1、Mechanism Of Placental 11β-hydroxysteroid Dehydrogenase Type 2 Contributing To The Pathogenesis Of Preeclampsia 2、Hydrogen Sulfide Is A Physiological Regulator Of Hemoglobin-oxygen Binding Affinity In Erythrocytes

First part:Preeclampsia,a major human pregnancy-specific disorder,leads to maternal and fetal morbidity and mortality.Placenta is believed to be the principal contributor to the pathogenesis of preeclampsia,however,the placental factors linked to preeclampsia remain elusive.The most effective solution of PE,so far,is delivery of placenta,which supports that the placenta is the principal contributor to the pathogenesis of PE.The pathogenesis of PE remains largely unknown,however,it is widely accepted that preeclampsia is initiated by shallow invasion of trophoblast cells into the uterine wall and thereafter results in abnormal placentation followed by increased release of placenta-produced factors into the maternal circulation.These in turn cause dysfunction of the maternal endothelium,leading to the preeclamptic clinical symptoms.Glucocorticoids（GCs）are involved in many events during pregnancy including embryo implantation,growth and development of the fetus and placenta as well as initiation of parturition.The gestational tissues including placentas,fetal membranes and deciduas are the targets of GCs.Interestingly,most of target tissues of GCs usually express11b-hydroxysteroid dehydrogenase（11b-HSD）type 1 and 2 by which inactive GCs（cortisone（E）/prednisone in humans）are interconverted with their active counterparts（cortisol（F）/prednisolone in humans）,thereby controlling local availability of GCs.The11b-HSD1 enzyme is intrinsically bidirectional but in vivo predominantly acts as an activating enzyme.By contrast,the 11b-HSD2 enzyme powerfully inactivates F to E.Both of 11b-HSD1 and 2 have been identified in human placentas,however,11b-HSD2 is predominant.Thus,11b-HSD2 maintains a concentration gradient between the F levels in the mother’s and the child’s compartments and constitutes a specific barrier protecting the fetus from the maternal GCs.A number of studies have demonstrated that expression and activity of placental 11b-HSD2 are significantly reduced in PE patients,which is associated with higher F level in placentas of these patients.F has a detrimental proapoptotic effect when it is in excess,therefore,downregulated 11b-HSD2 in placenta is implicated to be associated with intrauterine growth retardation（IUGR）in PE patients.However,placental trophoblasts are the targets of GCs as they express the receptors of GCs.Moreover,some studies have shown that synthetic GCs can modulate the function of trophoblasts including invasion and proliferation in vitro.Thus,it would be of great interest to investigate whether excessive F caused by reduced 11b-HSD2 in placenta is involved in the development of PE.The objectives of the present study were to explore the role of placental 11b-HSD2in the pathogenesis of PE using animal and human models.At first,we examined whether reduced 11b-HSD2 expression and activity in placenta leads to the hallmark of PE in the pregnant rat model.Then,we investigated involvement of 11b-HSD2 in the release of the placental factors linked to PE clinical features using the models of human placental cells.Finally,we elucidated the molecular mechanisms by which 11b-HSD2 modulates the release of the factors linked to PE in human placental cells.Results:1 The pregnant rats were administrated（s.c）with three different dosages of CBX（180μg/d,360μg/d and 720μg/d）from GD7.5 to GD19.5.the expression level and activity of 11β-HSD2 in the placenta was significantly decreased in the group of CBX（360,720μg/d）compared with vehicle group.However,CBX at 180μg/d had no impact on 11β-HSD2 expression and activity.Meantime,11β-HSD1 expression had on change in the placentas among CBX（180μg/d）,CBX（360μg/d）,CBX（720μg/d）and vehicle groups.The pregnant rats of CBX（360,720μg/d）group displayed PE-like features.2 Administration of the CBX-CSA every two days from GD7.5 to GD19.5 caused a significant decrease in 11b-HSD2 expression and activity,whilst administration of CBX-SCR,CBX-NPs and CBX180μg/2d did not affect 11b-HSD2 expression and activity compared with control group.Moreover,the pregnant rats of CBX-CSA displayed PE-like features.3 We injected dexamethasone（s.c.）200μg/kg into pregnant SD rats on D7.5 to D19.5 of their gestation period.The pregnant rats injected DEX displayed PE-like features.4 As mentioned,PE is often linked to impaired