Published online before print April 9, 2009, doi: 10.1161/CIRCRESAHA.108.192229
© 2009 American Heart Association, Inc.
Molecular Medicine |
Human Paraoxonase Gene Cluster Transgenic Overexpression Represses Atherogenesis and Promotes Atherosclerotic Plaque Stability in ApoE-Null Mice
From the National Laboratory of Medical Molecular Biology (Z.-G.S., W.Z., Y.-S.W., H.-Z.C., A.-B.W., H.-L.L., G.L., R.Z., J.-J.L., D.-P.L., C.-C.L.), Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; Burnham Institute for Medical Research (Z.-G.S., W.B.S.), Cancer Center, La Jolla, Calif; and Department of Biochemistry (Z.Z.), The University of Hong Kong, China.
Correspondence to De-Pei Liu, PhD, National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, People’s Republic of China. E-mail liudp{at}pumc.edu.cn
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The paraoxonase (PON) gene cluster consists of the PON1, PON2, and PON3 genes, each of which can individually inhibit atherogenesis. To analyze the functions of the PON gene cluster (PC) in atherogenesis and plaque stability, human PC transgenic (Tg) mice were generated using bacterial artificial chromosome. The high-density lipoprotein from Tg mice exhibited increased paraoxonase activity. When crossed to the ApoE-null background and challenged by high-fat diet, PC Tg/ApoE-null mice formed significantly fewer atherosclerotic lesions. However overexpression of the PC transgene had no additive effect on atherosclerosis compared to the overexpression of the single PON1 or PON3 transgene. Plaques from PC Tg/ApoE-null mice exhibited increased levels of collagen and smooth muscle cells, and reduced levels of macrophages and lipid, compared with those from ApoE-null mice, indicating lesions of PC Tg/ApoE-null mice had characteristics of more stable plaques than those of ApoE-null mice. PC transgene enhanced high-density lipoprotein ability to protect low-density lipoprotein against oxidation in vitro. Serum intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 were also repressed by PC transgene. Proatherogenic reactions of Tg mouse peritoneal macrophages induced by oxidized low-density lipoprotein were inhibited by PC transgene, as indicated by reduced reactive oxygen species generation, inflammation, matrix metalloproteinase-9 expression, and foam cell formation. Our results demonstrate that the PC transgene not only represses atherogenesis but also promotes atherosclerotic plaque stability in vivo. PC may therefore be a useful target for atherosclerosis treatment.
Key Words: atherosclerosis • plaques stability • macrophages • paraoxonase cluster
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Atherosclerosis constitutes the single most important contributor to cardiovascular diseases, which are the leading cause of death and illness in developed societies.1,2 Atherosclerosis-related ischemic symptoms, especially acute cardiovascular events that result in myocardial infarction and stroke, are generally thought to result from plaque rupture and thrombosis other than narrowing of the vessel lumen.3 New strategies for preventing and treating plaque rupture are very much needed based on understanding of factors that contributed. Oxidized low-density lipoprotein (oxLDL) plays a pivotal role in triggering proinflammatory events that initiate and exacerbate atherogenesis,1,4 which induces the expression of adhesion molecules, chemokines, and cytokines in vascular endothelial cells,5 triggering recruitment of monocytes into the subendothelial space of the arterial wall, where they differentiate into macrophages. Recruited macrophages ingest and further oxidize LDL, transforming into foam cell after excessive oxLDL accumulation. These transformed macrophages not only constitute early fatty streak lesions and the core of atherosclerotic plaques but also become active inflammation centers because of secretion of inflammatory molecules and matrix metalloproteinases (MMPs), which promote atherosclerotic plaques deterioration and ultimate rupture.6,7
