The Research Of Angiotensin Ⅱ Induces EMMPRIN Expression In THP-1 Macrophages Via The NF-κB Pathway

Background and Objective: Both clinical evidences and pathological researches have shown that the main cause of death in acute coronary Syndrome (ACS) is the rupture of an advanced atherosclerotic lesion. In this rupture event, the"culprit"coronary plaque,which is characterized as the pathological feature of vulnerable plaque, plays a vital role. Vulnerable plaque, also termed as thin cap fibroatheroma(TCFA),is recognized as a necrotic core with an overlying fibrous cap measuring <65μm and containing numerous macrophages at shoulder region in morphology.However, this culprit plaque can not rupture without any stimuli. Extant researches have revealed that it is the destruction of the arterial extracellular matrix (ECM) that contributes to the rupture. Metalloproteinases (MMPs), which is a family of more than 20+ related endopeptidases sharing a common Zn2+-based catalytic mechanism, is now considered as the main demolisher in plaque disruption. MMPs can completely degrade collagen and other ECM components, which is the major structure of the plaque. Galis has discovered a strong matrix-degrading activity and local MMPs high expression in the vulnerable shoulders of human atheroma. The results from MMPs knockout mice and from clinical study also found a strong relationship between MMPs level and plaque stability. MMPs are mainly secreted by macrophages in the atheroma, and it is regulated by many cytokines in the transcription level and is kept inactivating by being bound with its natural inhibitor tissue inhibitor of MMPs (TIMP) in the plaque. Yet, obscurities remain in the MMPs regulation.CD147 or basigin, a 57KD transmembrane glycoprotein, was first found expressing on the tumor cell membrane, which involves in tumor invasion in 1982. Afterwards, this novel protein was found to be able to induce the de novo synthesis of MMPs, and thereby was renamed as EMMPRIN(extracellular matrix metalloproteinase inducer). EMMPRIN was found to stimulate the production of many MMPs including MMP-1, MMP-2, MMP-3, MMP-9 on the oncology. Interestingly, EMMPRIN is shown to be expressed in human atheroma and is upregulated upon monocyte\marcophage differentiation which is the notable event in the atherosclerosis advancement. Emerging evidence has indicated that EMMPRIN participates in the plaque rupture by inducing the MMPs. EMMPRIN is mainly regulated in the transcription level; some redox-sensitive transcription factors as NF-κB, AP-1 and some intracellular proteins as p38 mitogen activated protein kinases ERK are shown involved.AngII (AngiotensinⅡ) is the critical component in the RAS (Renin-angiotensin system) which is widely considered possessing atherogenic properties, indeed, this very hormone participates in the whole atherosclerosis processes, not only in the pro-inflammation, angiogenesis, inducement of cells propagation and adhesion, but also involving in the final plaque rupture. It has been proved that angII is the key factor that modulates the express of MMPs. Our previous findings have suggested that the NF-κB is an important pathway in angII modulating MMPs.The aim of this study is to evaluate the effect of AngiotensinⅡon EMMPRIN expression in THP-1 macrophage and its potential mechanisms.Methods1. Human monocytic leukemia cell line, THP-1 were seeded into 6-well plate at a density of 1x106 cells/ml. Phorbol 12-myristate 13-acetate (PMA) was added to the culture for macrophage differentiation. After PMA treated for 48hours, Flow cytometry analysis was applied to identify the macrophage differentiation. AngII in dose(1*10-8 -7 -6 -5M) and time( 6 12 24h) courses stimulation was carried out, The effects of AT1Rand AT2R antagonist irbesartan(1*10-5M) and PD123319(1*10-5M),NF-κB inhibitor PDTC (1*10-5M) were also examined on EMMPRIN expression induced by angII on THP-1 macrophages by pre-incubated 30min before angII stimulation. EMMPRIN mRNA and protein expression were examined by RT-PCR and Western Blot.2. RNA interference (RNAi) targeted against NF-κB subunit P65 were used for NF-κB pathway study. SiRNA fragment were successful transfected into THP-1 macrophages. The transfection efficiency was measured by