Knockdown of Hepatic ABCA1 by RNA Interference Decreases Plasma HDL Cholesterol Levels and Influences Postprandial Lipemia in Mice
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动脉硬化血栓血管生物学 2005年第7期
From the Institute for Biochemistry and Molecular Biology II, Molecular Cell Biology (S.R., A.N., A.L., U.B., J.H.), and the Departments of Orthopedics (A.N.) and Internal Medicine (B.L., M.M.), University Hospital Hamburg-Eppendorf, Hamburg, Germany.
Correspondence to Dr Joerg Heeren, IBM II, Molecular Cell Biology, University Hospital Hamburg-Eppendorf Martinistrasse 52 D-20246 Hamburg, Germany. E-mail heeren@uke.uni-hamburg.de
Abstract
Objective— To investigate the impact of hepatic ABCA1 on systemic lipoprotein metabolism in vivo by an adenovirus-mediated RNA interference approach.
Methods and Results— Efficiency of plasmid-based small interference RNA (siRNA)-induced knockdown of cotransfected murine ATP binding cassette transporter A1 (mABCA1) in HEK-293 cells was judged by RT-polymerase chain reaction, immunofluorescence, and Western blot analysis. The most effective plasmid was used to generate a recombinant adenovirus as a tool to selectively downregulate ABCA1 expression in mouse liver (C57BL/6). In comparison to controls, Western blot analysis from liver membrane proteins of Ad-anti-ABCA1 infected mice resulted in an 50% reduction of endogenous ABCA1 and a clear upregulation of apolipoprotein E. Fast protein liquid chromatography analysis of plasma revealed that hepatic ABCA1 protein reduction was associated with an 40% decrease of HDL cholesterol and a reduction of HDL-associated apolipoprotein A-I and E. In the fasted state, other lipoprotein classes were not affected. To analyze the influence of ABCA1 downregulation on postprandial lipemia, infected mice were given a gastric load of radiolabeled trioleate in olive oil. In Ad-anti-ABCA1 infected mice, the postprandial increase of chylomicrons and chylomicron-associated apolipoproteins B and E was significantly reduced as compared with controls.
Conclusion— Hepatic ABCA1 contributes to HDL plasma levels and influences postprandial lipemia.
Here we describe the application of RNA interference in vivo by using an adenoviral vector to elucidate the liver-specific role of ABCA1 with regard to its influence on systemic lipoprotein metabolism. Knockdown of hepatic ABCA1 leads to a reduction of both plasma HDL levels and postprandial lipemia.
Key Words: ATP binding cassette transporter A1 ? high-density lipoproteins ? RNA interference ? apolipoprotein E ? postprandial lipemia
Introduction
ATP binding cassette transporter-1 (ABCA1) initiates the formation of mature HDL by facilitating apolipoprotein A-I (apoA-I) lipidation. Macrophages express ABCA1 and play an important role in reverse cholesterol transport from the periphery to the liver.1–3 The absence of functional ABCA1 protein in Tangier disease and in knockout mice results in very low plasma levels of high-density lipoprotein (HDL) cholesterol.4–6 This was traditionally thought to be caused by reduced peripheral HDL formation in macrophages and consequently impaired reverse cholesterol transport. Recent studies, however, have shown that macrophage ABCA1 expression has only a minimal effect on HDL plasma levels.7 In addition, transgenic as well as adenoviral overexpression suggest a prominent role for hepatic ABCA1 in the formation of mature HDL.8–12 To specifically investigate the role of hepatic ABCA1 for HDL metabolism, we suppressed expression selectively in the liver by adenoviral delivery of small interfering RNA (siRNA) targeting ABCA1 (Ad-anti-ABCA1). The downregulation of gene expression by siRNA has been established as an important tool for gene silencing to study the biological function of specific genes.13,14 Only few studies have been described that successfully used an adenovirus to apply siRNA in vivo.15–17
In contrast to its role in HDL metabolism, relatively little is known about the function of ABCA1 in the metabolism of apoB-containing lipoproteins. ABCA1 deficiency in Tangier patients and in the knockout mouse model result in an 40% decrease in total apoB plasma levels.4,5 Furthermore, a decrease in plasma triglyceride levels was observed in ABCA1 knockout mice.5 In addition, by capillary electrophoresis, a complete absence of chylomicrons was found in the plasma of ABCA1-deficient mice as compared with wild-type mice.6 Correspondingly, overexpression of ABCA1 resulted in elevated apoB and triglyceride levels.8,10,11 These observations could neither be explained by changes in hepatic apoB secretion nor by an accelerated low-density lipoprotein (LDL) uptake.8,18 Therefore, it is conceivable that ABCA1 regulates postprandial lipoprotein concentrations: higher ABCA1 expression would lead to a retardation, whereas lower ABCA1 expression would result in an acceleration of chylomicron clearance, which might account for the above described alterations in apoB and triglyceride levels in genetically modified mice.
In the current study, we describe that adenoviral delivery of siRNA targeting ABCA1 is a useful tool to investigate the liver-specific role of ABCA1 in lipoprotein metabolism in vivo. We demonstrate that hepatic ABCA1 is required to maintain HDL plasma concentrations and influences postprandial triglyceride metabolism.
Methods
For the method section, including primer sequences used to generate pALsh vectors and for quantitative RT-PCR, please see Tables I and II (available online at http://atvb.ahajournals.org).
