Studies have demonstrated that atherosclerosis (AS) is a chronic inflammatory disease, which contributes significantly to the initiation, progression and rupture of atherosclerotic lipid-rich plaques.1,2 Agents involved in the inflammatory pathways have received considerable attention as potential predictors of cardiovascular disease (CVD) risk and as targets whose modulation might prove beneficial in reducing residual cardiovascular risk. Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme produced mainly by macrophages involved in the development of atherosclerosis, is an example of such a marker.3 In humans, most circulating Lp-PLA2 is bound to low-density lipoprotein (LDL) particles in plasma and the rest to high-density lipoprotein (HDL).4 Lp-PLA2 hydrolyzes sn2-ester bonds of fragmented or oxidized phospholipids at sites where atherosclerotic plaques are forming to produce lysophosphatidylcholine (LysoPC) and oxidized non-esterified fatty acids (oxNEFA), two key pro-inflammatory mediators implicated in AS.4 Emerging evidence suggests that Lp-PLA2 acts as a novel risk marker for coronary heart disease (CHD),5,6 exerting pro-atherogenic effects and playing an important role in plaque progression and instability.7,8
Population studies have shown that plasma HDL levels correlate inversely with cardiovascular disease risk.9 In recent years, there has been intense interest in exploiting HDL cardioprotective properties. It has been shown that HDL exerts its cardiovascular protection by promoting the reverse cholesterol transport (RCT) and other pleiotropic beneficial effects.10 Anti-inflammatory activity represents one of the important anti-atherogenic properties of HDL. Previous studies have reported that HDL not only inhibits the chemotaxis of monocytes, the adhesion of leukocytes to the endothelium, endothelial dysfunction and apoptosis, LDL oxidation, complement activation, platelet activation and factor X activation, but also stimulates the proliferation of endothelial cells and smooth muscle cells.11 In macrophages, with functional HDL, fats and cholesterol can be adequately removed to antagonize AS progress, implying its synergistic actions with Lp-PLA2 in cardiovascular disease control. However, little is known regarding how HDL impacts Lp-PLA2 in macrophages, hindering the deep understanding to the underlying mechanism of AS.
To offer further insight into the influence of HDL on Lp-PLA2 in human macrophages, this study was performed to investigate the effects of HDL on Lp-PLA2 expression and activity in human monocyte-derived macrophages, and then explore the underlying mechanisms of these effects.
Cell isolation and culture
Human monocyte-derived macrophages were isolated and cultured as described in our previous study.12 In brief, mononuclear cells were isolated from the peripheral blood of healthy volunteers using Ficoll density gradient centrifugation. Cells were suspended and cultured in 6-well culture dishes ((1.0–1.5)×107/well) with RPMI-1640 medium (GIBICO, Grand Island, NY, USA) containing 10% fetal bovine serum (GIBICO) at 37°C in a humidified atmosphere with 5% CO2.. Twenty-four hours after incubation, non-adherent cells were removed by washing the dishes twice with fresh medium, and the remaining adherent monocytes were further cultured to induce differentiation into macrophage with medium replacement every three days.
HDL (density range (1.063–1.210) g/ml) was isolated from the plasma of healthy volunteers by discontinuous density gradient centrifugation.13 The purity of isolated HDL was analyzed by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining,14 and the protein content of the HDL was determined with the method described by Lowry et al.14
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from cultured cells with Trizol reagent (Invitrogen Corp., CarIsbad, CA, USA) according to the manufacturer's instructions. Two micrograms of total RNA was subjected to reverse transcription using a commercially available cDNA synthesis kit (MBI Fermentas, St. Leonrod, Germany). Human Lp-PLA2 and GAPDH cDNAs were amplified using the following primer pairs and parameters: Lp-PLA2, 5′-GAACAC- ACTGGCTTATGGGCAAC-3' (sense) and 5'-GAGTC-TGAATAACCGTTGCTCCACC-3' (antisense) at 94°C for 30 seconds, 57°C for 60 seconds, 72°C for 120 seconds and 30 cycles; GAPDH, 5'-ACGCATTTGGT-CGTATTGGG-3' (sense) and 5'-TGATTTTGGAGG- GATCTCGC-3' (antisense) at 94°C for 30 seconds, 57°C for 60 seconds, 72°C for 60 seconds and 25 cycles. The PCR products were subjected to electrophoresis on 2% agarose gels and visualized with ethidium bromide. The signal intensity of the Lp-PLA2 band was semiquantified by Quantity One imaging software (Bio-Rad, Hercules, CA, USA), and normalized by comparison with that of GAPDH band.