Paraoxonase (PON)1, originally recognized for its ability to hydrolyze paraoxon, is present in circulating high-density lipoprotein (HDL) and has been shown to inhibit LDL oxidation in vitro.8 Compared to wild-type (Wt) counterparts, PON1-deficient mice develop significantly larger atherosclerotic lesions in their aortas,9,10 whereas PON1-overexpressing mice possess atheroprotective properties.11 In humans, low PON1 activity has been shown to be an independent risk factor for coronary heart diseases.12,13 However, PON1 is only a member of the PON gene cluster (PC), which also includes PON2 and PON3. PON2 is ubiquitously expressed in mammalian tissues, and is a cellular antioxidant, retarding the oxidation of mildly oxidized LDL.14 PON2-deficient mice develop significantly larger atherosclerotic lesions compared to Wt controls.15 A role for PON2 in the development of human atherosclerosis is suggested by its relationship with intima–medial thickness in white patients with heterozygous familial hypercholesterolemia.16 PON2 can also reduce oxidative stress in vascular cells by decreasing stress-induced caspase activation in the endoplasmic reticulum.17 PON3 is also an HDL-associated enzyme with biological activity similar to PON1.18 PON3 transgenic (Tg) mice exhibit decreased atherogenesis compared to their littermates.19 Moreover, PON2 and PON3 are also reported being able to reduce the accumulation of macrophages in the atherosclerotic plaques.15,19 Based on these findings, Reddy et al concluded that all members of the paraoxonase gene family could be used as therapeutic targets for the treatment of atherosclerosis.20
Nevertheless, as a gene cluster, the role of the entire PC in atherogenesis still remains to be elucidated. Moreover, the danger of an atherosclerotic plaque is not related to its size but rather to its tendency to rupture.21 Given that high plasma and plaque levels oxLDL are correlated with vulnerability to rupture of atherosclerotic lesions in human,22 whether PC can exert its oxLDL- and macrophage-inhibiting function to promote atherosclerotic plaque stability deserves exploration. Using PC Tg mice, we demonstrate for the first time that the PC transgene repressed atherogenesis and promoted atherosclerotic plaque stability in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. All protocols regarding the care and use of animals were approved by the institutional guidelines.
Construction of Human PON Cluster Transgenic Mice
A 170-kb DNA fragment containing the PON1, PON2, and PON3 genes and their flanking sequences was prepared for microinjection as described.23
Mouse Breeding, Diet, and Analysis of Atherosclerotic Lesions
PC Tg mice were crossed with ApoE-null mice to obtain PC Tg/ApoE-null mice and ApoE-null littermates, which were maintained on chow diet for 6 weeks and then switched to a Western diet containing 10% fat and 1.25% cholesterol to induce atherogenesis. En face aorta atherosclerotic lesions were analyzed as described.24,25
Plaque Morphology Histomorphometric Analysis
Atherosclerotic plaques at the aortic valve level were sectioned as described.26 Plaque morphological histomorphometric characters were analyzed via hematoxylin/eosin (H&E) staining. Plaque composition was detected by oil red O staining (lipid-rich cores), Picric and Sirius staining (collagen), anti–
-smooth muscle cells (SMC)-actin antibody (smooth muscle cells) staining, and anti–MOMA-2 antibodies (macrophages) staining. Plaques stability was evaluated by comparing the percentages of the above plaque components in the entire plaques. The histological plaque stability score was calculated as follows26: (plaque stability score)=(SMC area+collagen area)/(macrophage area+lipid area).
PCR Analysis, Southern Blotting, RT-PCR, Western Blot, and ELISA
Target gene expression levels were probed either with RT-PCR using corresponding primers (Online Table I) or Western blotting. Plasma monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 levels were evaluated by solid-phase sandwich ELISA kits.
Analysis of Serum Lipid, Glucose, and PON Activity
Mice were fasted for 16 hours before being bled for analysis of serum total cholesterol (TC), HDL-cholesterol (HDL-C), very-low-density lipoprotein (VLDL)/LDL-cholesterol (VLDL/LDL-C), triglycerides, and glucose. Mouse HDL was isolated by ultracentrifugation of serum samples as described.10 HDL-associated PON activity was measured spectrophotometrically using phenylacetate as the substrate.27
LDL Oxidation Protection Assay
Human LDL was isolated by ultracentrifugation of serum as described.10 The LDL oxidation assay was performed as described,11 and the lipid hydroperoxide content of samples was then determined using the Fox assay.28
Mouse Peritoneal Macrophage Isolation and Assays of Oxidative Stress, Inflammation, and Foam Cell Formation
Mouse peritoneal macrophages (MPMs) were isolated as described.15 Intracellular oxidative stress induced by oxLDL was detected using 2',7'-dichlorofluorescein.15 Tumor necrosis factor (TNF)- and interleukin (IL)-6 levels were analyzed after 6 hours of 50 µg/mL oxLDL stimulation by RT-PCR using listed primers (Online Table I). Foam cell formation of MPMs was determined using oil red O staining after 24 and 48 hours of 50 µg/mL oxLDL stimulation.
