Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways

Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.


INTRODUCTION
Atherosclerosis is a chronic inflammatory disease that is associated with hypertriglyceridemia, hypercholesterolemia and vascular cell dysfunction. Apolipoprotein CIII (apo CIII), a small glycoprotein, is synthesized by the liver and to a lesser extent by the intestines, is one of the major components of triglyceride-rich lipoproteins (TRL) (Aalto-Setälä et al., 1992). The overexpression of human apo CIII in mice, rabbits and pigs has been shown to reduce lipoprotein lipase (LPL) activity and lead to hypertriglyceridemia, which is associated with atherosclerosis (Ding et al., 2011;Ito et al., 1990;Wei et al., 2012). Numerous in vitro studies have demonstrated that apo CIII can promote proatherogenic responses in endothelial cells (ECs) and macrophages. For example, apo CIII increases vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) expression in ECs, causing the adhesion of THP-1 cells to ECs. In addition, apo CIII-enriched apolipoprotein B lipoproteins enhance the adhesion of human monocytes to ECs (Caron and Staels, 2008;Kawakami et al., 2006a;Kawakami et al., 2006b).
Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) is a calcium-independent, secreted phospholipase A 2 that binds to circulating lipoproteins and catalyzes the hydrolysis of oxidized LDL with a truncated sn-2 acyl chain to release inflammatory products, oxidized fatty acids and lysophosphatidylcholine (LysoPC) (Rosenson and Stafforini, 2012;Stafforini et al., 1987;Tew et al., 1996;Zalewski and Macphee, 2005). These products have been shown to cause pro-inflammatory and pro-apoptotic effects in vitro studies (Matsumoto et al., 2007;Wang et al., 2010). In addition, increased Lp-PLA 2 expression has been observed in necrotic cores and in macrophages of vulnerable and ruptured plaques from human and rabbit atherosclerotic lesions (Häkkinen et al., 1999;Lavi et al., 2007). The WOSCOPS (West of Scotland Coronary Prevention Study) was the first report that demonstrated the plasma concentration of Lp-PLA 2 has a strong and positive association with the risk of coronary events, even when age, systolic blood pressure, and lipoprotein levels were put into consideration (Packard et al., 2000); these results were confirmed in subsequent investigations (Hatoum et al., 2011;Sabatine et al., 2007). The importance of Lp-PLA 2 as an independent biomarker of CVD remains controversial (Mallat et al., 2010;Thompson et al., 2010) due to recent clinical investigations (White et al., 2014) that contradict the positive association reported by the WOSCOPS. However, Darapladib, an Lp-PLA 2 inhibitor, decreased the levels of interleukin (IL)-6 and high-sensitivity C-reactive protein (hs-CRP) by 12.3% and 13%, respectively, after the oral administration of 160 mg daily for 12 weeks (Mohler et al., 2008) and significantly halted the necrotic core volume increase compared to a placebo at 12 months (Serruys et al., 2008). Therefore, investigation of the factors that regulate Lp-PLA 2 levels is needed.
Here, we investigated the effects of apo CIII on Lp-PLA 2 expression. In addition, we previously developed a genetically modified, human apo CIII overexpression porcine model, which has a longer triglyceride absorbance and clearance time than the wild types and exhibits a 2.5-fold and 2.3-fold increase in plasma triglycerides in the postprandial and fasting states, respectively (Wei et al., 2012). In these models, plasma Lp-PLA 2 activity and expression are also investigated. These data may benefit to understanding the regulation of Lp-PLA 2 and the relationship with apo CIII in vitro and in vivo.

RESULTS AND DISCUSSION
Effects of apo CIII on Lp-PLA 2 gene expression Lp-PLA 2 , as an independent biomarker and regulator of atherosclerosis, the level may be regulated by lipid associated factors. Apo CIII level is closely related with hypertriglyceridemia. To investigate the effect of apo CIII on Lp-PLA 2 expression level, we treated human monocytic THP-1 cells with apo CIII. As shown in Fig. 1, the levels of Lp-PLA 2 mRNA and protein expression were increased in THP-1 cells incubated with apo CIII in serum-free medium in a dose-and time-dependent manner ( Fig. 1A-E). Furthermore, apo CIIItransfected THP-1 cells also exhibited increased Lp-PLA 2 expression and activity (Fig. 1F,G). These data suggest that apo CIII can induce Lp-PLA 2 expression in vitro.

