The Role Of Regulation Of SFlt-1 Expression In Trophoblast In Placenta Vessel Development

Objective1. The study was aimed to investigate the effect of PAR-1 on sFlt-1 expression in trophoblast.2. The study was also aimed to investigate the effect of oxidative stress on sFlt-1 expression in trophoblast.Methods1. PAR-1 regulation of sFlt-1 expression in trophoblast1.1. Extravillous trophoblast line HTR-8/Svneo (H8) culture;1.2. H8 cells was transfected with PAR-1 interference plasmid and overexpression plasmid respectively, the transfection efficiency were detected by qRT-PCR and Western blot assay;1.3. SFlt-1, PlGF and VEGF mRNA expression in H8 cells were determined by qRT-PCR;1.4. SFlt-1, PlGF and VEGF secretion in H8 cells were tested with ELISA;2. Oxidative stress regulation of sFlt-1 expression in trophoblast2.1. H8 cells was treated with CoCl2 and/or eda, and the cell viability was detected by MTT assay;2.2. Intercellular ROS level in H8 cells was determined by Flow cytometry;2.3. SFlt-1, PlGF and VEGF mRNA expression in H8 cells were determined by qRT-PCR;2.4. SFlt-1, PlGF and VEGF secretion in H8 cells were tested with ELISA.Results1. PAR-1 regulation of sFlt-1 expression in trophoblast1.1. Successfully transfected cells expressed strong green fluorescent which showed high transfection efficiency;1.2. Compared to control, overexpression and inhibition group PAR-1 mRNA expression were:570.60±19.2(P<0.05),0.19±0.03(P< 0.05),and protein expression were 1.89±0.20 (P<0.05),0.63±0.05 (P<0.05),respectively;1.3. Compared with control group, overexpression and inhibition and group sFlt-1 mRNA were 2.60±0.26 (P<0.05) and 0.22±0.04, respectively (P<0.05), protein secretion were 1.49±0.13 (P<0.05) and 0.52±0.04 (P<0.05);1.4. Compared with control group, overexpression and inhibition group PIGF mRNA were 0.61±0.13 (P>0.05) and 2.30±0.18 (P<0.05), the protein expression were 1.06±0.03 (P>0.05) and 1.11±0.04 (P>0.05) respectively;1.5. Compared with control group, overexpression and inhibition group VEGF mRNA were 1.44±0.24 (P>0.05) and 2.67±0.45 (P<0.05),the protein expression were 0.93±0.21 (P >0.05) and 2.08±0.05 (P<0.05).respectively.2. Oxidative stress regulation of sFlt-1 expression in trophoblast2.1. Compared to control group, cell viability was reduced in hypoxia group with a concentration dependent, the optimal dose was 500?M (P<0.05), but no change in treatment control group (P>0.05), and was increased in treatment group with a dose dependent, the optimal dose was 100?uM (P<0.05);2.2. Compared to control group, hypoxia increased ROS (2.41±0.27, P<0.05) while treatmet and treatment control groups inhibited ROS production (1.44±0.11, P<0.05), respectively;2.3. Compared to control, hypoxia induced increase of sFlt-1 mRNA (2.71±0.35, P<0.05) and protein expression (3.15±0.12, P<0.05), treatment group (1.22±0.12) and treatmet control group (1.12±0.17) apparently inhibited sFlt-1 production (P<0.05);2.4. Compared to control group, hypoxia, treatment, treatment control groups PIGF mRNA expression are 2.06±0.47,3.12±0.32,2.50±0.82, respectively, (P>0.05), hypoxia slightly increased PIGF protein secretion(1.32±0.10, P>0.05), treatment group decreased PIGF expression (0.93±0.04, P<0.05);2.5. Compared to control, treatment group promoted VEGF mRNA expression(5.61±1.05, P<0.05), apparently higher than hypoxia group (2.36±0.68, P<0.05), there is no difference between three groups in VEGF protein expression (0.95±0.07,0.94±0.03, 1.02±0.06,P>0.05).Conclusion1. Upregulation of PAR-1 promoted sFlt-1 expression in HTR-8/SVneo cells, in the contrary, downregulation of PAR-1 could significantly inhibit sFlt-1 expression which indicated that PAR-1 could regulate sFlt-1 in trophoblast;2. Oxidative stress promoted sFlt-1 expression but oxygen radical scavenging significantly inhibited