Statistical Analyses
All values are expressed as means±SEM. Statistical differences between two data sets were determined via the Students t test. A value of P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human PC Tg Mice
Five independent Tg mice lines carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences (Figure 1A) were established, as identified by routine PCR analysis (data not shown) and Southern blotting (Online Figure I, A). P2 PC Tg mouse line carried highest (
8) copies of the transgene (Online Figure I, A), expressed the highest human PON1 level in the liver (Online Figure I, B), and was chosen for the following analysis. Studies with another independent line gave similar results (data not shown). The tissue distributions of the human PON1, PON2, and PON3 transgenes were similar to those of intrinsic mouse PONs, with PON1 expressed primarily in the liver and PON2 and PON3 expressed more universally (Figure 1B). Human PON1, PON2, and PON3 proteins were also detected in the aortas of PC Tg mice, with PON1 exhibiting the highest, PON2 the intermediate, and PON3 the lowest (Figure 1C). We also confirmed the presence of human PON1 protein in the HDL of P2 PC Tg mice (Figure 1D). Subsequent paraoxonase activity analysis revealed that the HDL of P2 PC Tg mice exhibited 60% higher paraoxonase activity than that of Wt mice (Figure 1E). When maintained on the chow diet, both male and female PC Tg mice were healthy and exhibited normal body weight, similar plasma TC, HDL-C, VLDL/LDL -C, triglyceride, and glucose levels compared with their Wt littermates (Online Table II).
|
PC Tg Represses Atherogenesis in ApoE-Null Mice
To investigate atherogenesis, PC Tg mice were crossed onto the ApoE-null background. Both male and female PC Tg/ApoE-null mice, together with control ApoE-null mice, were switched to the high-fat diet at the age of 6 weeks. After 10 weeks on the high-fat diet, male and female mice in both groups exhibited similar dramatic increases in plasma TC, HDL-C, and VLDL/LDL-C levels and similar decrease in serum triglycerides, with the same normal glucose levels (Online Table II). However, the incidence of atherosclerotic lesions in the entire aortas of female PC Tg/ApoE-null mice was 23.5% lower than that of ApoE-null littermates (Figure 2A). Among males, the reduction in atherosclerotic lesions was 33.9% (Figure 2B). To assess the effect of extended high-fat diet, the induction was prolonged to 16 weeks. Plasma TC, HDL-C, and VLDL/LDL-C remained at high levels in both sexes of the 2 groups (Online Table II). However, lesion areas in PC Tg/ApoE-null mice remained less than those in ApoE-null controls, with decreases of 33.3% (Figure 2C) and 27.7% (Figure 2D) for females and males, respectively. These results demonstrate that PC Tg repressed atherogenesis on both female and male ApoE-null mice.
|
PC Tg Promotes Atherosclerotic Plaque Stability
To further investigate whether PC Tg can influence atherosclerotic plaque stability, hearts of female PC Tg/ApoE-null and ApoE-null mice were collected after 16 weeks of high-fat diet induction and sectioned at the aorta sinus level for histological analysis. H&E staining of these sections indicated that plaque area and the ratio of blank area (indicating necrotic core) to entire plaque area of PC Tg/ApoE-null mice (Figure 3B) were 21.1% (Figure 3C) and 13.1% (Figure 3D) less than those of control ApoE-null mice (Figure 3A), respectively. Such results, on the one hand, indicated that PC transgene did repress atherogenesis but, on the other hand, suggested that plaques from PC Tg/ApoE-null mice exhibited thicker fibrous caps and smaller necrotic cores than plaques from control ApoE-null mice. Further stability investigation by detecting plaque composition indicated that plaques from PC Tg/ApoE-null mice exhibited increased percentages of collagen (76.9%; Figure 4C, 4D, and 4I) and smooth muscle cells (15.8%; Figure 4G, 4H, and 4I) and reduced percentages of macrophages (22.3%; Figure 4E, 4F, and 4I) and lipid area (9.5%; Figure 4A, 4B, and 4I) compared with those from ApoE-null mice. These serial alterations in the plaque composition indicated that PC transgene also promoted stability of plaques generated in ApoE-null mice besides repressing atherogenesis. Accordingly, the histological plaque stability score was also improved by PC transgene (Figure 4J).