invasion of trophoblasts.Interstitial trophoblast invasion was determined by calculating the total area occupied by cytokeratin-positive trophoblast cells in mesometrial triangle（MT）.Systemic administration of CBX（360,720μg/d）significantly decreased trophoblast invasion and impaired spiral artery remodeling compared with saline group,whereas treatment with CBX（180μg/d）did not have impact on trophoblast invasion.Further more,the animals with administration of CBX-CSA also showed a significant decrease in trophoblast invasion in uterus and impaired spiral artery remodeling.5 Decreased placental 11β-HSD2 by CBX alters uteroplacental perfusion in the pregnant rats.PSV of the umbilical artery,maternal channel,spiral artery was decreased in the 360,720μg/d group and CBX-CSA group.6 Systemic administration of CBX（360,720μg/d）led to a significant increase in sFlt-1and the sFlt-1/PlGF ratio but not PlGF level in circulation.Meanwhile,CBX-CSA resulted in elevated sFlt-1 level and the sFlt-1/PlGF ratio but not PlGF level as well.7 HTR8-S/Vneo cells were used to study the roles of 11b-HSD2 in the regulation of migration and invasion.Cortisol(10^-9M-10^-6M)treatment showed no significant effect on the migration and invasion function in HTR8-S/Vneo cells.However,cortisol(10^-6M)treatment suppressed migration and invasion in the cells transfected with 11b-HSD2siRNA.8 Syncytiotrophoblasts were the major cells of sFlt-1 synthesis.In the model of cultured primary syncytiotrophoblasts,cortisol(10^-6M)treatment increased sFlt-1 release in the cells transfected with 11b-HSD2 siRNA.9 A disintegrin and metalloprotease（ADAM）10 and ADAM17 are the sheddases for shedding of sFlt-1.Cortisol(10^-6M)treatment increased ADAM17 but not ADAM10expression in syncytiotrophoblasts in the presence of CBX.Further,we showed that cortisol(10^-6M)enhanced ADAM17 expression in syncytiotrophoblasts transfected with11b-HSD2 siRNA.10 The sFlt-1 secretion was significantly decreased in the cells with ADAM17knockdown.11 We constructed a reporter gene containing ADAM17 promoter and the ADAM17promoter with mutant GRE.We showed that synthetic GC,dexamethasone,stimulated ADAM17 reporter activity but not the activity of the gene containing mutant GRE promoter.12 Systemic administration of CBX（360,720μg/d）resulted in a significant increase in ADAM17 expression compared with vehicle treatment.Administration of CBX-CSA also led to increased ADAM17 expression in placentas.Meantime,the expression levels of ADAM17 were significantly increased in PE placentas compared with that in the placentas of normotensive patients.Level of 11β-HSD2 is significantly downregulated in PE placentas compared with normal ones.Correlation analysis showed that 11β-HSD2 level inversely correlated to ADAM17 level.Conclusion:Impaired function of trophoblasts during placentation and imbalance of proangiogenic and antiangiogenic factors in placenta are the mechanisms by which 11b-HSD2 contributes to PE development.Second part:In the past decade,the biological functions of gaseous transmitters including nitric oxide（NO）,carbon monoxide（CO）and hydrogen sulfide（H₂S）have received considerable attention because they are involved in wide ranges of physiological and pathophysiological processes.H₂S is involved in numerous physiologic and pathologic processes including vasodilation,angiogenesis,and inflammation.Its synthesis from L-cysteine occurs naturally in wide ranges of mammalian tissues mainly through the activityoftheenzymesincludingcystathionine-g-lyase（CSE）and cystathionine-b-synthase（CBS）.In particular,hypoxic conditions result in suppression of H₂S production in various tissues and cell types including brain and lung tissue,endothelium,cardiomyocytes,renal proximal tubular cells,pheochromocytoma cells and mesenchymal stem cells.Our previous research has shown that hypoxic conditions result in deregulation of placental CBS and CSE expression.The research also showed that decreased H₂S level suppressed the angiogenesis of HUEVCs.Red blood cells（RBCs）,the most abundant type of blood cell,constitute approximately75%of the human body’s total cell count.The principal function of RBCs is the transport of oxygen（O₂）between the respiratory system and peripheral tissues as RBCs have high intracellular concentration of hemoglobin（Hb）as O₂ carrier.In the lungs,O₂ diffuses across the alveolar barrier from inspired air into blood,where the majority is bound by Hb to form oxy-Hb.In the peripheral tissue O₂ is released from