After twice washing with ice-cold phosphate-buffered saline (PBS), cells were harvested and lysed with ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 μg/ml leupeptin). The protein concentration was determined by the Bradford method. Eighty microgram of crude proteins was fractionized by 10% SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (Pall Corp., Glen Cove, NY, USA). The membrane was then blocked in 1×TBST buffer (50 mm Tris-HCl, pH 7.5, 250 mm NaCl, 0.1% Tween 20) containing 5% non-fat dry milk, and then probed with goat anti-human Lp-PLA2 polyclonal antibody (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-human β-actin polyclonal antibody (1:400 dilution, Santa Cruz Biotechnology), rabbit anti-human peroxisome proliferator-activated receptor γ (PPARγ) polyclonal antibody (1:200, Abcam, Cambridge, United Kingdom), or rabbit anti-human phospho PPARγ polyclonal antibody (1:200, Abcam) at 4°C overnight. After three washes with 1×TBST, the membrane was probed with secondary antibody at room temperature for 1 hour. After extensive washing, the antigen-antibody signals were visualized using ECL substrate (Pierce Biotechnology, Rockford, IL, USA). The signal intensity of the Lp-PLA2 blot was semiquantified using Quantity One imaging software (Bio-Rad) and corrected by normalizing with that of the β-actin blot.
Determination of Lp-PLA2 activity
Lp-PLA2 activity was measured in the culture supernatant of human monocyte-derived macrophages using the colorimetric method with a platelet-activating factor (PAF) acetylhydrolase assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocol. Results were read on a Bio-Rad 550 Microplat Reader.
First, human monocyte-derived macrophages were incubated with 0, 12.5, 25, 50, and 100 μg/ml HDL for 24 hours, or with 25 μg/ml HDL for 0, 6, 12, 24, and 48 hours to observe the influence of HDL on the expression of Lp-PLA2 mRNA and protein, and the enzymatic activity in monocytes/macrophages. Second, pioglitazone (kindly provided by Beijing TaiYang Pharmaceutical Industry Co. Ltd, China) alone or in combination with HDL was added to the culture media to assess the role of PPARγ in the HDL mediated effects. Third, the phosphorylation of PPARγ was measured in the presence of pioglitazone alone or pioglitazone in combination with HDL.
All experiments were carried out in triplicate and results are presented as mean±standard deviation (SD). Statistical analyses of the data were performed by SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Comparisons of continuous variables among multiple groups were conducted by one-way analysis of variance (ANOVA) followed by S-N-K post-hoc analyses. A P value less than 0.05 was considered statistically significant.
HDL inhibits Lp-PLA2 in human monocyte-derived macrophages
To investigate how HDL impacts the action of Lp-PLA2, we initially introduced HDL to human monocyte-derived macrophages and then examined the different profiles of Lp-PLA2 in response to HDL stimulation. With HDL treatment for 24 hours, Lp-PLA2 mRNA levels (Figure 1A) and protein expression (Figure 1B) were significantly suppressed (P <0.05) in a dose-dependent manner compared with the control group without HDL. Furthermore, the PAF acetylhydrolase assay showed that Lp-PLA2 activity was also markedly suppressed in response to different final concentrations of HDL (Figure 1C).
To evaluate whether the Lp-PLA2 suppression by HDL stimulation is also time dependent, we next examined Lp-PLA2 mRNA and protein levels as well as secreted activity at a final concentration of 25 μg/ml of HDL for different durations. Similar to the profiles observed in Figure 1, Lp-PLA2 mRNA level, protein expression and secreted enzyme activity were substantially inhibited (P <0.05) in a time dependent manner with HDL treatment. All together, these results clearly demonstrated that HDL regulates Lp-PLA2 in macrophages.
PPARγ ligand pioglitazone upregulates Lp-PLA2 in human monocyte-derived macrophages
In cardiovascular cells, PPARγ decreases the inflammatory response by activating the PON1 gene, which decreases the risk of AS.15,16 It has been shown that activation of PPARγ is implicated in the activation of macrophages and suppresses production of various inflammatory cytokines.17 Pioglitazone is a well-known ligand for PPARγ, which selectively stimulates PPARγ. To examine the involvement of PPARγ in regulation of Lp-PLA2 expression and activity, pioglitazone was introduced to culture medium and then the Lp-PLA2 mRNA level, protein expression and secreted enzyme activity were determined. The results showed that low concentrations of pioglitazone (1–5 ng/ml) upregulated the Lp-PLA2 mRNA level (Figure 3A), protein expression (Figure 3B), and activity of secreted enzyme (Figure 3C) in human monocyte-derived macrophages (P <0.05). These results indicated that the PPARγ pathway may regulate Lp-PLA2-mediated AS protection.