|
|
PC Tg Reinforces the Ability of Mouse HDL to Protect Against Human LDL Oxidation In Vitro
Reported individually, PON1, PON2, and PON3 inhibited atherosclerosis mainly by inhibiting LDL oxidation.11,15,19 Here, HDL from PC Tg mice and Wt mice were isolated by ultracentrifugation and analyzed in a 12-hour LDL copper–induced oxidation system at final concentrations of 0.25 mg/mL HDL and 0.5 mg/mL HDL, respectively. Relative hydroperoxide levels were detected every 2 hours. At the dose of 0.25 mg/mL, the PC Tg HDL exhibited more obviously consistent reductions in the lipid hydroperoxide production after incubation than PBS control compared to the Wt HDL. For example, after 4 or 6 hours of induction, lipid hydroperoxide production of human LDL incubated with PC Tg HDL were 40% (P=0.0007) or 57% (P=0.0001) less than human LDL incubated with Wt HDL, respectively (Figure 5A). When the dose of the HDL was raised to 0.5 mg/mL, both sets of sample exhibited much lower levels of lipid hydroperoxide production than PBS control, with no apparent difference between the 2 groups (Figure 5B). Thus, although both sets of HDL are effective in preventing LDL oxidation, PC Tg HDL is more effective than the Wt HDL, especially when HDL concentration is lower.
|
Reduction of ICAM-1 and MCP-1 in Tg Mice
OxLDL stimulates endothelial cells to secrete inflammatory and attractant molecules such as ICAM-1 and MCP-1.29 Thus, using ELISA, we quantified serum ICAM-1 and MCP-1 levels in female and male PC Tg/ApoE-null and ApoE-null mice after 16 weeks of high-fat diet induction to detect whether the effect of in vitro oxLDL inhibition by PC transgene can translate into serum adhesion molecules reduction. Serum levels of ICAM-1 in female and male PC Tg/ApoE-null mice were 31% and 51% lower, respectively, than those of ApoE-null mice (Figure 6A). Serum levels of MCP-1 in female and male PC Tg/ApoE-null mice were 36% and 34% lower, respectively, than those of ApoE-null mice. These results indicate that PC transgene represses the production of ICAM-1 and MCP-1 in mice (Figure 6B).
