Differential Role of Smad2 and Smad3 in the Acquisition of an Endovascular Trophoblast-Like Phenotype and Preeclampsia

Abstract

During placental development, cytotrophoblast progenitor cells differentiate into the syncytiotrophoblast and invasive extravillous trophoblasts (EVTs). Some EVTs further differentiate into endovascular trophoblasts (enEVTs) which exhibit endothelial-like properties. Abnormal placental development, including insufficient enEVT-mediated remodeling of the uterine spiral arteries, is thought to be a precipitating factor in the onset of preeclampsia (PE), a pregnancy-related hypertensive disorder. Several members of the transforming growth factor-β (TGF-β) superfamily, such as TGF-βs, Nodal, and Activin have been reported to either promote or inhibit the invasive EVT pathway. These ligands signal through serine/threonine receptor complexes to activate downstream signaling mediators, Smad2 and Smad3. In this study, we determined Smad2 and Smad3 expression pattern in placenta and their effects on trophoblast invasion and differentiation. Total Smad2/3 levels were relatively constant across gestation while the ratio of active phosphorylated forms to their total levels varied with gestational stages, with a higher pSmad2/total Smad2 in later gestation and a higher pSmad3/total Smad3 in early gestation. Immunofluorescent staining revealed that pSmad3 was localized in nuclei of EVTs in anchoring villi. On the other hand, pSmad2 was mostly absent in this invasive EVT population. In addition, pSmad3/total Smad3, but not pSmad2/total Smad2, was significantly lower in both early onset and late onset PE cases, as compared to gestational age-matched controls. Functional studies carried out using a first trimester trophoblast cell line, HTR-8/SVneo, and first trimester human placental explants showed that Smad2 and Smad3 had differential roles in the invasive pathway. Specifically, siRNA-mediated knockdown of Smad2 resulted in an increase in trophoblast invasion and an upregulation of mRNA levels of enEVT markers while the opposite was observed with Smad3 knockdown. In addition, Smad2 siRNA accelerated the EVT outgrowth in first trimester placental explants while the Smad3 siRNA reduced the outgrowth of EVTs when compared to the control. Furthermore, knockdown of Smad2 enhanced, whereas overexpression of Smad2 suppressed, the ability of trophoblasts to form Brkić et al. Differential Role of Smad2 and Smad3 in Placenta endothelial-like networks. Conversely, Smad3 had opposite effects as Smad2 on network formation. These findings suggest that Smad2 and Smad3 have opposite functions in the acquisition of an enEVT-like phenotype and defects in Smad3 activation are associated with PE.

Overexpression of miR-210-3p Impairs Extravillous Trophoblast Functions Associated with Uterine Spiral Artery Remodeling

“…Experiments were conducted using an immortalized human first trimester trophoblast cell line, HTR8/SVneo, kindly provided by Dr. Charles Graham (Queen's University, Kingston, ON, Canada) and were maintained as previously described [40,73]. Briefly, HTR8/SVneo cells were cultured in HyClone™ classical liquid media RPMI 1640 with L-glutamine (GE Healthcare Life Sciences, Ottawa, ON, Canada) supplemented with 10% fetal bovine serum (FBS) (GIBCO ® Life Technologies, Mississauga, ON, Canada) and grown in humidified environment of 5% CO 2 and 37 • C. For oxygen tension experiments, cells were incubated in Sanyo tri-Gas incubators set at 21%, 8%, 3% or 1% O 2 levels and grown in humidified environment of 5% CO 2 and 37 • C.…”

“…Transient transfection of miR-210-3p mimics or inhibitor or small interfering RNA oligomers (100 nM) (Table 2) was conducted using Lipofectamine RNAiMAX ® reagent (Invitrogen, Life Technologies, Mississauga, ON, Canada). A modified protocol was used to optimize transfection efficiency and cell survival using a Lipofectamine volume of 2.4 µL per well in OMEM medium [40,73]. Cells were transfected 24 h post-seeding for 5 h in 6-well plates seeded at 6.0 × 10 4 cells per well.…”