HDL mediates pioglitazone-induced upregulation of Lp-PLA2 in human monocyte-derived macrophages
To further explore the link between HDL, PPARγ and Lp-PLA2, we examined the roles of HDL in pioglitazone-induced Lp-PLA2 activation. To this end, human monocyte-derived macrophages were simultaneously treated with 5 ng/ml pioglitazone and 25 μg/ml HDL for 24 hours and then Lp-PLA2 expression and activity were determined. As shown in Figure 4, solo pioglitazone treatment increased the Lp-PLA2 mRNA level (Figure 4A), protein expression (Figure 4B) and the activity of secreted enzyme (Figure 4C) (P <0.05). With the addition of HDL, the increased mRNA and protein level as well as enzyme activity were significantly reduced to the basal level. Therefore, we conclude that HDL regulates PPARγ-mediated Lp-PLA2 activation in macrophages.
HDL inhibits pioglitazone-induced phosphorylation of PPARγ in human monocyte-derived macrophages
To assess the effects of HDL on pioglitazone-induced phosphorylation of PPARγ, human monocyte-derived macrophages were treated with pioglitazone (5 ng/ml) in the presence or absence of HDL (25 μg/ml) for 24 hours. As shown in Figure 5, pioglitazone resulted in a significant increase in PPARγ phosphorylation in human monocyte-derived macrophages, which could be inhibited by HDL.
In this study, we found that HDL reduced the expression and activity of Lp-PLA2 in human monocyte-derived macrophages in both a dose-and time-dependent manner. The mechanism underlying these effects may be related to the PPARγ pathway.
Lp-PLA2 is a specific marker of vascular inflammation, and HDL plays an important role in cardiovascular protection by anti-inflammatory activity. However, in the process of atherosclerosis, the biological role of Lp-PLA2 has been controversial; with initial reports supporting its atheroprotective effects as a consequence of degrading platelet-activating factor and the removal of polar phospholipids in modified LDL. Recent studies, however, focused on the pro-inflammatory role of Lp-PLA2 mediated by products of the Lp-PLA2 reaction (LysoPC and oxNEFA). LysoPC and oxNEFA are proposed to play an important role in homing of inflammatory cells into lesion-prone areas and local increases in inflammatory mediators.18 Furthermore, the use of an inhibitor of Lp-PLA2 in cell culture systems has also been shown to significantly decrease the concentration of both LysoPC and oxNEFA oxLDL-mediated monocyte chemoattraction and to attenuate monocyte-macrophage apoptosis.19,20 In addition, Lp-PLA2 has also been suggested to have a role in promoting plaque instability. Multiple studies have shown that Lp-PLA2 is strongly expressed within the necrotic core and surrounding macrophages of vulnerable and ruptured plaques in coronary arteries, with relatively weaker expression in less advanced lesions.21,22 And Lp-PLA2 activity has also been shown to be upregulated in atherosclerotic lesions and in rupture-prone fibrous caps.23 The proinflammatory action of Lp-PLA2 is also supported by a number of epidemiology studies showing that Lp-PLA2 is an independent predictor of various cardiovascular events, over and above the traditional risk factors and inflammatory biomarkers, including high-sensitivity CRP.24–26 Thus, Lp-PLA2 acts as a specific marker of vascular and plaque inflammation, and plays an important role in the development of AS, and represents a potentially promising target for fighting atherosclerosis.
HDL particles possess multiple antiatherogenic activities. The central role of HDL in cellular cholesterol efflux and RCT is considered to form a basis for the capacity of HDL to attenuate atherogenesis. However, compelling evidence has emerged that additional dimensions of the antiatherogenic action of HDL, such as anti- inflammatory, antioxidant, endothelial-protective effects, and antithrombotic properties, may be of major physiological and pathological relevance.27 Satoh et al28 observed that HDL inhibited Lp-PLA2 protein expression and its activity in the human hepatic line HepG2 in a concentration-dependent manner, and 100 μg/ml of HDL inhibited the production by 40%. In addition, 50, 100, and 200 μg/ml of HDL inhibited Lp-PLA2 activity by 61%, 82%, and 88%, respectively. Consistent with these studies, our study also showed that Lp-PLA2 mRNA expression, protein expression and secreted enzyme activity were significantly inhibited in a dose-dependent manner by HDL treatment. The consistent effect of HDL on Lp-PLA2 in different cells may mean that there is a common mechanism by which Lp-PLA2 is regulated by HDL. Previous studies indicated that the major proteins of HDL, apoA-I and apoA-II, as well as other proteins such as paraoxonase that cotransport with HDL in plasma, are well-known to have anti-inflammatory properties.29 Thus we speculated that the mechanisms which can account for the inhibition of Lp-PLA2 by HDL may be associated with these HDL associated proteins as well.
