| Background and ObjectiveIn the past few years, research has focused on the detailed mechanisms of action of AngII in atherosclerotic plaques. Experiments have demonstrated that irrespective of whether in early or advanced atherosclerosis, or if transiently or constantly exposed to AngII, atherogenesis is promoted. The three clinical trials mentioned above also demonstrated that using selective AT1R antagonists can prevent atherogenesis. AngII therefore has an important pathophysiological role in the formation and development of atherosclerosis.Atherosclerosis is a complex disease, and the detailed mechanisms of its pathogenesis are incompletely defined. Studies have shown that inflammation may have a critical role in all the phases of atherosclerosis progression from the early formation of lesions to the rupture of advanced plaques. A review by Cipollone demonstrated that the inflammatory factors cyclo-oxygenase (COX) and prostaglandin (PG) synthases have an important role in atherosclerosis, particularly cyclooxygenase-2 (COX-2). COX-2 participates in an extensive inflammatory reaction process and can evoke different functions in different inflammatory courses. Immunohistochemical studies have shown that COX-2 can co-localize with prostaglandin-E (PGE) synthase and macrophages in symptomatic lesions, and related the induction of the COX-2/PGE synthase pathway to an acute ischemic event caused by rupture of atherosclerotic plaques. Further research by Cipollone revealed the AT1 receptor blockade can reduce the production of COX-2 and PGE2 and thus lead to the reduction of production of MMPs. It suggested that COX-2/PGE2 plays an important part in maintaining plaque stabilityExtracellular matrix metalloproteinase inducer (EMMPRIN) is expressed on the surface of many tumor cells. It could stimulate fibroblasts and carcinoma cells to secrete several matrix metalloproteinase proteins (MMPs) and thus facilitate the invasion and metastasis of tumor cells. Recent studies have shown that EMMPRIN can co-localize with MMPs and macrophages in atherosclerotic lesions. Other studies have reported that inflammatory factors such as tumor necrosis factor (TNF)-αand C-reactive protein (CRP) can promote EMMPRIN expression, suggesting that EMMPRIN plays an important part in the emergence of stroke and acute myocardial infarction caused by plaque rupture. Research has also suggested that the mechanism of EMMPRIN-regulated expression of MMPs may play a key part in plaque disruption.The effect of AngII on MMPs is one of the main research focuses on the mechanism of acute ischemic events. MMP family members are thought to be involved in acute coronary syndrome and stroke by degrading the extracellular matrix. AngII induced different MMPs via different pathways; it induced MMP-2 in a p47phox-dependent manner and MMP-9 in the NF-ΚB signal pathway in smooth muscle cells and MMP-9 via the COX-2/PGE2 pathway-dependent in macrophages. However, the effect of AngII on EMMPRIN co-localized with MMPs in macrophages at atherosclerotic plaques has scarcely been studied. In light of the AT1-driven instability of atherosclerotic plaques and as the basis of COX-2/PGE2-dependent plaque rupture, the present study was the first to: (1) demonstrate that AngII can enhance the expression of EMMPRIN in THP-1 macrophages; (2) show the enhancement of EMMPRIN through the AT1R and inhibition by an AT1R antagonist; and (3) relate the inhibition of COX-2/PGE2 to the reduction of EMMPRIN observed after intervention by an AT1R antagonist.Methods1. The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100μg/mL streptomycin at 37°C in an atmosphere of 5% CO2. According to the literature, THP-1 monocytic cells (1×106 cells) differentiate into macrophages in six-well plates containing 1 mL of RPMI 1640 medium and 5 ng/mL PMA over 48 h. After culture, plates were washed thrice with 1 mL PBS. Cells were further cultured in serum-free medium for an additional 24 h. Cells were then incubated with the corresponding stimuli. For the inhibition experiments, cells were preincubated with losartan, PD123319, and NS-398 for 1 h before stimulation with AngII. 2. The cytotoxicity of AngII was measured by MTT in 96-well plates. In brief, PMA-induced THP-1 macrophages were seeded at 1×105 cells per well with 100μL RPMI-1640 medium. Cells were then treated with AngII at various concentrations (0.01–50μΜ) for 24 h, whereas the control group was treated with 0.1% dimethyl sulfoxide (DMSO). Four hours before the end of the treatment, cells were cultured with 5 mg/mL MTT. The medium was then removed and replaced with DMSO. The optical density was measured atλ=550 nm.3. Total RNA from THP-1 macrophages was isolated with Trizol reagent according to the manufacturer's protocol. The quality of RNA was examined by an Experion Automated Electrophoresis System. First-strand cDNA was synthesized using ReverTra Ace reverse transcriptase. The primers used for PCR are listed in Table 1. The mRNA was measured by SYBR Green1 fluorescence, and the PCR reaction monitored by an iQ?5 Real-Time PCR Detection System (BioRad). The results were analyzed using BioRad iQ?5 software.4. For western blot analyses, cells were washed twice with ice-cold PBS and lysed by adding 100μL of ice-cold