Results
Knockdown of ABCA1 by siRNA in HEK-293 Cells
RNA interference was used for the downregulation of ABCA1 by the expression of small hairpin RNA (shRNA) molecules, which are intracellularly processed into active siRNA.14 We used the recently described pALsh system19 to generate 5 different plasmids targeting ABCA1 mRNA at different positions. To determine the ability of each plasmid to knock down ABCA1 in cell culture, human HEK-293 cells were cotransfected with pEGFP-N1 and the recombinant murine ABCA1-FLAG (pmABCA1-FLAG) construct together with pALsh (empty vector) or pALsh-anti-ABCA1. This experimental setup was chosen because murine ABCA1 is well detectable by immunofluorescence in human HEK-293 cells through the FLAG-tag, whereas cotransfection with EGFP was necessary to normalize mRNA expression and to visualize transfected cells. Three days after transfection, the relative mRNA expression level of ectopic murine ABCA1 was reduced down to 25% with one construct only, namely pALsh-anti-ABCA1 (V) (Figure I, available online at http://atvb.ahajournals.org). The capacity of pALsh-anti-ABCA1 (V) to inhibit murine ABCA1 expression was confirmed by immunofluorescence and Western blotting (Figure 1). HEK-293 cells were cotransfected with pEGFP-N1, pmABCA1-FLAG together with pALsh (Figure 1A) or pALsh-anti-ABCA1 (V) (Figure 1B). Recombinant ABCA1 was visualized with an antibody against the FLAG-tag (red fluorescence), whereas EGFP expression indicates transfected cells (green fluorescence). Human HEK-293 cells cotransfected together with the empty pALsh vector showed the typical membranous staining pattern for ABCA1 (Figure 1A) as described before.20 In contrast, in cells cotransfected together with pALsh-anti-ABCA1 (V) the red signal was markedly suppressed, indicating near-complete absence of recombinant ABCA1 protein synthesis (Figure 1B). In parallel experiments, cell lysates were subjected to SDS-PAGE, followed by a fluorescence scan for EGFP detection before Western blotting against ABCA1 (Figure 1C). The EGFP fluorescence, shown in the lower panel of the blot, indicates similar efficiency for each transfection. In comparison to the pALsh control, pAlsh-anti-ABCA1 (V) led to a strong reduction in the expression of murine ABCA1 protein down to background signal intensity (Figure 1C). Taken together, an effective plasmid-based system for the siRNA-mediated knockdown of murine ABCA1 expression was established.
Figure 1. Knockdown of murine ABCA1 protein expression by pALsh-anti-ABCA1 (V) in human HEK-293 cells. A and B, HEK-293 cells were cotransfected with pmABCA1-FLAG and with pEGFP-N1 as a transfection control and either pALsh (A) or pALsh-anti-ABCA1 (B) (V). Three days after transfection, cells were fixed and stained with an antibody against ABCA1-FLAG. Nuclei were stained with DAPI (blue), and confocal images were taken for EGFP (green) and ABCA1 (red). The merged images are shown in the respective lower right corner. Bars=20 μm. C, For Western analysis, HEK-293 cells were transfected as described above. Membrane proteins (50 μg) were separated by SDS-PAGE followed by fluorescence scans for EGFP (bottom). EGFP fluorescence was detected at 30 kDa. Subsequent Western blotting was performed with a specific antibody against ABCA1 (top).
Liver Specific ABCA1 Knockdown by siRNA Influences HDL Metabolism in Mice
To investigate the role of hepatic ABCA1 in mice, recombinant adenovirus (Ad) was prepared from the pALsh-anti-ABCA1 (V) construct. Hepatoma cells were used to confirm that Ad-anti-ABCA1 was functional to efficiently downregulate endogenous ABCA1 mRNA levels in vitro (data not shown). Next, to evaluate the ability of the Ad-anti-ABCA1 to suppress murine hepatic ABCA1 in vivo, recombinant vectors expressing anti-ABCA1 siRNA (Ad-anti-ABCA1) or EGFP (Ad-EGFP) as a control were injected intravenously into male C57BL/6 mice. By this approach almost 100% of the adenoviral dose ended up in the liver, which has been described before21 and was confirmed by confocal microscopy (Figure II, available online at http://atvb.ahajournals.org).
Seven days after infection, liver membrane proteins prepared from Ad-EGFP and Ad-anti-ABCA1 injected mice were evaluated by Western blotting against ABCA1, ApoE, LDL-R, SR-B1, and LRP1 (Figure 2A), which represent important molecules in HDL and postprandial chylomicron metabolism. As determined by densitometric analysis (Figure 2B), hepatic ABCA1 expression was significantly reduced down to 50%. In addition, apoE protein levels were markedly elevated as a response to Ad-anti-ABCA1 administration. Compared with the control, LDL-R was slightly reduced. No differences were found for SR-B1 and LRP1 expression.
Figure 2. Expression levels of murine liver plasma membrane proteins after infection with Ad-EGFP or Ad-anti-ABCA1. A, C57BL/6 mice were infected with 2.5x109 ifu Ad-EGFP or Ad.anti-ABCA1 (five animals per group). Seven days after infection, mice were perfused after a fasting period of 5 hours during daytime before organs were taken. Liver membrane proteins were prepared, 50 μg per animal were separated by 10% SDS-PAGE, followed by Western blotting with specific antibodies against ABCA1, apoE, LDL-R, SR-B1, and LRP185. B, Densitometric quantification of the Western blots presented in A was performed for ABCA1, apoE, and LDL-R. Gray bars represent Ad-anti-ABCA1– and white bars Ad-EGFP-infected mice. Data are given in % of protein levels from Ad-EGFP infected mice. *P<0.05, **P<0.01 by student t test.
