Roleof AMPK Signal Pathway In The Pathogenesis Of Abdominal Aortic Aneurysms

BackgroundAAA(Abdominal Aortic Aneurysm)is a kind of aortic degenerative disease which is fairly common in the elderly male population,with estimated prevalence of 5%to 10%in men older than 65 years.The incidence and severity of aneurysms increases with increasing age.AAA is potentially life-threatening with over 90%mortality after rupture.In general,AAA is a common,significant and potentially life-threatening medical problem nowadays.Since rupture is the major cause of death in AAA patients,the management of AAA focuses on the evaluation and prevention of rupture.Open surgery and endovascular aneurysm repair remained to be the only two established managements of AAA in high risk patients.These two strategies were recommended when the maximal aortic diameter was greater than 5 cm.On the other hand,systematical follow up is strongly recommended to the patients with maximal aortic diameter smaller than 5 cm.However,previous studies showed that AAA will grow larger and eventually need OR EVAR in nearly 60%follow-up small AAA patients.Therefore,pharmacological therapies to retard the growth of aneurysm were assumed to be the ideal management of small AAAs.Although many studies have been carried out to investigate the pathogenesis of AAA,effective pharmacological therapies were still not available.In this study,we aimed at the pathogenesis of AAA and trying to find out potential therapeutic target to modify the progression of AAA.Infiltration of inflammatory cells,neovascularization and elastic degradation are the main pathological features of aneurysm.Chronic inflammation plays a central and consistent role in the AAA development.Exuberant adventitial inflammatory infiltrate is the most prominent feature of the aneurysmal wall,and macrophages,T cells and neutrophils are the major inflammatory cells in the adventitia.These inflammatory cells secrete various cytokines,including interleukin-1?(IL-1?),monocyte chemoattractantprotein-1(MCP-1),interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a).These cytokines can incur tissue damage and recruit other inflammatory cells.Macrophages can secrete matrix metalloproteinases(MMPs),majorly MMP-2 and MMP-9,degrading the elastic fiber of aortic wall.The degradation of elastic fiber is another important feature of aneurysm what distinguishes this disease from other arterial pathologies.The impaired tensile strength of aortic wall leads to the progressively enlargement of aortic lumen under the pulsive blood flow.On the other hand,macrophages can secrete vascular growth factors,such as the vascular endothelium growth factor A(VEGFA),promoting neovascularization in the aneurysmal wall.On the other hand,neovascularization and elastic degradation can facilitate macrophage's infiltration into the AAA portion.These pathological changes are interdependent and of vital importance in the formation and progression of AAAs.AMPK(Adenosine monophosphate-activated protein kinase)is a member of a metabolite-sensing protein kinase family,which coordinates multiple metabolic pathways to balance energy demand and supply.AMPK is a trimer consisting of one catalytic subunit(a)and two regulatory subunits(? and ?).In mammals,each subunit has multiple isoforms and expresses in different tissues.For example,there are two isoforms(al and a2)of catabolic ? subunit,whereas two(?1 and ?2)and three(?1,y2 and y3)were present in ? and ? subunit respectively.Activation of AMPK attenuates anabolic processes such as the synthesis of proteins,fatty acids and cholesterol,and stimulates ATP(Adenosine Triphosphate)generating catabolic pathways such as respiration of glucose and fatty acids.Objective:Part I:Evaluate the activity of AMPK signal pathway in the AAA patients and investigate the relationship between the activity of AMPK signal pathway and aneurysmal size and inflammatory factors.Part II:Investigate the activity of AMPK signal pathway in the formation of Angiotensin II(Ang ?)infusion model.We also try to