|
PC Transgene Inhibits Proatherosclerotic Activities of MPMs
ICAM-I and MCP-1 mediate recruitment of macrophages, which play key roles in atherogenesis and plaque stability by participating reactive oxygen species (ROS) generation, inflammation, MMP production, and foam cell formation.2,30 To detect whether functions of macrophages were affected by PC transgene, MPMs were isolated and analyzed. Macrophages from PC Tg mice can express high level of human PON2 besides intrinsic mouse PON2 and PON3 (Figure 7A). When stimulated by oxLDL at 50 µg/mL, PC Tg macrophages maintained significantly lower levels of ROS for at least 2 hours than macrophages from Wt mice (Figure 7B), indicating that human PON2 overexpression inhibits ROS generation in MPMs. Inflammatory factors such as TNF- and IL-6 are expressed at lower levels in PC Tg MPMs compared with Wt MPMs. When stimulated by oxLDL at 50 µg/mL for 6 hours, both Wt and PC Tg MPMs express elevated levels of TNF- and IL-6. However, these increases were significantly lower in PC Tg MPMs (Figure 7C). These results suggest that inflammatory responses of MPMs are also prominently attenuated by human PON2 overexpression, even under oxLDL stimulation. In addition, MMP-9 expression in PC Tg MPMs was significantly lower than that in Wt MPMs, even after stimulation with 50 µg/mL oxLDL for 6 hours (Figure 7D), suggesting that human PON2 overexpression can inhibit MMP-9 expression in MPMs even under oxLDL stimulation, which was further confirmed by the fact that weaker immunohistochemical staining was detected in the plaques from PC Tg/ApoE-null female mice than that of control Wt/ApoE-null female littermates after 16 weeks of high-fat diet feeding (Figure 7E). Finally, foam cell formation in PC Tg MPMs was markedly less compared with that seen in Wt MPMs after stimulation with 50 mg/mL oxLDL for 24 hours or 48 hours (Figure 8A), as indicated by evaluation of cellular area (Figure 8B) and foam component (Figure 8C), respectively. The abovementioned results proved that proatherosclerotic activities of MPMs were extremely inhibited by PC transgenic overexpression.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
While PC members, PON1, PON2, and PON3 have been individually reported to inhibit atherosclerosis.11,15,19 The present study demonstrates for the first time that the intact human PC transgenic overexpression not only repressed the development of atherosclerosis but also promoted the stability of atherosclerotic plaques that did form in vivo. Furthermore, such functions are independent of alterations of parameters of lipid metabolism, with respect to the present results, along with previous genomic modification researches of PONs.9–11,15,19
It has been reported that PON1-deficient HDL lacks the ability to prevent LDL oxidation in a cell culture system,9,10 whereas HDL isolated from human PON1 Tg mice has an enhanced ability to prevent LDL oxidation in vitro.11 In present study, we evaluate the effect of HDL on copper-induced LDL oxidation. The results demonstrated that PC Tg HDL possessed elevated paraoxonase activity (Figure 1E) and enhanced the ability of the HDL in protecting LDL from being oxidized in vitro (Figure 5). Because oxLDL can stimulate endothelial cell secretion of MCP-1 and ICAM-1 and other adhesion molecules to mediate atherogenic recruitment of macrophages,29 this suggests a mechanism by which HDL-associated PON1 can reduce atherosclerosis. Indeed, it was reported that aortic MCP-1 expression was reduced 44% in PON1 Tg/ApoE-null mice when compared with ApoE-null littermates.11 Here, we found that, besides serum MCP-1, serum ICAM-1 levels were also lower in PC Tg/ApoE-null mice on high-fat diet than those in ApoE-null mice (Figure 6). Both MCP-1 and ICAM-1 have been well documented as having functions of mediating macrophage recruitment and migration to atherosclerotic lesion.31,32 Thus, by inhibiting MCP-1 and ICAM-1, PC transgene should decrease macrophage recruitment into local lesion. Then, we detected the recruitment of macrophages in the plaques and found that macrophages recruitment was dramatically reduced in the atherosclerotic plaques of female PC Tg/ApoE-null mice after 16 weeks on the high-fat diet, compared to plaques in female ApoE-null mice (Figure 4E, 4F, and 4I). It has been reported that recruited macrophages contribute to atherogenesis by triggering inflammation and forming foam cell.2,30 Previous research indicated that MPMs from PON2-null mice generate more ROS and exhibit more serious inflammation than Wt MPMs.15 In the present research, lower levels of ROS were produced in MPMs from PC Tg mice than in MPMs from Wt mice when challenged by oxLDL (Figure 7B). Inflammatory responses of MPMs were also remarkably inhibited by PC transgene, as indicated by reduced expression of TNF- and IL-6 (Figure 7C). Furthermore, oxLDL-induced foam cell formation of MPMs was dramatically attenuated by transgenic overexpression of human PON2 (Figure 8). As we have known, oxLDL can affect vascular cells by both internal and external mechanisms.4 Results of our present research suggest that transgenic human PONs can exert their antiatherosclerotic functions at 2 different levels, ie, externally in the serum (PON1 and PON3 effects on HDL function) and internally in the cells (PON2 effects on macrophages function).
