Mechanism Of Endothelial Injury In Preeclampsia: Oxidative Stress And Immune Activation

Chapter 1 Study on Oxidative Stress and Activation Immune Inflammation in Preeclamptic Patients and Its Clinical SignificanceAIM: To investigate the relations between hypoxia injury in placenta, and PMNs activation, oxidative stress, immune inflammation, and its clinical significance.Methods: To research hypoxia injury in placenta from preeclampsia by histochemical method. H₂-DCFDA, a ROS probe combined with flow cytometric detected level of ROS within PMNs from preeclampsia circulation, and then level of PMNs activation were evaluated; To evaluate level of oxidative stress in preeclampsia, concentration of H₂O₂ in their serum were detected by specific assay kits and spectrophotometer. Percentages of CD3⁺/HLA-DR⁺ or CD3⁺/CD62L⁺ T cells, and mean fluorescence values of CD62L within PMNs in full blood from preeclampisa were determined by multicolor flow cytometric analysis. Concentration of IL-6,IL-8,MCP-1,TNF-α,VEGF,NT-proBNP,IFN-γ,IL-1βand IL-10 in preeclampsia serum were determined by LUMINEX assay kit. Besides, corelations between concentration of H₂O₂ in preeclampsia serum and level of ROS within preeclampsia PMNs, concentration of TNF-αor unie acid in preeclampsia serum were determined respectively by SPSS statistical software.Results: 1. Compared to normal pregnancy, placentas from preeclampsia showed distinct features of hypoxie stress injury; 2. Compared with normal nonpregnancy (n=12) or normal pregnancy (n=19), concentration of H₂O₂ in preeclampsia serum (n=16) and level of ROS within preeclampsia PMNs were increased (p＜0.01); 3. Preeclamptic women (n=16) had significantly increased percentage of CD3⁺/HLA-DR⁺ T cells, Compared to normal nonpregnaney (n=12) or normal pregnancy women (n=19) (p＜0.01); Compared to normal nonpregnancy women (n=12), both preeclarnptic women (n=16) and normal pregnancy (n=19) women had significantly increased percentage of CD3⁺/CD62L⁺ T cells (p＜0.01), but compared to normal pregnancy women (n=19), preeclamptic women (n=16) had decreased percentage of CD3⁺/CD62L⁺ T cells (p＜0.01); 4. Mean fluorescence value of CD62L within PMNs decreased significantly in preeclamptic full bloods (n=16) when compared to its in normal pregnancy (n=19) (p＜0.01), but increased significantly when compared to normal nonpregnancy (n=12) (p＜0.01); 5. Compared between preeclamptic and normal pregnancy women, or between normal nonpregnancy (n=12) and normal pregnancy women (n=19), levels of IL-6, IL-8, MCP-1, TNF-αand VEGF were significantly increased in preeclamptic (n=16) and normal pregnancy women (n=19) (p＜0.01); Compared between normal nonpregnancy (n=12) or normal pregnancy women (n=19), levels of IFN-γ,IL-1βand IL-10 in preeclamptic serum (n=16) were no changes (p＞0.05); 6. There were positive corelations between levels of H₂O₂ in preeclamptic serum and levels of ROS within preeclarnptic PMNs(r=0.506), or levels of HLA-DR in preeclamptic T cells(r=0.728), or uric acid in preeclamptic serum(r=0.607) (0＜r＜1, p＜0.05).Conclusion: Hypoxic stress placenta and active PMNs could be sources of high levels of H₂O₂ in circulation; Hypoxic injury in placenta may be the essential causes which induced high state of oxidative stress in preeclampsia women; There were distinct evidences about activation of immune inflammation in preeclamptic women, and positive correlations between oxidative stress and immune activation, or severity of preeclarnpsia; Oxidative stress could play key roles in the translation from stage 1 to stage 2 of preeclampsia; Shedding of CD62L from preeclamptic PMNs may be one of mechanisms of negative feedback regulation in preeclampsia, and have potential values in disease prognosis.Chapter 