Lp-PLA 2 involves apo CIII-induced inflammation
Apo CIII treatment significantly increases TNF-a, IL-6 and MCP-1 release from monocytic THP-1 cells ( Fig. 2A-C). In circulation, Lp-PLA 2 can act on oxidized LDL and produce oxidized fatty acids and lysophosphatidylcholine (LysoPC) which are proinflammatory factors. To address the question that if Lp-PLA 2 mediates apo CIII's proinflammatory effect, the Lp-PLA 2 inhibitor 1-linoleoyl glycerol was used to pretreat the cells before apo CIII treatment. As shown in Fig. 2D-F, the pro-inflammatory effects of apo CIII were attenuated by 1-linoleoyl glycerol, and no inhibitory effect was observed in the cells that lack apo CIII. These results suggest that Lp-PLA 2 at least partially mediates the effects of apo CIII on TNF-a, IL-6 and MCP-1 release in monocytic THP-1 cells.

Apo CIII stimulates Lp-PLA 2 expression is regulated by MAPK and NFkB pathways
The nuclear factor (NF)-k B and mitogen-activated protein kinase (MAPK)-dependent pathways were closely related to the inflammatory response in cells. Other researcher and our previous report demonstrated that NF-k B and MAPK pathway mediate the regulation of Lp-PLA 2 gene expression. Therefore, here we assessed the possibility that both pathways involve in regulating Lp-PLA 2 expression by apo CIII in THP-1 cells. Apo CIII stimulated the phosphorylation of p65 NFkB and p42/44 MAPK and upregulated Lp-PLA 2 expression ( Fig. 3A-D). The MAPK kinase inhibitor PD98059 and NFkB inhibitor PDTC can completely block the increase in Lp-PLA 2 mRNA and protein expression induced by apo CIII (Fig. 3D-G). These results indicate that the p65 NFkB and p42/44 MAPK pathways mediate the upregulation of Lp-PLA 2 expression induced by apo CIII. (E) THP-1 cells were incubated with 30 mg apo CIII for the indicated durations. Lp-PLA 2 mRNA was subsequently extracted and detected using quantitative PCR (n53). (F,G) THP-1 cells were transfected with a human apo CIII vector for 48 h (n53). The Lp-PLA 2 activity was assayed using the PAF-AH assay kit, and the Lp-PLA 2 mRNA level was determined using quantitative PCR. *P,0.05, **P,0.01, ***P,0.0001. The relationship between apo CIII and Lp-PLA 2 expression in vivo and Lp-PLA 2 expression and activity in Apo CIII transgenic pigs Twenty pigs samples of blood and liver tissues were collected to observe the expression of apo CIII and Lp-PLA 2 , the results show that the mRNA level of apo CIII and Lp-PLA 2 are correlated (Fig. 4E). The previously developed apo CIII transgenic pigs specifically overexpress human apo CIII in the liver and intestines (Wei et al., 2012). The model was used to confirm the correlation of apo CIII and Lp-PLA 2 expression. When fed a normal chow diet, the apo CIII transgenic pigs exhibited a 4-fold increase in plasma Lp-PLA 2 activity and a 10-fold increase of Lp-PLA 2 mRNA in macrophage compared to the wild-type controls (Fig. 4A,B). There was no change in the plasma Lp-PLA 2 activity in wild-type pigs in the fasting or fed (olive oil) states (Fig. 4C). Interestingly, the Lp-PLA 2 activity can be increased in the apo CIII transgenic pigs after ingesting olive oil for 2 h (Fig. 4C). These findings demonstrate that apo CIII may be responsible for the changes in Lp-PLA 2 expression at the transcriptional level.
In addition, we observed that exogenous Lp-PLA 2 stable expressed in HepG2 cells could increase apo CIII mRNA level and the increased effects could be inhibited by 1-linoleoyl glycerol (Fig. 4D). Together these results suggested that increased apo CIII in liver could upregulate Lp-PLA 2 expression via p65 NFkB and p42/44 MAPK pathways in macrophage in circulation. The increased Lp-PLA 2 could stimulate macrophage inflammation independent with lipoproteins and the inflammatory factors in turn stimulate apo CIII expression in liver (Fig. 4F). The detailed mechanisms in the circle need to be further investigated.

