Increased Oxidative Stress in Scavenger Receptor BI Knockout Mice With Dysfunctional HDL

【摘要】  Objective— In the current study the effect of disruption of SR-BI, a prominent regulator of HDL metabolism, on the activity of the HDL-associated antioxidant enzymes PON1 and PAF-AH as well as in vivo oxidative stress were investigated.

Methods and Results— SR-BI deficiency resulted in 1.4-fold ( P <0.001) and 1.6-fold ( P <0.01) lower serum paraoxonase and arylesterase activity of PON1, respectively. Furthermore, a trend to slightly lower PAF-AH activity was observed. In vivo oxidative stress was evaluated by measuring isoprostane F2 -VI (iPF2 -VI) and protein carbonyls. Compared with wild-type animals, SR-BI knockouts had 1.4-fold ( P <0.05) higher levels of plasma iPF2 -VI, whereas urinary excretion was increased 2-fold ( P <0.0001). Plasma carbonyls were 1.5-fold ( P <0.05) higher in SR-BI knockout animals. Furthermore, iPF2 -VI and carbonyl levels were 2.1-fold ( P <0.01) and 1.4-fold ( P <0.01), respectively, increased in livers of SR-BI knockout mice, and in reaction to the increased oxidative stress the expression of several endogenous antioxidant systems was upregulated. On challenging the SR-BI knockout mice with an atherogenic Western-type diet, a further increase in oxidative stress in these animals was observed.

Conclusion— SR-BI deficiency results in a reduced activity of the antioxidant enzyme PON1 and a significant increase in oxidative stress, potentially contributing to the proatherogenic effect of SR-BI deficiency.

HDL reduces atherosclerosis by its antioxidant properties. In the current study we show that disruption of SR-BI, a prominent regulator of HDL metabolism, decreases the activity of the HDL-associated antioxidant enzyme paraoxonase 1 and leads to increased oxidative stress, potentially contributing to the proatherogenic effect of SR-BI deficiency.

【关键词】  HDL cholesterol antioxidant enzymes oxidative stress isoprostanes mouse models

Introduction

HDL levels are inversely correlated with the risk for atherosclerosis. An important mechanism by which HDL inhibits the development and progression of atherosclerosis is its facilitating role in reverse cholesterol transport, a process by which excess cholesterol from peripheral tissues is transferred from the plasma to the liver for either recycling or excretion from the body via the bile. 1 However, HDL also has multiple additional endothelial and antithrombotic actions that may provide cardiovascular protection. 2,3 Furthermore, HDL may protect by inhibiting the oxidative modification of LDL, which plays a central role in the initiation and propagation of atherosclerosis. 4,5 Several lines of evidence suggest that HDL can act as an antioxidant through the activity of the HDL-associated proteins, including paraoxonase 1 (PON1) 6 and platelet-activating factor acetylhydrolase (PAF-AH). 7

PON1 protects against LDL oxidation, reverses the biological effects of oxidized LDL, and preserves the function of HDL by inhibiting its oxidation. 8–10 The protective role of PON1 in atherosclerosis is clearly illustrated by the fact that PON1/apoE double knockout mice develop significantly larger atherosclerotic lesions as compared with apoE knockout mice with functional PON1. 11 PAF-AH attenuates the potent proinflammatory activity of PAF by hydrolyzing its sn-2 ester bond. 12 In addition, it also functions as an antioxidant enzyme by hydrolyzing oxidized phospholipids, such as F2-isoprostanes, formed during the oxidative modification of LDL. 13 Increasing plasma PAF-AH levels in apoE knockout mice reduces injury-induced neointima formation and spontaneous atherosclerotic lesion development. 14,15

