Published online before print November 6, 2008, doi: 10.1161/ATVBAHA.108.164723
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© 2009 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Transient Increase in Plasma Oxidized LDL During the Progression of Atherosclerosis in Apolipoprotein E Knockout Mice
From the Department of Biological Chemistry (R.K., C.M., K.K., T.O., K.T., T.A., H.I.), Center of Biotechnology (S.A.), Showa University School of Pharmaceutical Sciences, Japan; the Department of Molecular Pathology, Faculty of Pharmaceutical Sciences (M.M., T.T., H.I.), Teikyo University, Japan; and the Department of Neuronal Surgery (K.K.), Institute of Health Biosciences, and University of Tokushima Graduate School, Physiological Chemistry Research Laboratory (K.T.), Hoshi University, Japan.
Correspondence to Hiroyuki Itabe, PhD, Department of Biological Chemistry, Showa University School of Pharmaceutical Sciences, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail h-itabe{at}pharm.showa-u.ac.jp
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Abstract |
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Background— Plasma level of oxidized low-density lipoprotein (OxLDL) is a risk marker for cardiovascular diseases. The behavior of plasma OxLDL before disease progression has not been studied previously.
Methods and Results— In this study, we developed a sensitive ELISA procedure for detecting mouse circulating OxLDL using a monoclonal antibody that recognizes oxidized phosphatidylcholine and a rabbit antimouse apolipoprotein B-48 polyclonal antibody. Apolipoprotein E knockout mice were fed on a chow diet for 40 weeks. Oil red O–positive lesions developed gradually by 20 weeks, and the percentage area covered by the lesions increased dramatically after 28 weeks; it covers 33.4% of the surface area by 40 weeks. The OxLDL level, measured after LDL fraction was isolated from each mouse, at 10 weeks was 0.015 ng/µg LDL. It increased 3-fold at 20 weeks of age and then decreased to the basal level by 40 weeks of age, suggesting that OxLDL appears before the development of atherosclerotic lesions. The occurrence of lipid peroxidation products, acrolein and oxidized phosphatidylcholines, in aortic tissue were revealed by immunohistochemical staining as early as 10 weeks.
Conclusion— These results suggest that OxLDL might be involved in the early stages of progression of atherosclerotic lesions.
We successfully measured murine circulating OxLDL using sandwich ELISA. ApoE knockout mice showed a significant rise in the plasma OxLDL level at 20 weeks of age, whereas the atherosclerotic lesions were still small. This suggests an increase in the oxidative stress and the appearance of OxLDL before the progression of atherosclerotic lesions.
Key Words: oxidized low-density lipoprotein • atherosclerosis • apoE-knockout mouse • ELISA • oxidized phosphatidylcholine
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Introduction |
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Previous studies have indicated that oxidized low-density lipoprotein (OxLDL) is an important marker for cardiovascular diseases.1–4 OxLDL accumulation in macrophage-derived foam cells in atherosclerotic lesions has been revealed by immunohistochemical studies using anti-OxLDL monoclonal antibodies (mAbs).5–8 Thus, it is hypothesized that OxLDL plays a role in the development of the atherosclerotic lesions. However, questions concerning the behavior of OxLDL in vivo remain unanswered.
We have measured OxLDL present in human circulation by the sandwich ELISA procedure using an anti-OxLDL mAb, DLH3, which recognizes the oxidized phosphatidylcholine (OxPC),5 and an antihuman apolipoprotein B-100 (hapoB-100) polyclonal antibody (pAb).9 Other groups have also used mAbs against OxLDL for measuring OxLDL in human circulation.10–12 Several clinical studies using these mAbs have demonstrated that elevated OxLDL levels were observed in the plasma of patients with cardiovascular diseases.3–6,12–16
Most cardiovascular patients experience significant symptoms at middle or old age. Atherosclerosis develops over years through build up of advanced lesions with severe narrowing of arteries. Such long-term development of lesions is one of the hurdles faced in the study of atherosclerosis. The apolipoprotein E knockout mice (apoE-KO) are used universally as a good animal model for atherosclerosis.17 Depletion of apoE reduces the rate of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) uptake by peripheral tissues, resulting in severe hypercholesterolemia. Moreover, lack of apoE secretion by macrophages, significantly reduced reverse cholesterol transport from atherosclerotic lesions to the liver.18
To further investigate the oxidative stress in vivo and occurrence of OxLDL during atherogenesis, we developed a procedure for measuring murine circulating OxLDL by modifying our previous ELISA method for measuring human OxLDL. OxLDL levels in apoE-KO were measured, and the changes were monitored during aging. Our present data show that circulating OxLDL levels increased temporarily at the age of 20 weeks, which occurred before the extensive development of atherosclerotic lesions in apoE-KO.
