Twenty-four patients with unstable angina (UA) and twenty-four patients with non-ST-segment elevation myocardial infarction (NSTEMI) were recruited in Fuwai Hospital (Beijing, China) between March 2013 and January 2014 (Table 1). The diagnoses of UA/NSTEMI were all in line with the standards of the Chinese Medical Society of Cardiology. Twenty-four healthy persons served as a control group with normal echocardiograms (ECGs), negative exercise stress ECGs based on physical examination and laboratory tests and without coronary heart disease history. Patients with inflammatory diseases, acute myocardial injury, severe heart failure, malignant disease, impaired liver function, renal failure, or recent surgery were excluded.

The coronary atherosclerotic plaques were collected from explanted hearts donated by three patients undergoing heart transplantation and stored in liquid nitrogen in Fuwai Hospital. According to histomorphologic features, atherosclerotic plaques were classified as follows: unstable plaques were defined as the lesions with thin fibrous cap (<65 μm) covering large lipid core (>40% plaque volume); stable plaques were described as the lesions with well-matured necrotic core (<40% plaque volume) covered by thick fibrous cap (>65 μm). All specimens were carefully perfusion-fixed in 10% buffered formalin.

The study protocol conformed to the Declaration of Helsinki and was approved by the ethics review board of Fu Wai Hospital and all the patients gave written informed consent.

Formalin-fixed paraffin embedded tissue were cuted into 4-μm-thick sections and immersed in xylene and rehydrated with graded alcohols and subjected to H&E staining before examination by light microscopy.

IHC was performed by the standard protocol. Briefly, antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0), followed by quenching of endogenous peroxidase activity with 3% H₂O₂ in methanol. Sections were then incubated with MMP-9 (Abcam, Cambridge, UK) and ATX (as described Li and Zhang, 2009) primary antibodies overnight at 4°C. Powervision horseradish peroxidase-IgG was used as secondary antibody. Sections were visualized using diaminobenzidine and counter stained with hematoxylin.

Human monocytic cell line THP-1 (a gift from Prof. Junjie Zhang, Beijing Normal University, China) and maintained at a density of 10⁶/ml in RPMI 1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/Streptomycin, and then incubated at 37°C with 5% CO². Cells were cultured in six-well plates for 48 h in the presence of phorbol 12-myristate 13-acetate (PMA) (100 ng/mL, Sigma, St Louis, USA), which induced the differentiation into adherent macrophages (Aldo et al., 2013). After serum-starvation for 18–24 h, cells were treated with 0 to 50 μM LPA (oleoyl C 18:1, Avanti Polar Lipids, Alabaster, AL), or pretreated 0.1 μM Actinomycin D (Act.D), 0.1 μM Cycloheximide (CHX), 10 μM GW9662 (PPARγ inhibitor), 10 μM PDTC (NF-κB inhibitor) and 10 μM Tan II A (AP-1 inhibitor) for 1 h.

Isolation of peripheral blood mononuclear cells (PBMCs) and culture of macrophages were performed using classical methods (van Grevenynghe et al., 2003). Briefly, PBMCs were isolated through Ficoll-Paque™ PLUS (GE Healthcare, Piscataway, NJ, USA) gradient centrifugation within 1 h after blood collection. Cells were seeded in culture flasks with RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/Streptomycin for 2 h to adhesion. The adherent monocytes were then added recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) (PeproTech, New Jersey, USA) for 7 days.

Small interfering RNAs (siRNAs) for LPA₁, LPA₂, and LPA₆ were transfected using Lipofectamine™ RNAiMAX according to manufacturer's protocols. The target sequences of these siRNAs were listed as follow:

LPA₁ Stealth siRNA duplexes (LPA₁-siRNA) targeting sequences: 5′-GAA AUG AGC GCC ACC UUU A-3′and 5′-UAA AGG UGG CGC UCA UUU C -3′; LPA₂ Stealth siRNA duplexes (LPA₂-siRNA) targeting sequences: 5′-GGU CAA UGC UGC UGU GUA C-3′ and 5′-GUA CAC AGC AGC AUU GAC C-3′; LPA₆ Stealth siRNA duplexes (LPA₆-siRNA) targeting sequences: 5′-UCA GCA UGG UGU UUG UGC UUG GGU U-3′ and 5′-AAC CCA AGC ACA AAC ACC AUG CUG A-3′. NF-κB p65-siRNA targeting sequences: 5′-GCC CUA UCC CUU UAC GUC A-3′ and 5′-UGA CGU AAA GGG AUA GGG C-3′. The scrambled siRNA controls were used as a negative control.