Although it is generally accepted that HDL protects against the development of atherosclerosis, high levels of HDL, however, are not always protective. In the Framingham Heart Study 45% of all clinical events occurred in subjects with normal or elevated HDL cholesterol levels, 16 suggesting that not only the levels but also other HDL-associated factors are important. A prominent regulator of HDL metabolism is the scavenger receptor class B, type I (SR-BI), which mediates the selective uptake of cholesteryl esters from HDL without internalization of the HDL particle. 17,18 Selective disruption of SR-BI in mice results in highly increased plasma cholesterol levels due to the accumulation of large cholesteryl ester-rich HDL and is associated with an increased susceptibility to atherosclerosis. 19–21 The proatherogenic effects of SR-BI deficiency have primarily been attributed to disruption of the flux of cholesterol through the reverse cholesterol transport pathway. In the present study, we demonstrate that SR-BI deficiency is also associated with reduced activity of the important endogenous antioxidant enzyme PON1 and a significant increase in oxidative stress in vivo, both of which could contribute to the increased susceptibility to atherosclerosis reported in these mice.

Materials and Methods

For detailed methodology, please see the data supplement, available online at http://atvb.ahajournals.org. Briefly, PAF-AH activity and the arylesterase and paraoxanase activity of PON1 were determined in serum of SR-BI knockout mice using substrate-based assays. PAF-AH and PON1 serum protein levels were determined by Western blotting, whereas the mRNA expressions of PAF-AH and PON1 were determined in spleen and liver respectively, using real-time Quantitative polymerase chain reaction (PCR). Urinary, EDTA-anticoagulated plasma, and tissue levels of the isoprostane iPF2 -VI were measured by gas chromatography-mass spectrometry and total protein carbonyls were determined in plasma and organs by using the Zenith PC test kit (Zenith Technology). The serum decay and liver uptake of oxidized cholesterol esters from HDL was studied after injection of 200 µg [ 3 H-CEOH]-HDL in anesthetized mice.

Results

Decreased PON1 and PAF-AH Activity in SR-BI Knockout Mice

Selective disruption of SR-BI in mice results in the accumulation of large cholesterol ester-rich HDL particles. 17,18 The activity of the HDL-associated protein PAF-AH was 599±41 nmol/mL/min in wild-type mice (n=12), whereas in SR-BI knockout mice PAF-AH activity was 508±20 nmol/mL/min (n=12, P =0.11; Figure 1 A). Both in SR-BI knockout and in wild-type mice, the peak of the PAF-AH activity was mainly associated with large HDL, whereas there was no difference in HDL-associated PAF-AH activity (supplemental Figure I). PAF-AH protein levels in serum were reduced, probably as a result of reduced PAF-AH mRNA production as evidence by reduced PAF-AH mRNA expression in spleens of SR-BI knockout mice ( Figure 1 A). Interestingly, the activity of the HDL-associated protein PON1 was significantly reduced in absence of SR-BI ( Figure 1 B). The paraoxonase activity of PON1 was 1.4-fold ( P <0.001) lower in SR-BI knockout mice (71±3 nmol/mL/min; n=12) as compared with wild-type controls (100±6 nmol/mL/min; n=12), whereas the arylesterase activity was 1.6-fold ( P <0.01) lower (47±4 µmol/mL/min and 76±7 µmol/mL/min, respectively). In both groups of mice <1% of the PON1 activity was associated with apoB-containing lipoproteins (supplemental Figure I). In wild-type mice, 44% of the activity was associated to small HDL, as compared with only 26% in SR-BI knockouts. Large HDL contained 26% and 24% of the activity in wild-type and SR-BI knockout mice, respectively. Thus, the decrease in PON1 activity was primarily caused by a decrease in the activity of small HDL. Analysis of PON1 protein in serum showed that the reduced activity coincided with lower circulating protein levels ( Figure 1 B). Because liver is the primary site for the production of PON1, we determined the effect of SR-BI deficiency on the hepatic mRNA expression of PON1 ( Figure 1 B). A trend to slightly reduced PON1 mRNA expression was observed, but this difference failed to reach statistical significance (1.5±0.2 and 1.3±0.1 for wild-type and SR-BI knockout mice, respectively).