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Materials and Methods |
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Detailed descriptions of the methods are provided in the supplemental materials (available online at http://atvb.ahajournals.org).
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Results |
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Detection of OxLDL in Mouse Plasma
We previously developed a sandwich ELISA procedure using an anti-OxPC mAb and antihapoB-100 pAb for determining OxLDL in human plasma.9 However, the method was successful in case of humans but not mice (Figure 1A). Although the anti-OxPC mAb DLH3 reacts with both mouse and human OxLDL, antihapoB-100 pAb used in the assay is species specific. Because mapoB-48, a short version of apoB protein, is the major component in LDL as well as VLDL from apoE-KO and wild-type C57BL/6 mice (supplemental Figure II), a rabbit pAb for mapoB-48 was raised and used in this study. When this pAb was used in combination with DLH3, instead of antiapoB-100 pAb, mouse OxLDL but not human OxLDL was detected by ELISA (Figure 1B).
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A standard curve for the measurement of mouse OxLDL is shown in Figure 2A. By modifying the ELISA procedure to maximize the sensitivity, we were able to measure 0.1 ng mouse OxLDL per microtiter well—a sensitivity approximately 5 times that of the original method for human OxLDL.9 ELISA was dependent on DLH3 antibody, because no increase in absorbance was observed when isotype-matched nonimmune murine IgM(
) was used instead of DLH3 (Figure 2B). A slightly high background absorbance was obtained because of nonspecific reactivity to the IgM molecule. The composition of the buffers had a substantial effect on the sensitivity of the assay. Under our conditions, TBS containing 2% BSA was used as the blocking solution, which provided better results than the other reagents tested. TBS containing 2% skim milk was used to dilute the antibodies.
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), or PBS without antibodies (
). The nonimmune IgM gave a slight absorbance that could explain the background level. Data indicated are expressed as the mean±SD (n=3).In addition to LDL, the VLDL fraction is present in high quantities in apoE-KO. The amount of oxidized particles in VLDL and LDL fractions in 25-week-old apoE-KO was 0.006±0.001 ng/µg and 0.020±0.011 ng/µg, respectively (supplemental Table I). Because cholesterol content in the VLDL fraction in apoE-KO is twice that in the LDL fraction between 20 and 28 weeks (supplemental Table II), VLDL might partially contribute to the oxidative modification of lipoproteins in this strain.
Because LDL concentration in the wild-type mice plasma is low, we treated the 8-week-old mice with high-fat–high-cholesterol diet for 4 weeks to collect enough amounts of lipoproteins. OxLDL levels in VLDL and LDL fractions in wild-type mouse plasma were 0.008±0.006 ng/µg and 0.005±0.03 ng/µg, respectively (supplemental Table I). The OxLDL level in the wild-type mice was similar to that of young apoE-KO (4 to 10 weeks).
Plasma OxLDL and Development of Atherosclerosis in ApoE-Knockout Mice
ApoE-KO was maintained on a chow diet for up to 40 weeks after birth. The change in plasma OxLDL levels and atherosclerotic lesions in the aorta was examined over time. Total plasma cholesterol and triacylglycerol (TG) concentrations, blood pressure, and body weights of the mice are summarized in supplemental Table II. The mice gained weight consistently until 28 weeks. Blood pressure was constant throughout the experiment. The plasma TG concentration did not change significantly during the experiment, although the deviation observed in an individual mouse was large. The plasma total cholesterol concentration was about 340 mg/dL at 4 weeks, and it increased up to 924 mg/dL at 28 weeks. The increase in total cholesterol at 28 and 40 weeks was mainly attributable to the increase in the VLDL cholesterol.
