| Background:Aortic dissection(AD)is the most common and dangerous catastrophic event affecting the aorta,which has a high fatality rate.With the aging of the society and the improvement of people’s living standard,the incidence of the disease is increasing gradually.Nowadays,the treatment of aortic dissection mainly relies on surgical treatment,but its rapid onset and low treatment rate often fail to kill the dangerous events such as dissection rupture in the beginning.Therefore,early benign intervention and treatment of aortic dissection is particularly important.As a precursor of vascular Endothelial cells(VECs),EPCs have been shown in many studies to be involved in the repair of damaged blood vessels and the formation of new blood vessels.Therefore,whether EPCs have a clear and benign repair effect on aortic dissection remains to be verified.Objective:1.To identify the correlation between EPCs and aortic dissection,as well as the differences in the number and distribution of EPCs in different layer of vascular wall in clinical samples.2.To isolate primary EPCs,and the role of EPCs in angiogenesis of aorta will be observed at the tissue level.3.To prove whether EPCs can promote the vascular remodeling of aortic dissection in living animal models and further clarify the specific ways or mechanisms of the role of EPCs,so as to provide new strategies and prospects for the early intervention and treatment of aortic dissection.Methods :1.Study on clinical samples: collect aortic dissection pathological tissue and normal aorta tissue when the research was approved by the medical ethics committee and patients’ informed consent was granted.The specific antibodies CD34 and VEGFR2 of EPCs in tissues were detected by immunofluorescence assay.The difference in the number of EPCs in different layer of vessel wall of two groups were quantitatively analyzed.2.Co-culture of primary endothelial progenitor cells and aortic tissues in mice: Several wild-type male C57BL/6 mice aged 4-8 weeks were selected to obtain the femur bone marrow single-cell suspension,and the mononuclear cells and a small number of EPCs were extracted by density gradient centrifugation method.They were induced to differentiate into primary EPCs in EGM-2 medium;The surface markers sca-1,flk-1 and CD133 of the EPCs were detected by flow cytometry to identify the cellular immunophenotype and determine the purity of the EPCs.Immunofluorescence method was used to observe the ability of EPCs to phagocytosis of acetylated low density lipoprotein and binding of gorse lectin 1,so as to complete its functional identification.Mouse aorta was cut into 1mm long vascular ring and randomly divided into two groups,which were cultured in medium containing endothelial progenitor cells and control medium respectively.The number and length of aortic ring budding after 7 days were observed,and tissue composition of sprouts was observed by immunofluorescence method.3.Animal experiments: 36 3-week-old wild-type male C57BL/6 mice were randomly divided into 3 groups,namely A Model control group(MC)group,B.Aortic dissection modeling +EPCs treatment(EPC)group and C.Shame operation(SO)group.Both A and B were fed with β-aminopropanitrile(1g/kg/d)drinking water for 4 weeks and permeated with angiotensin II micropump for 3 days to establish a mouse aortic dissection model,and the formation of aortic dissection was monitored under ultrasound;CM-dii dye was used to label the EPCs after culture and identification,and make the single cell suspension,then 2X106 EPCs were injected into the tail vein of mice in group B;Groups A and C were injected with normal saline of the same volume;3 days after EPCs’ transplantation,mice in group B were placed under live imager to observe the trace and distribution of EPCs;7 days after EPCs’ transplantation,the diameters of different sections of the aorta in groups A and B were measured under ultrasound;7 days after EPCs’ transplantation,six mice in each of the three groups were injected with Evans blue dye at the apex of the heart to observe the degree of aortic reendothelialization;The rest of the mice were sacrificed and dissected 7 days after EPCs’ transplantation.Complete pathological or normal aortic tissues were taken out,and compare the formation and vascular morphology of aortic dissection in each group;Frozen sections of aortic tissue were performed to trace Dil-EPCs and observe its homing in vascular wall;Paraffin sections were made and the