Figure 1. Effect of SR-BI deficiency on the HDL-associated enzymes PAF-AH and PON1. PAF-AH (A) and arylesterase/paraoxanase PON1 (B) activities were determined in serum of SR-BI +/+ (open bars) and SR-BI –/– (closed bars) mice (top). Values represent the mean±SEM of 12 mice. Statistically significant difference of ** P <0.01 and *** P <0.001 as compared with SR-BI +/+ mice. Protein levels of PAF-AH and PON1 were determined by Western blotting (middle), whereas the effect of SR-BI deficiency on mRNA expression of PAF-AH and PON1 was determined using real-time PCR with SYBR-green detection in spleen and liver, respectively (bottom). Values represent the mean±SEM of 6 mice. No statistically significant differences were observed.

Increased Lipid and Protein Oxidation in SR-BI Knockout Mice

Next, we investigated the effect of SR-BI deficiency on lipid and protein oxidation by measuring 2 distinct and specific markers, isoprostane F2 (iPF2 ) and protein carbonyls, respectively. 22,23

As shown in Figure 2 A, compared with wild-type mice, SR-BI knockout mice had 1.4-fold higher levels of circulating plasma iPF2 -VI (269±23 pg/mL versus 367±37 pg/mL, P <0.05). Urinary excretion of iPF2 -VI was increased 2-fold in absence of SR-BI (1.95±0.14 versus 0.98±0.08 ng/mg creatinine, P <0.0001; Figure 2 A). In addition to increased lipid oxidation, protein oxidation was also increased. Circulating levels of carbonyls in plasma were 0.247±0.023 nmol/mL ( P <0.05) in SR-BI knockouts and 0.167±0.019 nmol/mL in wild-type animals.

Figure 2. SR-BI deficiency induces oxidative stress in plasma, urine, and organs. As a measure of lipid oxidation total iPF2 -VI were determined in plasma and urine (A) as well as in homogenates of the indicated organs (B) of SR-BI +/+ (open bars) and SR-BI –/– (closed bars) mice. Carbonyls were determined in plasma and the indicated organs as a measure of protein oxidation. Values represent the mean±SEM of 10 mice. Statistically significant difference of * P <0.05, ** P <0.01, and *** P <0.001 as compared with SR-BI +/+ mice.

As shown in Figure 2 B, iPF2 -VI levels were 2.1-fold ( P <0.01) while carbonyls were 1.4-fold ( P <0.01) higher in livers of SR-BI knockout mice than wild-type animals. In spleen and kidney a 1.4-fold ( P <0.05) and 1.8-fold ( P <0.01) increase in iPF2 -VI levels was observed, whereas carbonyls were 1.3-fold ( P <0.01) and 1.5-fold ( P <0.05) higher, respectively. In brain and aorta a trend to increased iPF2 -VI levels ( P =0.059 and 0.051, respectively) was observed, whereas no effect was observed on carbonyl levels. In intestine, iPF2 -VI levels were unaltered, whereas carbonyls were 1.3-fold ( P <0.05) higher. No significant effect was observed on either iPF2 -VI levels or carbonyls in the ovaries ( Figure 2 B) or testes (data not shown) of SR-BI knockout mice.

Effect of SR-BI Deficiency on the Expression of Antioxidant Enzymes in Livers

Because liver of SR-BI knockout mice showed a significant increase in both lipid and protein oxidation, the antioxidant defense of this organ was evaluated by assessing different antioxidant systems. As shown in Figure 3, the mRNA expression of the glutathione peroxidases GPx1 and GPx4 were 1.7-fold ( P <0.001) and 1.2-fold ( P <0.05) higher in livers of SR-BI knockout than wild-type mice. Similarly, superoxide dismutase SOD1 and SOD2 levels were 1.2-fold ( P <0.05) and 1.3-fold ( P <0.05) higher in absence of SR-BI. Also the expression of the glutathione S-transferases GSTA2 and GSTA4, which reduce lipid peroxidation products, were 1.7-fold ( P =0.075) and 1.8-fold ( P <0.05) higher in mice lacking SR-BI, whereas no effect was observed on GSTA3. Heme oxygenase (HO), which is involved in the removal of free heme (a prooxidant) and the production of bilirubin (an antioxidant), showed a 3-fold increase in SR-BI knockout animals ( P =0.051). No effect was observed on the expression of catalase.