We visualized the atherosclerotic lesions in the en face aorta using Oil red O (ORO) stain and evaluated the percentage area covered by the lesions (Figure 3A and supplemental Figure IV). Several small lesions were observed on the surface of the aorta as early as 10 weeks after birth. However, the lesion size was very small, and the percentage lesion area was only 0.5%. The lesion area was less than 3% at 20 weeks; however, it increased dramatically after that. It increased to 33.4% of the aorta at 40 weeks. The lesion size at the aortic root was evaluated by measuring the ORO-positive area present in the sections (Figure 3B). The lesion area in the root region was very small at 4 and 10 weeks, but after 20 weeks intimal thickening and accumulation of lipids became significant. Thickened and lipid-rich lesions were observed at 28 and 40 weeks. The pattern was similar to that of the en-face assay; however, lesion development at 20 weeks might have been slightly greater.
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) and the mean±SD (
) are indicated. Data were statistically evaluated using ANOVA and either Kruskal-Wallis, Games-Howell or Tukey-Kramer test. *P<0.05, **P<0.01.We found that the behavior of the OxLDL level in apoE-KO was different from that of lesion development. As shown in Figure 3C, the plasma OxLDL level was gradually increased from 4 weeks to 20 weeks. The plasma OxLDL level at 20 weeks was 3 times higher than that at 10 weeks; it then reduced by 60% at 28 weeks. The experiments were conducted using 3 or 4 mice per time point, and were repeated several times to obtain observation on 6 to 11 mice. At 20 weeks, all mice demonstrated significantly high levels of plasma OxLDL. This observation suggests that the oxidative modification of LDL occurs under normal conditions in vivo and that the plasma OxLDL level might increase before the extensive development of atherosclerotic lesions in apoE-KO.
We measured the concentrations of anti-OxLDL IgG and IgM in the apoE-KO plasma at the edge of 20 and 40 weeks, and did not find any changes in the autoantibody concentrations between the two groups (supplemental Figure V). Therefore, we speculated that the autoantibodies against OxLDL are unlikely to decrease the plasma OxLDL level under the current experimental conditions.
Oxidative Changes in Atherosclerotic Lesions
Acrolein is one of the major oxidation products formed during lipid peroxidation of lipoproteins. Because acrolein is a highly reactive aldehyde compound, it readily reacts with side chains of several amino acids, such as lysine, histidine, and cysteine. It has been reported that OxLDL prepared in vitro is modified by acrolein.19,20 Uchida et al21 demonstrated the presence of acrolein modified proteins in human atherosclerotic lesions. To examine whether oxidative changes occurred in the aortic tissues of the apoE-KO, thin sections of aorta root regions were immunohistochemically stained using antiacrolein mAb. As shown in Figure 4A, acrolein-positive materials were clearly detected in the thickening intima at 20 and 28 weeks, but scattered pattern is observed at week 40. Figure 5 shows the distribution of the immunostained spots in the whole aortic sections. The aorta at 40 weeks has enlarged lesion area and the aclorein-positive products tend to scatter over the lesions, however its total immunostained area does not seem to decrease during week 28 to 40. Furthermore, OxPC deposition was observed throughout the intimal thickening area at 20 and 28 weeks, and a scattered pattern is observed at week 40 (Figure 4B). Accumulation of cells and extracellular matrix was evaluated by Azan staining (Figure 4C). Compared with 28 weeks, acellular space enlarged and collagen fibers accumulate in the lesion at 40 weeks. It is suggested that OxLDL may be deposited in the vessel wall as the plasma OxLDL levels decline during the progression of the lesions.
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The appearance of oxidative products in the aortic root at the very early stages was examined by comparing the sections at 4, 10, and 20 weeks (Figure 6). Positive immunostaining with antibodies against acrolein or OxPC was observed in medial layers at 10 and 20 weeks, and very faintly at 4 weeks of age. These observations suggest that lipid peroxidation reactions may occur in the vessel wall tissue during the early stages of atherogenesis.
