| Backgroud and Purpose: It is well-known that cerebral aneurysm is a common and frequently-occurring disease, which is harmful to human health. It is the first cause of spontaneous subarachnoid hemorrhage that causes death or deformity highly.So far, it has been not clear that the mechanism of formation and progression in cerebral aneurysms.Previous studies have shown that atorvastatin can attenuate the formation and progression of CAs by supressing the inflammation response.In our prophase research, the results were illustrated that the number of EPCs in patients with CAs is decreased and the function of EPCs is impaired. Moreover, there was a mazing phenomenon the incidence of CAs is reduced and the progression is attenuated by up-regulating EPCs.It has been shown that atorvastatin can increase the number and function of EPCs.Therefore,we hypothesized that EPCs-mediated neovascularization is considered as a innovative target and mechanism, which atorvastatin attenuat the progression in CAs. We wish to get the objective in this study, as follows.(1)It is further clarified that EPCs defects play an important role in CAs progression, as to perfect the pathophysiology of intracranial aneurysm formation and development. It is expected to enrich the new clinical idea of the CAs prevention and treatment,which EPCs as targets.(2) To clarify the mechanism that EPCs mediated by atorvastatin delay the the development of CAs and put forward the specific regulating strategy. With translational medicine as a guide, we explore the establishment of a new clinical treatment opinion that the mobilization of endogenous EPC can home to injured site, using in the treatment of intracranial aneurysms.(3)To get the evidence of the pre-clinical study that atorvastatin can attenuat the progression of CAs, in order to provide a new ideas for the prevention and treatment of CAs.Methods: This study is divided into two section. The first section is about animal model experiment and the second section is about clinical investigation.The first part:the preliminary study for mechanism of atorvastatin can attenuate the progression in CAs by EPCs mobilization. In this section,we established a stable Hashimoto rat model for cerebral aneurysm by ligation of the left common carotid artery and bilateral renal artery. According to the research purpose, all experimental animals were divided into four groups,namely normal control group(n=24), cerebral aneurysm model group(n=24),low-dose atorvastatin group(3mg/kg/d) and high-dose atorvastatin group(10mg/kg/d).The blood pressure values in rats was evaluated by mouse tail sleeve method under anesthesia.The circulating blood EPCs levels were measured by flow cytometry at the end of 1 month and 3 month in each group.VG staining and electron microscope observation was done by spinning off on the right right anterior cerebral artery/olfactory artery(ACA/OA) in bifurcation vessel, in order to evaluate pathological finding in the Willis rings of rat aneurysm tissue.It is detected that circulating blood and vessel wall of the repair/inflammatory factor levels and gene m RNA expression in CAs by the ELISA method and quantitative RT-PCT, The vascular inflammation/repair/apoptosis related proteins differentially expressed were analyzed between different dose atorvastatin CAs groupwall by western blot test. Finally, we determine the experimental rats blood routine and biochemical indexes by the blood cell analyzer and fully automatic biochemical analyzer,to evaluate the safety of atorvastatin for the treatment of intracranial aneurysms.The second section:the potential clinical significance of circulating EPCs for reducing the recurrence of the aneurysm and the changes of circulating EPCs in patients with CAs after embolization by oral atorvastatin.In this section, we included 19 patients with unruptured intracranial aneurysm patients from two neurosurgical centers in tianjin. All the patients were performed interventional embolization and were randomly divided into two groups,as follows. Oral atorvastatin group(11cases) and non oral atorvastatin group(8cases).We also included 18 healthy persons as control group. Blood samples were collected at admission, first 4th, 14 th, 21 th, and 30 th days after admission,respectively. We detect the number of circulating EPCs by flow cytometry.The levels of vascular endothelial growth factor(VEGF) and stromal cell derived factor alpha(SDF- 1 alpha) were measured by ELISA.We measured the circulating lipid, levels of liver and kidney function in all subjects by the automatic biochemical analyzerResults:(1)In the first section, we successfully modify the Hashimoto CAs rat model.Based on the data analysis, rats blood pressure is significantly elevated between CA group and ATR group in 1 months after modifing the animal model compared to normal group.Aneurysm after 3 months of building, CA and ATR treatment group rats blood pressure values is back to normal in CAs and ATR group after 3 months in establishment of animal model.The EPCs levels in ATR groups were significantly higher than CA groups in 3 months.The above results showed that atorvastatin can greatly to mobilize the blood circulating EPCs.The degenerative change of blood vessel walls is not obvious in vascular branch of right ACA/OA in normal control goup. The right ACA/OA bifurcate in rats after 3 months have the obvious degenerative changes in CAs group. The above patholical changes conform to the pathological progession of intracranial aneurysms.There is lower IEL score,thicker tunica media and sizes of the mature in ATR group. The results in TEM showe that, as follows. There are interesting results that vascular endothelial cells is complete, the elastic without fracture, smooth muscle cell apoptosis, vascular deconstruction outer membrane integrity. 