PPARγ is a ligand-activated transcription factor that is trans-activated by binding its ligands. PPARγ is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation.30 In this study, we showed that the PPARγ ligand pioglitazone upregulated Lp-PLA2 mRNA and protein levels and enzymatic activity in human peripheral blood macrophages, however, the effects of pioglitazone can be reversed by HDL. In addition, the increase in phosphorylation of PPARγ induced by pioglitazone could be markedly inhibited by HDL in human monocyte-derived macrophages. These findings suggest that the PPARγ pathway may mediate the inhibition of Lp-PLA2 expression and activity through HDL. Consistent with our findings, Sumita et al31 reported that pioglitazone increased plasma platelet activating factor-acetylhydrolase, also referred as Lp-PLA2, in THP-1 macrophages. These data indicate that the expression of Lp-PLA2 is partly regulated by PPARγ. In addition, a previous study32 also showed that HDL inhibited CD36, a receptor for oxidized low density lipoprotein, via the PPARγ pathway.
In this study, we found that pioglitazone significantly upregulated Lp-PLA2 in human monocyte-derived macrophages. However, previous studies found that activation of PPARγ played an important role in combating inflammation.31,33–34 The explanation for these seeming conflicting results is as follows. In vivo studies showed that the anti-inflammatory or pro-inflammatory roles of Lp-PLA2 were closely related to the lipoprotein which is binding to Lp-PLA2. The present study only observed the increase in Lp-PLA2 expression and activity in vitro, and its function needs to be investigated by further studies.
In summary, this study observed that HDL inhibited the expression and activity of Lp-PLA2 in human monocyte-derived macrophages, and the underlying mechanisms are probably mediated through the PPARγ pathway. These results provide novel evidence for the anti-inflammatory effects of HDL and may contribute, at least in part, to explain the beneficial role of HDL in cardiovascular protection.
1. Hansson GK, Hermansson A. The immune system in atherosclerosis. Nat Immunol 2011; 12: 204-212.
2. Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med 1999; 340: 115-126.
3. Packard CJ. Lipoprotein-associated phospholipase A2 as a biomarker of coronary heart disease and a therapeutic target. Curr Opin Cardiol 2009; 24: 358-363.
4. Silva IT, Mello AP, Damasceno NR. Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A2 (Lp-PLA2): a review. Lipids Health Dis 2011; 10: 170.
5. Liu CF, Qin L, Ren JY, Chen H, Wang WM, Liu J, et al. Elevated plasma lipoprotein-associated phospholipase A2 activity is associated with plaque rupture in patients with coronary artery disease. Chin Med J 2011; 124: 2469-2473.
6. Ikonomidis I, Michalakeas CA, Lekakis J, Parissis J, Anastasiou-Nana M. The role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in cardiovascular disease. Rev Recent Clin Trials 2011; 6: 108-113.
7. Herrmann J, Mannheim D, Wohlert C, Versari D, Meyer FB, McConnell JP, et al. Expression of lipoprotein-associated phospholipase A(2) in carotid artery plaques predicts long-term cardiac outcome. Eur Heart J 2009; 30: 2930-2938.
8. Munzel T, Gori T. Lipoprotein-associated phospholipase A(2), a marker of vascular inflammation and systemic vulnerability. Eur Heart J 2009; 30: 2829-2931.
9. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am J Med 1977; 62: 707-714.
10. Rye KA, Bursill CA, Lambert G, Tabet F, Barter PJ. The metabolism and anti-atherogenic properties of HDL. J Lipid Res 2009; 50 Suppl: S195-S200.
11. Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis 2002; 161: 1-16.
12. Ren J, Jin W, Chen H. oxHDL decreases the expression of CD36 on human macrophages through PPARgamma and p38 MAP kinase dependent mechanisms. Mol Cell Biochem 2010; 342: 171-181.
