Renin - Angiotensin System On THP-1 Derived Macrophages ATP Binding Cassette Transporter Expression And Its Mechanism Study

Background:The rennin-angiotensin-aldosterone system (RAS) plays a key role in the development and pathophysiology of hypertension and cardiovascular disease (CVD). Angiotensin (Ang) Ⅱ is a potent vasoactive peptide and the main effector of the RAS. Besides its hemodynamic effect, it can also act as a proinflammatory factor, inducing the expression of many inflammatory and adherence factors, leading to vascular injury and AS. Formation of macrophage derived foam cell plays a key role in the development and progression of artherosclerosis. In vitro, AngⅡ is now proved to be able to promote foam cell formation through multiple mechanisms, including: enhancing macrophages’uptake of ox-LDL, down-regulating the expression of ABCA1, up-regulating the expression of ACAT1. Application of angiotensin type1blocker (ARB) can reverse this effect of AngⅡ. However, detailed mechanism and signal pathway involved in this effect is still little known.ARB is one of the most frequently used first-line anti-hypertension drugs, with well documented anti-hypertension effect and many other benefits beyond blood pressure control. And its physiological functions are mainly mediated by blockade of AT1receptors, together with compensatory overstimulation of AT2receptors as angiotensin Ⅱ levels rise during chronic AT1blockade. Recently, it was reported that telmisartan and irbesartan, besides their AT1receptor blocking action, have the capacity to activate PPARγ, and might have an important role to play in the prevention and treatment of the metabolic syndrome, diabetes and atherosclerosis. Considering that PPARγ/LXRα is one of the most essential signal pathways that can up-regulate macrophages’ABCA1expression, here raise the question:do angiotensin Ⅱ receptor blockers with or without PPARy agonist action bear different mechanisms in reversing angiotensin Ⅱ’s down-regulation of ABCA1?So, the aim of this study is to investigate the mechanism and signal pathway involved in AngⅡ suppressing the ABCA1expression of macrophage. Then, we investigate whether the two kinds of angiotensin Ⅱ receptor blockers have different mechanisms in reversing angiotensin Ⅱ’s down-regulation of ABCA1.Objectives:1. To investigate the effect of AngⅡ on ABCG1expression of macrophages; to confirm the suppression effect of AngⅡ on the expression of ABCA1in macrophage, and to find whether there is a time-and dose-dependent manner in this effect.2. To investigate the mechanism and signal pathway involved in this suppression effect of AngII on ABCA1expression of macrophages.3. Under high level of angiotensin II, to investigate whether the two kinds of angiotensin II receptor blockers have different mechanisms in reversing angiotensin II’s down-regulation of ABCA1.Methods:1. The THP-1cell lines are cultured in RPMI1640supplemented with10%fetal bovine serum.2. The monocytes are differentiated into macrophages using PMA. And we use anti-CD68antibody to identify macrophages immunohistochemistrically.3. In vitro, we incubated macrophages with different concentrations of AngII for different durations. Then we use real-time PCR and Western Blot to detect mRNA and protein expression level of ABCA1and ABCG1respectively.4. We pre-incubated macrophages with TPCK (NF-κB antagonist), valsartan(ATlR blocker), PD123319(AT2R blocker) or both of the last two reagents, then detect mRNA and protein expression level of ABCA1by real-time PCR and Western Blot.5. We pre-incubated macrophages with telmisartan, pioglitazone, valsartan and GW9662(PPARy antagonist), then detect mRNA and protein expression level of ABCA1by real-time PCR and Western Blot.6. Statistic analysis—Data for all experiments were analyzed using the SPSS17.0software program. Comparisons between groups were performed using ANOVA methods. Data are graphically represented as mean±S.E.Results:1. After incubated with PMA, THP-1cells differentiated into macrophages, which are CD68positive.2. Incubation of different concentrations (0.01μM,0.1μM and1μM) of AngII and incubation of different durations (24h,36h and48h) of1μM AngII could all remarkably suppress macrophages’s ABCA1expression both in mRNA and protein levels(p<0.01). And the largest suppression effect was achieved by either the highest concentration or the longest duration. No effect by AngII on ABCG1expression was observed (p>0.05).3. No effect on ABCA1expression was observed by PD123319pre-incubation. However, pre-incubation of valsartan could remarkably reverse the suppression effect of AngII on ABCA1expression in macrophage(p<0.05vs. AngII1μM), and so did TPCK on mRNA level. However, on protein level, TPCK showed the trend to reverse the suppression effect of AngII on ABCAl expression, but there was no statistically significance (p=0.088vs. AngII1μM). Pre-incubation of both valsartan and PD123319could also recover the expression level of ABCA1(p<0.05vs. AngII1μM), and the degree of recovery was of no significant difference compared with single AT1receptor block.4. Pre-incubation of telmisartan, valsartan and pioglitazone could all remarkably reverse the suppression effect of AngII on ABCA1expression in macrophage. Based on these treatment, pre-incubation of GW9662could remarkably suppress the expression recover acquired by pioglitazone, but had no effect on either telmisartan lOμM, or valsartan lOμM.Conclusion:1. In macrophages, AngII could remarkably suppress ABCA1expression both in mRNA and protein levels, and a time-and dose-dependent manner of suppression seemed existed. There was no effect on ABCG1expression by AngII.2. In macrophages, AngII suppressed ABCA1expression through AT1receptor and NF-κB signal pathway.3. Angiotensin II receptor blockers could remarkably reverse the suppression effect of AngII on ABCA1expression in macrophages, predominantly by its AT1receptor blocking action, even though a PPARy agonist action exists.