sFlt-1 elevation, which showed oxidative stress play an effect on sFlt-1 expression in trophoblast.Objective1. The work was aimed to investigate the effect of PAR 1-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling;2. The work was also aimed to investigate the effect of oxidative stress-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling;Methods1. The effect of PAR1-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling1.1. Cell apoptosis was detected by Hoechst 33258 stainning;1.2. BAX and BCL2 protein expression was texted by Western blotting assay;1.3. Cell migration ability was measured by Transwell migration assay;1.4. Cell invasion capacity was measured by Matrigel invasion assay;1.5. MMP2 and TIMP2 expreesion were measured by qRT-PCR and ELISA;1.6. Tube formation assay was used to determined the role of PAR-1-regulated sFlt-1 expression on tube formation ability;1.7. Villous and deciduals culture system was established to clarify the role of PAR-1-regulated sFlt-1 expression on spiral artery remodeling process.2. The effect of oxidative stress-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling2.1. Cell apoptosis was detected by Hoechst 33258 stainning;2.2. BAX and BCL2 protein expression was texted by Western blotting assay;2.3. Cell migration ability was measured by Transwell migration assay;2.4. Cell invasion capacity was measured by Matrigel invasion assay;2.5. MMP2 and TIMP2 expreesion were measured by qRT-PCR and ELISA;2.6. Tube formation assay was used to determined the role of oxidative stress-regulated sFlt-1 expression on tube formation ability;2.7. Villous and deciduals culture system was established to clarify the role of oxidative stress-regulated sFlt-1 expression on spiral artery remodeling process.Results1. The effect of PAR1-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling1.1. Compared to control group, overexpreesion group promoted H8 cell apoptosis (246.8±15.3%, P< 0.05) inhibition group have no influence on cell apoptosis (121.4±14.8%, P>0.05);1.2 Compared to control group, overexpression group promoted BAX production (1.29±0.08, P<0.05), reduced BCL2 expression (0.79±0.05, P<0.05); while inhibition group downregulated BAX protein (0.72±0.03, P<0.05), upregulated BCL2 expression (1.21±0.06,P<0.05);1.3. Compared to control group, overexpression group inhibited cell migration(32.7±7.1%, P<0.05), while inhibition group promoted cell migration (156.9±23.4%, P<0.05);1.4. Compared to control group, overexpression group inhibited cell invasion (46.1±4.4%, P<0.05), inhibition group promoted cell invasion (121.0±7.5%, P<0.05);1.5. Compared to control group, overexpression group inhibited MMP2 mRNA(0.53±0.08, P<0.05) and protein secretion (0.78±0.04, P<0.05), promoted TIMP2 expression (1.61±0.17,1.53±0.05, P<0.05), while inhibition group significantly increased MMP2 mRNA (1.90±0.17, P<0.05) and protein (1.20±0.06, P<0.05) expression, but decreased TIMP2 expression (0.35±0.12,0.65±0.04, P<0.05);1.6. Compared to control group, overexpression inhibited tube formation ability (0.38±0.04, P<0.05) while inhibition group promoted this capacity (1.28±0.09, P<0.05);1.7. Overexpression group meaningfully inhibited uterine spiral artery recasting, in contrast, inhibition group improved artery remodeling disorder.2. The effect of oxidative stress-regulated sFlt-1 expression on placenta angiogenesis and artery remodeling2.1. Compared with control, relative apoptosis rate in hypoxia group was 336.1±21.7%, (P <0.05), treatment group was (175.0±14.4%, P<0.05) compared to hypoxia group; treatment control was 80.5±22.7%, (P<0.05);2.2. Compared to control, hypoxia upregulated BAX protein expression (3.47±0.74, P< 0.05) but downregulated BCL2 expression (0.58±0.08, P<0.05), treatment group inhibited increase of BAX (0.88±0.02, P<0.05) and promoted BCL2 production (1.38±0.12, P< 0.05); treatment control group BAX and BCL2 expression were (0.86±0.03,1.07±0.06, P >0.05);2.3. Compared to control group, migration rate in hypoxia group was 30.0±5.6%(P<0.05), treatment group was 100.1±7.5%, (P<0.05), treatment control was 122.9±7.6%(P>0.05);2.4. Compared to control, invasion rate in hypoxia group and treatment control group were (12.8±3.6%, P<0.05) and (111.5±17.3%, P>0.05), respectively, treatment group was higher than hyupoxia group (68.9±4.7%, P<0.05);2.5. Compared to control group, hypoxia group inhibited MMP2 mRNA(0.28±0.05, P< 0.05) and protein secretion (0.73±0.05, P<0.05), promoted TIMP2 protein secretion (1.20±0.04,P<0.05), treatment group significantly increased MMP2 mRNA (1.22±0.23, P <0.05) and protein (1.08±0.06, P<0.05) expression, but decreased TIMP2 expression (0.12±0.04,0.71±0.05, P<0.05), treatment control group MMP2 were (1.41±0.21, 1.12±0.05) and TIMP2 were (0.41±0.16,0.69±0.07);2.6. Compared with control group, hypoxia group inhibited tube formation (0.55±0.10, P< 0.05), compared to hypoxia, treatment group promoted tube formation (98±0.08, P<0.05), treatment control was (0.93±0.07 (P>0.05);2.7. Compared to control group, artery remodeling was significantly inhibited in hypoxia group while improved in treatment and treatment groups.Conclusion1. Upregulation of PAR-1 induced HTR-8/SVneo cells apoptosis, inhibited cell migration, invasion and tube formatin as well as artiry remodeling, in the contrary, downregulation of PAR-1 could significantly enhanced cell biological function and improve tube formation capacity and artery remodeling which indicated that PAR-1-regulated sFlt-1 expression play an important effect in agiogenesis and artery remodeling.2. Oxidative stress promoted HTR-8/SVneo cell apoptosis, inhibited migration, invasion and tube formation, caused artery remodeling obstacle, while oxygen radical scavengeing significantly improved cells dysfunction, vessle formation and artery recasting disorder which may have an effect on vessel formation and artery remodeling in placenta.Objective1. To investigate the effect of PAR-1-regulated sFlt-1 expression on placenta angiogenesis in pregnant rat.2. To investigate the effect of oxidative stress-regulated sFlt-1 expression on placenta angiogenesis in pregnant rat.Methods1. PAR-1-regulated sFlt-1 expression improve placenta angiogenesis in preeclampsia rat.1.1. Preeclampsia model was established by intraperitoneal injection of L-NAME in pregnant rat;1.2.18 model preeclampic rats were randomly devided into three groups, treatment groups were transfected with shPAR-1 in placenta;1.3. After transfection in vivo, the transfected placenta was observed with frozen section under fluorescence microscope; and PAR-1 expression was detected by RT-PCR and Western boltting assay;1.4. On day 20 day of pregnancy, fetus and placenta were collected, and whose size and weight were detected;1.5. After transfected, placenta morphological change and microvessel density was measured by immunohistochemical assay.1.6. SFlt-1, VEGF and PLGF expression in placenta were detected by RT-PCR;1.7. SFlt-1, VEGF and PLGF expression in serum of pregnant rats were tested by ELISA.2. Oxidative stress-regulated sFlt-1 expression improve placenta angiogenesis in preeclampsia rat2.1. Preeclampsia model was established by intraperitoneal injection of L-NAME in pregnant rat;2.2.20 model of preeclampic rats were randomly devided into four groups, treatment groups were treated with injection of eda;2.3. On day 20 day of pregnancy, placenta and fetus were collected and the weight and size of which were measured;2.4. Placenta morphological change and placenta microvessel density was detected by immunohistochemical assay.2.5. SFlt-1, VEGF and PlGF expression in