Next, the effect of the hepatic ABCA1 knockdown on the plasma lipoprotein profile was evaluated by fast protein liquid chromatography (FPLC) analysis. Pooled plasma samples (200 μL) derived from Ad-EGFP– or Ad-anti-ABCA1–treated (Figure III, available online at http://atvb.ahajournals.org) C57BL/6 mice were separated by gel chromatography at day 0 versus day 5 and day 7 after infection. Compared with day 0, the cholesterol levels in HDL-containing fractions were considerably reduced five and seven days after Ad-anti-ABCA administration. In addition, HDL-associated phospholipids were slightly reduced by ABCA1 knockdown (data not shown). No differences in the cholesterol concentrations of apoB-containing lipoproteins were observed, indicating that the knockdown of hepatic ABCA1 did not alter hepatic VLDL secretion or apoB particle clearance in the fasted state. Alterations of the lipoprotein profile induced by the adenoviral infection as such can be ruled out, as cholesterol levels before and after adenoviral administration in Ad-EGFP injected mice were not changed. In accordance to the results obtained with plasma of ABCA1 knockout mice,5,22 decreased HDL levels in Ad-anti-ABCA1 were associated with a concomitant remarkable reduction in HDL-associated apoE and apoA-I particle content 5 and 7 days after infection. To a lesser extent this phenomenon was observed also for apoC-III. (Figure III).
Taken together, these data clearly demonstrate that the function of ABCA1 in the liver is closely connected to systemic HDL metabolism.
Liver Specific ABCA1 Knockdown Influences Postprandial Lipoprotein Metabolism in Mice
Because ABCA1 deficiency is characterized by reduced apoB and triglyceride levels, which cannot be explained by decreased secretion or increased clearance of apoB containing particles,8,18 one possible explanation could be found in alterations in the postprandial metabolism of ABCA1 deficient mice. Therefore, we compared the postprandial response of Ad-anti-ABCA1 with Ad-EGFP infected animals by an intragastic fat load (olive oil supplemented with radiolabeled triolein). The gavage was administered 7 days after infection (Figure 3). In the fasted state (Figure 3A, 0 minutes) we did not observe any differences in triglyceride levels between the 2 groups. Cholesterol concentrations, however, were significantly reduced in the fasted state and at all indicated time points after the oral gavage in ABCA1-deficient mice (Figure 3A). Furthermore, the postprandial increase in triglyceride levels was less pronounced in Ad-anti-ABCA1 mice than in Ad-EGFP controls (Figure 3A; Table III, available online at http://atvb.ahajournals.org). Radiolabeled fatty acids in the plasma represent the intestinally derived chylomicrons. The determination of plasma radioactivity revealed that chylomicron clearance is accelerated significantly by hepatic ABCA1 knockdown (Figure 3B). Furthermore, the plasma content of the characteristic apolipoproteins of HDL and triglyceride-rich lipoproteins (TRL) were consistently reduced by hepatic ABCA1 knockdown (Figure 3C). To rule out potential differences in intestinal lipid absorption, the production of chylomicrons in Ad-anti-ABCA1 and Ad-EGFP infected mice was measured by the concomitant administration of an oral gavage with radiolabeled triolein and intravenous injection with tyloxapol, which completely blocks plasma lipoprotein clearance (Figure 3D). No differences were detected in plasma triglyceride levels as well as in the appearance of radiolabeled lipids, indicating that intestinal chylomicron synthesis is not affected by Ad-anti-ABCA1 treatment.
Figure 3. Postprandial response of Ad-anti-ABCA1– vs Ad-EGFP-treated mice after an oral fat load. C57BL/6 mice were treated with Ad-EGFP or Ad-anti-ABCA1. Seven days after infection, the animals were administered an oral fat load composed of radiolabeled (14C) triolein in 100 μL olive oil. Triglyceride and cholesterol levels (A) as well as chylomicron-associated radioactivity (B) were measured before (0 minutes) and 30 minutes, 60 minutes, and 120 minutes after the fat load. C, Plasma samples of 4 individual animals per group were analyzed for apoB, apoE, and apoA-I content by Western blotting. D, To measure chylomicron synthesis, Tyloxapol was injected intravenously two minutes after the oral gavage. Plasma triglycerides and 14C-radiolabeled lipids were determined before (0 minutes) and 60 minutes, 120 minutes, and 180 minutes after the fat load. Data points represent mean values±SD of 7 mice per group. **P<0.01 by student t test.
To further define the lipoproteins which contribute to the observed changes in the total plasma apolipoprotein and lipid concentrations in the pre- and postprandial state, FPLC analysis from Ad-anti-ABCA1 versus Ad-EGFP mice was performed (Figure 4A). While in the preprandial state triglyceride levels were indistinguishable, the hepatic ABCA1 knockdown resulted in a significant reduction of triglyceride content in the TRL fractions8–10 in the postprandial state. Western blot analysis revealed a clear postprandial increase of both apoE and apoB48 in pooled TRL fractions8–10 independent of the adenoviral treatment, whereas Ad-anti-ABCA1 treatment resulted in a marked reduction of apoE and apoB48 (Figure 4B). A similar reduction of HDL apoE content by Ad-anti-ABCA1 was observed in the pre- and postprandial state.
Figure 4. Pre- and postprandial lipoprotein profile apolipoprotein composition of TRL and HDL after infection with Ad-EGFP and Ad-anti-ABCA1. A, Pre- and postprandial pooled plasma samples from 5 mice per group were separated by FPLC 7 days after infection and cholesterol and triglyceride concentrations were determined. B, After FPLC, TRL fractions 8 to 10 and HDL fractions 22 to 26 were pooled and separated by SDS-PAGE, followed by immunblotting against apoB and apoE.
In conclusion, these data demonstrate that the hepatic ABCA1 knockdown not only reduces systemic HDL concentrations, but also positively influences postprandial lipemia.