Because of these 2 levels of antiatherosclerotic effects of PC transgenic overexpression, atherosclerotic lesions in the entire aortas of PC Tg/ApoE-null mice were evidently less than that of ApoE-null littermates (Figure 2). However, no evidently additive effect on atherogenesis could be found when comparing atherosclerosis reduction caused by PC transgene with those caused by PON1 or PON3 single transgene. One of the reasons could be that any single PON gene overexpression could independently exert saturated oxLDL-inhibiting function, which is partly suggested by our results that even sufficient Wt HDL could attenuate the lipid hydroperoxide production of human LDL induced by CuSO4 as strong as equal amounts of PC HDL (Figure 5B).
Moreover, reducing size of atherosclerotic plaque is not enough to cure atherosclerosis, because the main cause of complications of atherosclerosis is the rupture of atherosclerotic plaque.33 Stabilizing plaque is becoming an important focus for atherosclerosis treatment. Although each member of PON gene cluster has been individually reported as being able to inhibit atherogenesis, little information is available on the function of PONs in the atherosclerotic plaque stability. Vulnerable plaques have a larger lipid core and thinner fibrous cap, more infiltration of macrophage and less SMC, and more active inflammation and less extracellular matrix compared with stable plaques.7 Here, we analyzed and compared the plaques of female Tg/ApoE-null mice and their ApoE-null littermates. H&E staining indicated that plaques from PC Tg/ApoE-null mice exhibited thicker fibrous caps and smaller necrotic cores (as evidenced by less blank area inside the plaques) than those from control ApoE-null mice (Figure 3), suggesting that plaques in PC Tg/ApoE-null mice were more stable than those of control ApoE-null mice. Further analysis revealed that presence of macrophages along with the lipid area in the plaques of PC Tg/ApoE-null mice (Figure 4B, 4F, and 4I) were obviously reduced compared to ApoE-null mice (Figure 4A, 4E, and 4I). Infiltrated macrophages are the main contributors to foam cell and necrotic core formation, both of which promote plaques rupture.34 Inhibiting macrophage infiltration therefore should help to stabilize atherosclerotic plaques. Local macrophages can also express several species of MMPs for degradation of extracellular matrix generated in the plaques. Among these, MMP-9 is highly expressed and plays a pivotal role in the process of matrix degradation via cleavage of elastin and type IV collagen.35 Thus, inhibition of MMPs, especially MMP-9, is a pivotal strategy for stabilizing plaques.36 It has also been reported that oxLDL could upregulate MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages.37 In the present research, we found that PC transgene, which dramatically inhibited LDL oxidation in vitro, could effectively inhibit MMP-9 in vitro (expression of MMP-9 was significantly lower in MPMs of PC Tg compared with Wt mice; Figure 7D) and in vivo (expression of MMP-9 in the plaques of PC Tg/ApoE-null mice was lower than that in ApoE-null mice; Figure 7E), which resulted more abundant collagen presented in PC Tg/ApoE-null plaques than Wt/ApoE-null plaques (Figure 4C, 4D, and 4I) at least in part. These abovementioned scenarios promoted the composition of the plaques to a more consolidated state. Calculated plaque stability score of PC Tg/ApoE-null mice was 70% higher than that of ApoE-null mice (Figure 4J), which further indicated that PC transgene promoted plaque stability formed in ApoE-null mice. However, whether the plaque stability–promoting function of PONs can eventually inhibit plaque rupture needs to be determined in future developed models, because the present mouse aorta sinus atherosclerotic plaque models seldom rupture. Here, the evidence of the plaque stability–promoting functions of PONs was based on the entire gene cluster overexpression instead of on any single PON member. Further investigations are needed to explore whether any single PON member gene has such function or which member plays a central role in this process. Still, our results indicate the possible usefulness of PONs for atherosclerosis treatment, especially for promoting plaque stability.