Figure 3. Effect of SR-BI deficiency on mRNA expression of antioxidant enzymes in liver. mRNA levels of the indicated genes in livers of SR-BI +/+ and SR-BI –/– mice were quantified using real-time PCR with SYBR-green detection. Values represent the mean±SEM of 6 mice. Statistically significant difference of * P <0.05, *** P <0.001 as compared with SR-BI +/+ mice.

Importance of SR-BI for the Removal of HDL-Associated Oxidized Cholesterol Esters by the Liver

Previous studies have suggested a role for SR-BI in the removal of oxidized cholesterol esters from HDL. 24,25 To study the direct role of SR-BI in the removal of oxidized cholesterol esters from HDL in vivo, the kinetics of the serum decay and liver uptake of [ 3 H-CEOH] labeled HDL were determined in SR-BI–deficient mice and wild-type littermates. At 45 minutes after injection 67±2% of the injected trace amount of [ 3 H-CEOH]-HDL was cleared from the circulation in SR-BI +/+ mice, as compared with 58±4% in SR-BI –/– mice ( Figure 4 ). In both wild-type and SR-BI–deficient mice the maximum association value for [ 3 H-CEOH]-HDL to the liver was reached at 20 minutes after injection. At this time point 27±3% of the injected dose of [ 3 H-CEOH]-HDL was taken up by the liver in wild-type mice, as compared with only 17±1% ( P <0.05) in SR-BI–deficient mice, indicating that SR-BI expression in the liver facilitated the removal of oxidized cholesterol esters from HDL.

Figure 4. SR-BI deficiency reduces serum decay and liver association of [ 3 H-CEOH]-HDL in mice. An amount of 200 µg (2.2*10 6 cpm) of [ 3 H-CEOH]-HDL was injected into SR-BI +/+ (; n=3) and SR-BI –/– (; n=3) mice, and the serum decay and liver uptake were followed in time. Values represent the mean±SEM of 3 mice. Statistically significant difference of * P <0.05 as compared with SR-BI +/+ mice.

Challenging SR-BI Knockout Mice With an Atherogenic Western-Type Diet, Containing 0.25% Cholesterol and 15% Total Fat, Further Increases Oxidative Stress

To analyze the possible relationship between the increased oxidation status in SR-BI knockout mice and their susceptibility to atherosclerosis, the effect of Western-type diet feeding on lipid and protein oxidation was determined after 4 weeks diet feeding. On chow diet monocytes chemoattractant protein 1 (MCP-1) levels, indicative of inflammation were 36±6 pg/mL and 37±5 pg/mL in SR-BI knockout and wild-type mice, respectively, indicating that the observed increased oxidative stress in the SR-BI knockout animals is independent from the presence of systemic inflammation. Furthermore, no increase in MCP-1 levels was observed in SR-BI knockout animals on Western-type diet (36±6 pg/mL).

Isoprostanes are excreted in the urine, and urine thus provides a global measure of oxidative stress. Western-type diet feeding induced a 1.5-fold ( P <0.05) increase in urinary iPF2 -VI in wild-type mice ( Figure 5 A). Although basal urinary iPF2 -VI were already higher in SR-BI knockout mice under chow conditions, Western-type diet feeding induced a 2-fold ( P <0.0001) increase in urinary iPF2 -VI levels. Under these conditions urinary excretion of iPF2 -VI was thus 2.6-fold ( P <0.001) higher in SR-BI knockout mice as compared with wild-types (4.00±0.15 versus 1.53±0.20 ng/mg creatinine, respectively; Figure 5 A). Western-type diet feeding did not further increase iPF2 -VI and carbonyls in plasma. Of the analyzed organs, especially brain, spleen, kidney, intestine, aorta, and ovary displayed enhanced accumulation of iPF2 -VI on Western-type diet feeding ( Figure 5 B). In addition, carbonyls were further increased in liver, kidney, intestine, and aorta.