use AMPK specific activators and inhibitors to delineate the role and effect of AMPK signal pathway in the pathogenesis of aneurysm.In the end,the mechanism and signal pathways were discussed in this portion.Method:Part ?:1.Patients with aortitis,connective tissue disorders,or ruptured aneurysm were excluded.During surgery,aortic tissues were collected from the largest portion of the aneurysm,which were routinely excised and discarded during repair.Control abdominal aortic tissue was collected from 8 age-matched organ donors w.ithout aortic aneurysm,dissection,coarctation or previous aortic repair in several transplantation center of Shandong Province.2.Western Blot technique was used to evaluated the activity of AMPK signal pathways in AAA tissue and common aortic tissue.Immunohistological staining(IHC)was used to localize the activated AMPK(P-AMPK)in AAA tissue and control aortic tissue.3.RT-PCR was used to detect the expression of IL-1?,MCP-1 in AAA tissue and control aortic tissue.Spearson regression analysis was used to evaluate the correlation between the activity of AMPK.signal pathway and the expression of inflammatory cytokines.Part ?:1.Ang ? infusion model was used in this study.Briefly,mice were implanted with Alzet osmotic minipumps(Model 1004,Durect Corporation),filled with Ang II solutions(Abcam,Cambridge,United Kingdom.Ab 120183,1,000 ng/kg/min).The infusion persisted for up to 4 weeks.At 1d,3d,7d,14d,28d,the aortic diameter was examined by ultrasound examination.At 28 days,mice were sacrificed for image acquisition and tissue harvest.All procedures were approved by the Animal Care and Use Committee of Shandong Provincial Hospital and were conducted following the institutional guidelines.2.To evaluate the relationship between AMPK activation and AAA formation,both AMPK activator and inhibitor were used.AICAR(5-aminoimidazole-4-carboxamide-1-p-d-ribofuranoside)was used as AMPK activator and Compound C was used as AMPK inhibitor.Mice were divided into four groups(n=18 in each group),one group was used as normal control(Sham group).Rest AAA mice were randomly given AICAR(500 mg/kg,i.p.q.d.AAA+ACIAR group),Compound C(300 mg/kg,i.p.q.d.AAA+C.C group)or equal volume of PBS(Phosphate Buffer solution,AAA group).These injections were started once the implantation of pumps and persisted 28 days.At 28 days,mice were sacrificed for image acquisition and tissue harvest.3.Mice specimen were evaluated by HE staining,EVG staining,RT-PCR,Western Blot and gelatin zymography.The pathological feature,inflammation,and MMPs'activity were evaluated.ResultsPart ?:1.Pathological features of AAA patients.HE staining revealed destruction of the media and thickening of the adventitia in the AAA group compared with the normal aortic tissue.EVG staining showed the disrupted elastic lamellae in the AAA tissue in contrast to the wavy appearance in the normal aortic tissue.As chronic inflammation of the aortic wall is a main feature of AAA,we examined the expression of proinflammatory cytokines,such as IL-1p,IL-6,MCP-1 and TNF-?,using the RT-PCR method.Expression of these cytokines in the AAA patients was significantly higher than those in the control aorta.The abundance of zymographic active form of MMP-2 and MMP-9 was increased in AAA patients compared with control aorta.2.AMPK signal pathway in human AAA.Western blot analysis showed that there was no significant difference in total AMPKa protein levels between control aortic tissues and AAA tissues.On the other hand,phospho-AMPKa level was significantly lower in AAA tissues than in control aortic tissues,indicating the reduced activation of AMPKa signal pathway in human AAA.The expression of phospho-AMPKa was readily detectable in the media of control aorta,whereas it was barely detectable in the AAA tissues.3.The correlation of CIRP and AAA inflammation.Spearson correlation analysis revealed the activity of AMPK signal pathway was negatively correlated with the expression of TNF-a in the aortic wall.