In conclusion, our present research has demonstrated that PC transgene not only represses high-fat diet–induced atherogenesis but also leads to formation of more stable plaques in ApoE-null mice. Considering that treatment of atherosclerosis is somewhat disappointing because of a lack of effective ways of preventing atherosclerotic plaque rupture, the present work sheds light on this challenge. The PC Tg mice model will also be useful for investigating potent strategies aiming to reinforce PONs to inhibit atherogenesis and stabilize atherosclerotic plaques.
|
Acknowledgments |
|---|
We thank Baosheng Chen (Peking Union Medical College) for providing human LDL and oxLDL and Guoqing Liu and Zhongwu Li (Peking University) for assistance in isolating mouse HDL and in processing sides, respectively.
Sources of Funding
This work was supported by National Basic Research Program of China grants 2005CB522507 and 2006CB503801; National Natural Science Foundation of China grants 30500297, 30529002, and 30721063; and National 863 project grant 2006AA02A406.
Disclosures
None.
|
Footnotes |
|---|
Original received December 8, 2008; revision received March 31, 2009; accepted April 1, 2009.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.[CrossRef][Medline] [Order article via Infotrieve]
2. Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell. 2001; 104: 503–516.[CrossRef][Medline] [Order article via Infotrieve]
3. Lee RT, Libby P. The unstable atheroma. Arterioscler Thromb Vasc Biol. 1997; 17: 1859–1867.
4. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997; 272: 20963–20966.
5. Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.[CrossRef][Medline] [Order article via Infotrieve]
6. Galis ZS. Vulnerable plaque: the devil is in the details. Circulation. 2004; 110: 244–246.
7. Schwartz SM, Galis ZS, Rosenfeld ME, Falk E. Plaque rupture in humans and mice. Arterioscler Thromb Vasc Biol. 2007; 27: 705–713.
8. Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M. Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin Invest. 1995; 96: 2882–2891.[Medline] [Order article via Infotrieve]
9. Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG, Fogelman AM, Lusis AJ. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature. 1998; 394: 284–287.[CrossRef][Medline] [Order article via Infotrieve]
10. Shih DM, Xia YR, Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF, Berliner JA, Witztum JL, Lusis AJ. Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J Biol Chem. 2000; 275: 17527–17535.
11. Tward A, Xia YR, Wang XP, Shi YS, Park C, Castellani LW, Lusis AJ, Shih DM. Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice. Circulation. 2002; 106: 484–490.
12. Mackness B, Durrington P, McElduff P, Yarnell J, Azam N, Watt M, Mackness M. Low paraoxonase activity predicts coronary events in the Caerphilly Prospective Study. Circulation. 2003; 107: 2775–2779.
13. Ayub A, Mackness MI, Arrol S, Mackness B, Patel J, Durrington PN. Serum paraoxonase after myocardial infarction. Arterioscler Thromb Vasc Biol. 1999; 19: 330–335.
14. Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S, Grijalva VR, Navab M, Fogelman AM, Reddy ST. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein. J Biol Chem. 2001; 276: 44444–44449.
15. Ng CJ, Bourquard N, Grijalva V, Hama S, Shih DM, Navab M, Fogelman AM, Lusis AJ, Young S, Reddy ST. Paraoxonase-2 deficiency aggravates atherosclerosis in mice despite lower apolipoprotein-B-containing lipoproteins: anti-atherogenic role for paraoxonase-2. J Biol Chem. 2006; 281: 29491–29500.
16. Leus FR, Zwart M, Kastelein JJ, Voorbij HA. PON2 gene variants are associated with clinical manifestations of cardiovascular disease in familial hypercholesterolemia patients. Atherosclerosis. 2001; 154: 641–649.[CrossRef][Medline] [Order article via Infotrieve]
17. Horke S, Witte I, Wilgenbus P, Kruger M, Strand D, Forstermann U. Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation. 2007; 115: 2055–2064.
18. Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, Shih DM, Lusis AJ, Navab M, Fogelman AM. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vasc Biol. 2001; 21: 542–547.
19. Shih DM, Xia YR, Wang XP, Wang SS, Bourquard N, Fogelman AM, Lusis AJ, Reddy ST. Decreased obesity and atherosclerosis in human paraoxonase 3 transgenic mice. Circ Res. 2007; 100: 1200–1207.
