A Study Of The Inhibitory Effects Of Benazepril On Abdominal Aortic Aneurysm Formation In A Rabbit Model

BackgroundAbdominal aortic aneurysm(AAA) is the most common arterial degenerative disease that threatens human life.There is a new understanding of the pathology and pathophysiology for this disease recent years.The traditional atherosclerosis theory could not elucidate the main characters of AAA.Now it is admitted that AAA involves a complicated degenerative process induced by several factors,which is characterised by chronic inflammatory infiltration and extracellular matrix remodeling.The AAA treatments involved open surgical treatment and newly endovascular repair.However,it is considerd that AAA needs no treatment until the aorta diameter reached the threshold of 5.5 cm,which is a guide line that AAA rupture becomes more frequent.Yet there is no effective methods to forestall small AAA development,patients could do nothing but wait for surgical or endovascular treatment.Now the research hotspot all over the world is to find effective drugs that could delay or even block AAA development aiming at interfering with pathophysiological process.Renin-angiotensin system inhibitors(RAS) such as Angiotensin-converting enzyme inhibitors(ACE-I) and Angiotensin receptor blockers(ARB) have been applied to various heart diseases and vascular diseases with good results.They can not only control blood pressure but also directly inhibit the remodelling process in the diseases.An large retrospective research (n=15326) made in Canadian found that patients ever took ACE-I suffered a marked decreased rate of AAA rupture ,whileβ-receptor blockers, Calcium channel blockers,α-receptor blockers, Diuretic agents and ARB have no such effects.Before the retrospective research it was found in some animal experiments that ACE-I could inhibit AAA developments without changing blood pressure ,yet ARB did not have the effect. There is still no further investigation on the mechanism how could ACE-I inhibit AAA formation.In our study we expect to establish an animal AAA model,and further investigate the effects and mechanisms of ACE-I towards AAA formation according to the main pathophysiological process.Our study may help supply new idea and theory of AAA drug therapy. This article is composed of two parts.Part one A study on establishing experimental models of infrarenal abdominal aortic aneurysm in rabbitsObjective we aim to establish an infra-renal AAA on rabbits that is characterized by facility, stabilization and similar pathological process to human AAA.Methods Thirty-two healthy male rabbits were randomly divided into four equal groups:two control groups:saline perfusion group that was killed two days after operation(group A) and seven days after operation(group B),and two experimental group: elastase perfusion group that was killed two days after operation(group C) and seven days after operation(group D). Rabbits were underwent perfusion of infra-renal aorta with porcine pancreatic elastase or saline respectively. Surviving rabbits were underwent a second laparotomy according to groups.The diameter of the aorta was measured, and the aortic tissue was excised for histologic examination. MMP-9 was detected by immunohistology.Results 1.The aortic diameters of each group dilate to approximately 150% after 30 minutes perfusion (Kruskal-Wallis H test, P=0.114)2.There are six rabbits survival in group D and all the aortic diameter exhibit more than 100% dilation.The rate of AAA formation is 100% and the successful rate is 75%.The aortic diameter of the other three groups could not reach AAA standard(exceed 100% dilation). (Mann-Whitney U test, P<0.01)3.The AAAs exhibit a rough arterial wall,intima losing normal smooth structure and obvious thrombosis attached to arterial wall.There is a great amount of inflammatory cells across aortic wall under microscope,accompanied by degeneration and rupture of elastic fibre.Two elastase perfusion groups exhibit enhanced MMP-9 expression compared to two saline perfusion groups respectively (P<0.01) .Group D is stronger than Group B in MMP-9 expression (P<0.01) .Conclusion Using elastase perfusion to establish the model of abdominal aortic aneurysm in rabbit required no micro-operation.The operation was simplified and the time needed for operation was shortened along with a higher successful rate. This model exhibited several histologic similarities with human abdominal aortic aneurysm.Part two A Study of the Inhibitory Effects of Benazepril on abdominal aortic aneurysm formation in a rabbit modelObjective we observe if benazepril influences AAA formation and investigate how it reacts with Nuclear factor-κB (NF-κB ) which induces chronic inflammatory infiltration and Matrix metalloproteinases-9 (MMP-9) which induces extracellular matrix degeneration. We expect to elucidate the mechanism how could ACE-I inhibit AAA formation. Methods Forty-two healthy male rabbits were randomly divided into six groups:the A to D group is the same to described above. Benazepril-treated elastase perfusion group that was killed two days after operation(group E) and seven days after operation(group F). Surviving rabbits were underwent a second laparotomy according to groups.The diameter of the aorta was measured, and the aortic tissue was excised for histologic examination.The amount of elastic fibre in aorta was caculated using analytic programme system. Total protein of MMP-9,plasma protein and nuclear protein of NF-κB were measured by Western Blot test. MMP-9 mRNA of aorta was detected by RT-PCR. MMP-9 protein activity was detected by Gelatin zymography. Serum IL-6 was measured by Elisa method.Results 1 .The aortic diameters of the three groups killed two days after perfusion all turned back.The average dilation rate of the three groups were 19.18%,29.58%and 24.94% respectively (Kruskal-Wallis H test, P=0.065) Diameters of group B turned back nearly to nomal leval.Group D all form AAA,while the dilation rate of group F using benazepril was only 39.20%,showing obvious inbitiory effect of AAA formation.2.After benazepril intervention the aortic wall exhibited fewer inflammatory cells infiltration.The elastic fibre content conserved better in group F than in group D (P<0.01) .3.Two saline perfusion groups had no MMP-9 protein and mRNA expression. Group C showed a high level of MMP-9 expression,and in group D it was much stronger than in group C(P<0.01). Benazepril-treated elastase perfusion groups showed weakened MMP-9 protein and mRNA expression than elastase perfusion groups (P<0.01) .The difference of MMP-9 activity in each group was similar to protein and mRNA expression.4.The plasma protein of NF-κB p65 in saline perfusion groups maintained a low level while the nuclear protein of NF-κB p65 could be hardly detected.In two elastase perfusion groups plasma protein expression of NF-κB p65 enhanced and nuclear protein expression obviously strengthened.But comparison between the two elastase perfusion groups was insignificant (plasma: P=0.090, nuclear: P=0.092). In two benazepril-treated groups the result was reversed compared to elastase perfusion groups.5.Serum IL-6 content of group C was 2.4 times higher than group A,while group D was approximately 3.4 times higher than group B. Group E made a 39.8% decline of serum IL-6 content compared to group C,and the number was 66.5% between group F and D,which was near normal level.Conclusion Benazepril could obviously inhibit AAA formation in rabbit models.The mechanism may relate to inhibition of inflammation infiltration,down-regulation of extracellular matrix degeneration in multi levels and preservation of elastic fibre.