13. Chung BH, Wilkinson T, Geer JC, Segrest JP. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 1980; 21: 284-291.
14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
15. Hamblin M, Chang L, Fan Y, Zhang J, Chen YE. PPARs and the cardiovascular system. Antioxid Redox Signal 2009; 11: 1415-1452.
16. Khateeb J, Gantman A, Kreitenberg AJ, Aviram M, Fuhrman B. Paraoxonase 1 (PON1) expression in hepatocytes is upregulated by pomegranate polyphenols: a role for PPAR-gamma pathway. Atherosclerosis 2010; 208: 119-125.
17. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391: 82-86.
18. Khakpour H, Frishman WH. Lipoprotein-associated phospholipase A2: an independent predictor of cardiovascular risk and a novel target for immunomodulation therapy. Cardiol Rev 2009; 17: 222-229.
19. MacPhee CH, Moores KE, Boyd HF, Dhanak D, Ife RJ, Leach CA, et al. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor. Biochem J 1999; 338: 479-487.
20. Carpenter KL, Dennis IF, Challis IR, Osborn DP, Macphee CH, Leake DS, et al. Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages. FEBS Lett 2001; 505: 357-363.
21. Kolodgie FD, Burke AP, Skorija KS, Ladich E, Kutys R, Makuria AT, et al. Lipoprotein-associated phospholipase A2 protein expression in the natural progression of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 2006; 26: 2523-2529.
22. Häkkinen T, Luoma JS, Hiltunen MO, Macphee CH, Milliner KJ, Patel L, et al. Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1999; 19: 2909-2917.
23. Koenig W, Twardella D, Brenner H, Rothenbacher D. Lipoprotein-associated phospholipase A2 predicts future cardiovascular events in patients with coronary heart disease independently of traditional risk factors, markers of inflammation, renal function, and hemodynamic stress. Arterioscler Thromb Vasc Biol 2006; 26: 1586-1593.
24. Ballantyne CM, Hoogeveen RC, Bang H, Coresh J, Folsom AR, Heiss G, et al. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident coronary heart disease in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Circulation 2004; 109: 837-842.
25. Koenig W, Khuseyinova N, Lowel H, Trischler G, Meisinger C. Lipoprotein-associated phospholipase A2 adds to risk prediction of incident coronary events by C-reactive protein in apparently healthy middle-aged men from the general population: results from the 14-year follow-up of a large cohort from southern Germany. Circulation 2004; 110: 1903-1908.
26. Gerber Y, McConnell JP, Jaffe AS, Weston SA, Killian JM, Roger VL. Lipoprotein-associated phospholipase A2 and prognosis after myocardial infarction in the community. Arterioscler Thromb Vasc Biol 2006; 26: 2517-2522.
27. Cutri BA, Hime NJ, Nicholls SJ. High-density lipoproteins: an emerging target in the prevention of cardiovascular disease. Cell Res 2006; 16: 799-808.
28. Satoh K, Imaizumi T, Yoshida H, Takamatsu S. High-density lipoprotein inhibits the production of platelet activating factor acetylhydrolase by HepG2 cells. J Lab Clin Med 1994; 123: 225-231.
29. Heinecke JW. The protein cargo of HDL: implications for vascular wall biology and therapeutics. J Clin Lipidol 2010; 4: 371-375.
30. Tontonoz P, Hu E, Spiegelman BM. Regulation of adipocyte gene expression and differentiation by peroxisome proliferators activated receptor gamma. Curr Opin Genet Dev 1995; 5: 571-576.
31. Sumita C, Maeda M, Fujio Y, Kim J, Fujitsu J, Kasayama S, et al. Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage. Biochim Biophys Acta 2004; 1673: 115-121.
32. Han J, Hajjar DP, Zhou X, Gotto AM Jr, Nicholson AC. Regulation of peroxisome proliferator-activated receptorgamma-mediated gene expression. A new mechanism of action for high density lipoprotein. J Biol Chem 2002; 277: 23582-23586.
33. Kapadia R, Yi JH, Vemuganti R. Mechanisms of anti-inflammatory and neuroprotective actions of PPAR-gamma agonists. Front Biosci 2008; 13: 1813-1826.
34. Orasanu G, Ziouzenkova O, Devchand PR, Nehra V, Hamdy O, Horton ES, et al. The peroxisome proliferator-activated receptor-gamma agonist pioglitazone represses inflammation in a peroxisome proliferator-activated receptor-alpha- dependent manner in vitro
and in vivo
in mice. J Am Coll Cardiol 2008; 52: 869-881.