20. Reddy ST, Devarajan A, Bourquard N, Shih D, Fogelman AM. Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis? Curr Opin Lipidol. 2008; 19: 405–408.[CrossRef][Medline] [Order article via Infotrieve]
21. Webb NR. Getting to the core of atherosclerosis. Nat Med. 2008; 14: 1015–1016.[CrossRef][Medline] [Order article via Infotrieve]
22. Nishi K, Itabe H, Uno M, Kitazato KT, Horiguchi H, Shinno K, Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler Thromb Vasc Biol. 2002; 22: 1649–1654.
23. Gao J, Wei Y, Huang Y, Liu D, Liu G, Wu M, Wu L, Zhang Q, Zhang Z, Zhang R, Liang C. The expression of intact and mutant human apoAI/CIII/AIV/AV gene cluster in transgenic mice. J Biol Chem. 2005; 280: 12559–12566.
24. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995; 36: 2320–2328.[Abstract]
25. Zhang QJ, Wang Z, Chen HZ, Zhou S, Zheng W, Liu G, Wei YS, Cai H, Liu DP, Liang CC. Endothelium-specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice. Cardiovasc Res. 2008; 80: 191–199.
26. Ni W, Egashira K, Kitamoto S, Kataoka C, Koyanagi M, Inoue S, Imaizumi K, Akiyama C, Nishida KI, Takeshita A. New anti-monocyte chemoattractant protein-1 gene therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2001; 103: 2096–2101.
27. Shih DM, Gu L, Hama S, Xia YR, Navab M, Fogelman AM, Lusis AJ. Genetic-dietary regulation of serum paraoxonase expression and its role in atherogenesis in a mouse model. J Clin Invest. 1996; 97: 1630–1639.[Medline] [Order article via Infotrieve]
28. Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem. 1992; 202: 384–389.[CrossRef][Medline] [Order article via Infotrieve]
29. Dwivedi A, Anggard EE, Carrier MJ. Oxidized LDL-mediated monocyte adhesion to endothelial cells does not involve NFkappaB. Biochem Biophys Res Commun. 2001; 284: 239–244.[CrossRef][Medline] [Order article via Infotrieve]
30. Rader DJ, Pure E. Lipoproteins, macrophage function, and atherosclerosis: beyond the foam cell? Cell Metab. 2005; 1: 223–230.[CrossRef][Medline] [Order article via Infotrieve]
31. Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaudet AL. P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med. 2000; 191: 189–194.
32. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998; 394: 894–897.[CrossRef][Medline] [Order article via Infotrieve]
33. Rothwell PM, Villagra R, Gibson R, Donders RC, Warlow CP. Evidence of a chronic systemic cause of instability of atherosclerotic plaques. Lancet. 2000; 355: 19–24.[CrossRef][Medline] [Order article via Infotrieve]
34. Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Nerem RM. The pathogenesis of atherosclerosis: an overview. Clin Cardiol. 1991; 14: I1–I16.[Medline] [Order article via Infotrieve]
35. Palombo D, Maione M, Cifiello BI, Udini M, Maggio D, Lupo M. Matrix metalloproteinases. Their role in degenerative chronic diseases of abdominal aorta. J Cardiovasc Surg (Torino). 1999; 40: 257–260.[Medline] [Order article via Infotrieve]
36. Gough PJ, Gomez IG, Wille PT, Raines EW. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest. 2006; 116: 59–69.[CrossRef][Medline] [Order article via Infotrieve]
37. Xu XP, Meisel SR, Ong JM, Kaul S, Cercek B, Rajavashisth TB, Sharifi B, Shah PK. Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages. Circulation. 1999; 99: 993–998.
This article has been cited by other articles:
 |
|
 |
|
  D. De Keyzer, S.-A. Karabina, W. Wei, B. Geeraert, D. Stengel, J. Marsillach, J. Camps, P. Holvoet, and E. Ninio Increased PAFAH and Oxidized Lipids Are Associated With Inflammation and Atherosclerosis in Hypercholesterolemic Pigs Arterioscler Thromb Vasc Biol, December 1, 2009; 29(12): 2041 - 2046. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||