title: Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.
abstract: OBJECTIVE
  This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae.
  
  
  METHODS
  Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant.
  
  
  RESULTS
  Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested.
  
  
  CONCLUSIONS
  These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.
authors.affiliation: Department of Natural Sciences, Middlesex University, London, United Kingdom. f.hills@mdx.ac.uk
authors.forename: Frank A
authors.initials: FA
authors.lastname: Hills
authors.forename: Huseyin
authors.initials: H
authors.lastname: Mehmet
authors.forename: Mark H
authors.initials: MH
authors.lastname: Sullivan
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002585:Cesarean Section; D005260:Female; D006493:Heparin / Q000502:physiology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology; D007335:Insulin-Like Growth Factor II / Q000502:physiology*; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011263:Pregnancy Trimester, Third; D019311:Staurosporine / Q000494:pharmacology; D014327:Trophoblasts / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D014409:Tumor Necrosis Factor-alpha; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D006493:Heparin; D019311:Staurosporine