Discussion
The aim of this study was to investigate the relevance of hepatic ABCA1 expression for HDL and postprandial lipoprotein metabolism. An adenovirus-mediated siRNA approach, a new technique for specific gene silencing in vivo,14,15 was used to establish an effective knockdown of hepatic ABCA1 in vivo. Next to the liver, ABCA1 is expressed in a variety of different organs including lung, brain, intestine, and macrophages.23,24 The technique of adenovirus-mediated delivery enabled us to investigate the liver-specific role of ABCA1 in the systemic lipoprotein metabolism. The first report in the literature using adenovirus-mediated expression of siRNA in vivo to decrease the expression of endogenous genes described a 12% reduction in the activity of ?-glucuronidase, an enzyme which is highly expressed in murine liver.15 Recently, a similar adenovirus-based method allowed the creation of a near-complete knockdown of a nuclear hormone receptor coactivator with low endogenous expression in rat liver.17 In the current study, with Ad-anti-ABCA1, we achieved an 50% reduction of endogenous hepatic ABCA1 protein levels in mice (Figure 2). From the present and the few previous reports using this technique, it can be concluded that adenovirus-mediated siRNA delivery in vivo has the potential to significantly decrease the expression of target proteins. Attenuation of protein expression allows studying liver-specific gene function in vivo as an alternative method to the time-consuming and cost-intensive tissue-specific knockout systems, which can be created by Cre/loxP recombination.
We used this siRNA approach to study the role of hepatic ABCA1 because several recent reports discuss that hepatic ABCA1 expression considerably contributes to plasma HDL cholesterol. This discussion is based on data from transgenic mice overexpressing ABCA1 predominantly in liver and macrophages, but also in lung and intestine, as well as from mice with adenovirus-mediated hepatic ABCA1 overexpression. These studies revealed a dose-dependent increase of HDL cholesterol and HDL apoE content.8,10–12 In the current study the 50% reduction of ABCA1 protein levels in the liver (Figure 2) resulted in an 40% decrease in HDL cholesterol (Figures III and 4), suggesting that indeed hepatic ABCA1 is responsible for the regulation of plasma HDL cholesterol. Furthermore, the accumulation of apoE in the liver (Figure 2) and the decrease of HDL-derived apoE (Figures IIIC and 4B) imply an important role of intracellular apoE for the formation of mature HDL through ABCA1 in vivo. This hypothesis receives support from 2 recent studies with ABCA1-deficient mice with focus on the central nervous system.22,25 In agreement with our data (Figure 3C), the lack of ABCA1 resulted in considerably lower apoE levels in plasma as well as cerebrospinal fluid, an impaired lipidation of astrocyte-derived apoE,22 and an intracellular lipid accumulation in astrocytes.25 Thus, the results are suggestive of an intracellular pathway in lipoprotein-synthesizing cells, in which apoE may be tethered to lipids within endosomal compartments.26 These lipid-laden vesicles probably serve as a pool for the lipidation of extracellular apoA-I in a process which is regulated by the gatekeeper ABCA1.
Since the discovery of ABCA1, much effort has been made to develop agonists which increase ABCA1 expression with the idea to generate an antiatherogenic lipoprotein profile by raising HDL. However, the potentially beneficial effects of increased HDL levels might be overcome by the concomitant increase in atherogenic apoB-containing lipoproteins which has been observed in ABCA1 overexpressing mice.8,11 To analyze the underlying mechanism by which ABCA1 influences the metabolism of apoB-containing lipoproteins, several studies have been performed. Based on these reports, an altered secretion of VLDL can be excluded because no differences in the synthesis of triglyceride-rich apoB-containing lipoproteins were found in transgenics8 or in ABCA1-deficient mice.18 Correspondingly, ABCA1-deficient mice display decreased apoB levels.5,6 This is even true in the combined ABCA1/LDL-R–deficient background, suggesting an alternative pathway independent of LDL-R–mediated LDL catabolism.18,27 Intestinally derived chylomicrons also contribute to apoB plasma levels. With some variations, depending on diet, sex, and genetic background, apoB levels correlate with triglycerides in ABCA1 knockout and transgenic mice.5,6,8,11 Altogether, these data suggest that at least in genetically engineered mice the association of apoB levels with ABCA1 expression is not necessarily explained by an altered LDL concentration but also at least partially by the plasma level of remnant particles. Therefore we evaluated the postprandial response in dependence of ABCA1 expression by the administration of an oral fat load by gavage. Because the chylomicron synthesis was not affected by the hepatic ABCA1 knockdown, whereas the increase and number of TRL particles were clearly reduced in the postprandial phase (Figures 3 and 4), the current data are strongly indicative of an accelerated postprandial clearance of chylomicrons and their remnants. These results could help to explain the low apoB levels and the absence of chylomicrons observed in ABCA1-deficient mice.5,6 Because the liver-specific knockdown in this study rules out intestinal ABCA1 involvement, we assume that the accelerated postprandial lipemia is an effect of the changes in HDL composition. However, to elucidate the underlying mechanism, further studies will be required. For instance, the activity of lipoprotein lipase and other enzymes which modify HDL and chylomicron composition will have to be studied in these mice.
In conclusion, we describe the application of RNA interference in vivo by using an adenovirus-mediated siRNA approach to elucidate the liver-specific role of ABCA1. The efficient knockdown of ABCA1 was accompanied by a reduction of both plasma HDL levels and postprandial triglyceridemia, demonstrating an important role of hepatic ABCA1 for systemic lipoprotein metabolism.
