The Role And Mechanism Of Endothelial Microparticles In Vascular Leakage After Sepsis In Rats

Sepsis,a severe critical illness,is characterized by systemic inflammation syndrome,and induced by bacterial infection,which can cause severe tissue damage and organ failure,even multiple organ dysfunction syndrome and death.Sepsis has always been a prevalent severe syndrome in the intensive care unit(ICU),withhigh mortality rate.Vascular leakage is the important pathological process of sepsis,which is characterized by the endothelial barrier dysfunction and increased vascular permeability.Vascular leakage will contribute to the abnormal diffusion of proteins and other molecules from the blood to the tissues,thereforelead to tissue edema and decrease of effective circulating blood volume.Microparticles(MPs)are described as membrane-derived particles that are secreted into the extracellular environment by almost every cell type under both normal and stress conditions.The size of the MPs is usually defined as 0.1-1?m in diameter.MP can bring many bioactive molecules such as proteins,DNA,RNA and so on.Studies have shown that MPs can play an important role in many diseases,for instance atherosclerosis,myocarditis,pre-eclampsia,acute pulmonary injury,chronic nephritic syndrome,and so on.Recent studies show that the concentration of MPs is increased after sepsis,and it can influence the vascular relaxation and contraction response by modulation of the expression of NO,i NOS and ROS.Nevertheless,whether MPs play a role in vascular leakage after sepsis is not known.Therefore,using the sepsis rat models and rat pulmonary venous endothelial cells,we investigated the role of MPs in vascular leakage after sepsis and the underling mechanisms in the present study.Main research contents and methods:Part I: Effect of EMP in vascular leakage after sepsis.1.By using sepsis rat models,vascular leakage of lung,kidney and intestine was measured.Different origins of MPs were collected from the rats.2.Rats were stimulated with different origins of MPs(Endothelial microparticle,Neutrophil microparticle,Platelet microparticle),vascular leakage of lung,kidney and intestine was measured.3.The endothelial cells cultured in the Transwell were stimulated with EMP,and the Trans Electric Resistance,the penetration rate of Fluorescein isothiocyanate labeled Bovine Serum Albumin(FITC-BSA)and the expression of ZO-1 were measured.Part II: The mechanism of EMP in vascular leakage.1.Micro RNAs(miR)in MPs from sepsis rats and EMPs from LPS-stimulated endothelial cells were measured and screened by RT-q PCR.2.The endothelial cells cultured in the Transwell were stimulated with miR-23b(-)EMP,and the TER,the penetration rate of FITC-BSA and the expression of ZO-1were measured.Part III: Role of MPs collected from sepsis patients in the vascular leakage.1.MPs in blood of 10 health adults and 10 sepsis patients were collected,and the concentration was measured.2.Effect of MPs collected from sepsis patients in the vascular leakage in the rats and endothelial cells including: vascular leakage of lung,the TER,the penetration of FITC-BSA and the expression of ZO-1 were measured.Main results:Part I:Effect of EMP in vascular leakage after sepsis1.The vascular leakage of lung,kidney and intestine and the concentration of MPs were all significantly increased after sepsis.2.MPs and EMPs from sepsis rats and LPS-stimulated endothelial cells significantly increased the vascular leakage,while PMPs and NMPs had no obvious effect on vascular leakage.3.After stimulated with EMP,the TER of endothelial cells was significantly decreased,and the penetration rate of FITC-BSA was obviously enhanced,meanwhile,the expression of ZO-1 was significantly declined.Part II: The mechanism of EMP in vascular leakage.1.The expression of miR-23 b,miR-29,miR-125 a,miR-126 a,miR-181 a,miR-199 a,miR-203,miR-374 were significantly increased in MPs from sepsis rats,while miR-23 b was the main miR in EMPs.2.Inhibition of the expression of miR-23 b in EMP significantly decreased the increased permeability of endothelial cells.Part III: Role of MPs collected from sepsis patients in vascular leakage.1.The concentration of MPs in sepsis patients was significantly higher than that in health adults.2.MPs collected from sepsis patients significantly increased the vascular leakage of lung in rats and increased the permeability of cultured endothelial cells.Conclusion:1.The concentration of MPs is significantly enhanced after sepsis.EMP plays the most important part in vascular leakage among different types of MPs.2.Many types of miR have higher expression in EMP,and miR-23 b is the main miR which regulates the vascular leakage.3.Sepsis patients study confirms the animal study result that sepsis patients have higher MP production,MP plays an important role in vascular leakage after sepsis.