Lysophosphatidic Acid Is Associated with Atherosclerotic Plaque Instability by Regulating NF-κB Dependent Matrix Metalloproteinase-9 Expression via LPA2 in Macrophages

Abstract

Lysophosphatidic acid (LPA), one of the simplest phospholipid signaling molecules, participates in formation and disruption of atherosclerotic plaque. Matrix metalloproteinases (MMPs) contribute to atherosclerotic plaque rupture by involving in extracellular matrix (ECM) degradation and then thinning fibrous cap. Our previous study demonstrated that macrophage-derived MMP-9 was associated with coronary plaque instability, but the relationship between LPA and MMP-9 remains unclear. The present work therefore aimed at elucidating association between LPA and MMP-9 and the regulation mechanism of LPA on MMP-9 in macrophages. We found that plasma LPA and MMP-9 levels were correlated positively (r = 0.31, P < 0.05) and both elevated significantly in patients with acute myocardial infarct (AMI). Consistent with peripheral blood levels, histochemical staining indicated that autotaxin (ATX), LPA-producing ectoenzyme, and MMP-9 were expressed frequently in the necrotic core and fibrous cap of human unstable plaques, which might increase the instability of plaque. Experiments in vitro were done with THP-1-derived macrophages and showed that LPA enhanced the expression, secretion and activity of MMP-9 in a time- and dose-dependent manner. Induction of LPA on pro-MMP-9 and active-MMP-9 was confirmed in human peripheral blood monocyte-derived macrophages. PDTC, NF-κB inhibitor, but not inhibitor of AP-1 and PPARγ, effectively prevented LPA-induced MMP-9 expression and NF-κB p65 siRNA decreased MMP-9 transcription, confirming that LPA might induce MMP-9 elevation by activating NF-κB pathway. In addition, knockdown of LPA₂ attenuated LPA-induced MMP-9 expression and nucleus p65 levels. These findings revealed that LPA upregulated the expression of MMP-9 through activating NF-κB pathway in the LPA₂ dependent manner, hence blocking LPA receptors signaling may provide therapeutic strategy to target plaque destabilization.

Keywords: LPA2; coronary atherosclerotic plaques; lysophosphatidic acid; macrophages; matrix metalloproteinase-9.

Figure 1

Changes of plasma LPA and MMP-9 levels among control, unstable angina and acute myocardial infarction groups. Plasma LPA (A) and MMP-9 (B) levels were detected by mass spectrometry and ELISA, respectively. The levels of MMP-9 and LPA in the group with AMI were both higher than the control and UA groups (P < 0.05). (C) Pearson correlation analysis showed that there is a positive correlation between LPA and MMP-9 (r = 0.31, P < 0.05). ^*P < 0.05, compared with control.

Figure 2

Expression of ATX and MMP-9 in human coronary plaque tissues. (A,D,G) for normal arteries; (B,E,H) for stable plaques; (C,F,I) for unstable plaques; (A–C) HE staining; (D–F) Immunohistochemical staining of MMP-9; (G–I) Immunohistochemical staining of ATX.

Figure 3

LPA induced the expression, secretion and activity of MMP-9 in a dose- and time- dependent manner. Macrophages were treated with the pointed concentrations of LPA for 24 h or with 10 μM LPA for the pointed time course. The mRNA levels (A,E), protein levels (B,F), secretion (C,G) and activity (D) of MMP-9 were detected by RT-PCR, Western Blot, ELISA and gelatin zymography, respectively. LPA induced MMP-9 expression in human peripheral blood monocyte-derived macrophages (H). Data were expressed as mean ± SD of three independent experiments. ^*P < 0.05, compared with control.

Figure 4

NF-κB activation was required for LPA-induced MMP-9 expression. After pretreating with 0.1 μM Act.D (A,B) and 0.1 μM CHX (C) for 1 h to inhibit mRNA and protein synthesis, and then cells were incubated with 10 μM LPA for 24 h to detect MMP-9 expression. (D,E) Cells were pretreated with 10 μM GW9662 (PPARγ inhibitor), 10 μM PDTC (NF-κB inhibitor) and 10 μM TanIIA (AP-1 inhibitor) respectively, and then incubated with 10 μM LPA for 24 h. The expression of MMP-9 was determined. (F) The NF-κB and AP-1 promoter activity was measured. The mRNA expression of NF-κB p65 (G) and MMP-9 (H) were determined after interference of NF-κB p65-siRNA. Data were expressed as mean ± SD of three independent experiments. ^*P < 0.05, compared with control; ^#P < 0.05, compared with inhibitor treatment groups.

Figure 5

LPA₂ mediated LPA-induced MMP-9 expression. (A) The expression of LPAR was detected in THP-1-derived macrophages. The mRNA expression of LPAR (B) and MMP-9 (C) were determined after interference of LPA₁-siRNA, LPA₂-siRNA and LPA₆-siRNA, respectively. The expression of MMP-9 (D), nucleus and cytoplasm p65 (E) were determined after interference of LPA₂-siRNA. Data were expressed as mean ± SD of three independent experiments. ^*P < 0.05, compared with control. ^#P < 0.05, compared with negative control.