Angiotensin ? Accelerates Atherosclerosis In Ckd Apoe-deficient Mice And The Effects In Early And Late Inflammation

Background and ObjectiveAtherosclerosis is one kind of high incidence complications in patients who are suffering chronic kidney disease?CKD?on dialysis.It was confirmed that CKD accelerated atherosclerosis.Angiotensin II?Ang ??and nicotinamide adenine dinucleotide phosphate oxidases?NOXs?are both tightly associated with CKD and atherosclerosis.However,the reports are far from enough about the early stage of atherosclerosis and the connections with Ang ? and NOXs under the circumstances of CKD.With the application of losartan,one kind of Ang ? type 1 receptor,it assisted us to observe the the effects of Ang ? during the process of atherosclerosis.This research was aimed to take knowledge of the time-dependence of these changes about immune cells and inflammatory factors during this process.With the usage of Ang ? and human umbilical vascular endothelial cells?HUVECs?to explore the time-dependence and the mechanisms of signal pathways happened in this situation.Methods1.Experiments in vivo?1?CKD with atherosclerosis animal model was established by 5/6 nephectomy?5/6 Nx?on apoE-deficient mice.?2?Mice were divided into three groups randomly,sham-operated control group,5/6 Nx group,5/6 Nx with losartan gavage group.After two weeks of the whole procedure of model establishment finished,the mice in 5/6 Nx with losartan group were gavage with losartan under the dosage of 15mg/kg/d,and the rest of mice were given 1% carboxymethyl cellulose solution with the same volume.?3?Data were collected at four stages,before model establishment,after model establishment,early stage of atherosclerosis,late stage of atherosclerosis.Peripheral blood were collected from caudal vein,and detected the levels of Scr,BUN,Ang ?,neutrophil ratio,monocyte ratio,VCAM-1,CINC-1 and MCP-1.?4?After four weeks of gavage,half of the mice in each group were sacrificed for hearts and aortas harvest.Hearts were used to make sections of aortic roots with three aortic valves presents,and aortas were used for PCR and Western Blot experiments.The rest mice were sacrificed after sixteen weeks gavage by the ways mentioned above.?5?The atherosclerotic plaque in aortic roots were observed and detected by hematoxylin and eosin stain.Neutrophil and monocyte infiltration in atherosclerotic plaque were observed and detected by Ly6 G and CD68 antibodies stains.The expression of NOX1,NOX2 and NOX4 in aortic roots were also marked and stained by antibodies.Each aortic and plaque data were measured by software Image Pro Plus 6.0,the positive data were calculated through the ratio of positive area to aortic root vascular wall area.All the statistics were calculated by software SPSS 19.0.?6?PCR and Western Blot were used to detect the mRNA and protein expression of NOX1,NOX2 and NOX4 in mice aortas.All the statistics were calculated by software SPSS 19.0.2.Experiments in vitro?1?Screen out one appropriate working concentration of Ang ? for next experiments by CCK-8.?2?Ang ? stimulates HUVECs for 10?20?30?40?50?60min and 1?2?4?8?12?24h.Two fluorescent probes,DHE and DCFH-DA were applied to detect the ROS production.Real-time PCR and Western Blot were applied to detect the mRNA and protein expression of NOX1,NOX2 and NOX4.Western Blot was used to detect the protein expression of phosphorated p38,ERK1/2 and JNK.The supernatants of HUVECs stimulated by Ang ? for different hour time points were collected to measure the concentrations of IL-8,MCP-1,TNF-? and IL-6 by ELISA.?3?With the application of apocynin,we explored the mechanisms and signal pathways participated in the expression of NOXs and inflammatory factors after the stimulation of Ang ? on HUVECs.Results1.Results in vivo1.1 General parametersThe levels of Scr,BUN and Ang ? increased after 5/6 nephrectomy?p<0.05?,losartan decreased these levels mentioned above?p<0.01?.1.2 Immuno cell ratios in PBMCs and the levels of the chemokines?1?The ratio of neutrophil in PBMCs and the level