Figure 5. Increased oxidative stress in SR-BI knockout mice challenged with an atherogenic Western-type diet. As a measure of lipid oxidation total iPF2 -VI were determined in plasma and urine (A) as well as in homogenates of the indicated organs (B) of SR-BI +/+ (open bars) and SR-BI –/– (closed bars) mice after 4 weeks feeding an atherogenic Western-type diet, containing 0.25% cholesterol and 15% total fat. Carbonyls were determined in plasma and the indicated organs as a measure of protein oxidation. Values represent the mean±SEM of 4 mice. Statistically significant difference of * P <0.05, ** P <0.01, and *** P <0.001 as compared with SR-BI +/+ mice.

Western-type diet feeding induced a 2.4-fold increase in the paraoxonase activity of PON1 in both SR-BI knockout and wild-type mice. As a result, also under these conditions the paraoxonase activity of PON1 was 1.3-fold ( P <0.01) lower in SR-BI knockout mice (175±4 nmol/mL/min; n=6) as compared with wild-type controls (234±10 nmol/mL/min; n=6). In addition, the arylesterase activity of PON1 was 1.3-fold ( P <0.01) lower in SR-BI knockout mice compared with wild-type animals. No significant difference was observed in PAF-AH activity under these conditions.

Discussion

Oxidative stress is the result of an imbalance between increased generation of reactive oxygen species (ROS) and the decreased ability of endogenous antioxidant systems to scavenge them. ROS induce cell, tissue, or organ damage and are involved in the pathogenesis of several diseases, including atherosclerosis and diabetes. In the current study we show for the first time a marked enhancement of oxidative stress in SR-BI knockout mice. SR-BI is a multifunctional receptor capable of binding a wide array of native and oxidatively modified lipoproteins. It is a prominent regulator of HDL metabolism, and selective disruption of SR-BI in mice results in increased plasma cholesterol levels attributable to the accumulation of large cholesteryl ester-rich HDL particles and an increased susceptibility to atherosclerosis. 19–21 In addition, SR-BI knockouts develop reticulocytosis, 27,28 and female mice lacking SR-BI are infertile because of a defective maturation of oocytes. 26 Interestingly, the pathologies observed in SR-BI knockout mice can all be reversed by treatment with the HDL-lowering antioxidant probucol. 29,30

To protect the cells and organ systems of the body against ROS, a highly complex antioxidant protection system has evolved, including enzymes such as SOD and catalase, and dietary antioxidants, like -tocopherol and beta carotene. In the current study, we show that as a reaction to the increased oxidative stress several endogenous antioxidant systems in the SR-BI knockout mice are upregulated, including glutathione peroxidases, superoxide dismutases, and glutathione S-transferases. Interestingly, by microarray expression profiling on livers of SR-BI transgenic animals, lacking HDL, Callow et al also observed an increased expression of gluthatione S-transferase. 31 This finding supports the hypothesis that in SR-BI knockout mice with dysfunctional HDL and possibly also in SR-BI transgenic mice without any HDL the amount of ROS produced exceeds the capacity of those enzyme systems to counteract them or that the removal system for oxidants from the circulation is hampered. This concept is corroborated by the observation that the activity of the important HDL-associated antioxidant enzyme PON1 was markedly reduced in SR-BI knockout mice. The reduced PON1 activity in SR-BI knockout mice coincided with lower circulating protein levels and a trend to slightly reduced mRNA levels of PON1 in livers of SR-BI knockout mice, indicating that the reduced activity might be partly explained by lower production of PON1. In an elegant report by Deakin et al it was recently shown that oxidation of HDL decreases its ability to remove PON1 from cells and compromises its ability to stabilize the enzyme activity. 32 Thus, the increased oxidation status of circulating HDL in SR-BI knockout mice, as evidenced by increased plasma iPF2 -VI levels, will most likely have attributed to the decreased PON1 protein and activity in the circulation.