Total RNA was isolated from THP-1 derived macrophages using Trizol according to the manufacture's instruction and then quantified by NANODROP 2000 spectrophotometer. cDNA was generated from 2 μg total RNA using M-MLV reverse transcriptase and oligo (dT) 15 primer. qRT -PCR was performed using SYBR GREEN PCR Master Mix in the Applied Biosystems 7300 (Foster city, CS, USA). All gene specific extron primers used in the present study were as follows:

LPA₁: 5′-GCT GCC ATC TCT ACT TCC AT-3′ and 5′-CCA TTC TGT GGC AAG ATG CT-3′; LPA₂: 5′-CAG CGC ATG GCA GAG CAT GT-3′ and 5′-CCA GGA CAT TGC AGG ACT CAC A-3′; LPA₃: 5′-AAC GTG AGC GGA TGT TCA CT-3′ and 5′-CCG CGA TGA CCA GAG AAT TA-3′; LPA₄: 5′-TTC GAA CTA ATG TGG AGG AA-3′ and 5′-TGG AAT TGG AAG TCA ATG AA-3′; LPA₅: 5′-CAG AGC AAC ACG GAG CAC AG-3′ and 5′-CAC CAG AAT CAT GGC ATG GC-3′; LPA₆: 5′-CTT CAC AAC ACG GAA TTG GC-3′ and 5′-AGT TAA CCA CAC GCC AGT GC-3′; MMP-9: 5′-TGG AAA ATT ATT GCG CCT CT-3′ and 5′-CCT CGA TGA GAC CAT CAA CA-3′; GAPDH: 5′-ACT AGG CGC TCA CTG TTC TC-3′ and 5′-GCC CAA TAC GAC CAA ATC CG-3′; NF-κB p65: 5'-GCG AGA GGA GCA CAG ATA CC-3' and 5'-CTG ATA GCC TGC TCC AGG TC-3'. The relative expression of each gene was normalized to GAPDH by subtracting the corresponding GAPDH threshold cycle (Ct) values using comparative 2^−ΔΔCt method.

The treated cells were washed with ice-cold phosphate-buffered saline (PBS) twice and then lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF and 1% sodium deoxycholate) for 30 min on ice. The BCA assay was used to quantify the extract protein concentration. Equal amounts of protein (20 μg/lane) were separated on 10% SDS-PAGE gels by electrophoresis and then transferred to nitrocellulose membranes using semi-dry electroblotting apparatus. The membranes were blocked for 2 h at room temperature in 5% skim milk and subsequently incubated with primary antibodies overnight at 4°C. In the following day, the members were washed and incubated with secondary antibody for 2 h. Protein bands were visualized by FluorChem M system (Protein Simple, USA) and quantified by densitometry with Quantity One software. The target signals were normalized to the GAPDH levels.

THP-1 derived macrophages were cultured on 96-well plates (10⁵ cells/well) for 48 h and stimulated with 10 μM LPA for 24 h. Reporter plasmid REPO™NF-κB and REPO™AP-1 (Genomeditech, Shanghai, China) were transfected using Lipofectamine 2000. Luciferase activity, quantified as relative luminescence units (firefly luminescence/Renilla luminescence), was measured with the Dual-Glo® Luciferase Assay System (Promega, Madison, WI) according to the manufacturer's instructions using a microplate reader (Infinite 200, Tecan).

The MMPs activities were assayed as described (Zhang et al., 2014). Briefly, 2 × 10⁶ cells in a 6-well plate were cultured in serum-free medium and 10 μL supernatants were loaded on 8% SDS-PAGE gels containing 2 mg/ml gelatin under non-denaturing conditions. The gels were washed with 2.5% Triton X-100 and incubated overnight in buffer (150 mM NaCl, 5 mM CaCl₂, 50 mM Tris-HCl, pH 7.6) at 37°C. Following staining with Coomassie brilliant blue R250, the gelatinolytic activities were detected as clear bands against a blue background. The scan of the gel was analyzed by densitometry using FluorChem M system (Protein Simple, USA).

Levels of MMP-9 in plasma or conditioned medium from THP-1 derived macrophages were quantified by the human MMP-9 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. All plasma samples were diluted 1:100 and the conditioned mediums were diluted 1:10 when testing.

Detection of plasma LPA levels were carried out by Prof. Zhenwen Zhao (Institute of Chemistry Chinese Academy of Science, Beijing, China) through high performance-liquid-chromatography electrospray ionization tandem mass spectrometry (HPLCESI-MS/MS) as described (Zhao and Xu, 2009).

All statistical data were presented as mean ± SD and categorical variables were represented as percentages. Comparisons among the groups were performed using the chi-square test or one-way ANOVA. Bivariate correlation analysis was used to analyze the relations of plasma LPA and MMP-9 levels. Two-sided P-values were used and P < 0.05 were considered statistically significant. All the statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).