fluorescence microscope,and cells were collected after 48h for western blot and EMSA analysis to determine the RNAi efficiency.3. Conditioned medium was electrophoresed in a polyacrylamide gel containing 0.1% (w/v) gelatin for Gelatin zymography assay to examine the activity of MMP-9. Result1. Assessment of THP-1 macrophages after differentiated from THP-1 by PMATo identify the THP-1 macrophages, flow cytometry was applied. On the morphology, THP-1 cells are round and do not adhere on the plastic surface, after PMA treatment, THP-1 macrophages become flat and spreading. On the phenotype,THP-1 macrophages were shown highly CD14 expression on the surface.The Flow cytometry result showed after THP-1 induced by PMA 48hours,THP-1 macrophages expressed highly CD14 compared with the untreated group. The result demonstrated THP-1 macrophage is successful established.2. EMMPRIN expression was strongly induced by angII stimulated on THP-1 macrophagesTo determine the changes of EMMPRIN expression upon angII stimulated on THP-1 macrophages, angII treatment on dose and time courses tests were performed. Considering the effect of PMA on EMMPRIN expression, PMA was completely removed by washing for 3 times, and then THP-1 macrophages were cultured for additional hours to attenuate PMA effect.AngII at the four tested concentrations did not significantly produce cytotoxicity, MTT shows 95% cell vitality. Four doses angIItreatment groups were examined by RT-PCR and western bloting. AngII can induce EMMPRIN expression in dose-dependent manner. The common dose (1*10-6M) was applied in the subsequent experiments.Three time-courses of THP-1 macrophages with and without angII treatment were performed. RT-PCR and western blot showed EMMPRIN can be induced by angII treatment in 6 hours compared with the untreated group, this effect is persistence in 24hs.3. EMMPRIN expression induced by angII were effected by angII two types receptor blockersTo test the roles of AT1R and AT2R in EMMPRIN expression induced by angII, two respective antagonist irbesartan and PD123319 were added 30 minutes before angII stimulation. Datas from RT-PCR and western blot showed that it was AT1R, not AT2R, participating in the angII upregulating EMMPRIN expression.4. EMMPRIN expression induced by angII was NF-κB pathway dependentTo investigate whether NF-κB pathway is involving in angII upregulating EMMPRIN expression, two strategies intervening NF-κB pathway were used. Antioxidant NF-κB inhibitor PDTC and P65 RNAi were applied in the experiment.P65 RNAi model was successful established. Photos were taken by fluorescence microscope which was used to defined the transfection efficiency. Results showed siRNA fragments were successful transfected into THP-1 macrophages, the efficiency was >70%.The effect of P65 RNAi was examined by western blot. Result showed the expression of P65 is remarkable knockdown in the RNAi group. To rule out the effect of the transfection itself, cells were transfected with a control siRNA. There was no apparent effect of the control siRNA transfection on p65 expression.EMSA was applied to test the activity of transcription factor NF-κB. THP-1 macrophages treated by TNF-α(Tumor necrosis factor-α) was used as positive control, the results showed that angII can induce NF-κB activity in THP-1 macrophages, and this effect was restrained by RNAi-mediated knockdown of p65.The effect of NF-κB on EMMPRIN expression induced by angII was examined by RT-PCR and Western Blot. Data have shown when NF-κB pathway was inhibited by PDTC or P65 was knockdown, EMMPRIN expression induced by angII was notable suppressed.5. EMMPRIN expression is coincidence with the MMP-9 activity.Conditioned mediums of THP-1 macrophages treated in different groups (positive control, angII,angII+irbersatan,angII+PD123319, angII+PDTC,angII+P65RNAi,angII+ negative RNAi) were subjected to the Gelatin zymography assay. The activity of MMP-9 increased significantly in angII treated group comparing with the control group. This regulation can be suppressed by irbesartan, PDTC, P65RNAi treatment.MMP-9 activity is coincidence with EMMPRIN expression.Conclusion1. AngII can stimulate EMMPRIN expression in THP-1 marcophages via AT1R.2. NF-κB pathway plays an important role in this response.