lysis buffer. Protein concentration was measured by Lowry's method using bovine serum albumin (BSA) as a standard. Protein extracts (50μg) underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels and were transferred onto nitrocellulose filters for 1.5 h at 150 mA. Filters were blocked in 5% non-fat milk solution in Tris-buffered saline (TBS) buffer with 0.5% Tween 20 for 1 h at room temperature. The filters were then allowed to incubate overnight at 4°C with the specific primary antibodies anti-COX-2 (1:500 dilution) and anti-EMMPRIN (1:500 dilution). Filters were then washed thrice in TBS with 0.5% Tween 20 for 10-min each followed by incubation 1 h at room temperature with the fluorescent-labeled secondary antibodies. After three (10-min each) washes with TBS and Tween-20, filter signals were scanned by Odyssey. Fluorescent signals were quantified by Odyssey 2.0 software.5. PGE2 in the culture medium was measured with a PGE2 enzyme immunoassay kit (Zhongshan Golden Bridge) according to manufacturer instructions. The amount of PGE2 was analyzed by measuring absorbance at 405 nm.Results1. Cell model establishment: THP-1 cells differentiate into macrophages in six-well plates containing 1 mL of RPMI 1640 medium and 5 ng/mL PMA over 48 h, then THP-1 macrophages become flat and spreading. The induced condition of macrophages was investigated by inverted phase-contrast microscopy.2. Effect of AngII on the viability of THP-1 macrophages: To characterize if AngII caused toxicity in THP-1 macrophages, the MTT assay was carried out to monitor cell viability. Result shows the number of THP-1 macrophages stimulated with AngII (0.01–50μΜ) after 24 h. Concentrations of 0.01–10μM AngII had no significant effect on THP-1 macrophages. These data demonstrated that 0.01–10μΜAngII did not cause significant cytotoxicity in THP-1 macrophages.3. AngII induces expression of COX-2 and EMMPRIN in THP-1 macrophages: Used real time RT-PCR and western blotting to examine if COX-2 and EMMPRIN mRNA and protein were up-regulated in THP-1 macrophages with AngII (10–6 mol/L) stimulation. Expression of COX-2 and EMMPRIN mRNA and protein increased significantly at 6 h compared with those expressions in the control group, and both reached a maximum at 12 h. After 24-h treatment, mRNA and protein levels declined with the levels in the 12-h group. The trends in the changes of expression of EMMPRIN mRNA and protein were in agreement with that of COX-2.4. Effects of AngII on PGE2 production: To examine the effect of AngII on PGE2 production in THP-1 macrophages, cells were stimulated with AngII (10–6 mol/L) alone. Treatment of cells with AngII (10-6mol/L) for 6 h led to a rapid increase in PGE2 secretion, and this induction reached a peak at 12 h.5. Losartan inhibits AngII-induced expression of COX-2 and EMMPRIN in THP-1 macrophages: To determine if the type-1 angiotensin II receptor (AT1R) or the type-2 angiotensin II receptor (AT2R) are involved in the AngII-induced up-regulation of expression of COX-2 and EMMPRIN, losartan (AT1R antagonist) and PD123319 (AT2R antagonist) were used to inhibit the effect of AngII. After pretreatment for 1 h with losartan (10–5 mol/L) and PD123319 (10–5 mol/L), THP-1 macrophages were stimulated with AngII (10–6 mol/L) for an additional 12 h. Levels of COX-2 and EMMPRIN mRNA were measured by real-time RT-PCR and protein expression measured by western blotting.Losartan could effectively inhibit the expression of COX-2 and EMMPRIN, but PD123319 could not . This result was in accordance with that of Cipollone. This result suggested that AngII-induced up-regulation of expression of COX-2 and EMMPRIN via the AT1R pathway, and that up-regulation of expression of EMMPRIN may be relevant to COX2 enhancement in THP-1 macrophages.6. AngII induces EMMPRIN expression via the AT1/COX-2/PGE2 pathway: In this experiment, after pretreatment for 1 h with losartan (10–5 mol/L) and NS-398 (10–5 mol/L), THP-1 macrophages were stimulated with AngII (10–6 mol/L) or AngII and PGE2 (10–7 mol/L) for an additional 12 h. The PGE2 level was increased by AngII and decreased by Losartan and NS-398. Interestingly, when Losartan or NS-398 were co-pretreated with PGE2, significant reversal of the inhibitory effects of losartan or NS-398 on AngII-induced EMMPRIN expression were observed. From the results stated above, we inferred that the expression of EMMPRIN in THP-1 cells increased due to an increase in the expression of PGE2, whereas losartan or NS-398 suppressed the expression of PGE2. After adding PGE2, the inhibition was overturned. We therefore hypothesized that PGE2 restored the levels of EMMPRIN inhibited by AT1 antagonists or COX-2 inhibitors. In addition, expression of the COX-2 gene and protein induced by AngII could also be inhibited by Losartan. These observations indicated that AngII up-regulated the expression of EMMPRIN via the AT1/COX-2/PGE2 signal pathway.Conclusion1.AngII could induce the COX-2,PGE2,EMMPRIN expression obviously in macrophages.2. The enhancement of EMMPRIN through the AT1R and inhibition by an AT1R antagonist.3. The effect of the expression of angiotensin II on extracellular matrix metalloproteinase inducer (EMMPRIN) in macrophages is mediated via the AT1/COX-2/PGE2 pathway.
|
PDF Full Text Request