Amlodipine Down-regulates MCP-1 Expression In Human Umbilical Vein Endothelial Cells Induced By OxLDL And Inhibits THP-1 Monocyte Chemotaxis

BACGROUNDMitogen-activated protein kinase(MAPK) is serine threonine kinase that performs important functions as mediators of cellular responses to a variety of extracellular stimuli. P38MAPK subfamily, a more recently described member of the MAPK family, is strongly activated in response to proinflammatory cytokines such as tumor necrosis factor-a and oxidizelow density lipoprotein(oxLDL). Activation of p38MAPK cascades can trigger Inflammatory reaction, which has been implicated to play an important role in the development of atherosclerosis(As).It has been shown that monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte chemoattractant and largely responsible for the recruitment of monocytes/macrophages to the vessel wall in the early stage of atherogenesis. Studies have showed that the xepression of MCP-1 is significantly increased in atherosclerosis lesions.It has become evident that atherosclerosis is an inflammatory reaction processes, which are key components of atherosclerosis, from fatty streak formation to plaque rupture and thrombosis. Amlodipine is a long-acting dihydropyridine-type thirdgeneration calcium channel blocker(CCB), which is well tolerated and widely used in the treatment of hypertension. There is growing evidence that amlodipine limit the progression of arteriosclerosis in animals and humans. Although the mechanisms underlying the direct protective effects of calcium channel blockades on endothelial cell injury are not fully understood, such vasoprotective effects as the amelioration of endothelial dysfunction and decreases in the proliferation of vascular smooth muscle cells, oxidative stress, are at least in part independent of the blood pressure-lowering effect of the agent. The study is aimed to observe the effet of amlodipine on expression of MCP-1 and the THP-1 monocyte chemotaxis in human umbilical vein endothelial cell induced by oxLDL.OBJECTIVETo investigate the effects of amlodipine on the expression of MCP-1 in human umbilical vein endothelial cell by oxLDL and THP-1 chemotaxis, and explore their mechanism, which would provide experimental base for suppresseing the development of atherosclerosis.METHODS1. HUVEC-12 endothelial cells were treated with 0,25,50,100 mg/L oxLDL for 24 hours,and HUVEC-12 endothelial cells were treated with 50 mg/L oxLDL for 0, 3, 6, 12, 24h respectively. And then choose a proper concentration and time of oxLDL for treating HUVEC-12 endothelial cells through the determining MCP-1 mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR) and p-p38MAPK level by western blot respectively.2. HUVEC-12 endothelial cells were pretreated with amlodipine at different concentrations (0, 0.1, 1.0, 10.0μM) for 1h and then incubated with oxLDL at the same concentration(50mg/L) for 24h. P-p38MAPK level and MCP-1 mRNA expression were detected by RT-PCR and Western blot respectively. Expressoin of MCP-1 protein was determined by enzyme linked immunosorbent assay in medium.3. HUVEC-12 endothelial cells were pretreated with 10.0μM amlodipine for 1h and p38MAPK inhibitor SB203580 for 30mintues, P-p38MAPK level and MCP-1 mRNA expression were detected by Western blot and RT-PCR respectively. Expressoin of MCP-1 protein was determined by enzyme linked immunosorbent assay in medium.4. Treat HUVEC-12 endothelial cells in lower compartment of transwell by oxLDL in proper concentration for 24 hours. And then, put the THP-1 monocyte in upper compartment for 4 hours until counting the THP-1 cells in the lower compartment. Moreover, 10.0μM amlodipine and 20μM SB203580 was added in the lower compartment, and count the THP-1 monocyte in lower compartment to observe the chemotaxis[0]. RESULTS1. OxLDL strongly evoked phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner within 0-50mg/L for 3h and enhanced significantly expression of monocytes chemotactic protein-1 mRNA in a time-dependent manner within 0-24h in human umbilical vein endothelial cells, peaked at 50mg/L for 24h.2. MCP-1 expression in both protein and mRNA level and p-p38MAPK level induced by oxLDL were obviously up-regulated in a dose-dependent manner within 0.1-10μM for 24h, most effect at 10μM after pretreated HUVEC-12 endothelial cells with Amlodipine at different concentration for 1 h.3. Activation of p38 MAPK and expression of MCP-1 by oxLDL was significantly decreased by pretreatment with p38MAPK inhibitor SB203580(20uM) and was effectively reduced by calcium channel blocker amlodipine(10.0uM) no effect on normal endotheliocyte.4. The effect of THP-1 monocyte chemotaxis[0] to HUVEC-12 endothelial cells was also enhanced significantly by culturing the HUVEC-12 endothelial cells in lower compartment and treating it by oxLDL. When HUVEC-12 endothelial cells were pretreated with 10.0uM amlodipine and 20uM SB2035801, the effect of THP-1 monocyte chemotaxis was obviously attenuated.CONCLUSIONS1. OxLDL may evoke phosphorylation of p38MAPK and upregulate expression of MCP-1 mRNA in HUVEC-12 endothelial cell;2. Amlodipine may downregulate expression of MCP-1 induced by oxLDL in HUVEC-12 endothelial cell, which may be part through p38MAPK singal pathway;3. Amlodipine may inhibiter the chemotaxis[0] THP-1 monocyte to migrate to HUVEC-12 endothelial cells induced by oxLDL.