We measured the plasma levels of LPA and MMP-9 in patients with AMI (n = 24), UA (n = 24) and asymptomatic controls (n = 24) and the data showed that both LPA and MMP-9 with higher levels in plasma in AMI group compared with that in the control group (LPA: 0.66 ± 0.23 vs. 0.42 ± 0.16, P < 0.05; MMP-9: 775.5 ± 242.4 vs. 532.1 ± 204.1, P < 0.05), but no significant difference was found between patients with UA and controls (Figures 1A,B). Pearson correlation analysis showed that there was a positive correlation between plasma LPA and MMP-9 levels (r = 0.31, P < 0.05) (Figure 1C).

The positive correlation between LPA and MMP-9 in the peripheral blood was found, but the presence and localization of them in the local tissue are unclear. ATX, LPA-producing ectoenzyme, were detected because LPA is a bioactive phospholipid molecules and it is difficult to detect in the tissue. As shown in Figures 2A–C, normal artery was characterized by uniform intima-media thickness (IMT) and smooth vascular lumen, stable plaque with large lipid core but thick fibrous cap and the large lipid core and rupture-prone fibrous cap in unstable plaques. The positive staining for MMP-9 was lacked in normal artery (Figure 2D) and mainly located in the shoulder of necrotic core and cap region in stable plaque (Figure 2E). In contrast, unstable plaque showed strong staining for MMP-9 in and surrounding the lesion (Figure 2F). The positive staining for ATX was found in normal artery, stable and unstable plaque and it seemed somewhat a change in distribution. ATX is mainly distributed in the vascular smooth muscle layer and accumulated in the necrotic core and fibrous cap with the increase of plaque instability (Figures 2G–I). Furthermore, distribution patterns of MMP-9 and ATX overlapped in unstable plaque although were different.

To evaluate the effect of LPA on MMP-9, we first treated THP-1-derived macrophages with different concentrations of LPA (0, 1, 5, 10, 25, 50 μM) for 24 h. The expression and secretion of MMP-9 increased from 1 μM of LPA in dose-dependent manner and reached maximum at 10 μM of LPA (Figures 3A–C). Meanwhile, Figure 3D revealed that the ability of MMP-9 to degrade collagen was enhanced with the strongest at 10 μM of LPA, while MMP-2 made no responses to LPA. As shown in Figures 3E–G, mRNA levels, protein syntheses and secretion of MMP-9 increased time-dependently with 10 μM LPA for 4–48 h. Additionally, the protein levels of pro-MMP-9 and active-MMP-9 were both increased with LPA treatment in human peripheral blood monocyte-derived macrophages (Figure 3H).

MMPs are regulated at the levels of transcription, secretion, activation and physiological suppression of tissue inhibitor of matrix metalloproteinases (TIMPs). To determine the regulation at transcriptional or post transcriptional, Act.D and CHX were used to inhibit synthesis of nascent mRNA and protein of MMP-9, respectively. As shown in Figures 4A–C, the expression of MMP-9 failed to increase after pretreatment with Act.D or CHX followed by LPA exposure. It indicated that up-regulation of LPA-induced MMP-9 occurred in the de novo synthesis of mRNA, namely transcription. Thus, we analyzed promoter region of MMP-9 and filtered out three possible transcription factors: PPARγ, NF-κB and AP-1. As shown in Figures 4D,E, after pretreatment with PPARγ inhibitor GW9662 and AP-1 inhibitor, the basic levels of MMP-9 reduced, but LPA-induced MMP-9 still increased, indicating that PPARγ and AP-1 were not involved in the regulating process of LPA on MMP-9. In contrast, LPA-induced MMP-9 expression was markedly attenuated by NF-κB inhibitor PDTC and siRNA for NF-κB p65 (Figures 4E,G,H), and transcriptional activity of NF-κB was up-regulated by LPA as expected (Figure 4F).

LPA exerts various biological effects through different G protein-coupled receptors: LPA₁–LPA₆, and therefore we investigated which subtype of LPA receptors mediated MMP-9 expression. Since LPA₁, LPA₂, and LPA₆ were highly expressed in THP-1-derived macrophages (Figure 5A), we separately knocked down the three LPA receptors using specific siRNAs and detected the MMP-9 expression with or without LPA treatment. As shown in Figure 5B, LPA₁, LPA₂, and LPA₆ were effectively knocked down and LPA-induced MMP-9 expression and nucleus p65 levels were attenuated significantly by inhibition of LPA₂ (Figures 5C–E). These data suggested that LPA₂ was involved in LPA-induced upregulation of MMP-9 and activation of NF-κB.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.