for the lack of effect on macrophage efflux to HDL3, an acceptor for ABCG1-mediated efflux.28,29 Despite reductions in SR-BI mRNA and protein, the lack of effect of lipopolysaccharide on HDL3 efflux also argues against a significant role for SR-BI in human macrophage–foam cell efflux, as has been suggested by recent studies in rodents.28–30 These findings suggest that inflammation may chronically impair ABCA1-mediated cholesterol efflux from arterial foam cells, thus accelerating atherosclerosis.
Reduced 3H-cholesterol in plasma may also reflect reduced capacity of inflammatory HDL particles to efficiently accept cholesterol from macrophages. HDL particles undergo remodeling during acute inflammation through activation of lipases such as secretory phospholipase A231 and endothelial lipase,31,32 induction of and HDL association with SAA,33 attenuation of lecithin cholesterol acyltransferase34 (and cholesteryl ester transfer protein activity in humans35), as well as oxidant modification of apoA-I.36 In fact, such changes have been shown to modify HDL efflux27,34 and anti-inflammatory functions.37 In our in vivo rodent studies, the marked reduction of HDL 3H-cholesterol/cholesterol mass suggests an inflammatory-mediated loss of HDL acceptor function in vivo coincident with impaired RCT. In parallel, our ex vivo studies of HDL function demonstrated that inflammatory remodeling of HDL impairs its capacity to serve as an acceptor for macrophage ABCA1 cholesterol efflux.
Van der Westhuyzen and colleagues38 have shown that SAA can accept cholesterol from ABCA1 and SR-B1 efflux pathways and, contrary to our findings, demonstrated increased efflux to HDL from mice overexpressing SAA and acute phase HDL. Differences in cell models (J774 macrophages versus hepatocytes and CHO cells overexpressing SR-BI) and differences in HDL preparation may account for discrepant findings. Our studies do not directly address SAA efflux function, whereas Van der Westhuyzen et al did not assess the impact of inflammation on other aspects of HDL function or on RCT in vivo. It is not clear that increased HDL-SAA in vivo improves HDL acceptor function. For example, SAA may displace apoA-I from HDL and, in conjunction with other documented inflammatory HDL changes,39–41 may not improve HDL efflux functions. In fact, our mouse in vivo RCT and ex vivo HDL efflux data as well as our human HDL ex vivo efflux studies strongly suggest that the integrated inflammatory effect is to retard multiple steps of RCT in vivo including HDL cholesterol acceptor function.
During rodent endotoxemia, we observed increased numbers of larger HDL particles that were apoA-I poor and apoE/SAA enriched. Increased HDL-associated apoE has been reported previously42,43 and is likely attributable to reduced clearance via the LDL receptor rather than increased production as we and others42 found reduced hepatic apoE mRNA levels during endotoxemia. We also found that lipopolysaccharide increased total cholesterol and apoB levels, as has been reported in rodents.4,22–24 Mechanisms of apoB lipoprotein changes include reduced LDL clearance due to downregulation of LDL receptor and upregulation of PCSK9 and increased 3-hydroxy-3methylglutaryl coenzyme A reductase activation.4,22,23 Our findings, however, suggest that increased apoB plays a trivial role, if any, in modulating RCT changes during inflammation.
Despite species differences in lipoprotein metabolism,4,44 several inflammatory-mediated changes in HDL structure, composition, and efflux function appear consistent across human and rodent species. In humans, endotoxin reduced plasma and HDL phospholipids, possibly as a result of activation of inflammatory HDL lipases, endothelial lipase, and secretory phospholipase A2.31,45 We recently demonstrated that reduced HDL phospholipids coincided with induction of endothelial lipase during human endotoxemia.32 Despite only modest changes in human HDL cholesterol and apoA-I levels, endotoxin markedly increased plasma and HDL-associated SAA. Furthermore, the functional capacity of human acute phase HDL to efficiently efflux cholesterol from macrophages ex vivo was impaired, in concordance with our rodent studies. Our human studies provide evidence for consistent effects, across species, of endotoxin on HDL function.
In rodent studies, the greatest impact of lipopolysaccharide was on movement of 3H-cholesterol through liver to bile and feces, indicating that inflammation targets hepatic RCT-related lipoprotein metabolism. Despite the consistent reduction in hepatic SR-BI mRNA expression, little difference in SR-BI protein levels and hepatic 3H-cholesterol counts was observed, suggesting that hepatic cholesterol uptake is not affected during inflammation. In contrast, the striking reduction in bile and fecal counts suggests attenuation of cholesterol elimination from liver to bile. A remarkable and consistent finding was early and sustained suppression of the biliary cholesterol transporters (ABCG5 and ABCG8) at both the mRNA and protein levels. These changes correlated with reduced bile counts and fecal elimination of 3H-cholesterol, the final step of RCT. In fact, low-dose endotoxin also reduced bile and fecal counts and expression of bile-acid and cholesterol transporters in the absence of significant systemic inflammation and changes in plasma or hepatic 3H-cholesterol levels. Thus, even modest hepatic inflammation may result in selective attenuation of these final steps of RCT.
The endotoxemia model used in our rodent and human studies provides proof of principle that inflammation attenuates RCT while being broadly relevant to human inflammatory pathophysiologies, especially infections, sepsis, and acute rheumatologic disorders. Although subacute in nature, the low-dose rodent studies may be of relevance to chronic conditions associated with hepatic inflammation including nonalcoholic steatohepatitis,46 hepatic insulin resistance, and metabolic syndrome.47,48 We emphasize, however, the need for additional work to assess the specific impact on RCT of chronic low-grade human inflammation in obesity, metabolic syndrome, and atherosclerosis.
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Conclusions |
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We present the first in vivo functional evidence to support the hypothesis that inflammation impairs RCT and does so at numerous steps in the pathway from initial macrophage efflux to HDL acceptor function and the final step of cholesterol flux through liver to bile and feces. This study strengthens the hypothesis that impaired RCT may be an important link between the low-grade inflammation of insulin-resistant conditions and the development and acceleration of atherosclerosis.
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Acknowledgments |
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We would like to gratefully acknowledge Aisha Wilson, Maosen Sun, Edwige Edouard, Anna DiFlorio, Linda Morrell, and Leticia Pruscino for their technical expertise.
Sources of Funding
This work was supported by a Clinical and Translational Science Award (RFA-RM-06-002) from the National Center for Research Resources, RO1 HL-073278 (Dr Reilly), P50 HL-083799-SCCOR (Dr Reilly), and the Alternative Drug Discovery Initiative award to the University of Pennsylvania from GlaxoSmithKline.
Disclosures
Dr Reilly is the recipient of research grants from GlaxoSmithKline and Merck Research Laboratories. The other authors report no potential conflicts of interest.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.810721/DC1.
Related Article:
Circulation 2009 119: 1067-1068.
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