UCP-2 Is Involved In Angiotensin-?-Induced Abdominal Aortic Aneurysm In Apolipoprotein E-knockout Mice

objective: The present study suggests that AAA formation mechanism including inflammation,matrix degradation and remodeling have a certain degree of relationship with ROS,mitochondrial uncoupling protein?UCPs?is considered as antioxidants,anion carriers located in the mitochondrial inner membrane,in a number of organizations have expressed,to prevent oxidative damage by maintaining the balance of active oxygen,UCP-2 shows an important role in modulating of mitochondrial membrane potential.Besides,UCP-2 has been well-established as an apoptosis suppressor in different cell systems while vascular smooth muscle cells apoptosis has been documented in the aortic aneurysms and dissections Thus,we supposed that UCP-2 could been a critical factor in preventing AAA formation via anti-oxidants and anti-apoptosis.To determine this hypothesis,a UCP-2 and apolipoprotein E?apoE?double-knockout mice was used to determine the effect of UCP-2 to the pathology of AAA.Methods :1?The UCP-2^-/-ApoE^-/-mice were generated by crossbreeding UCP-2 null mice with ApoE null mice.The genotyping in the knockout mice was verified by PCR.And DNA samples were obtained from tails of the mice.2?Groups: Wild mice?WT??Single gene knockout mice(ApoE^-/-)?Double knockout mice(UCP-2^-/-ApoE^-/-).3?Abdominal aortic aneurysm modeling:the AAA of UCP-2+/+ApoE^-/-or UCP-2^-/-ApoE^-/-mice was induced by chronic infusion of 1000 ng/kg/min angiotensin ??Ang-?,sigma,St.Louis,USA?via mini-osmotic pumps?Model 2004,Durect,Cupertino,CA?in 8-week-older mice for 4 weeks.4?q RT PCR and Western blotting were used to detect the expression of UCP2 mRNA and protein in abdominal aorta of WT,ApoE^-/-?ApoE^-/-+ Ang-?.5 ? After deparaffinizing and rehydrating by xylene and different concentration ethanol,the sections were stained with hematoxylin and eosin?H&E?under standard protocol,or treated with rabbit anti-?-SMA antibody for immunohistochemistry.Moreover,the studies applied the Verhoeff-Van Gieson?VVG?stain for elastin measurement.6 ? Western blotting was used to detect the expression of MMP2 and MMP9 in abdominal aortic aneurysm.7?The ROS level was also evaluated with the dihydroethidium?DHE,Beyotime,China?staining.The frozen sections of aortic tissue were stained with DHE for 20 min and observed with a fluorescence microscope at excitation wave-lengths of 490 nm and emission of 590 nm.The fluorescence intensity of ROS was quantified by Image J?NIH,Bethesda,MD?.the level of oxidative stress was determined via measurement of NADPH oxidase activity,malondialdehyde?MDA?concentration and SOD activity.The tissue homogenates were used to detect NADPH oxidase activity withdiphenyleneiodonium?DPI?,detect MDA with thiobarbituric acid?TBA?and detect SOD with nitroblue tetrazolium?NBT?.8 ? Detection of apoptosis cells of abdominal aortic aneurysm by TUNEL during apoptosis,activates the enzyme to some DNA genomic DNA fracture,exposed 3 '-OH in terminal deoxynucleotidyl transferase?TdT?catalyzed with green fluorescent probe fluorescein?FITC?labeled dUTP,detected by fluorescence microscopy;Caspase-3?caspase 3?is a key enzyme involved in the execution of apoptosis,which is activated in the early stage of apoptosis,and breaks down the corresponding nuclear substrate,leading to apoptosis.The expression of caspase 3 protein was detected by Western blot;Results:1?The data found that the UCP-2 protein and mRNA expression were significantly higher in Ang-?-induced AAA of mice,indicating that the changes of UCP-2 expression in AAA occurred at both post-translational and transcriptional levels.2 ? The incident rate of AAA in UCP-2^-/-ApoE^-/-mice after Ang-?treatment was 83.9%?26 of 31?,higher than the rate in the UCP-2+/+ApoE^-/-mice?55 %,11 of 20?.And the aortic expansion of UCP-2^-/-ApoE^-/-mice was significantly increased as compared with the UCP-2+/+ApoE^-/-mice.The histological studies showed the medial hypertrophy,fragmentation of elastic lamellas and depletion of ?-SMA in abdominal aortafrom UCP-2^-/-ApoE^-/-mice3?The superoxide production,determined by fluorescent dye DHE staining,was increased in aorta from UCP-2^-/-ApoE^-/-mice.While the NADPH oxidase activity and level of MDA was significantly higher in UCP-2^-/-ApoE^-/-mice than UCP-2+/+ApoE^-/-or WT mice.Besides,the SOD activity is increased in UCP-2+/+ApoE^-/-mice as compared with WT mice,whereas deficiency of UCP-2 decreased the increasing SOD activity in Ang-? treated ApoE^-/-mice,our data found a protective effect of UCP-2 against Ang-? induced oxidative stress and AAA.our results also showed UCP-2 knockout up-regulated the MMP2 and MMP9 expression in aortic aneurysm.4?further studies checked the apoptosis by TUNEL assay,and the result found that Ang-?induced apoptosis of VSMCs was increased in UCP-2^-/-ApoE^-/-mice.And the effect of UCP-2 in prevention of apoptotic VSMC death was further evident in the immunoblot of caspase 3,a key enzyme involved in execution of apoptosis.Clearly,the cleaved and active form caspase-3 was significantly increased in vascular tissue from UCP-2^-/-ApoE^-/-mice.Conclusion: our studies showed that UCP-2 protein and mRNA expression were significantly higher in Ang-?-induced AAA of mice,and deficiency of UCP-2 increased susceptivity and severity of AAA via elevated ROS level and VSMCs apoptosis,indicating UCP-2 could been an anti-oxidants and anti-apoptosis factor in preventing AAA.