placenta were measured by RT-PCR;2.6. SFlt-1, VEGF and PlGF secretion in serum of pregnant rats were tested by ELISA.Results1. PAR-1-regulated sFlt-1 expression improve placenta angiogenesis in preeclampsia rat.1.1. In all 18 rats, one rat was died from anesthesia accident, other rats were alive through the whole gestation;1.2. Strong green fluorescence expression was observed in transfected placenta under fluorescence microscop and PAR-1 expresssion was significantly inhibited;1.3. The blood pressure were gradually increased in preeclampsia and treatment groups (P <0.05) bfeore the day 17 of gestation, and that is lower in treatment group than in preeclampsia group at day 19 of pregnancy (P<0.05);1.4. Compared to control group, the intervillous in placenta were narrow and the number of microvessel was decreased in preeclampsia group (53.33±4.26> P<0.05), in treatment group, the intervillous was improved and microvessel was (68.67±2.40, P<0.05)1.5. Compared to control group, the mean weight and size of placenta and fetus were significantly smaller in preeclampsia group while were improved in treatment group compared with preeclampsia group (P<0.05);1.6. Compared to control group, sFltl expression (1.87±0.12, P<0.05) was increased in preeclampsia placenta and was apparently reduced in treatment group (1.01±0.14, P>0.05), the expression of VEGF and P1GF in preeclampsia were 0.44±0.05 and 0.49±0.06, (P< 0.05) while in treatment placenta were 1.48±0.21 and 1.12±0.05, (P<0.05);1.7. SFlt-1 protein secretion in serum of pregnant rats in preeclampsia group (2.02±0.03, P <0.05) were significantly higher than that in treatment (0.64±0.06, P<0.05) and control group, while VEGF (0.73±0.01) and P1GF (0.60±0.04) expressin were lower (0.96±0.02 and 0.77±0.04, P<0.05) than two groups.2. Oxidative stress-regulated sFlt-1 expression improve placenta angiogenesis in preeclampsia rat2.1.In all 20 rats, one rat was died from unclear disease, other all rats were alive through the whole gestation;2.2. The blood pressure were gradually in preeclampsia and treatment groups (P<0.05) before the day 15 of gestation, while was decreased in treatment group than in preeclampsia group on day 19 of pregnancy (P<0.05);2.3. Compared to control group, intervillous in preeclampsia placenta was narrowed and the microvessel was decreased (54.40±.25, P<0.05), in treatment group, the intervillous was improved and the number of microvessel was increased (73.07±1.31,P<0.05);2.4. The mean weight and size of placenta and fetus in preeclampsia were significantly smaller than control group and treatment group (P<0.05)2.5. In preeclamsia placenta, sFlt-1 expression was increased (1.86±0.14, P<0.05) while VEGF (0.95±0.12, P>0.05) and PIGF (0.72±0.04, P<0.05) were decreased, in treatment group sFlt-1 expression (1.12±0.12, P<0.05) was reduced and VEGF (1.79±0.36, P< 0.05) and P1GF (0.96±0.01, P<0.05) expression were increased compared with preeclampsia group;2.6. Compared to control group, sFlt-1 secretion in serum was increased (3.42±0.19, P< 0.05) but VEGF (0.68±0.06, P<0.05) and P1GF(0.50±0.05, P<0.05) were decreased in preeclampsia group, in treatment group, sFlt-1 expression was significantly decreased (1.37±0.03), meanwhile VEGF (0.84±0.03) and PLGF (0.83±0.04) expression were increase.Conclusion1. Inhibition of PAR-1 in placenta could reduce sFlt-1 production and enhanced placental angiogenesis, improve pregnant outcome in preeclamptic rats, it implied that regulation of PAR-1 on sFlt-1 expression may be contributed to placenta vasoculogenesis.2. Administration of oxygen radical scavenger inhibitd sFlt-1 expression which was promoted placental agiogenesis and improve pregnant outcome in preeclamptic rats, it suggested the effect of improvement of oxidative stress play an role in placenta vasoculogenesis.