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Original article

High density lipoprotein suppresses lipoprotein associated phospholipase A2 in human monocytes-derived macrophages through PPARγ pathway

Guan-ping, HAN; Jing-yi, REN; Li, QIN; Jun-xian, SONG; Lan, WANG; Hong, CHEN

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doi: 10.3760/cma.j.issn.0366-6999.2012.24.028
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Abstract

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.

METHODS

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 isolation

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.

Western blotting

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.

Experimental protocol

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.

Statistical analysis

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.

RESULTS

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).

Figure 1.
Figure 1.:
HDL inhibits Lp-PLA2 in macrophages with dose-dependent manner. A: HDL inhibited Lp-PLA2 mRNA expression in a dose-dependent manner. Human monocyte-derived macrophages were incubated with various concentrations of HDL (0, 12.5, 25, 50, 100 μg/ml) for 24 hours. Then RT-PCR was performed to amplify Lp-PLA2 and GAPDH mRNA and PCR products were visualized by agarose electrophoresis (upper panel). Bar graph shows the relative band intensities of Lp-PLA2 mRNA with densitometry analysis and normalized to GAPDH mRNA expression (lower panel). The values are mean ± SD of three independent experiments performed in duplicate. * P <0.05, compared with control. B: HDL inhibited Lp-PLA2 protein expression in a dose-dependent manner. Same experimental protocol with A, except the Western blotting analysis (upper panel) and densitometry analysis (lower panel) were performed to evaluate Lp-PLA2 protein expression upon HDL treatment with different final concentration. Bar graph shows the relative band intensities of Lp-PLA2 protein after densitometry and normalized to control group. The values are mean ± SD of three separate experiments. * P <0.05, compared with control. C: HDL inhibited activity of secreted Lp-PLA2 in a dose-dependent manner. With the same experimental protocols in A, Lp-PLA2 activity was analyzed using the colorimetric method with a PAF acetylhydrolase assay kit according to the manufacturer's protocol. Bar graph shows the relative band intensities of Lp-PLA2 activity after densitometry and normalized to control group. The values are mean ± SD of three separate experiments. * P <0.05, compared with control.

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.

Figure 2.
Figure 2.:
HDL inhibits Lp-PLA2 in macrophages with time-dependent manner. A: HDL inhibited Lp-PLA2 mRNA expression in a time-dependent manner. Human monocyte-derived macrophages were incubated with 25 μg/ml of HDL for different times (0, 6, 12, 24, and 48 hours). Then RT-PCR was performed to amplify Lp-PLA2 and GAPDH mRNA and PCR products were visualized by agarose electrophoresis (upper panel). Bar graph shows the relative band intensities of Lp-PLA2 mRNA with densitometry analysis and normalized to GAPDH mRNA expression (lower panel). The values are mean ± SD of three independent experiments performed in duplicate. * P <0.05, compared with control. B: HDL inhibited Lp-PLA2 protein expression in a time-dependent manner. Same experimental protocol with A, except the Western blotting analysis (upper panel) and densitometry analysis (lower panel) were performed to evaluate Lp-PLA2 protein expression upon HDL treatment with different time durations. The values are mean ± SD of three separate experiments. * P <0.05, compared with control. C: HDL inhibited the activity of secreted Lp-PLA2 in a time-dependent manner. With the same experimental protocol in A, Lp-PLA2 activity was analyzed using the colorimetric method with a PAF acetylhydrolase assay kit according to the manufacturer's protocol (See Methods). Bar graph shows the relative band intensities of Lp-PLA2 activity after densitometry and normalized to control group. The values are mean ± SD of three separate experiments. * P <0.05, compared with control.

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.

Figure 3.
Figure 3.:
Pioglitazone upregulates Lp-PLA2 expression and secreted activity in human macrophages. A: Pioglitazone treatment increased Lp-PLA2 mRNA levels in a dose-dependent manner. Human monocyte-derived macrophages were incubated with different concentrations of pioglitazone (0, 1, 5, 10 ng/ml) for 24 hours. Then RT-PCR was performed to amplify Lp-PLA2 and GAPDH mRNA and PCR products were visualized by agarose electrophoresis (upper panel). Bar graph shows the relative band intensities of Lp-PLA2 mRNA with densitometry analysis and normalized to GAPDH mRNA expression (lower panel). The values are mean ± SD of three independent experiments performed in duplicate. * P <0.05, compared with control. B: Pioglitazone treatment increased Lp-PLA2 expression in a dose-dependent manner. Same experimental protocol with A, except the Western blotting analysis (upper panel) and densitometry analysis (lower panel) were performed to evaluate Lp-PLA2 protein expression upon pioglitazone treatment with different final concentration. The values are mean ± SD of three separate experiments. * P <0.05, compared with control. C: Pioglitazone inhibited Lp-PLA2 secreted activity with dose-dependent manner. With the same experimental protocol in A, Lp-PLA2 activity was analyzed using the colorimetric method with a PAF acetylhydrolase assay kit according to the manufacturer's protocol. Bar graph shows the relative band intensities of Lp-PLA2 activity after densitometry and normalized to control group. The values are mean ± SD of three separate experiments. * P <0.05, compared with control.

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.

Figure 4.
Figure 4.:
HDL compromises pioglitazone-induced upregulation of Lp-PLA2 expression and secreted activity in macrophages. A: HDL compromised pioglitazone-induced upregulation of Lp-PLA2 mRNA level. Human monocyte-derived macrophages were incubated with 25 μg/ml of HDL in either the absence or presence of pioglitazone (5 ng/ml) for 24 hours. Then RT-PCR was performed to amplify Lp-PLA2 and GAPDH mRNA and PCR products were visualized by agarose electrophoresis (upper panel). Bar graph shows the relative band intensities of Lp-PLA2 mRNA with densitometry analysis and normalized to GAPDH mRNA expression (lower panel). The values are mean ± SD of three independent experiments performed in duplicate. * P <0.05, compared with control; P <0.05, compared with pioglitazone group. B: HDL compromised pioglitazion-induced upregulation of Lp-PLA2 protein expression. Same experimental protocol with A, except the Western blotting (upper panel) and densitometry analysis (lower panel) were performed to evaluate Lp-PLA2 protein expression. The values are mean ± SD of three separate experiments. * P <0.05, compared with control; P <0.05, compared with pioglitazone group. C: HDL compromised pioglitazone-induced upregulation of activity of secreted Lp-PLA2. Same experimental protocol with A, Lp-PLA2 activity was analyzed using the colorimetric method with a PAF acetylhydrolase assay kit according to the manufacturer's protocol. Bar graph shows the relative band intensities of Lp-PLA2 activity after densitometry and normalized to control group. The values are mean ± SD of three separate experiments. Pio: pioglitazone. * P <0.05, compared with control; P <0.05, compared with pioglitazone group.

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.

Figure 5.
Figure 5.:
HDL inhibits pioglitazone-induced phosphorylation of PPARγ in human monocyte-derived macrophages. Human monocyte-derived macrophages were treated with pioglitazone (5 ng/ml) in the presence or absence of HDL (25 μg/ml) for 24 hours. PPARγ expression and phosphorylation were measured by Western blotting. The values are mean ± SD of three separate experiments. Pio: pioglitazone. * P <0.05, compared with control; P <0.05, compared with pioglitazone group.

DISCUSSION

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.

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Keywords:

atherosclerosis; high-density lipoprotein; lipoprotein-associated phospholipase A2; peroxisome proliferator-activated receptor

© 2012 Chinese Medical Association