Acknowledgments
This work was supported by the DFG research grants Ni-637/1-2, Me-1507/2-4, and the GRK 336 "Molecular Endocrinology and Metabolism." A. Laatsch is supported by a fellowship from the Studienstiftung des deutschen Volkes. We are grateful to S. Ehret and D. Niedzielska for excellent technical assistance.
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Correspondence to Dr Joerg Heeren, IBM II, Molecular Cell Biology, University Hospital Hamburg-Eppendorf Martinistrasse 52 D-20246 Hamburg, Germany. E-mail heeren@uke.uni-hamburg.de
Abstract
Objective— To investigate the impact of hepatic ABCA1 on systemic lipoprotein metabolism in vivo by an adenovirus-mediated RNA interference approach.
Methods and Results— Efficiency of plasmid-based small interference RNA (siRNA)-induced knockdown of cotransfected murine ATP binding cassette transporter A1 (mABCA1) in HEK-293 cells was judged by RT-polymerase chain reaction, immunofluorescence, and Western blot analysis. The most effective plasmid was used to generate a recombinant adenovirus as a tool to selectively downregulate ABCA1 expression in mouse liver (C57BL/6). In comparison to controls, Western blot analysis from liver membrane proteins of Ad-anti-ABCA1 infected mice resulted in an 50% reduction of endogenous ABCA1 and a clear upregulation of apolipoprotein E. Fast protein liquid chromatography analysis of plasma revealed that hepatic ABCA1 protein reduction was associated with an 40% decrease of HDL cholesterol and a reduction of HDL-associated apolipoprotein A-I and E. In the fasted state, other lipoprotein classes were not affected. To analyze the influence of ABCA1 downregulation on postprandial lipemia, infected mice were given a gastric load of radiolabeled trioleate in olive oil. In Ad-anti-ABCA1 infected mice, the postprandial increase of chylomicrons and chylomicron-associated apolipoproteins B and E was significantly reduced as compared with controls.
Conclusion— Hepatic ABCA1 contributes to HDL plasma levels and influences postprandial lipemia.
Here we describe the application of RNA interference in vivo by using an adenoviral vector to elucidate the liver-specific role of ABCA1 with regard to its influence on systemic lipoprotein metabolism. Knockdown of hepatic ABCA1 leads to a reduction of both plasma HDL levels and postprandial lipemia.
Key Words: ATP binding cassette transporter A1 ? high-density lipoproteins ? RNA interference ? apolipoprotein E ? postprandial lipemia
Introduction
ATP binding cassette transporter-1 (ABCA1) initiates the formation of mature HDL by facilitating apolipoprotein A-I (apoA-I) lipidation. Macrophages express ABCA1 and play an important role in reverse cholesterol transport from the periphery to the liver.1–3 The absence of functional ABCA1 protein in Tangier disease and in knockout mice results in very low plasma levels of high-density lipoprotein (HDL) cholesterol.4–6 This was traditionally thought to be caused by reduced peripheral HDL formation in macrophages and consequently impaired reverse cholesterol transport. Recent studies, however, have shown that macrophage ABCA1 expression has only a minimal effect on HDL plasma levels.7 In addition, transgenic as well as adenoviral overexpression suggest a prominent role for hepatic ABCA1 in the formation of mature HDL.8–12 To specifically investigate the role of hepatic ABCA1 for HDL metabolism, we suppressed expression selectively in the liver by adenoviral delivery of small interfering RNA (siRNA) targeting ABCA1 (Ad-anti-ABCA1). The downregulation of gene expression by siRNA has been established as an important tool for gene silencing to study the biological function of specific genes.13,14 Only few studies have been described that successfully used an adenovirus to apply siRNA in vivo.15–17
In contrast to its role in HDL metabolism, relatively little is known about the function of ABCA1 in the metabolism of apoB-containing lipoproteins. ABCA1 deficiency in Tangier patients and in the knockout mouse model result in an 40% decrease in total apoB plasma levels.4,5 Furthermore, a decrease in plasma triglyceride levels was observed in ABCA1 knockout mice.5 In addition, by capillary electrophoresis, a complete absence of chylomicrons was found in the plasma of ABCA1-deficient mice as compared with wild-type mice.6 Correspondingly, overexpression of ABCA1 resulted in elevated apoB and triglyceride levels.8,10,11 These observations could neither be explained by changes in hepatic apoB secretion nor by an accelerated low-density lipoprotein (LDL) uptake.8,18 Therefore, it is conceivable that ABCA1 regulates postprandial lipoprotein concentrations: higher ABCA1 expression would lead to a retardation, whereas lower ABCA1 expression would result in an acceleration of chylomicron clearance, which might account for the above described alterations in apoB and triglyceride levels in genetically modified mice.
In the current study, we describe that adenoviral delivery of siRNA targeting ABCA1 is a useful tool to investigate the liver-specific role of ABCA1 in lipoprotein metabolism in vivo. We demonstrate that hepatic ABCA1 is required to maintain HDL plasma concentrations and influences postprandial triglyceride metabolism.
Methods
For the method section, including primer sequences used to generate pALsh vectors and for quantitative RT-PCR, please see Tables I and II (available online at http://atvb.ahajournals.org).
