Thalidomide Inhibits The Growth Of Experimental Abdominal Aortic Aneurysms In Rabbits

BackgroundAbdominal aortic aneurysm (AAA) is a common degenerative condition that causes progressive aortic expansion and eventual rupture with high mortality. For large AAAs (≥5.5cm in diameter) with an increased risk of rupture,current treatments are preventive open or endovascular repair to prevent rupture. However, risk of rupture is negligible in small AAAs with a diameter<5.5cm, and early open or endovascular repair does not offer a survival advantage for them, the current management recommendations for these patients usually propose interval measurements of aneurysm size until elective repair is indicated based on rapid expansion (≥1cm/year) or size criteria (≥5.5cm). Obviously, the management based on such a "watchful-waiting" approach might not be sufficient for small AAAs.A more proactive and non-invasive measure is cried for to inhibit or slow down the expansion and risk of rupture, which might be a significant advance for a subset of patients with small AAAs.The pathogenesis of AAAs represents a complex and multi-factorial process. Pathologically, AAAs are characterized by chronic aortic wall inflammation, destructive remodeling of the elastic media, neovascularization, and depletion of vascular smooth muscle cells (VSMC). Especially, AAA typically involves tissue inflammation as evidenced by the transmural infiltration of inflammatory cells mainly including macrophages and lymphocytes. On the one hand the infiltrating inflammatory cells secrete metalloproteinases (MMPs) and other proteases that degrade the aortic media and then weaken the aneurismal wall and subsequent thrombosis and AAA progression,on the other hand they release various inflammatory factors,such as tumor necrosis factor-a(TNF-a), interleukin-1β(IL-1β), interleukin-6(IL-6), interferon-y(IFN-y) and so on,that further promote the inflammation.Medial neovascularization and decreased vascular smooth muscle cells also characterize AAA lesions. The intraluminal thrombus may contribute to the process by causing a functional hypoxia at the luminal intima and inner media, thus inducing subsequent neovascularization and inflammation. Inflammatory cells in the thrombus also release active proteases such as matrix metalloproteinase (MMP)-9and urokinase-type plasminogen activator (u-PA).In view of the improved understanding above, pharmacological strategies on modulating the pathological processes may represent a potential means for the stabilization of small AAAs. More recently, some of pharmacological agents have demonstrated an effective suppression of induced AAA formation or expansion in experimental models,such as statins,angiotensin-converting enzyme inhibitors, matrix metalloproteinase inhibitors,c-Jun N terminal kinase(JNK) inhibitor, and so on.Thalidomide was introduced in the late1950's for morning sickness in pregnancy, but was withdrawn from the market in1961after its association with teratogenesis was documented.However,it has more recently demonstrated anti-inflammatory, anti-angiogenesis, and immunomodulatory properties with new clinical applications in oncology and the treatment of inflammatory disease. Recent evidence has shown that thalidomide is capable of inhibiting atherogenesis in apoE-deficient mice and rabbits with high lipid diet, presumably by inhibition of TNF-alpha secretion.As the properties of thalidomide might be expected to inhibit the progression of small AAAs, this study was focused on thalidomide influencing aneurysm growth in a rabbit elastase-induced AAA model.ObjectivesAnimal models are necessary to develop and test innovations in aneurysm therapy before clinical introduction.The establishment of animal model is the indispensable research approach to explore the pathogenesis and treatment of abdominal aortic aneurysm. To establish an elastase-induced rabbit abdominal aortic aneurysm model, we sought to examine the effect of thalidomide on the growth of AAA, and explain the mechanism.MethodsMale New Zealand white rabbits (2.0-2.5kg) were used.An isolated segment of rabbit abdominal aorta was infused with type I pancreatic elastase. The diameter of the infused aortic segment was measured using a micrometer pre-infusion and while collecting aorta samples. One week after infusion with elastase, dilatation of the aorta was confirmed by ultrasonography, and only rabbits that developed AAA were used in the next regression study. Aneurysm formation was defined as a50%increase in aortic diameter compared to the uninfused aorta just proximal to the infused segment.The rabbits that developed AAA were randomly divided into thalidomide treatment group (AAA/Tha, n=5) and model control group (AAA/Con, n=5), which received daily intragastric administration of thalidomide suspension (100mg/kg/day in5ml of normal saline) and the same volume of normal saline alone respectively from1week until they were sacrificed at4weeks after operation. In addition, developed AAA rabbits (n=5) and normal rabbits(n=5) did not undergo aortic infusion with elastase and saline or thalidomide administration, which were sacrificed at1weeks after infusion and normally feeding. Ultrasonography was used to assess dilatation of the abdominal aorta before and1,2and4weeks after operation. Angiography was also performed to evaluate dilatation of the abdominal aorta before and1and4weeks after infusion with elastase. All aortas were harvested and cut into two equal segments,one was fixed in fresh cold4%polymerisatum for Hematoxylin-Eosin (HE) for inflamatory cell infiltration and neovascularization, for Elastic Van-Gieson(EVG) stain for elastin and for immunohistochemistry for qualitative study of TNF-a, MMP-9, VEGF. The second segment was immediately snap-frozen at-80℃for western blot analysis for quantitative analysis of TNF-α,MMP-9,VEGF. Blood samples were taken before and1weeks,4weeks after operation. Samples were centrifuged to separate the serum, and the levels of TNF-a and IL-1β were measured with ELISA.Results1.18rabbits received modeling operation in batches. Within1week,2died and1developed paraplegia.At1week post-operation,15developed AAA confirmed by ultrasonography, the model success rate was up to85%. Before operation, the mean aortic diameter was2.9mm,5.0mm afer1week, the average aortic dilation rate were71%. Morphologically, the segments of infused aorta exhibited wall thickening, thrombosis could be seen in some samples.2.Four weeks later the infusion with pancreatic elastase, aortic diameter was enlarged furtherly in AAA/CON by gross morphology, ultrasonography and arteriography, but in AAA/Tha the expansion was inhibited markedly, the expansion rate of aorta in AAA/Tha group was decreased by an average of46%compared to the rate in AAA/Con group(AAA/Tha,5.2±0.4mm vs. AAA/Con,7.6±0.9mm,P<0.001).3. At4weeks post-operation, HE staining showed that infiltration of inflamatory cells and neovascularization in aneurismal wall was prominent in AAA/Con,however,it was attenuated in AAA/Tha.EVG stain exibited progressive destruction of the elastic lamellar wavy stucture with flattening, fragmentation and even disappearance of elastin fibers accompanies aortic expansion in AAA/Con, whereas the wavy structure of the elastic lamellar is largely preserved in AAA/Tha.4. Immunohistochemical staining for TNF-α, MMP-9, and VEGF in the rabbit aortic wall is shown. Protein expression of TNF-α, MMP-9, and VEGF was negative in normal aortic wall, and1week post-operation, the expression became positive, and the positive position was mainly observed in media and adventitia of the aneurismal wall.At4weeks post-operation, whereas their further expression evaluated was extensive and transmural in AAA/Con, the levels were detected slightly in AAA/Tha. Furtherly, western blot analysis indicated that thalidomide treatment attenuated markedly TNF-α,MMP-9,VEGF by an average of47%,48%,75%respectively. The increased serum IL-1β and TNF-a expression was reduced by thalidomide treatment compared to AAA/CON (P<0.001).Conclusions Infusion of an isolated segment of rabbit aorta with elastase provide a reproducible animal model of AAA.Treatment with thalidomide in rabbit prevented the development of elastase-induced AAA, in association with reduced inflammation, neoangiogenesis, and extracellular matrix disruption.These findings suggest therapeutic potential of thalidomide for AAA.