Peripheral Blood Exosomes Regulate The Mesenchymal Transformation Of Vascular Endothelial Cells In Moyamoya Disease

Objective: To clarify the key role of Endothelial to Mesenchymal Transition(End MT)in Moyamoya disease(MMD),and to explore the effect of peripheral blood exosomes of MMD on the mesenchymal transition of cerebrovascular endothelial cells.Methods:1 Blood samples: From December 2020 to September 2022,22 patients who were hospitalized in the Department of Neurosurgery,Affiliated Hospital of Jining Medical College and diagnosed with moyamoya disease were selected as the experimental group.At the same time,22 healthy people who underwent physical examination in the physical examination center of the Affiliated Hospital of Jining Medical College were selected as the control group.Venous blood 10 ml(fasting)was collected from the target object,and plasma was obtained by centrifugation of whole blood.Exosomes were isolated from plasma by exosome extraction and purification kit and stored in a refrigerator at-80 °C.2 Tissue samples: From December 2020 to September 2022,the diseased vascular tissues of 5 patients who were diagnosed with moyamoya disease and underwent surgical treatment in the Department of Neurosurgery,Affiliated Hospital of Jining Medical College were selected as the experimental group.The internal mammary artery vascular tissues of 3 patients who underwent vascular bypass surgery in the Department of Cardiac Surgery,Affiliated Hospital of Jining Medical College were selected as the control group.HE staining was used to observe the tissue morphology,and immunohistochemical staining was used to detect the expression of End MT-related molecular markers in the vascular tissue of moyamoya disease and internal mammary artery.3 Mouse brain-derived Endothelial Cells.3(b END.3)and Human Umbilical Vein Endothelial Cells(HUVECs)were routinely cultured.The same amount of exosomes from the experimental group and the control group were used to intervene b END.3 for 48 hours,and HUVECs were treated in the same way.The morphological changes of b END.3 after exosome intervention were observed by inverted phase contrast microscope.CCK-8 assay was used to detect the proliferation of b END.3and HUVECs after exosome intervention.Transwell assay was used to detect the migration of the two cells after intervention.The angiogenesis ability of the two cells in vitro was detected by angiogenesis assay.The expression of End MT-related molecular markers in b END.3 cells after intervention was detected by Western-blot,RT-q PCR and immunofluorescence staining.4 After the intervention of exosomes in b END.3,RT-q RCR was used to detect the expression of mi R-125b-5p,mi R-151a-3p,mi R-128-3p and mi R-200c-3p in cells(differentially expressed micro RNAs in exosomes screened in previous experiments).Liposome transfection technology was used to overexpress and knock down meaningful micro RNAs in cells,and Transwell assay was used to detect the effect on cell migration ability.Results:1 Exosomes were observed as disk-shaped and / or cup-shaped vesicles under transmission electron microscopy.The size was mostly distributed between 30-150 nm,and its specific proteins TSG101,CD63 and CD9 were significantly enriched.2.1 HE staining showed that the wall thickness of the vascular tissue of MMD lesions was uneven,the lumen was narrow,the intima was thickened,the endometrial cells were mesenchymal,and the middle smooth muscle was not significantly proliferated.The wall thickness of the internal mammary artery was uniform,the intima was neat,and the nucleus morphology was regular.2.2 The results of immunohistochemical staining showed that the protein expression levels of endothelial cell markers CD31 and VEcadherin in vascular tissues of MMD lesions were lower than those in internal mammary artery tissues(P < 0.01),while the protein expression levels of mesenchymal cell markers N-cadherin,α-SMA,Snail and Vimentin were higher than those in internal mammary artery tissues(P <0.01).The above results indicate that End MT exists in the vascular tissue of MMD.3.1 After 48 hours of intervention with b END.3 in peripheral blood exosomes of MMD group,the overall morphology of the cells changed,the cytoskeleton became longer,the pseudopods increased,the cells changed to irregular spindle shape,and the trend of interstitialization appeared.The results of CCK-8 and Transwell experiments showed that the proliferation rate of cells was accelerated and the number of migration was increased(P < 0.05).The results of vascularization assay showed that the number of mesh,branch and cross nodes between cells was similar to that of the control group.The results of Western-blot,RT-q PCR and immunofluorescence staining showed that the expression levels of endothelial cell markers CD31 and E-cadherin in the cells were lower than those in the control group(P < 0.01),and the expression levels of mesenchymal cell markers N-cadherin,Snail,Vimentin and α-SMA were higher than those in the control group(P < 0.01).The above results indicate that peripheral blood exosomes of MMD patients can promote abnormal proliferation and migration of cerebrovascular endothelial cells and promote their transformation into mesenchymal cells.3.2 CCK-8 and Transwell results showed that the proliferation rate and migration number of HUVECs were similar to those of the control group after 48 hours of exosome intervention in MMD group,indicating that exosomes from peripheral blood of MMD patients had no significant effect on the proliferation and migration of HUVECs.4 After overexpression and knockdown of mi R-151a-3p or mi R-125b-5p in b END.3,Transwell assay showed that the number of cell migration in mimics group was higher than that in NC group,and the number of cell migration in inhibitor group was lower than that in inhibitor-NC group,indicating that mi R-151a-3p or mi R-125b-5p can promote the migration of b END.3.Conclusion:End MT exists in the development of MMD,and MMD peripheral blood exosomes can promote the abnormal proliferation of cerebrovascular endothelial cells and induce their transformation into mesenchymal cells,but the specific mechanism affecting this process remains to be further elucidated.