Study On The Role Of Hemeoxygenase/Carbon Monoxide System On Pathogenesis Of Hypertension Disorder Complicating Pregnancy

Part I Study on the expression of Heme oxygenase and Carbon Monoxide in the placenta of hypertension disorder complicating pregnancyObjective : To investigate the expression of hemeoxygenase(HO) and endogenous Carbon Monoxide(CO) in the placenta of women with hypertension disorder complicating pregnancy (HDCP) and discusses its role in the pathogenesis of HDCP.Methods : Reverse transcript polymerase chain reaction (RT-PCR) was used to detect the expression of HO-1 and HO-2 mRNA in placental tissues on 45 HDCP patients (HDCPgroup) and 15 normal pregnant women (control group); HE staining observed the pathology changes in placental tissues; Immuno-histochemistry was used to detect the expression of HO-1 and HO-2 in placental tissues and IMEGRA-PRO 5.0 was used to determine the quantity of them; The activity of HO in placental tissues was assayed by spectrophotometry; Western blot analysis was applied to detect the expression of HO-1 and HO-2 protein; COHb in placental tissues were determined by spectrophotometry. The placental NO2^-/NO3^- , the stable metabolic end product of NO, was measured by nitrate reductase.Results: (1)The decreased villi,arange upset alinement, thickening and fibronoid necrosis of placental vessel were significantly higher in women with HDCP than in normal group.(2)The results of immunohistochemistry demonstrated that that HO-1 was predominantly localized trophoblast and in villous stroma cell,HO-2 predominantly localized in trophoblast capillaries with weak staining of villous stroma.(3) the expressive quantity of HO-1mRNA(1.13± 0.19),and HO-2 mRNA(1.05±0.68)was high in control group,HO-1 mRNA quantity of gestational hypertension, moderate , severe preeclampsia and eclampsia were 1.34±0.7,0.68 ± 0.4,0.72 ± 0.3. (4) The activity of HO was significantly decreased in women with HDCP(3.46 ± 1.2)noml.mg^-1.h^-1compared with the normal group (6.01±1.4) noml mg^-1.h^-1 (P<0.01).(5)Western blot analysis showed that both groups expressed 32kd (HO-1) and 36kd proteins (HO-2) ,but the HO-1 level was significantly higher in women with gestational hypertension than in the normal group (121.86 ± 4.2) ;HO-1 protein quantity of gestational hypertension, moderate , severe preeclampsia and eclampsia were (125.1 ± 3.9, 102.76 ± 3.4,91.1 ± 5.2,62.53 ± 2.7) (P<0.01) .(6) The plencental tissues COHb in women with HDCP patients was lower than in the normal group (P<0.01). (7) HO-1 mRNA ,proteins,HO activity and COHb was the highest in gestational hypertension ,but decreased to follow the progress of HDCP,it is significant among HDCP groups (P<0.05,P<0.01).(8) No significant difference among groups was observed in the expression of placental HO-2 mRNA and proteins (P>0.05).(9)The placental levels of NO2^-/NO3^- were significantly lower in women with HDCP (3.39±1.18) μmol/g than in the normal group (17.69±3.53) μmol/g(p<0.01).(10)A significant posetive correlation existed between the expression of HO-1 and the concentrations of CO in placental tissues of women with HDCP (r=0.42, P< 0.05).Conclusions: The expression level of HO-1 protein,transcript and HO activity descreased accompanying with endogenous CO in HDCP groups, The decreased level of CO and HO-1 level in placenta correlated with the progress of HDCP. It demonstrated that HO and CO may play an important role in the pathogenesis of HDCP.Part II Experimental study on effects of heme oxygenase and Carbon Monoxide in human umbilical vein endothelial cell Objective: To observe effects of hemin , CoCl₂ inducing hypoxia and hypoxia culture on the expression of hemeoxygenase and proliferation in cultural human umbilical vein endothelial cell (HUVEC) and to simulate hypertensive disorder complicating pregnancy (HDCP).To investigate the effect of cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_I) by hypoxia culture in HUVEC.Methods: Cultured in vitro HUVEC, hemin and zinc protoporphyrin--IXwere added to induce and inhibit the expression of HO-1.Inmunohistochemistry were resorted to demonstrate the expression and distribution HO-1 and HO-2 in HUVEC;The expression of HO-1 mRNA were detected using reversetranscription polymerase chain reaction (RT-PCR); The relative amount of CO released in to the media was quantitated as caron monoxide hemoglobin(COHb) by enzyme-linkd immunosorbent aasy;Flow cybomytry and MTT were performed the effect of cell proliferation;The time concentration relationship and time effect relationship between Cocl₂ and the expression of HO-1 mRNA were explored by using RT-PCR;The fluorescence Ca²⁺ indicator Fura-2/AM was used to measure [Ca²⁺]_I of HUVEC in normal and hypoxic condition.by spectrofluorometry.Results: (1) HO was localized in cytoplasm of HUVEC, HO-2 predominantly localized in cytoplasm with weak staining of HO-1. (2) The expression of HO-1 mRNA in HUVEC and the amount of COHb in the media were increased significantly by hemin;meanwhile the proliferation of endothelial cell was progressed markedly (P<0.05, P<0.01); (3)Promoting proliferation and endogenous CO were increased significantly by CoCl₂ simulated hypoxia and Hypoxia culture upregulated the expression of hemeoxygenase and promoted proliferation in HUVEC in a time dependent and dose dependent manner (P<0.05, P<0.01).