title: Blockade of c-Jun N-terminal kinase activation abrogates hyperglycemia-induced yolk sac vasculopathy in vitro.
abstract: OBJECTIVE
  Maternal hyperglycemia has an impact on both the function and morphology of the rodent visceral yolk sac; the objective of the present study was to determine whether hyperglycemia in vitro affects yolk sac vasculogenesis. Because maternal hyperglycemia triggers c-Jun N-terminal kinase (JNK) 1 and 2 activation in the yolk sac, we tested whether the inhibition of JNK activation would ameliorate hyperglycemia-induced yolk sac vasculopathy. In rodents, the yolk sac serves as the primitive placenta after implantation and before the formation of the chorioallantoic placenta. Furthermore, during this early stage, the nutrition from mother to embryo is considered to be facilitated by a tissue-to-tissue form of nutrition, referred to as histiotropic nutrition, and subsequently via yolk sac facilitation (hemotropic nutrition). In addition, during embryopathy, teratogen such as hyperglycemia is associated with concomitant injury to the yolk sac and embryo.
  
  
  METHODS
  Rat embryos at embryonic day 9 were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (500 mg /dL glucose) conditions. JNK activation was inhibited using a JNK1/2-specific inhibitor SP60025 at concentrations of 40, 400, and 800 nM. After 48 hours, the development of yolk sac vasculatures was evaluated by assigning to arbitrative scores on the basis of yolk sac vasculature morphology. The correlation between yolk sac vasculature and embryonic malformation rates was assessed. Levels of phosphorylated JNK1/2 and Bcl-2-associated X protein (Bax) in the yolk sacs from conceptuses of the euglycemic and hyperglycemic groups were determined by Western blotting with densitometric quantification.
  
  
  RESULTS
  Under hyperglycemic conditions, yolk sac development was morphologically impaired. The yolk sac vasculature score of the hyperglycemic group was significantly lower than that of the euglycemic group. Yolk sac vasculature morphologic scores were inversely correlated with embryonic malformation rates. Levels of phosphorylated JNK1/2 and Bax in yolk sacs of the hyperglycemic group were significantly higher than those in yolk sacs of the euglycemic group. JNK1/2-specific inhibitor, SP600125, ameliorated the adverse effect of hyperglycemia on yolk sac vasculature development. Whereas the vasculature morphologic score of yolk sacs in the hyperglycemic group was 54% lower than that of euglycemic group, the vasculature morphologic score of yolk sacs in hyperglycemic plus 800 nM SP600125 group was as same as that in the euglycemic group. Thus, SP600125 at 800 nM completely reversed hyperglycemia-induced vasculopathy as well as embryopathy.
  
  
  CONCLUSIONS
  Hyperglycemia in vitro induces yolk sac vasculopathy. Embryonic malformation is inversely correlated with the yolk sac vasculature development, suggesting that hyperglycemia-induced yolk sac vasculopathy may be one of the causative factors in hyperglycemia-induced embryonic malformation. Blockade of JNK1/2 activation restores hyperglycemia-induced vasculopathy and reduces the malformation rates. These findings indicate that JNK1/2 activation mediates the deleterious effect of hyperglycemia on yolk sac vasculature and embryonic development.
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Peixin
authors.initials: P
authors.lastname: Yang
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zhao
authors.forename: E Albert
authors.initials: EA
authors.lastname: Reece
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology*; D006943:Hyperglycemia / Q000150:complications*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014652:Vascular Diseases / Q000209:etiology* / Q000517:prevention & control*; D015017:Yolk Sac / Q000098:blood supply*
chemical_list: D000873:Anthracenes; C432165:pyrazolanthrone; D048031:JNK Mitogen-Activated Protein Kinases