changes of aortic structure and intimal growth in each group were observed and compared by HE staining;EVG staining was used to observe and compare the content of elastic fibers in each group;The collagen fiber content of the aorta in each group was observed and compared by Masson staining.Results:1.In the clinical sample experiment,7 cases of aortic dissection and donated normal tissues were collected,and the number of EPCs in each visual field was quantified.The results showed that the number of EPCs in aortic dissection was significantly higher than that in normal aortic dissection,with a significant statistical difference(P<0.05).Compared with normal aortic tissue,aortic dissection showed significantly higher EPCs content in the intima and intima-media junction area,with a significant statistical difference(P<0.05).There was no significant difference in the middle area between two groups(P>0.05).2.Co-culture of primary endothelial progenitor cells and aortic tissues in mice: the extracted EPCs grew in accordance with the morphological characteristics of adherent-colony formation-long spindle-"pebble" shape;Flow cytometry showed that the proportions of sca-1,flk-1 and CD133 were 95.8%,91.9% and 87.6%,respectively.The extracted EPCs had the function of phagocytic acetylation of LDL and binding of gorse lectin 1.The number of sprout in the primary EPC co-culture group(20.00±2.92)was significantly higher than that in the control group(11.67±6.08),and the difference was statistically significant(P<0.05).At the same time,it was found that the microvascular length(87.86±39.25 m)of the primary EPC co-culture group was also significantly higher than that of the control group(62.53±31.57 m),and the difference between the two groups was statistically significant(P<0.05),endothelial cells and smooth muscle cells are both found in the sprout tissue;3.Animal experiments: compared with MC and EPC group,the difference was statistically significant in section 2(1.27±0.08 mm VS 1.40± 0.13mm)which also is the section 1 of the aortic arch,and in section 4(1.28±0.13 mm VS 1.43± 0.13mm)which also is the junction of the aortic arch and the descending thoracic aorta(main site of dissection).However,compared with section 1(1.33±0.15 mm VS 1.38± 0.12mm)at the ascending aorta and section 3(1.22±0.16 mm VS 1.27± 0.14mm)which is the 2nd section of the aortic arch,the diameter of the MC group was wilder than that of the EPC group,but the difference was not statistically significant(P>0.05);The intimal growth area was quantified and normalized to 1 in the SO group,and the intimal area in the EPC group was 53.36 times that of the SO group,with statistically significant differences(P<0.05);The intimal area of the MC group was about 0.8 times that of the SO group,and the difference was not statistically significant(P>0.05);The difference between EPC group and MC group was also statistically significant(P<0.05);The reendothelialization degree of EPC group was significantly higher than that of MC group(83.18±4.22% VS 75.47±4.70%),and the difference was statistically significant(P<0.05);The aortic collagen volume fractionin of EPC group(45.50±7.21%)and in SO group(42.97±4.39%)were significantly higher than that in MC group(26.38±4.96%),and the difference was statistically significant(P<0.05);There was no significant difference in aortic collagen volume fraction between EPC group and SO group(45.50±7.21% VS 42.97±4.39%,P>0.05);The content of aortic elastic fiber in EPC group(27.97±4.56%)and SO group(31.90±2.91%)was significantly higher than that in MC group(21.05±7.11%),and the difference was statistically significant(P<0.05);The content of aortic elastic fiber in EPC group was not significantly different from that in SO group(27.97±4.56% VS 31.90±2.91%,P>0.05).Conclusion:1.Endothelial progenitor cells(EPCs)are involved in the pathophysiological process of aortic dissection,and the potential targets are the intima and mesenchymal layers of the aorta.2.The isolated cells are the iconic EPCs.The aortic sprout experiment confirmed that EPCs could promote angiogenesis in the aorta of mice.3.Exogenous EPCs will homing to the damaged site of aortic dissection,which can reduce the vascular dilatation caused by aortic dissection;EPCs can promote the growth of intima of aorta and promote the reendothelialization of blood vessels.EPCs can improve the content of collagen and elastic fibers in the dissected tissue and protect the aorta. |
PDF Full Text Request