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Discussion |
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In this study, we measured the amount of OxLDL in circulating plasma in apoE-KO. To measure mouse OxLDL, we raised a rabbit pAb against mapoB-48 protein and used it in combination with an anti-OxPC mAb, DLH3. Using this method, we found that plasma OxLDL levels in apoE-KO increased transiently at 20 weeks, although the atherosclerotic lesion area was still less than 3% of the aortic surface. To the best of our knowledge, this is the first observation of increased OxLDL production in vivo preceding the progression of atherosclerotic lesions in the aorta.
The basal plasma OxLDL levels in male apoE-KO ranges between 0.006 and 0.018 ng/µg LDL. A comparison with OxLDL levels in human control subjects reported previously (approximately 0.10 ng/µg LDL)4,7,9 indicated that OxLDL level in mice is much lower than that in humans. The reason for the difference between mouse and human basal OxLDL levels is still unclear; however, there are several possibilities such as differences in overall age (40 weeks at most in mice compared with nearly 50 years in humans) or lifestyle factors (mice maintained at 25°C under a germ-free atmosphere). Alternatively, the contribution of Lp(a) for the OxLDL levels in human plasma may explain this issue. It is reported recently that Lp(a) is a major carrier of OxPC in human plasma, but mouse does not have Lp(a).22 In addition, OxPC is likely to accumulate more preferentially in Lp(a) particles containing hapoB than those with mapoB, when comparing the hapoB-transgenic mice and hapoB/apo(a)-double transgenic mice.23
The occurrence of oxidation reactions in the vessel wall tissue was supported by immunohistochemical detection of acrolein and OxPC, which are major lipid peroxidation products. Acrolein is a highly reactive 
, β-unsaturated aldehyde, which generates adducts with any protein.21,24,25 OxPC is also able to modify various proteins.5,9 Therefore, the antigens detected in the tissues are not necessarily OxLDL; however, they are good indicators of the oxidative modification of proteins.
The behavior of the plasma OxLDL level during the 40 weeks was quite different from the progression of atherosclerotic lesions. Plasma OxLDL increased significantly at 20 weeks. The immunohistochemical study using antiacrolein mAb showed the presence of lipid peroxidation products in the aortic tissues in this stage of atherogenesis although the lesion area was very small. These observations suggest 2 interesting points. First, oxidative modification of LDL occurs under certain conditions in vivo. Note that apoE-KO are hypercholesterolemic; however no experimental stress was introduced in this study. Second, because the increase in OxLDL precedes the extensive development of atherosclerotic lesions, oxidative events might be involved in the early development of atherosclerotic lesions in apoE-KO. For decades, it has been unclear whether OxLDL is a cause of atherosclerosis or a result of lesion development,25,26 and it is difficult to investigate the oxidative status of patients before the appearance of vascular diseases. Our current data provides an intriguing observation of the possible involvement OxLDL in atherogenesis in apoE-KO.
If the appearance of OxLDL precedes the lesion progression, then the next question would be where OxLDL is generated. At 4 weeks the plasma OxLDL levels was lower than any other time points, aortic lesion size was very small but the oxidation epitopes in vessel wall were observed. It was reported earlier that OxLDL was accumulated in lesions before monocyte recruitment in human fetal aortas.27 These is a possibility that medial tissues under oxidative stress may be a site of LDL oxidation.
Transient increases in plasma OxLDL levels were observed in several studies on patients with acute myocardial infarction7,11,15 and cerebral infarction,6,16 and on patients receiving percutaneous coronary intervention.12 These results suggest a possibility that a transient increase in plasma OxLDL levels at the acute phase after infarction could be attributed to the release of OxLDL into the circulation from atherosclerotic plaques. However, because the lesion size in the 20-week-old apoE-KO was very small, the increase in plasma OxLDL at an early stage of atherogenesis is most likely attributable to a mechanism different from that of the rupture of advanced plaques. Because oxidation products accumulated in the lesions by 28 weeks, it is possible that the endothelial function or permeability of intima might be unstable during the early stages. Further studies are needed to elucidate the occurrence of critical changes at the early stages of atherogenesis.