3 months after aneurysm model establishment, a serious degradation of hemal wall deconstruction is show that of the loss of endothelial cells, the elastic layer fracture, visible were severely damaged and degradation of smooth muscle cells. At the same time, the outer membrane deconstruction of vascular disorders. The base of Willis in blood vessel walls can see parts of endothelial cells and fracture of the elastic layer, part of the smooth muscle, part of the outer membrane organization deconstructs complete in ATR group, hemal wall degeneration lighter than non-medicine intervention group obviously.VEGF,TGF-beta level in ATR group is obviously higher than that of CAs group. VEGF, TGF-beta is the chemokines of atorvastatin EPCs mobilization and atorvastatin can make associated with vascular repair gene m RNA expression level(VEGF, TGF- beta). Atorvastatin can make associated with CAs wall inflammation m RNA expression of cut(NF-k B, MMP-2 and MMP-9). High-dose atorvastatin group(10mg/kg/d) can increase the expression of vascular factors(VEGF) and promote the expression angiogenesis inhibiting factor(VEGI). There is no difference between Different doses of simvastatin on statins inhibit CAs wall inflammation(MMP-2 and MMP-9).High-dose atorvastatin increase the apoptosis protein expression(Caspase-9). Howere,it is damaged to the vessel wall. The RBC, hemoglobin, white blood cells, platelets, blood lipid and liver and kidney function of dirty is no obvious difference among groups.(2)In the second section of this study, we enrolled 19 patients with unruptured intracranial. Also, we enrolled 18 age and gender matched healthy objective as control group.Compared with healthy people, hypertension, smoking and hyperlipidemia is higher in the proportion of patients with aneurysm. There was no significant difference in the tension between each drug use(including aspirin, beta blockers and renin vascular angiotensin converting enzyme inhibitors, etc.), previous history of cerebral vascular disease(coronary artery disease, diabetes and cerebrovascular disease) and fasting blood glucose level. After embolization, no oral atorvastatin in patients with atorvastatin calcium circulating endothelial progenitor cells number began to rise slowly, peaked at 14 days after operation and then decreased gradually, until the 30 th postoperative day, declined to near the level before embolization. Embolization of aneurysm after 3 weeks, the blood level of EPCs was higher than that before embolization / on admission. After oral administration of atorvastatin calcium, within 30 day follow-up, patients of circulating EPCs water continued to rise, at each time point were significantly higher than those of the admission level, and on the 14 th day, the highest peak of EPCs is not oral atorvastatin statin group of 1.4 times. And after 14 days of treatment, the blood EPCs level remains high. The plasma level of VEGF also showed the same trend with the dynamic changes of circulating EPCs, namely after treatment began to rise slowly, until the 14 day peak. After oral administration of atorvastatin calcium, within 30 days of follow-up, the plasma VEGF water continued to rise, at each time point were significantly higher than those of the admission level, and on the 14 th day, the highest peak of VEGF is not oral atorvastatin statin group of 1.8 times. And 14 days after treatment, the plasma VEGF level remains high. After interventional embolization, the plasma levels of CRP were lower at each time point compared with the non oral atorvastatin group at all time points. We found that VEGF showed a significant upward trend after oral administration of atorvastatin, while SDF-1α levels were suppressed.In this study, through regression analysis shows: in the atorvastatin in the treatment of patients, circulating endothelial progenitor cells, and vascular endothelial growth factor was positively correlated. The blood lipid level of the patients with oral atorvastatin was decreased, but there was no significant difference between the two groups. But there was no significant difference between the liver kidney function indexes(ALT, Cr and CK).Conlusion:(1)In this animal experiment, it is illustrated that the atorvastatin can attenuate the progression in cerebral aneurysms by suppressing inflammation and by promoting angiogenesis/vascular repair.(2)It is advantaged that the high-dose atorvastatin(10mg/kg/d) can promote the vascular repair of cerebral aneurysms. However, there is no favorable that this dosage can inhibit the local inflammation in inner wall of the aneurysm cavity.On the contrary,this dosage can increases apoptosis in the vessel wall.Also, it is so destructived for vessle wall that it is not the appropriate dose for attenduating the progression in cerebral aneurysm.(3)It is safe and effective that the low dose atorvastatin(3mg/kg/d) can attenduate the progression in cerebral aneurysms. The main pharmacologic effect of this dosage is promoting vascular repair and suppressing inflammation, rather than lipid-decreasing.(4)Based on the preliminary research data in second section, we come up with a hypotheses, as follows. The endothelial progenitors(EPCs) can be mobilized from the bone marrow of the patients in cerebral aneurysm after embolization and induced by oral atorvastatin. Circulating EPCs can home to the neck of cerebral aneusysms stimulated by the the chemokines of SDF-1 alpha and involve in the neointima formation.(5)There is great clinical application value and good prospect, which the atorvastatin can reduce the recurrence of cerebral aneurysm after embolization. However, it is needed that the further basic experiment and prospective randomized controlled trial should be done in the future. |
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