| Background and ObjectivePreeclampsia(PE)is a human-specific pregnancy complication.It is characterized by the occurrence of new-onset hypertension,accompanied by proteinuria or other end-organ dysfunction after 20 weeks of gestation in previously normotensive women.Preeclampsia is one of the leading causes of maternal and perinatal morbidity and mortality in the whole world.To date,the pathogenesis of this morbidity is poorly understood.The effects of clinical treatment are unsatisfactory,and the only treatment available is to terminate the pregnancy and remove the placenta.Thus,the prognosis of preeclamptic patients are poor.Fibrosis is an important histological change occurring in preeclamptic placentas.Some studies showed that the weight of preeclamptic placenta were reduced compared with normal placenta,combining with villous fibrosis.Previous study once found that high production of collagen I was detected in preeclamptic placenta.However,the similar studies are limit.Fibrosis is well-known pathogenic process resulting in the progressive loss of organ structure and function.It is defined by overproduction of the extracellular matrix(ECM)in connective tissues and plays a significant role in the impairment of organ function,such as during liver cirrhosis and cardiac fibrosis.It is noteworthy that diverse diseases in different organs are associated with common pathogenic pathways,as excessive ECM deposition causes physical organ deformation,which impairs organ function and ultimately induces organ failure.collagen I is well known as one of the main member of the collagen family,constituting over 90%of the collagen of the body.Excesive deposition of collagen Ⅰ enchanes and leads to fibrosis directly.To date,detailed investigation of fibrosis in preeclamptic placentas is lacking.Whether collagen Ⅰ is the characteristic collagen deposited in preeclamptic placenta call for further studies.Whether placental fibrosis is involved in the pathogenesis of preeclampsia is still unclear.In this study,we hypothesize that exacerbation of fibrosis leads to impairment of placental function and is involved in the pathogenesis of preeclampsia.Therefore,we performed in vivo and in vitro experiments to clarify the relationship between placental fibrosis and preeclampsia.Methods1.To identify the characteristic collagen deposited in preeclamptic placenta(1)Women with PE and normotensive pregnant(NP)women without previous treatments were recruited in the third trimester from March 2018 to 2019 in the Department of Obstetrics of the Nanfang Hospital,Southern Medical University,China.According to the current American College of Obstetricians and Gynecologists criteria.(2)A sample size of 10 placentas in each group was used for the visualization of placenta architecture by HE staining and the identification of fibrosis degree by Masson staining.Sirius red staining were performed to identify the type of collagen.(3)On the basis of results of staining,tissue sections showing collagen Ⅰ maybe the Characteristic collagen deposited in preeclamptic placenta.Protein level of E-Cadherin、N-Cadherin、MMP-9、Vimentin were analyzed by Western blot.2.To investigate the effect of collagen Ⅰ on pregnant mice(1)Pregnant female mice were randomly divided into five groups on E0.5d(six mice per group):pregnant control group,0.5 mg/kg collagen Ⅰ-treated group,5 mg/kg collagen Ⅰ-treated group,IFA-treated group,and NG-nitro-L-arginine methyl ester(L-NAME)-treated group.In the collagen Ⅰ-treated groups,100 μL of collagen Ⅰ(0.5 mg/kg and 5 mg/kg)was injected intradermally at the base of the tail of each mouse on E0.5d.The mice in the L-NAME group were treated with continuous administration of 125 mg/kg/d of L-NAME,a common vasoconstrictor,starting at 10.5 days of gestation via subcutaneous injection on the nucha[[1]8].Control mice were not injected with any solution.SBP(systolic blood pressure)was recorded every four days.(2)To evaluate the preeclampsia symptom of animal,blood pressure was measured.We determined the blood pressure in conscious mice by tail cuff plethysmography using the Softron BP 2010(Softron Biotechnology,Beijing,China).All the mice were habituated to the measurement procedure 10 times a day before caging with male mice.At least 9 consecutive measurements were recorded,but only when the condition of the mice was stable.Six time point(pre-pregnancy,E0.5,E4.5,E8.5.E12.5,and E16.5)were selected for assessing systolic blood pressure level throughout the pregnancy.(3)All mice were sacrificed,and placental tissues were harvested on gestational day 17.5(E17.5d).H&E staining were performed for morphologic observation in labyrinth layer.Masson staining were used to assess the degree of fibrosis in both labyrinth and junctional zones.(4)The expression of E-Cadherin、N-Cadherin、MMP-9 and Vimentin protein in mice placenta were detected by Western blot.3.To investigate the effect of collagen Ⅰ on trophoblast and the potential signal pathway(1)HTR-8SV/neo cells were seeded in six-well plate which were incubated with different concentration of collagen Ⅰ for 48h.CCK-8 assay was conducted to detect cell viability and cell cycle analysis cell cycle stage was analyzed using flow cytometry.