Previously, we have shown that feeding SR-BI knockout mice an atherogenic Western-type diet for 20 weeks induces atherosclerotic lesion development, whereas no lesion development was observed in wild-type animals. 20 On challenging the SR-BI knockout mice with this Western-type diet, a further increase in oxidative stress in these animals was observed, particularly evidenced by an increased urinary iPF2 -VI secretion, a global measure of oxidative stress. Interestingly, under these conditions also a dramatic increase in oxidative stress in the aorta was observed. This increased oxidative stress was observed after only 4 weeks Western-type diet feeding. MCP-1 levels were not induced in the SR-BI knockout animals, suggesting that oxidative stress precedes this inflammatory marker in the pathogenesis of atherosclerosis. A significant negative correlation has been demonstrated between HDL-PON1 activity and the levels of lipid hydroperoxides associated with HDL from healthy subjects. 33 The physiological significance of this HDL-associated enzyme with antioxidative activity is further emphasized by the association between low plasma PON1 activity and the risk for cardiovascular disease 34 and by the fact that overexpression of PON1 35 decreases oxidative stress and reduces atherosclerosis in mice.

In addition to the observed reduced PON1 activity also other factors might contribute to the increased oxidative stress in SR-BI knockout mice. Oxidized lipoproteins are known ligands for SR-BI. 36 Previously we have shown that the selective uptake of HDL-associated oxidized cholesterol esters could be efficiently blocked by oxidized LDL and phosphatidylserine liposomes to a similar extent as native cholesterol ester uptake, suggesting an important role for SR-BI in the removal of oxidized cholesterol from HDL. 24,25 Furthermore, oxidized cholesterol esters were selectively taken up by Chinese hamster ovary cells transfected with SR-BI. 25 Absence of SR-BI might thus also directly have impaired the removal of oxidized lipids from the circulation. In agreement, we now provide definite proof that SR-BI deficiency results in an impaired uptake of oxidized cholesterol esters from HDL. The effect of SR-BI deficiency on oxidized cholesterol ester clearance from HDL, however, was less dramatic as previously shown for native cholesterol esters, 18 suggesting that in addition to SR-BI additional HDL binding sites on the liver are involved in the removal of oxidized cholesterol esters from HDL. Furthermore, although the impaired clearance of oxidized cholesterol esters from HDL in absence of SR-BI could provide an alternate explanation for the accumulation of oxidized lipids in plasma of these animals, it does not explain the enhanced oxidation status of the liver.

Finally, SR-BI has been implicated in the intestinal absorption of the lipophylic dietary antioxidants beta carotene 37 and -tocopherol. 38 Furthermore, SR-BI mediates the uptake of -tocopherol by tissues. 39 As a result, biliary secretion and the levels of -tocopherol in selected tissues such as brain, lung, and gonads were decreased, whereas circulating -tocopherol levels were increased in SR-BI knockout mice. 40 Thus, reduced bioavailability of these dietary antioxidants might also have contributed to the increased oxidative stress observed in SR-BI knockout mice.

In conclusion, SR-BI deficiency results in a pronounced oxidative imbalance in vivo, which culminates in increased oxidative stress. This increase is, at least in part, the result of a reduced activity of its HDL-associated antioxidant enzyme PON1 and may ultimately contribute to the proatherogenic effect of SR-BI deficiency. Interestingly, in humans coronary heart disease risk associated with a C1050T polymorphism in exon 8 of CLA-1 (C8C8), the human homologue of SR-BI, was confined to a subset of individuals with Gln192Arg and Met55Leu polymorphisms in PON1, indicating that also in humans a clear association exists between SR-BI, PON1, and coronary heart disease. 41

Acknowledgments

Sources of Funding

This work was supported by The Netherlands Heart Foundation (grant 2001T041) and the National Institute of Health (grant AG-11542).

Disclosures

None.

【参考文献】
  Von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001; 21: 13–27.