Animals and cells
The apo CIII transgenic pigs were developed using Chinese experimental miniature pig fibroblasts according to a previously described method, and they were fed a chow diet (Wei et al., 2012). For the experiments, wildtype and apo CIII transgenic pigs were administered olive oil orally (fat 23%) for fat load at 10 ml/kg body weight after a 16-h fast, and blood was collected for analysis. Human monocytic THP-1 cells were donated by Dr. Yang's lab (Jilin University) and maintained in RPMI-1640 medium with 10% fetal bovine serum (PAA, Austria). Pig monocytes were isolated from apo CIII transgenic or wild-type pigs using Histopaque-1077 (Sigma-Aldrich, USA). All of the animal experiments were conducted according to Jilin University Animal Care and Use Committee protocol no. 2008-11.

Reagents
Apolipoprotein C-III was purchased from Sigma-Aldrich. The antibodies p42/44, p-p42/44, p65 and p-p65 were purchased from Cell Signaling. Fig. 3. Apo CIII stimulated Lp-PLA 2 increasing is regulated by MAPK and NFkB pathways. THP-1 cells were incubated with apo CIII and the indicated inhibitors. (A-C) After incubating with apo CIII for the indicated durations, THP-1 cell proteins were collected to detect p42/44 and p65 expression and phosphorylation using western blotting (n53). (D-G) THP-1 cells were incubated in PD98059 and PDTC for 1 h and then incubated with apo CIII, PD98059 and PDTC for an additional 12 h. The levels of Lp-PLA 2 , p42/44, p65 and their phosphorylated counterparts were determined by western blotting (n53). (H) THP-1 cells were treated as described in D, except that the incubation time was extended to 36 h. Quantitative PCR was performed to determine the levels of Lp-PLA 2 mRNA expression (n53). Error bars represent mean6s.e.m. *P,0.05, **P,0.01.
The antibodies Lp-PLA2 and b-actin were obtained from Bioss. PD98059 and PDTC were purchased from Beyotime, and 1-linoleoyl glycerol was obtained from Cayman. Human MCP-1, TNF-a, and IL-6 Elisa Kits were obtained from Boster.
Immunoblotting THP-1 cells were incubated with different combinations of reagents. For the expression analysis, the cells were lysed in cell lysis buffer (Beyotime, China) containing 1 mM PMSF and protein phosphatase inhibitor (Applygen, China) for 30 min on ice and were then centrifuged. The protein concentrations were determined using the Enhanced BCA protein assay kit (Beyotime, China). Equal amounts of protein were used for 12% SDS-PAGE and then transferred to nitrocellulose membranes. Immunoblots were performed with the indicated primary antibodies and the corresponding secondary antibodies. The signal was detected using BeyoECL Plus (Beyotime, China).

Lp-PLA2 activity
Blood was collected from pigs using anticoagulant EDTA tubes and centrifuged at 1000 g for 10 min. The plasma was analyzed using a PAF Acetylhydrolase kit (Cayman, USA) according to the manufacturer's instructions.

Quantification of proinflammatory cytokines
The levels of proinflammatory cytokines which stimulated by apo CIII and released in the culture media were evaluated by Human IL-6 ELISA Kit, Human TNFa ELISA Kit and Human MCP-1 ELISA Kit (Boster Inc., China), following the manufacturer's instructions.

Statistical analyses
The data are expressed as the mean6SEM and were analyzed using a two-tailed unpaired t test in the GraphPad Prism software. P,0.05 was considered to be statistically significant. Blood samples from apo CIII transgenic and wild-type pigs were collected, and the plasma was isolated for use in Lp-PLA 2 activity assays. Mononuclear cells were isolated for total RNA extraction. (A) The plasma Lp-PLA 2 activity in the wild-type and apo CIII transgenic pigs was assayed less than 2 h after plasma isolation. (B) Total RNA was extracted from the mononuclear cells, and quantitative PCR for Lp-PLA 2 mRNA level was conducted. (C) The wildtype and transgenic pigs were fasted for 16 h and fed olive oil for 2 h, the plasma was isolated, and the Lp-PLA 2 activity were measured. n58 in the wild-type pigs, and n54 in the apo CIII transgenic pigs. Error bars represent mean6s.e.m. (D) Lp-PLA 2 stable expressed HepG2 cells was incubated with 1-linoleoyl glycerol for 12 h. Quantitative PCR was performed to determine the levels of apo CIII mRNA expression (n53). (E) Blood and liver tissue were collected from slaughtered landraces, and quantitative PCR was performed to determine the levels of apo CIII mRNA expression in liver tissue and Lp-PLA 2 mRNA expression in monocytes isolated from blood. Linear regression analysis is shown. *P,0.05, **P,0.01, ***P,0.0001. (F) A schematic model of apolipoprotein CIII regulates Lp-PLA 2 expression.