(2)Cells were harvested after incubated with collagen Ⅰ,western blot were performed to accessed the protein level of E-Cadherin、N-Cadherin、MMP-9 and Vimentin.(3)After incubation with 100μg/mL collagen Ⅰ,cells were collected.The high throughput mRNA sequencing analysis was performed on Illumina sequencing platform to obtain the transcriptome.(4)To validate the transcriptome sequencing result,the expression of relative gene was verified by Western Blot.(5)Honokiol,which enhance the phosphorylation of ERK phosphorylation,dissolved at a concentration of 10μM.In the co-treatment group,HTR-8/SVneo cells were treated with Honokiol and collagen Ⅰ 48h.SKL-2001,an agonist of β-catenin which was used at a concentration of 5μM.Co-treament with collagen Ⅰ 24h.Cell viability,cell cycle and relative protein level were analysed by CCK-8,cycle cell and western blotting.Result(1)The degree of fibrosis of the various placental samples is shown in Figure 1A.Collagen deposited in villous tissues and wrapped in blood vessels.According to the Masson’s trichome staining the collagen expression,a high degree of fibrosis of stem villi and terminal villi was more frequent in preeclamptic placentas(p<0.01).(2)collagen Ⅰ appears red-yellow when observed under a polarizing microscope.Notably,the red-yellow colour density was much higher in preeclamptic placentas than in control placentas.Further,the relative gene and protein expression levels of collagen Ⅰ were higher in preeclamptic placentas.(3)At E4.5 d,collagen Ⅰ-injected mice exhibited significantly higher SBP than did control and IFA mice(p<0.05).Moreover,the SBP of the collagen Ⅰ-and L-NAME-treated groups was elevated from the second trimester to the end of gestation.Histomorphometric analysis revealed structural changes in the placenta of mice in the collagen Ⅰ-and L-NAME-treated groups;particularly,infarction was observed in the labyrinth layer.Moreover,in both the labyrinth and junctional zones,a higher degree of fibrosis was observed in the collagen Ⅰ-and L-NAME-treated groups than in the control group.(4)Fetal weight was decreased in both the collagen Ⅰ-and L-NAME-treated groups(p<0.01).However,the number of live pups only decreased in the group treated with 5 mg/kg of collagen Ⅰ(p<0.01).(5)The relative protein expression of MMP-9,E-cadherin,N-cadherin,and vimentin,which regulate trophoblast invasion,using western blotting.Upon exposure to collagen Ⅰ,the relative protein expression of MMP-9,N-cadherin,and vimentin in the placenta decreased compared to that of the control placentas,while E-cadherin expression increased.(6)At collagen Ⅰ doses of 100 and 1000μg/mL,the viability of cells decreased compared with that of cells grown in control plates(p<0.05).Particularly,collagen Ⅰsignificantly induced cell cycle arrest at the G2/M phase at a dose of 1000 μg/mL(p<0.01 in the 1000μg/mL group,Figure 3B).Further,cell invasion was significantly attenuated by collagen Ⅰ(p<0.01).(7 The expression of 801 genes increased while that of 436 genes decreased under collagen Ⅰ treatment compared with gene expression in the control group.The relative expression of upregulated and downregulated genes was illustrated in a heat map.Additionally,we found 2347 PE-related genes to be expressed in our dataset,among which 227 were differentially expressed.With the latter gene set,we carried out KEGG pathway enrichment analysis.Interestingly,genes involved in proteoglycans in cancer,cell cycle,and the PI3K-AKT signaling pathway(which is related to cell proliferation and invasion),were significantly downregulated in our dataset.Consistently,collagen Ⅰ treatment downregulated the expression of ERK2,MET,PI3K,β-catenin,and WNT5A,genes related to cell proliferation and invasion in trophoblasts)(8)Together with transcriptome sequencing analysis,western blotting was used to detect p-ERK/ERK and β-catenin expression in collagen I-treated and untreated mouse placentas and HTR-8SV/neo cells.In both types of samples,we observed decreased expression of p-ERK and β-catenin after collagen Ⅰ treatment.(9)The combination of the ERK activator,honokiol,with collagen I to treat HTR-8/SVneo cells,reversed the decline in proliferation and the G2/M arrest induced by collagen Ⅰ.(10)β-catenin levels decreased in both collagen I-treated mouse placenta and HTR-8/SVneo cells.Therefore,SKL-2001,an agonist of the Wnt/β-catenin pathway that can stabilize intracellular β-catenin levels,was used to clarify the role ofβ-catenin in collagen I-dependent remodulation of trophoblast properties.The relative protein levels of MMP-9,vimentin,N-cadherin,and E-cadherin were also recovered compared with those of collagen I-treated cells.Conclusion(1)Collagen Ⅰ is the characteristic collagen deposited in preeclamptic placenta.(2)Collagen Ⅰ induces preeclampsia-like symptoms in pregnant mice.(3)Collagen Ⅰ suppressed proliferation and invasion through inhibition of ERK/MAPK and Wnt/β-catenin signal pathway... |
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