Expression Of MitoMVs In The Plasma Of Patients With Sepsis And Its Effect On Vascular Endothelial Injury

Part 1 Detection of human plasma MVs and verification of mitochondrial content it carriesBackground and Object: Microvesicles(MVs)are phosphatidylserine exposing membrane vesicles with a diameter of 0.1 to 1 m that are released by cells.It can carry a variety of ingredients from the cytoplasm,nucleus and is related to the pathogenesis of sepsis.The mitochondrial damage-associated molecular patterns(mt DAMPs)are also currently considered to be related to the pathogenesis of sepsis,and have been shown to be carried by MVs released by platelets activated in vitro.However,it is not clear whether MVs in human plasma can also carry components similar to mt DAMP.Therefore,in the first part,we aim to construct a plasma MVs detection protocol and verification whether plasma MVs are rich in mitochondrial content,which can lay a foundation for the next step in the study of the role of MVs and their contents in sepsis.Methods: We first collected plasma samples from patients with sepsis,patients with infections,and healthy people,then separated and extracted plasma MVs with a high-speed centrifuge,then used known nano-particle standards to gate the flow cytometer and used a new "Logicle" display method to display the flow cytometry data,and finally human plasma MVs were detected with the gated flow cytometer and compared with the known characteristics of MVs.In addition,plasma MVs was labeled with mitotracker and anti-tom22 and detected by laser confocal and flow cytometry to determine whether plasma MVs were rich in mitochondrial content.Results: The Aria II flow cytometer with Logicle display method could clearly display standard particles with diameters of 1um,0.5um,and 0.2um,and the clusters were obvious.Flow cytometry analysis with the help of particle standards and logarithmic display showed that the diameter of human plasma MVs was mostly between 0.1 and 1um and the number of MVs peak situated between 0.2 ~ 0.5um.Laser confocal analysis showed that there were phosphatidylserine and “mitotracker” double-positive MVs in the plasma.Flow cytometry analysis showed that there were not only phosphatidylserine and "mitotracker" double-positive MVs,but also phosphatidylserine and tom22 double-positive MVs.Conclusion: Human plasma MVs are rich in mitochondrial content.Part 2 The expression of plasma MVs in patients with sepsis and its correlation with inflammatory markers and endothelial injury markerBackground and Object: MVs and mt DAMPs are currently considered to be related to sepsis-mediated inflammation.At the same time,the differentially expressed mito MVs have been found in the plasma of hepatitis mice.However,similar findings have not been found in the plasma of sepsis,which is also pathologically based on inflammation.Therefore,by detecting the plasma MVs and some inflammation markers and endothelial injury marker in the sepsis group and the related control groups,we aim to determine whether there is a difference in the expression of mito MVs between the above groups and to explore the relationship between mito MVs and the above markers.Methods: We first collected plasma and serum samples from patients with sepsis,patients with infections,and healthy people,and then used flow cytometry to detect the expression levels of MVs and tom22+MVs in the above plasma samples,and enzyme-linked immunosorbent assay(ELISA)to detect the expression levels of IL-6,IL-8,CRP,TNF-α and s VCAM-1 in the above serum samples.Finally,we conducted a correlation analysis of the expression levels of the two indicators in patients with sepsis.Results: A total of 20 healthy controls,34 patients with local infection,and 19 patients with sepsis were included in the study.There was no statistical difference in age,gender and past medical history between the three groups.Both sepsis patients and local infection patients had significantly higher levels of MVs and tom22+MVs compared to healthy control subjects.Even compared with infection patients,tom22+MVs were significantly higher in sepsis patients.Concentrations of IL-6,IL-8,s VCAM-1,CRP and TNF-α were significantly higher in sepsis patients than people in healthy control group.In terms of correlation analysis,in the sepsis group,the number of tom22+ MVs was not only related to the expression level of TNF-α,but also related to the expression level of s VCAM-1.Conclusion: There are significantly elevated MVs,mito MVs,inflammatory markers and endothelial injury marker in the blood of patients with sepsis.In sepsis patients,mito MVs are both related to inflammation and endothelial injury.It may be the cause of endothelial dysfunction.Part 3 The study of vascular endothelial injury mediated by plasma microvesicles of patients with sepsisBackground and Object: Sepsis is defined as a systemic inflammatory response syndrome induced by infection,and endothelial cells are an important component involved in the above-mentioned inflammatory response.MVs are increasingly recognized as important mediators between cells,and the cargo they carry are considered to define their biological activity.In addition,several components of mt DAMPs have the ability to induce the activation of type I IFN response.Therefore,on the basis of the previous two parts of research,we aim to clarify whether MVs in the plasma of patients with sepsis could also induce inflammation in endothelial cells,especially type I IFN response,and whether the mitochondrial content it carries is the cause of the activation of type I IFN response.Methods: First,we collected MVs(MVcontrol)extracted from the plasma of healthy people and MVs(MVsepsis)extracted from the plasma of patients with sepsis,and cultured human umbilical vein endothelial cells(HUVECs).Then,confocal laser-scanning microscopy was used to observe the fusion of MVs with HUVECs.Then,after HUVECs were treated with MVcontrol and MVsepsis,quantitative real-time polymerise chain reaction(q RT-PCR)analysis was used to detect the expressions of IL-8,2’-5’-oligoadenylate synthetase 2(OAS2),radical S-adenosyl methionine domain containing 2(RSAD2),C-X-C motif chemokine ligand 10(CXCL10)and NOS3 in HUVECs.ELISA was used to detect the concentration of IL-8 in the HUVECs supernatant.Flow cytometry analysis was used to evaluate the release of NO in HUVECs.After that,ultrasound was used to crush the mitochondrial contents that MVs may carry.Subsequently,these sonicated MVs were also used for co-incubation with HUVECs.QRT-PCR analysis was used to detect the expressions of OAS2,RSAD2 and CXCL10 in HUVECs.Results: Compared with MVcontrol,MVsepsis induced an increase in the expression of IL-8 in HUVECs.And,after MVsepsis were incubated with HUVECs,flow analysis showed that the red fluorescence intensity of HUVECs was significantly increased,and the laser confocal showed that mito MVs gathered around the nucleus.In addition,MVsepsis significantly promoted the m RNA expression levels of CXCL10,RSAD2,and OAS2,and the increase in CXCL10 was the most obvious.Subsequently,we sonicated MVsepsis and incubated them again with HUVECs,laser confocal analysis showed that the number of mito MVs gathered around the nucleus of HUVECs was significantly reduced.The gene expression levels of RSAD2,OAS2 and CXCL10 shown by q RT-PCR analysis were also significantly down-regulated.Finally,compared with MVcontrol,although MVsepsis can cause the reduction of the release of NO and the expression of NOS3 m RNA in insulin-stimulated HUVECs,the difference was not statistically significant.Conclusion: MVsepsis have a capacity of inducing inflammation in vascular endothelial cells,and the mitochondrial content carried by MVsepsis is the cause of inducing type I IFN response in vascular endothelial cells.