of the neutrophil chemokine,CINC-1,both increased high one shot at the early stage of atherosclerosis?p<0.01?.At the late stage of atherosclerosis,neutrophil ratio and CINC-1 levels dropped,the level of 5/6Nx group were even lower than the control group and 5/6 Nx with losartan group?p<0.05?.?2?5/6Nx increased the ratio of monocyte in PBMCs and the levels of monocyte chemokine,MCP-1,both increased gradually,reached to the top levels at the late stage of atherosclerosis?p<0.05?.1.3 The results detected on mice aortas?1?The atherosclerotic plaque was observed at the mice aortic roots by hematoxylin and eosin stain.The area of atherosclerotic plaque in 5/6 Nx group was boarder than the control group.Losartan taken could partly control the atherosclerotic plaque growing in 5/6Nx group mice.?2?5/6Nx helped activating the expression of NOX1,NOX2 and NOX4 in the aortic roots and aortas at the early stage of atherosclerosis.Losartan taken for four weeks could partly control the expression of NOX1 and NOX2 in aortas,but failed the control effects on the expression of NOX4 in aortas?p<0.05?.?3?The expressions of NOX1 and NOX2 in 5/6Nx aortas were still higher than the control group at the late stage of atherosclerosis.Sixteen weeks of losartan gavage for 5/6Nx still partly inhibited the effect on the expression of NOX1,but lost the effect on NOX2 expression control.The expression of NOX4 in three groups had no difference at the late stage of atherosclerosis?p<0.05?.2.Results in vitro2.1 The early and late inflammatory expression of NOXs,ROS and MAPK signal pathways after HUVECs stimulated by Ang ??1?The mRNA and protein expression of NOX1 and NOX2 reached to the top at 30 min and 40 min,but NOX4 reached to the top at 20 min and 30 min after Ang ? working on HUVECs for 10,20,30,40,50 and 60 min respectively?p<0.05?.The mRNA and protein expression of NOX1 and NOX2 reached to the top at 8h and NOX4 reached to the top at 4h after Ang ? working on HUVECs for 1,2,4,8,12 and 24 h respectively?p<0.05?.?2?O2·-expressed most at 30 and 40 min,and H₂O₂ expressed most at 20 and 30 min after Ang ? working on HUVECs for 10,20,30,40,50 and 60 min respectively?p<0.05?.However,the expression of two kinds of ROS had no significant difference after HUVECs were stimulated by Ang ? for 1,2,4,8,12 and 24 h respectively?p>0.05?.?3?The expression of phosphorated p38,ERK1/2 and JNK reached to the highest levels at 30-40 min,30min,20-30 min while Ang ? was working on HUVECs for 10,20,30,40,50 and 60 min respectively?p<0.05?.The highest protein expression points of phosphorated p38,ERK1/2 and JNK were 8h,8h and 4h respectively when HUVECs were activated by Ang ? for 1,2,4,8,12 and 24 h separately?p<0.05?.?4?4h of Ang ? stimulated on HUVECs made them produced these inflammatory factors most?p<0.001?.?5?With the application of apocynin,we found that H₂O₂ was produced mainly by NOX4 through JNK pathway,and O2·-was produced mainly by NOX1 and NOX2 through p38 and ERK1/2 pathways.Conclusions1.5/6Nx accelerated the formation of atherosclerosis in the aortas in ApoE^-/- mice,and assistant losartan intaken partly control the speed of plaue formation and the area of plaque.2.At the early stage of atherosclerosis,losartan partly control the levels of Ang ?,VCAM-1,CINC-1,the ratio of neutrophil,NOX1 and NOX2 expressions in aortas.3.The most important factor on accelerating and aggravating atherosclerosis at the late stage of atherosclerosis was not the level of Ang ?.4.Ang ? activated HUVECs expressed NOX1,NOX2 and NOX4 through p38,ERK1/2 and JNK signal pathways both at the early and late inflammatory phases.NOX4 was mainly activated through JNK pathway.NOX1 and NOX2 were mainly activated through p38 and ERK1/2 pathways.5.The expression of H₂O₂ was mainly based on NOX4 activation.The expression of O2·-was mainly based on NOX1 and NOX2 activation.The expression of H₂O₂ and O2·-acted as the second messangers for the activation of late inflammatory reaction.6.The expression of inflammatory factors was the effect of late inflammatory reaction.