“…To measure the levels of mRNA, 1.0 µg of total RNA was reversed transcribed into cDNA using M-MuLV Reverse Transcriptase (New England Biolabs), following the manufacturer's protocol. qRT-PCR was performed using EvaGreen qPCR 2X Master mix (Applied Biological Materials Inc., Richmond, BC, Canada) following the manufacturer's recommendation and samples were normalized to CYC1 internal control [40,73] using the 2 −∆∆CT method. Primers used were designed using NCBI primer blast and are listed in Table 3.…”

Integrated bioinformatics analysis reveals novel key biomarkers and potential candidate small molecule drugs in gestational diabetes mellitus

“…CEBPB (CCAAT enhancer binding protein beta) [38], ACSL4 [39], MBD2 [40], ULK1 [41], NUCB2 [42], TWIST1 [43], HOOK2 [44], CLDN7 [45], TBK1 [46], YIPF6 [47], TFRC (transferrin receptor) [48], ENPP2 [49], SLIT2 [50], MFGE8 [51], FAT1 [52], GPC4 [53], COL6A3 [54], EGFL6 [55], AOC3 [56], CCN2 [57], LYVE1 [58], RARA (retinoic acid receptor alpha) [59], COL18A1 [60], THY1 [61], CD36 [62], PEMT (phosphatidylethanolamine N-methyltransferase) [63], AIF1L [64], OXTR (oxytocin receptor) [65], LMNA (lamin A/C) [66], CXCL14 [67], DKK3 [68], ANGPTL2 [69] and CMTM7 [70] were reported to be associated with obesity, but these genes might be linked with progression of GDM. AHR (aryl hydrocarbon receptor) [71], STS (steroid sulfatase) [72], PLAC1 [73], CYP11A1 [74], PSG11 [75], STAT5B [76], TLR3 [77], FOLR1 [78], HSPB1 [79], HSP90AA1 [80], ANXA4 [81], ATF3 [82], DAPK1 [83], ENTPD1 [84], ABL1 [85], VSIG4 [86], CD99 [87], VWF (von Willebrand factor) [88], PODXL (podocalyxin like) [89], PDPN (podoplanin) [90], RND3 [91], VCAN (versican) [92], AXL (AXL receptor tyrosine kinase) [93], PIEZO1 [94], GAS6 [93], LAMA4 [95], CAV1 [96], DLL1 [97], CD44 [98], CD81 [99], SMAD3 [100], NES (nestin) [101], DCN (decorin) [102], AGTR1 [103], SLIT3 [104], B2M [105], STAT3 [106], STC1 [107] and ADAMTS1 [108] were shown to participate in facilitating the preeclampsia. Majchrzak-Celiń ka et al [109] ...…”

Integrated bioinformatics analysis reveals novel key biomarkers and potential candidate small molecule drugs in gestational diabetes mellitus

“…Potential pathways were obtained after GO and pathway enrichment analysis. Evidence suggests that CEBPB (CCAAT enhancer binding protein beta) [ [91], VCAN (versican) [92], AXL (AXL receptor tyrosine kinase) [93], PIEZO1 [94], GAS6 [93], LAMA4 [95], CAV1 [96], DLL1 [97], CD44 [98], CD81 [99], SMAD3 [100], NES (nestin) [101], DCN (decorin) [102], AGTR1 [103], SLIT3 [104], B2M [105], STAT3 [106], STC1 [107], ADAMTS1 [108], HSD11B2 [109] and HSD3B1 [110] in preeclampsia were specific, but these genes might be novel target for GDM. Increasing evidence indicates that the development of type 2 diabetes mellitus, due to the dysregulation of genes, such as CSNK2A2 [111], NFE2 [112], CAMK2G [113], RASGRP1 [114], S100P [115], SRR (serine racemase) [116], DHPS (deoxyhypusine synthase) [117], DYRK1A…”