(P=0.0342 R=0.751)Part ?:1.Pathological features of Ang ? infusion mice model.Ang ? infusion persists for 4 weeks.During the infusion,10%AAA group mice died because of AAA rupture.The enlargement of suprarenal aorta can be seen in 75%mice in AAA group.Mice in the sham group,on the other hand,do not reveal the enlargement of suprarenal aorta.These results indicate the success in the mice AAA model.HE and EVG staining revealed destruction of the media elastic lamellae,thickening of the adventitia and infiltration of inflammatory cells in the AAA group compared with the sham group.2.Effect of AMPK signal pathway on the pathogenesis of AAA.The death rate,AAA incidence and maximal aortic diameter decreased dramatically after the injection of AICAR,the AMPK activator.Compound C,the AMPK inhibitor,on the other hand,decreased the death rate,AAA incidence and maximal aortic diameter in AAA mice.HE staining revealed destruction of the media and thickening of the adventitia in the AAA group compared with the sham group.These pathological features were aggravated in the AAA+C.C group and alleviated in the AAA+AICAR group.The relative contents of collagen and SMCs in the aortic wall were substantially increased in the AAA+AICAR group,whereas the content of macrophages and microvessels was decreased in the AAA+AICAR group.In contrast,these parameters showed only insignificant difference between the AAA group and AAA+C.C group.3.Effect AMPK signal pathway on the inflammation and signal proteins duringAAA formation.As chronic inflammation of the aortic wall is a main feature of AAA,we examined the expression of proinflammatory cytokines,such as IL(interleukin)-1?,IL-6,MCP(monocyte chemotactic protein)-1 and TNF(tumor necrosis factor)-a.Expression of these cytokines in the AAA+AICAR group was significantly lower than those in the AAA group.While in the AAA+C.C group,expression of those proinflammatory cytokines was slightly higher.Neovascularization is also of vital importance in the AAA progression.VEGFA(vascular endothelial growth factor A),Flt(fms related tyrosine kinase)-1 and CD31(cluster of differentiation31)are the major vascular growth factors in the AAA progression.PCR analysis revealed the expression of these cytokines were elevated after AAA induction and this elevation could be attenuated by AICAR.To verify the signaling proteins,we examined the expression level of AMPK,STAT-3 and NF-?B signal pathway.In AAA mice,Phospho-AMPK production was significantly decreased while NF-?B and Phospho-STAT-3 were increased compared with the sham group mice.AICAR group activate AMPK phosphorylation in AAA mice.AAA+AICAR group showed a lower level of Phospho-STAT-3 and NF-?B expression compared with AAA group.AAA+C.C group downregulate AMPK phosphorylation while upregulate the NF-?B expression and STAT-3 phosphorylation.Conclusion:1.AMPK signal pathway was inhibited in human AAA tissue.The activity of AMPK signal pathway was negatively correlated with the expression of aneurysmal TNF-?.2.AICAR,the specific AMPK signal activator,suppressed the Ang ? induced mice aneurysm.Compound C,the AMPK inhibitor,tends to aggravate the Ang II induced mice aneurysm.3.AMPK activation can alleviate the inflammation in the AAA.This effect was correlated with downregulating NF-?B and STAT-3 activation.Background:ILT(Intraluminal thrombus)was proven correlated with the enlargement of aneurysm.Hypoxia conditions caused by ILT(intraluminal thrombus)may stimulate smooth muscle cells and macrophages to secrete MMPs(matrix metalloproteinase),and contribute to the development of AAA.HIF-1(Hypoxia inducible factor-1)is a major transcriptional factor in the cellular response to hypoxia.HIF-1 is a heterodimer consisting of an unstable alpha subunit and a stable beta subunit.HIF-1?