Mineo C, Deguchi H, Griffin JH, Shaul P. Endothelial and antithrombotic actions of HDL. Circ Res. 2006; 98: 1352–1364.

Seetharam D, Mineo C, Gormley AK, Gibson LL, Vongpatanasin W, Chambliss KL, Hahner LD, Cummings ML, Kitchens RL, Marcel YL, Rader DJ, Shaul PW. High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I. Circ Res. 2006; 98: 63–72.

Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM. Antiinflammatory properties of HDL. Circ Res. 2004; 95: 764–772.

Assmann G, Gotto AM Jr. HDL cholesterol and protective factors in atherosclerosis. Circulation. 2004; 109: III8–III14.

Ng CJ, Shih DM, Hama SY, Villa N, Navab M, Reddy ST. The paraoxonase gene family and atherosclerosis. Free Rad Biol Med. 2005; 38: 153–163.

Ninio E. Phospholipid mediators in the vessel wall: involvement in atherosclerosis. Curr Opin Clin Nutr Metab Care. 2005; 8: 123–131.

Mackness MI, Arrol S, Abbott C, Durrington PN. Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase. Atherosclerosis. 1993; 104: 129–135.

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.

Aviram M, Rosenblat M, Bisgaier CL, Newton RS, Primo-Parmo SL, La Du BN. Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. J Clin Invest. 1998; 101: 1581–1590.

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.

Wardlow ML, Cox CP, Meng KE, Greene DE, Farr RS. Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor. J Immunol. 1986; 136: 3441–3446.

Stremler KE, Stafforini DM, Prescott SM, McIntyre TM. Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates. J Biol Chem. 1991; 266: 11095–11103.

Hase M, Tanaka M, Yokota M, Yamada Y. Reduction in the extent of atherosclerosis in apolipoprotein E-deficient mice induced by electroporation-mediated transfer of the human plasma platelet-activating factor acetylhydrolase gene into skeletal muscle. Prostaglandins Other Lipid Mediat. 2002; 70: 107–118.

Quarck R, De Geest B, Stengel D, Mertens A, Lox M, Theilmeier G, Michiels C, Raes M, Bult H, Collen D, Van Veldhoven P, Ninio E, Holvoet P. Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2001; 103: 2495–2500.

Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study. JAMA. 1986; 256: 2835–2838.

Acton S, Rigotti A, Landschulz KT, et al. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996; 271: 518–520.

Out R, Hoekstra M, Spijkers JA, Kruijt JK, Van Eck M, Bos IS, Twisk J, Van Berkel Th JC. Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice. J Lipid Res. 2004; 45: 2088–2095.

Rigotti A, Trigatti BL, Penman M, et al. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc Natl Acad Sci U S A. 1997; 94: 12610–12615.

Van Eck M, Twisk J, Hoekstra M, et al. Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver. J Biol Chem. 2003; 278: 23699–23705.

Braun A, Trigatti BL, Post MJ, et al. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. Circ Res. 2002; 90: 270–276.

Pratico D, Rokach J, Lawson J, FitzGerald GA. F2-isoprostanes as indices of lipid peroxidation in inflammatory diseases. Chem Phys Lipids. 2004; 128: 165–171.

Nystrom T. Role of oxidative carbonylation in protein quality control and senescence. EMBO J. 2005; 24: 1311–1317.

Fluiter K, Vietsch H, Biessen EA, Kostner GM, van Berkel TJ, Sattler W. Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells. Biochem J. 1996; 319: 471–476.

Fluiter K, Sattler W, De Beer MC, Connell PM, van der Westhuyzen DR, van Berkel TJ. Scavenger receptor BI mediates the selective uptake of oxidized cholesterol esters by rat liver. J Biol Chem. 1999; 274: 8893–8899.

Trigatti B, Rayburn H, Vinals M, Braun A, Miettinen H, Penman M, Hertz M, Schrenzel M, Amigo L, Rigotti A, Krieger M. Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci U S A. 1999; 96: 9322–9327.