oxy-Hb（deoxygenation）and diffused into the cells.The relationship between O₂ affinity of Hb and O₂ partial pressure can be described as the Hb–oxygen dissociation curve.Erythrocytes can functionally adjust O₂ uptake,transport,and delivery through sophisticated regulation of Hb-O₂ affinity bypH,temperatureandendogenousallostericmodulators,suchas2,3-bisphosphoglycerate（2,3-BPG）.The pH and concentrations of these allosteric modulators in RBCs vary depending on cellular metabolism and changes in the extracellular milieu,leading to adjustments in Hb-O₂ affinity that may be adaptive in optimizing tissue O₂ supply.The formation of 2,3-BPG in erythrocytes is modulated by various signaling pathways and its levels is known to be induced under hypoxia.BPGM is the major enzyme to generate 2,3-BPG.However,it is unknown how H₂S regulate the formation of 2,3-BPG and O₂ affinity of Hb.To address these questions,we conducted mouse genetic,metabolomic,bone marrow transplantation and mass spectral analysis coupled with in vitro human and mouse erythrocyte culture to determine the specific function of H₂S in the erythrocytes and underlying mechanisms.Results:1 To better understand the role of H₂S in erythrocytes,we first examined the metabolic profiles of erythrocytes isolated from wild type（WT）mice and CSE knockout(Cse^-/-)mice reported with a remarkably decreased H₂S level.The results revealed that more than 30 metabolites in erythrocytes were differently distributed between wild type（WT）and Cse^-/-mice.Among the metabolites we detected,2,3-BPG was highly elevated in the erythrocytes of Cse^-/-mice compared to WT mice.2 Elevated 2,3-BPG level in erythrocytes of Cse^-/-mice was confirmed by spectrophotometric assay.As 2,3-BPG is a specific allosteric modulator that regulates Hb-O₂ affinity,we examined the oxygen dissociation curve of Cse^-/-mice.As expected,the50%oxygen saturation（P50）value was significantly increased in Cse^-/-mice,by 4 to 6mmHg compared with WT mice.Next,to ensure increased 2,3-BPG and P50 levels in Cse^-/-mice were due to decreased H₂S production,we treated the Cse^-/-mice with a slow-releasing hydrogen sulfide donor GYY4137 and monitored its effects on erythrocyte2,3-BPG and P50.We found that GYY4137（50 mg/kg）treatment significantly reduced2,3-BPG and P50 levels.Of note,GYY4137 reduced erythrocyte 2,3-BPG levels and P50in WT mice.3 Three groups of mice were generated by BMT including:（1）‘Cse^-/--to-WT’group was generated by transplanting BM of Cse^-/-mice to WT mice to examine if CSE deficiency only in BM-derived cells was sufficient to affect Hb-O₂ affinity;（2）‘WT-to-Cse^-/-’group was designed to critically determine if CSE expressed in peripheral tissues could have effects on Hb-O₂ affinity in WT erythrocytes;（3）‘WT-to-WT’group was WT mouse BM transplanted to WT mice and served as an experimental control group.CSE protein level in mature erythrocytes was then detected as an indicator of chimerism.Among these groups,H₂S level in circulation,erythrocyte 2,3-BPG and P50 levels were similar between‘WT to WT’and‘Cse^-/-to WT’mice.In contrast,the‘WT-to-Cse^-/-’mice showed significantly lower circulatory H₂S levels compared with that of‘WT-to-WT’and‘Cse^-/--to-WT’mice.Accordingly,erythrocyte 2,3-BPG and P50 levels in the‘WT-to-Cse^-/-’mice were significantly higher than those in‘WT-to-WT’and‘Cse^-/--to-WT’mice.The elevated erythrocyte 2,3-BPG and P50 in the‘WT-to-Cse^-/-’mice were reversed by H₂S donor GYY4137.4 Next,to extend our mouse studies,we tested the direct effects of H₂S on 2,3-BPG production and oxygen-affinity of Hb in erythrocytes isolated from WT mice and humans.Treatment of isolated mouse erythrocytes with H₂S donor GYY4137（500μM）significantly decreased 2,3-BPG concentration in the cells and reduced P50 levels.Similar effects of GYY4137 on 2,3-BPG production and P50 levels were obtained in isolated human erythrocytes.5 BPGM is the major enzyme to generate 2,3-BPG.Thus,we hypothesized that H₂S might regulate BPGM activity.As expected,Cse^-/-mice showed an increase in BPGM activity in erythrocytes compared with WT mice.GYY4137 treatment decreased BPGM activity in Cse^-/-mice.Similar effect of GYY4137 on erythrocyte BPGM activity occurred in WT mice.To extend our mouse studies,we tested the direct effects of GYY4137 on BPGM activity using cultured mouse and human erythrocytes.GYY4137 treatment inhibited BPGM activity in human and mouse erythrocytes.Conclusion:Here we report an important role of H₂S in regulating erythrocyte Hb-O₂ binding affinity.