(4)Hypoxic caused the rise in [Ca²⁺]_i of HUVEC(P<0.05).Conclusion: Our data showed that hemin,CoCl2 inducing hypoxia and hypoxia increased proliferation and upregulated the expression of hemeoxygenase-1;The change of HO-1 mRNA ,endogenous CO and the proliferation of HUVEC is the molecular base of the anti apoptosis of endothelial cell; Increase of [Ca²⁺]_i in HUVEC and endogenous CO might contribute to the angiotasis of cardiovascular diseases. The HO/CO system might play a important role in the development of hypertensive disorder complicating pregnancy and cardiovascular diseases.Part III Experimental study on protection of heme oxygenase and Carbon Monoxide against oxidative stress injury in human umbilical vein endothelial cellsObjective: The Experimental cell model of oxidative stress injury was made successfully by Hydrogen Peroxide(H₂O₂)in human umbilicus vein endothelial cell(HUVEC).The cultured cells were divided in to four groups:control group,H₂O₂ group in which different concentration H₂O₂ was given, hypoxia group in which 24h hypoxia cultured in vitro HUVEC previously and hemin group in which hemin was added previously.To study effects of HO/CO system against oxidative stress injury in HUVEC.Methods: Cell growth condition among groups were observed under the light microscope and the electron microscope;Trypan blue exclusion was used to detect the cell viability; The surviving cells scunted by MTT; The activity of cytoplasmic enzyme lactatede hydro -genase (LDH)that was released into the culture medium was assayed to indicate the integrity of the cell membrane; The expression of HO-1 mRNA were detected using reversetranscription polymerase chain(RT-PCR);Cellular injury was evaluated in terms of the changes of concentration frequency, LDH leakage and cell viability.Results: (1) When H₂O₂ concentration between 500～1000umol/L, there were significant differences between H₂O₂ group and other groups in cell growth condition, LDH leakage and cell viability (P<0.01) , when H₂O₂ concentration between 100～250umol/L, there were no significant differences among groups(P>0.05) .hydrogen peroxide with 500umol/L was acted as the damage group concentration of HUVEC by oxidative stress injury; (2)Hemin and hypoxia promoted proliferation and increased the expression of hemeoxygenase-1 in HUVECs time dependent manner (P<0.01) .(3)It could strengthen against oxidative stress injury of HUVEC ,Compare with damage group, other groups cell growth condition improved, cell viability increased and LDH leakage droped, its protection can be disrupt by specific inhibiter-zinc protoporphyrin-IX.Conclusion:The data demonstrated that the concentration of hydrogen peroxide with 500umol/L is the most suitable to HUVEC by oxidative stress injury,and that hemin and hypoxia pretreatment could protect the endothelial cell against oxidative stress injury.Part IV Study on the role between Heme oxygenase and Carbon Monoxide system and ERK MAPK Pathway in hypertensive disorder complicating pregnancy and human umbilical vein endothelial cellsObjective: In this study HO/CO system of induction by hypoxia culture and acidic fibroblast growth factor (aFGF) and in hypertensive disorder complicating pregnancy (HDCP) ,and the possible role of ERKmitogen-activated protein kinase (MAPK) in this process were evaluated.Methods: The expression of HO-1 mRNA were detected using reverse transcription polymerase chain(RT-PCR) in HDCP and HUVEC; Phosphorylated ERKs (extracellular signal-regulated kinases) proteins were detected by Western blotting;As caron monoxide of hemoglobin (COHb) in the placenta tissue homogenate and media were quantitated by enzyme linked immunosorbent aasy;MTT assay were performed the effect of cell proliferation.Results: (1)Hypoxia promoted proliferation and increased the expression of heme oxygenase-1 and endogenous CO in HUVEC;Phosphorylated ERK1/ ERK2 rocketed by 36h hypoxia, however, The activated forms of ERKs were blocked by PD98059, a specific inhibitor of ERK MAPK in cells.(2)The proliferation could improve significantly by aFGF,PD98059 could suppress this proliferation by aFGF induced compared with the control group (P<0.05, P<0.01).(3)The expressive quantity of HO-1 mRNA (1.13±0.19) was higher in gestational hypertension placenta than control group.(4)HO-1 and COHb was the highest in gestational hypertension, but decreased to follow the progress of HDCP,moderate and severe preeclampsia (1.34 ±0.7,0.68±0.4,P<0.05,P<0.01). (5)ERK1/ERK2 proteins was weak in control group,but accompanying with the HDCP pregress, it was increaed in gestational hypertension , moderate preeclampsia and severe preeclampsia (61.79 ± 1.87,116.2±2.29, 97.88±3.48),a significant difference were observed among groups (P<0.01).Conclusion: These results suggest that hypoxia and aFGF leads to induction of the HO/CO system, it might adjust the proliferation and active the ERK signal transductive pathway ,the PD98059 could block the transduction effectively .ERK MAPK pathway may play an important role in the pathogenesis of HDCP. The ERK MAPKs pathway is important in mediating this process.