Plasma OxLDL is removed from the circulation by clearance systems, and anti-OxLDL autoantibodies are thought to contribute to this phenomenon.28 Because the autoantibody concentrations did not change during the decrease in OxLDL level, the decrease in plasma OxLDL level in the apoE-KO after 20 weeks cannot be explained by the effect of autoantibodies. Alternatively, changes in oxidative stress and organization of vessel wall tissues could be responsible for the changes in plasma OxLDL levels. Further studies are needed to address this issue.
Circulating OxLDL had not been studied in animal models until recently. Recently, Tsimikas et al29 reported that plasma OxPL/apoB ratios in adult cynomolgus monkeys and New Zealand White rabbits decreased during the consumption of high-fat diets and increased during reversion to normocholesterolemic condition. Upregulation of plasma OxLDL under lipid-lowering conditions was also observed in healthy volunteers consuming a low fat diet30 and in patients having cardiovascular diseases treated with atorvastatin.31 Such OxLDL behavior might be attributable to a translocation of OxLDL or OxPL between the lesions and the circulation. It is interesting that the total immunostained area does not seem to decrease from 20 weeks to 40 week in apoE-KO, and note that the percent surface lesion area in aorta increased more than 10-fold during the same period. The translocation could be a possible explanation for the decrease in plasma OxLDL levels in our apoE-KO, during progression of atherosclerotic lesions after 20 weeks.
The plasma OxPC/apoB ratio in rabbits and monkeys was measured by a sandwich ELISA using E06 antibody.29 This method has many similarities with ours, but there are some critical differences. Whole plasma is used as a sample in their ELISA method whereas LDL fraction is separated from each plasma sample in our procedure, so that the profiles of OxLDL detected by these methods would have some difference.4
ApoB-48 is formed posttranscriptionally by a mechanism known as RNA editing.32 Human chylomicrons contain hapoB-48 and hapoB-100, whereas VLDL and LDL contain hapoB-100 almost exclusively, because apobec-1, an RNA editing enzyme, is active in the intestine but not in the liver. However, VLDL and LDL in the mouse plasma contain mapoB-48 and mapoB-100, because RNA editing is active in the liver of rodents.33 The editing pattern of the apoB protein depends on the mouse strains. Based on the literature34 and our data (supplemental Figure II), it appeared that mapoB-48 is the predominant subtype in apoE-KO and wild-type mice, however LDL-receptor knockout mice maintained on a normal chow diet express mapoB-100 more than mapoB-48.35 Because apoE-KO develop atherosclerotic lesions under normal chow diet and have been used in a number of studies, we decided to raise a pAb against mapoB-48 in this study.
Measurement of murine plasma OxLDL offers many possibilities for future studies, for example, in vivo assessment of antioxidative drugs and lipid-lowering therapy on LDL oxidation. It should be noted that the amount of LDL in a mouse is very small. We drew approximately 0.8 mL of blood from a mouse, which provided about 0.4 mL of plasma and 0.2 mg of LDL. Separating LDL fraction from plasma samples from each mouse is not easy. We are currently in process of improving the procedure for detecting oxidized lipoproteins in mouse and human plasma without separating the LDL fraction by introducing chicken anti-mouse apoB IgY antibody.
In conclusion, we found transient increase in the plasma OxLDL levels at an early stage of lesion progression with our ELISA protocol. This method can be useful for investigating the behavior and function of OxLDL in vivo, especially at the early stages of lesion development.
Study Limitations
This study tested the time course changes of plasma OxLDL levels by measuring 5 time points during 40 weeks after the birth. To find a critical timing of the OxLDL elevation, a finer time course stuffy is desired. Because apoE-KO maintained on normal chow diet were used throughout this study, whether the same is true for the LDL receptor knockout mice is to be studied.
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Acknowledgments |
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Sources of Funding
This study was supported in part by a grant-in-aid from the Ministry of Education, Technology, Sports, and Sciences of Japan (10590065), a Research on Health Science Focusing on Drug Innovation grant from The Japan Health Science Foundation (KH21011), the High-Tech Research Center Project for Showa University, matching fund subsidy from MEXT (2005–2007), and the Fugaku Fund.
Disclosures
None.
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Footnotes |
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Received February 9, 2008; revision accepted October 27, 2008.
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References |
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