Results
Knockdown of ABCA1 by siRNA in HEK-293 Cells
RNA interference was used for the downregulation of ABCA1 by the expression of small hairpin RNA (shRNA) molecules, which are intracellularly processed into active siRNA.14 We used the recently described pALsh system19 to generate 5 different plasmids targeting ABCA1 mRNA at different positions. To determine the ability of each plasmid to knock down ABCA1 in cell culture, human HEK-293 cells were cotransfected with pEGFP-N1 and the recombinant murine ABCA1-FLAG (pmABCA1-FLAG) construct together with pALsh (empty vector) or pALsh-anti-ABCA1. This experimental setup was chosen because murine ABCA1 is well detectable by immunofluorescence in human HEK-293 cells through the FLAG-tag, whereas cotransfection with EGFP was necessary to normalize mRNA expression and to visualize transfected cells. Three days after transfection, the relative mRNA expression level of ectopic murine ABCA1 was reduced down to 25% with one construct only, namely pALsh-anti-ABCA1 (V) (Figure I, available online at http://atvb.ahajournals.org). The capacity of pALsh-anti-ABCA1 (V) to inhibit murine ABCA1 expression was confirmed by immunofluorescence and Western blotting (Figure 1). HEK-293 cells were cotransfected with pEGFP-N1, pmABCA1-FLAG together with pALsh (Figure 1A) or pALsh-anti-ABCA1 (V) (Figure 1B). Recombinant ABCA1 was visualized with an antibody against the FLAG-tag (red fluorescence), whereas EGFP expression indicates transfected cells (green fluorescence). Human HEK-293 cells cotransfected together with the empty pALsh vector showed the typical membranous staining pattern for ABCA1 (Figure 1A) as described before.20 In contrast, in cells cotransfected together with pALsh-anti-ABCA1 (V) the red signal was markedly suppressed, indicating near-complete absence of recombinant ABCA1 protein synthesis (Figure 1B). In parallel experiments, cell lysates were subjected to SDS-PAGE, followed by a fluorescence scan for EGFP detection before Western blotting against ABCA1 (Figure 1C). The EGFP fluorescence, shown in the lower panel of the blot, indicates similar efficiency for each transfection. In comparison to the pALsh control, pAlsh-anti-ABCA1 (V) led to a strong reduction in the expression of murine ABCA1 protein down to background signal intensity (Figure 1C). Taken together, an effective plasmid-based system for the siRNA-mediated knockdown of murine ABCA1 expression was established.
Figure 1. Knockdown of murine ABCA1 protein expression by pALsh-anti-ABCA1 (V) in human HEK-293 cells. A and B, HEK-293 cells were cotransfected with pmABCA1-FLAG and with pEGFP-N1 as a transfection control and either pALsh (A) or pALsh-anti-ABCA1 (B) (V). Three days after transfection, cells were fixed and stained with an antibody against ABCA1-FLAG. Nuclei were stained with DAPI (blue), and confocal images were taken for EGFP (green) and ABCA1 (red). The merged images are shown in the respective lower right corner. Bars=20 μm. C, For Western analysis, HEK-293 cells were transfected as described above. Membrane proteins (50 μg) were separated by SDS-PAGE followed by fluorescence scans for EGFP (bottom). EGFP fluorescence was detected at 30 kDa. Subsequent Western blotting was performed with a specific antibody against ABCA1 (top).
Liver Specific ABCA1 Knockdown by siRNA Influences HDL Metabolism in Mice
To investigate the role of hepatic ABCA1 in mice, recombinant adenovirus (Ad) was prepared from the pALsh-anti-ABCA1 (V) construct. Hepatoma cells were used to confirm that Ad-anti-ABCA1 was functional to efficiently downregulate endogenous ABCA1 mRNA levels in vitro (data not shown). Next, to evaluate the ability of the Ad-anti-ABCA1 to suppress murine hepatic ABCA1 in vivo, recombinant vectors expressing anti-ABCA1 siRNA (Ad-anti-ABCA1) or EGFP (Ad-EGFP) as a control were injected intravenously into male C57BL/6 mice. By this approach almost 100% of the adenoviral dose ended up in the liver, which has been described before21 and was confirmed by confocal microscopy (Figure II, available online at http://atvb.ahajournals.org).
Seven days after infection, liver membrane proteins prepared from Ad-EGFP and Ad-anti-ABCA1 injected mice were evaluated by Western blotting against ABCA1, ApoE, LDL-R, SR-B1, and LRP1 (Figure 2A), which represent important molecules in HDL and postprandial chylomicron metabolism. As determined by densitometric analysis (Figure 2B), hepatic ABCA1 expression was significantly reduced down to 50%. In addition, apoE protein levels were markedly elevated as a response to Ad-anti-ABCA1 administration. Compared with the control, LDL-R was slightly reduced. No differences were found for SR-B1 and LRP1 expression.
Figure 2. Expression levels of murine liver plasma membrane proteins after infection with Ad-EGFP or Ad-anti-ABCA1. A, C57BL/6 mice were infected with 2.5x109 ifu Ad-EGFP or Ad.anti-ABCA1 (five animals per group). Seven days after infection, mice were perfused after a fasting period of 5 hours during daytime before organs were taken. Liver membrane proteins were prepared, 50 μg per animal were separated by 10% SDS-PAGE, followed by Western blotting with specific antibodies against ABCA1, apoE, LDL-R, SR-B1, and LRP185. B, Densitometric quantification of the Western blots presented in A was performed for ABCA1, apoE, and LDL-R. Gray bars represent Ad-anti-ABCA1– and white bars Ad-EGFP-infected mice. Data are given in % of protein levels from Ad-EGFP infected mice. *P<0.05, **P<0.01 by student t test.