2 Effects of ROS on Activation and Adhesive of T Cells and PMN from Normal pregnancyAIM: To investigate effects of extracellular H₂O₂ on activation and adhesive function of PMNs or T cells from normal pregnancy women, and its potential mechanism involved. To seek progressive knowledge about relations between high level of H₂O₂ in circulation and activation of PMNs and T cells of preeclampsia.Methods: 1. Coresponding to concentration in preeclamptic serum, PMNs from normal pregnancy women were stimulated by 40μmol/L extracellular H₂O₂, then levels of activation and CD62L expression of PMN were valued by flow cytometric analysis; 2. to research effects of 40μmol/L extracellular H₂O₂ on level of HLA-DR and CD62L expression of T cells from normal pregnancy women by flow cytometric analysis; 3. to research effects of PMNs pretreatd with 40μmol/L extracellular H₂O₂ on level of HLA-DR and CD62L expression of T cells from normal pregnancy women by flow cytometric analysis. Furthermore, investigate roles of monocytes or DCs in above case by ways of adhesive characteristic of these cells; 4. to research effects of 40μmol/L extracellular H₂O₂ on adhesive abilityof PMNs to HUVECs.Results: 1. activation level of PMNs from normal pregnancy women was significantly increased when stimulated with 40μmol/L extracellular H₂O₂ (p＜0.01, n=12), but level of CD62L expression decreased in the same conditions (p＜0.01, n=12); 2.40μmol/L extracellular H₂O₂ was no effects on level of HLA-DR and CD62L expression of T cells from normal pregnancy women; 3. 40μmol/L extracellular H₂O₂ promoted PMNs adhesive to HUVECs significantly (p＜0.01, n=12); 4. Pretreated with 40μmol/L extracellular H₂O₂, level of HLA-DR expression increased, but CD62L expression decreased significantly on T cells from notmal pregnancy (p＜0.01, n=12). But when adhesive cells, such as monocytes and DCs, were discarded by its adhesive characteristic, the effects of above were impaired apparently(p＜0.01, n=12).Conclusion: 40μmol/L extracellular H₂O₂ could promote activation of PMNs, and fartherly promote activation of T cells from normal pregnancy women by monocytes or dentritic cells assistants; High level of ROS in preeclamptic circulation could be facilitating factors of PMNs and T cells activation.Chapter 3 Role of Reactive Oxidative Species, Mitochondria Membrane Potential, and Calcium Homeostasis in HUVECs Apoptosis Induced by Server Preeclamptic SerumAIM: To investigate role of ROS, mitochondria membrane potential, and calcium homeostasis in HUVECs apoptosis induced by server preeclamptic serum.Methods: 1. To research HUVECs apoptosis induced by server preeclamptic serum through low cytometric analysis and transmission electron microscope; 2. Effects of server preeclamptic serum on ROS, mitochondria membrane motential, and malcium homeostasis of HUVECs were determined by laser confocal microscope. Results: 1. Compared to serum from normal pregnancy, HUVECs stimulated with server preeclamptic serum 24hs showed apparent apoptosis by ways of hochest staining; Server preeclamptic serum significantly increased levels of APO2.7 expression in HUVECs (p＜0.01, n=12); Extracellular catalase could impaired toxicity of server preeclamptic serum on HUVECs in the same time(p＜0.01, n=12). 2. Compared to serum from normal pregnancy, server preeclamptic serum could significantly increased levels of ROS and calcium within HUVECs(p＜0.01, n=12), and decreased level of mitochondria membrane potential in the same conditions. Extracellular catalase could impaired these effects of server preeclamptic serum on HUVECs(p＜0.01, n=12).Conclusion: Server preeclamptic serum had positive toxicity to HUVECs, and ROS, calcium, mitochondria membrane potential involved its mechanism.