title: Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.
abstract: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress?
  
  
  
  LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress.
  
  
  
  Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness.
  
  
  
  First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF.
  
  
  
  Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression.
  
  
  
  LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling.
  
  
  
  N/A.
  
  
  
  The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available.
  
  
  
  These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders.
  
  
  
  This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Bolnick
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Jay M
authors.initials: JM
authors.lastname: Bolnick
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Hamid-Reza
authors.initials: HR
authors.lastname: Kohan-Ghadr
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Kilburn
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Omar J
authors.initials: OJ
authors.lastname: Pasalodos
authors.affiliation: Department of Obstetrics and Gynecology, Good Samaritan Hospital Medical Center, West Islip, NY, USA.
authors.forename: Pankaj K
authors.initials: PK
authors.lastname: Singhal
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Dai
authors.affiliation: Department of Obstetrics and Gynecology, Augusta University, Augusta, GA, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Diamond
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.|Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI, USA.
authors.forename: D Randall
authors.initials: DR
authors.lastname: Armant
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
authors.forename: Sascha
authors.initials: S
authors.lastname: Drewlo
mesh_terms: D000028:Abortion, Induced; D019138:Antibodies, Blocking / Q000494:pharmacology; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017984:Enoxaparin / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D066257:Heparin-binding EGF-like Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D010920:Placenta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011247:Pregnancy; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D046509:Tissue Culture Techniques; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D000925:Anticoagulants; D017984:Enoxaparin; D005343:Fibrinolytic Agents; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D047428:Protein Kinase Inhibitors; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase
keywords: BCL-2α; HBEGFimplantation; LMWH; apoptosis; caspase; differentiation; integrins; placenta; trophoblast