Next, the effect of the hepatic ABCA1 knockdown on the plasma lipoprotein profile was evaluated by fast protein liquid chromatography (FPLC) analysis. Pooled plasma samples (200 μL) derived from Ad-EGFP– or Ad-anti-ABCA1–treated (Figure III, available online at http://atvb.ahajournals.org) C57BL/6 mice were separated by gel chromatography at day 0 versus day 5 and day 7 after infection. Compared with day 0, the cholesterol levels in HDL-containing fractions were considerably reduced five and seven days after Ad-anti-ABCA administration. In addition, HDL-associated phospholipids were slightly reduced by ABCA1 knockdown (data not shown). No differences in the cholesterol concentrations of apoB-containing lipoproteins were observed, indicating that the knockdown of hepatic ABCA1 did not alter hepatic VLDL secretion or apoB particle clearance in the fasted state. Alterations of the lipoprotein profile induced by the adenoviral infection as such can be ruled out, as cholesterol levels before and after adenoviral administration in Ad-EGFP injected mice were not changed. In accordance to the results obtained with plasma of ABCA1 knockout mice,5,22 decreased HDL levels in Ad-anti-ABCA1 were associated with a concomitant remarkable reduction in HDL-associated apoE and apoA-I particle content 5 and 7 days after infection. To a lesser extent this phenomenon was observed also for apoC-III. (Figure III).
Taken together, these data clearly demonstrate that the function of ABCA1 in the liver is closely connected to systemic HDL metabolism.
Liver Specific ABCA1 Knockdown Influences Postprandial Lipoprotein Metabolism in Mice
Because ABCA1 deficiency is characterized by reduced apoB and triglyceride levels, which cannot be explained by decreased secretion or increased clearance of apoB containing particles,8,18 one possible explanation could be found in alterations in the postprandial metabolism of ABCA1 deficient mice. Therefore, we compared the postprandial response of Ad-anti-ABCA1 with Ad-EGFP infected animals by an intragastic fat load (olive oil supplemented with radiolabeled triolein). The gavage was administered 7 days after infection (Figure 3). In the fasted state (Figure 3A, 0 minutes) we did not observe any differences in triglyceride levels between the 2 groups. Cholesterol concentrations, however, were significantly reduced in the fasted state and at all indicated time points after the oral gavage in ABCA1-deficient mice (Figure 3A). Furthermore, the postprandial increase in triglyceride levels was less pronounced in Ad-anti-ABCA1 mice than in Ad-EGFP controls (Figure 3A; Table III, available online at http://atvb.ahajournals.org). Radiolabeled fatty acids in the plasma represent the intestinally derived chylomicrons. The determination of plasma radioactivity revealed that chylomicron clearance is accelerated significantly by hepatic ABCA1 knockdown (Figure 3B). Furthermore, the plasma content of the characteristic apolipoproteins of HDL and triglyceride-rich lipoproteins (TRL) were consistently reduced by hepatic ABCA1 knockdown (Figure 3C). To rule out potential differences in intestinal lipid absorption, the production of chylomicrons in Ad-anti-ABCA1 and Ad-EGFP infected mice was measured by the concomitant administration of an oral gavage with radiolabeled triolein and intravenous injection with tyloxapol, which completely blocks plasma lipoprotein clearance (Figure 3D). No differences were detected in plasma triglyceride levels as well as in the appearance of radiolabeled lipids, indicating that intestinal chylomicron synthesis is not affected by Ad-anti-ABCA1 treatment.
Figure 3. Postprandial response of Ad-anti-ABCA1– vs Ad-EGFP-treated mice after an oral fat load. C57BL/6 mice were treated with Ad-EGFP or Ad-anti-ABCA1. Seven days after infection, the animals were administered an oral fat load composed of radiolabeled (14C) triolein in 100 μL olive oil. Triglyceride and cholesterol levels (A) as well as chylomicron-associated radioactivity (B) were measured before (0 minutes) and 30 minutes, 60 minutes, and 120 minutes after the fat load. C, Plasma samples of 4 individual animals per group were analyzed for apoB, apoE, and apoA-I content by Western blotting. D, To measure chylomicron synthesis, Tyloxapol was injected intravenously two minutes after the oral gavage. Plasma triglycerides and 14C-radiolabeled lipids were determined before (0 minutes) and 60 minutes, 120 minutes, and 180 minutes after the fat load. Data points represent mean values±SD of 7 mice per group. **P<0.01 by student t test.
To further define the lipoproteins which contribute to the observed changes in the total plasma apolipoprotein and lipid concentrations in the pre- and postprandial state, FPLC analysis from Ad-anti-ABCA1 versus Ad-EGFP mice was performed (Figure 4A). While in the preprandial state triglyceride levels were indistinguishable, the hepatic ABCA1 knockdown resulted in a significant reduction of triglyceride content in the TRL fractions8–10 in the postprandial state. Western blot analysis revealed a clear postprandial increase of both apoE and apoB48 in pooled TRL fractions8–10 independent of the adenoviral treatment, whereas Ad-anti-ABCA1 treatment resulted in a marked reduction of apoE and apoB48 (Figure 4B). A similar reduction of HDL apoE content by Ad-anti-ABCA1 was observed in the pre- and postprandial state.
Figure 4. Pre- and postprandial lipoprotein profile apolipoprotein composition of TRL and HDL after infection with Ad-EGFP and Ad-anti-ABCA1. A, Pre- and postprandial pooled plasma samples from 5 mice per group were separated by FPLC 7 days after infection and cholesterol and triglyceride concentrations were determined. B, After FPLC, TRL fractions 8 to 10 and HDL fractions 22 to 26 were pooled and separated by SDS-PAGE, followed by immunblotting against apoB and apoE.
In conclusion, these data demonstrate that the hepatic ABCA1 knockdown not only reduces systemic HDL concentrations, but also positively influences postprandial lipemia.