Holm TM, Braun A, Trigatti BL, Brugnara C, Sakamoto M, Krieger M, Andrews NC. Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI. Blood. 2002; 99: 1817–1824.

Meurs I, Hoekstra M, van Wanrooij EJ, Hildebrand RB, Kuiper J, Kuipers F, Hardeman MR, Van Berkel TJ, Van Eck M. HDL cholesterol levels are an important factor for determining the lifespan of erythrocytes. Exp Hematol. 2005; 33: 1309–13019.

Miettinen HE, Rayburn H, Krieger M. Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)-deficient mice. J Clin Invest. 2001; 108: 1717–1722.

Braun A, Zhang S, Miettinen HE, Ebrahim S, Holm TM, Vasile E, Post MJ, Yoerger DM, Picard MH, Krieger JL, Andrews NC, Simons M, Krieger M. Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse. Proc Natl Acad Sci U S A. 2003; 100: 7283–7288.

Callow MJ, Dudoit S, Gong EL, Speed TP, Rubin EM. Microarray expression profiling identifies genes with altered expression in HDL-deficient mice. Genome Res. 2000; 10: 2022–2029.

Deakin s, Moren X, James RW. HDL oxidation compromises its influence on paraoxonase-1 secretion and its capacity to modulate enzyme activity. Arterioscler Thromb Vasc Biol. In press.

Ferretti G, Bacchetti T, Busni D, Rabini RA, Curatola G. Protective effect of paraoxonase activity in high-density lipoproteins against erythrocyte membranes peroxidation: a comparison between healthy subjects and type 1 diabetic patients. J Clin Endocrinol Metab. 2004; 89: 2957–2962.

Jarvik GP, Hatsukami TS, Carlson C, Richter RJ, Jampsa R, Brophy VH, Margolin S, Rieder M, Nickerson D, Schellenberg GD, Heagerty PJ, Furlong CE. Paraoxonase activity, but not haplotype utilizing the linkage disequilibrium structure, predicts vascular disease. Arterioscler Thromb Vasc Biol. 2003; 23: 1465–1471.

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.

Calvo D, Gomez-Coronado D, Lasuncion MA, Vega MA. CLA-1 is an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins. Arterioscler Thromb Vasc Biol. 1997; 17: 2341–2349.

van Bennekum A, Werder M, Thuahnai ST, Han CH, Duong P, Williams DL, Wettstein P, Schulthess G, Phillips MC, Hauser H. Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol. Biochemistry. 2005; 44: 4517–4525.

Reboul E, Klein A, Bietrix F, Gleize B, Malezet-Desmoulins C, Schneider M, Margotat A, Lagrost L, Collet X, Borel P. Scavenger receptor class B type I (SR-BI) is involved in vitamin E transport across the enterocyte. J Biol Chem. 2006; 281: 4739–4745.

Mardones P, Rigotti A. Cellular mechanisms of vitamin E uptake: relevance in alpha-tocopherol metabolism and potential implications for disease. J Nutr Biochem. 2004; 15: 252–260.

Mardones P, Strobel P, Miranda S, Leighton F, Quinones V, Amigo L, Rozowski J, Krieger M, Rigotti A. Alpha-tocopherol metabolism is abnormal in scavenger receptor class B type I (SR-BI)-deficient mice. J Nutr. 2002; 132: 443–449.

Rodriguez-Esparragon F, Rodriguez-Perez JC, Hernandez-Trujillo Y, Macias-Reyes A, Medina A, Caballero A, Ferrario CM. Allelic variants of the human scavenger receptor class B type 1 and paraoxonase 1 on coronary heart disease: genotype-phenotype correlations. Arterioscler Thromb Vasc Biol. 2005; 25: 854–860.

作者单位：Division of Biopharmaceutics (M.V.E., M.H., R.B.H., J.K.K., Th.J.C.V.B.), Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands; INSERM UMRS525 (D.S., E.N.), Faculté de Médecine, Université Pierre et Marie Curie-Paris 6 and Faculté de Médeci