(hypoxia inducible factor-la)is rapidly degraded under normoxic conditions,but stabilized when oxygen supply is limited.Under hypoxia conditions,the activity of PHD-2(prolyl-hydroxylase-2),which is the main enzyme degrading HIF-1? was inhibited and HIF-1? accumulated in the tissue.However,growing evidence suggested that doses of HIF-1? protein could be modulated by under normoxia conditions.Ang ? and wall shear stress could stimulate HIF-1? expression in vascular smooth muscle cells under normoxia conditions.Ang II and wall shear stress play a vital important role in the pathogenesis of aneurysm.Studies of HIF-1? expression under normoxia conditions at the early phase of AAA induction was lack.In this study,we are aimed to clarify whether HIF-1? express at the early phase of aneurysm and how they affect the pathogenesis of aneurysm.In this study,HIF-la knockdown mice build by RNAi(RNA interference)technique was adopted.Objective:1.Build HIF-1? knockdown mice.2.Investigate whether HIF-1? knockdown retard the pathogenesis of abdominal aortic aneurysm in mice.3.Clarify the mechanism of HIF-1? knockdown on the the pathogenesis of abdominal aortic aneurysmMethod:1.Build lentivirus carrying the HIF-la shRNA.Three different HIF-1? shRNA were produced by the generated by Genomeditech Company(Shanghai,China).MOVAS transfection was carried out to evaluate the effect of HIF-la shRNA.The most effective sequences of murine/HIF-1? shRNA were:sense,5'-GCAGGAAUUGGAACAUUAU dTdT-3';antisense,3'-dTdTCGUCCUUAACCUUGUAAUA-5'.Lentivirus expressing scrambled shRNA used as control was also purchased from Genomeditech.2.Animal Experiment.Three groups of ApoE-/-mice were included:PBS infusion + scramble shRNA lentivirus(Sham group n=20),Ang ? infusion + scramble shRNA lentivirus[Con group(control-group),n=20],and Ang ? infusion+HIF-1? shRNA lentivirus(HIF-1? group,n=20).PBS or Ang ? was infused by ALZET mini osmotic pumps(Model 1004)as described above.Maximal aortic diameter was measured in all mice by ultrasound examination via ultrasound examination at 3d,7d,14d,28d after pump implantation.At 3 days and 28 days after implantation of pumps,murine HR(heart rate)and BP(blood pressure)were measured by tail-cuff method under conscious conditions.10 mice in each group were sacrificed at day 3;superenal aortic tissue was harvest for PCR,western blot and gelatin zymography analysis.Rest 10 mice in each group were sacrificed at day 28 for histological analysis.Results:1.HIF-1? knockdown mice were built by the RNAi technique.RT-PCR and Western Blot revealed that Ang ? could upregulate the HIF-1?mRNA and protein in the MOVAS under normoxia conditions.After lentivirus transfection,the HIF-la mRNA and protein was knockdown.After the implant of the osmotic pumps,an upregulated HIF-1? mRNA and protein was detected in the mice aorta.These over expression of HIF-1? mRNA and protein could also be knockdown after the injection of lentivirus carrying the HIF-1? shRNA.These in vivo and in vitro experiments showed that the HIF-la knockdow:n mice could be built by the lentivirus transfection method.2.Silencing of HIF-la gene attenuated Ang II-induced abdominal aortic aneurysm in ApoE-/-mice.The incidence and maximal aortic diameter was reduced after the HIF-la knockdown.HE stain showed the pathological features,such as the destruction of the media and thickening of the adventitia,were attenuated after the HIF-1? knockdown.The EVG staining showed preserved elastin after the injection of HIF-1? shRNA.The IHC staining showed a reduced microvessels and macrophages in the HIF-la knockdown group.The expression and activity of MMP-2 and MMP-9 were examined by the RT-PCR and Gelatin zymography experiment.These experiments showed the expression and activity of MMP-2 and MMP-9 were inhibited after the injection of HIF-la shRNA lentivirus.The RT-PCR also showed a reduced expression of proinflammatory cytokines such as IL-1,IL-6,MCP-1,TNF-a and VEGFA,CD31,FLT-1.As a result,HIF-1? knockdown attenuated Ang ?-induced abdominal aortic aneurysm in ApoE--/-mice.Conclusion:1.HIF-1? upregulation occurred at the early phase of AAA induction and independent of hypoxia conditions.2.HIF-la knockdown mice could be built by the RNAi technique.3.Silencing of HIF-1? knockdown could inhibit the inflammation and neovascularity in the aneurysm and inhibit AAA formation in Ang ? infusion model.