Discussion
The aim of this study was to investigate the relevance of hepatic ABCA1 expression for HDL and postprandial lipoprotein metabolism. An adenovirus-mediated siRNA approach, a new technique for specific gene silencing in vivo,14,15 was used to establish an effective knockdown of hepatic ABCA1 in vivo. Next to the liver, ABCA1 is expressed in a variety of different organs including lung, brain, intestine, and macrophages.23,24 The technique of adenovirus-mediated delivery enabled us to investigate the liver-specific role of ABCA1 in the systemic lipoprotein metabolism. The first report in the literature using adenovirus-mediated expression of siRNA in vivo to decrease the expression of endogenous genes described a 12% reduction in the activity of ?-glucuronidase, an enzyme which is highly expressed in murine liver.15 Recently, a similar adenovirus-based method allowed the creation of a near-complete knockdown of a nuclear hormone receptor coactivator with low endogenous expression in rat liver.17 In the current study, with Ad-anti-ABCA1, we achieved an 50% reduction of endogenous hepatic ABCA1 protein levels in mice (Figure 2). From the present and the few previous reports using this technique, it can be concluded that adenovirus-mediated siRNA delivery in vivo has the potential to significantly decrease the expression of target proteins. Attenuation of protein expression allows studying liver-specific gene function in vivo as an alternative method to the time-consuming and cost-intensive tissue-specific knockout systems, which can be created by Cre/loxP recombination.
We used this siRNA approach to study the role of hepatic ABCA1 because several recent reports discuss that hepatic ABCA1 expression considerably contributes to plasma HDL cholesterol. This discussion is based on data from transgenic mice overexpressing ABCA1 predominantly in liver and macrophages, but also in lung and intestine, as well as from mice with adenovirus-mediated hepatic ABCA1 overexpression. These studies revealed a dose-dependent increase of HDL cholesterol and HDL apoE content.8,10–12 In the current study the 50% reduction of ABCA1 protein levels in the liver (Figure 2) resulted in an 40% decrease in HDL cholesterol (Figures III and 4), suggesting that indeed hepatic ABCA1 is responsible for the regulation of plasma HDL cholesterol. Furthermore, the accumulation of apoE in the liver (Figure 2) and the decrease of HDL-derived apoE (Figures IIIC and 4B) imply an important role of intracellular apoE for the formation of mature HDL through ABCA1 in vivo. This hypothesis receives support from 2 recent studies with ABCA1-deficient mice with focus on the central nervous system.22,25 In agreement with our data (Figure 3C), the lack of ABCA1 resulted in considerably lower apoE levels in plasma as well as cerebrospinal fluid, an impaired lipidation of astrocyte-derived apoE,22 and an intracellular lipid accumulation in astrocytes.25 Thus, the results are suggestive of an intracellular pathway in lipoprotein-synthesizing cells, in which apoE may be tethered to lipids within endosomal compartments.26 These lipid-laden vesicles probably serve as a pool for the lipidation of extracellular apoA-I in a process which is regulated by the gatekeeper ABCA1.
Since the discovery of ABCA1, much effort has been made to develop agonists which increase ABCA1 expression with the idea to generate an antiatherogenic lipoprotein profile by raising HDL. However, the potentially beneficial effects of increased HDL levels might be overcome by the concomitant increase in atherogenic apoB-containing lipoproteins which has been observed in ABCA1 overexpressing mice.8,11 To analyze the underlying mechanism by which ABCA1 influences the metabolism of apoB-containing lipoproteins, several studies have been performed. Based on these reports, an altered secretion of VLDL can be excluded because no differences in the synthesis of triglyceride-rich apoB-containing lipoproteins were found in transgenics8 or in ABCA1-deficient mice.18 Correspondingly, ABCA1-deficient mice display decreased apoB levels.5,6 This is even true in the combined ABCA1/LDL-R–deficient background, suggesting an alternative pathway independent of LDL-R–mediated LDL catabolism.18,27 Intestinally derived chylomicrons also contribute to apoB plasma levels. With some variations, depending on diet, sex, and genetic background, apoB levels correlate with triglycerides in ABCA1 knockout and transgenic mice.5,6,8,11 Altogether, these data suggest that at least in genetically engineered mice the association of apoB levels with ABCA1 expression is not necessarily explained by an altered LDL concentration but also at least partially by the plasma level of remnant particles. Therefore we evaluated the postprandial response in dependence of ABCA1 expression by the administration of an oral fat load by gavage. Because the chylomicron synthesis was not affected by the hepatic ABCA1 knockdown, whereas the increase and number of TRL particles were clearly reduced in the postprandial phase (Figures 3 and 4), the current data are strongly indicative of an accelerated postprandial clearance of chylomicrons and their remnants. These results could help to explain the low apoB levels and the absence of chylomicrons observed in ABCA1-deficient mice.5,6 Because the liver-specific knockdown in this study rules out intestinal ABCA1 involvement, we assume that the accelerated postprandial lipemia is an effect of the changes in HDL composition. However, to elucidate the underlying mechanism, further studies will be required. For instance, the activity of lipoprotein lipase and other enzymes which modify HDL and chylomicron composition will have to be studied in these mice.
In conclusion, we describe the application of RNA interference in vivo by using an adenovirus-mediated siRNA approach to elucidate the liver-specific role of ABCA1. The efficient knockdown of ABCA1 was accompanied by a reduction of both plasma HDL levels and postprandial triglyceridemia, demonstrating an important role of hepatic ABCA1 for systemic lipoprotein metabolism.
Acknowledgments
This work was supported by the DFG research grants Ni-637/1-2, Me-1507/2-4, and the GRK 336 "Molecular Endocrinology and Metabolism." A. Laatsch is supported by a fellowship from the Studienstiftung des deutschen Volkes. We are grateful to S. Ehret and D. Niedzielska for excellent technical assistance.
References
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