title: Contrasting effects of chronic hypoxia and nitric oxide synthase inhibition on circulating angiogenic factors in a rat model of growth restriction.
abstract: OBJECTIVE
  We hypothesized that nitric oxide (NO) inhibition has synergistic effects with chronic hypoxia in altering maternal serum levels of soluble fms-like tyrosine kinase 1 (sFlt-1), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF). We tested our hypothesis in a rodent model of intrauterine growth restriction induced by chronic hypoxia and NO inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME).
  
  
  METHODS
  Timed pregnant adult Sprague-Dawley rats were assigned to the following groups: (1) 20% (oxygen) O2 + saline (n = 7); (2) 20% O2 + L-NAME (n = 8); (3) 14% O2 + saline (n = 5); (4) 14% O2 + L-NAME (n = 5); (5) 10% O2 + saline (n = 6); and (6) 10% O2 + L-NAME (n = 6). Seven nulliparous females served as nonpregnant controls. L-NAME (50 mg/rat/day) or saline was administered via subcutaneous osmotic pumps, inserted on day 17 of gestation. A hypoxic chamber was used to assure mild (14% O2) or severe (10% O2) hypoxic environment after surgical placement of the minipumps and until the animals were killed on day 21 of gestation before the onset of labor. Maternal blood was collected preceding death. Free serum levels of VEGF, PlGF, and sFlt-1 were measured by highly specific immunoassays. Two composite indices were calculated (sFV: log [(sFlt-1)/VEGF] and sFP: log [(sFlt-1)/PlGF] and compared among groups.
  
  
  RESULTS
  Fetal growth restriction was induced by both severe hypoxia (10% O2) and L-NAME infusion (2-way analysis of variance, P = .02 O2 levels, P < .001 L-NAME), whereas their combination proved to be the most damaging (P < .001). Pregnancy was characterized by higher maternal serum concentrations of VEGF (P < .001) and PlGF (P < .001), but lower levels of sFlt-1 (P = .037) compared with nonpregnant controls. Serum VEGF levels were not altered by either hypoxia or L-NAME infusion (P = .348 O2 levels, P = .205 L-NAME). In contrast, L-NAME significantly increased sFlt-1 serum levels independent of O2 levels (P = .032, L-NAME treatment, P = .991 O2 levels). Chronic hypoxia significantly decreases the circulating levels of PlGF (P < .001) independent of L-NAME treatment. The sFV ratio was neither altered by hypoxia nor by L-NAME infusion. In contrast, the sFP ratio was significantly increased by both L-NAME (P < .001) and severe hypoxia (P < .001), but the effect was not synergistic (P = .655).
  
  
  CONCLUSIONS
  Chronic NO inhibition as well as hypoxia induce fetal growth restriction and significantly change maternal circulating levels of sFlt-1 and PlGF, but not of VEGF. The primary effect of chronic hypoxia is in decreasing circulating levels of PlGF that contrasts with that of NO inhibition, which selectively increases sFlt-1 levels. Their effect is thus not synergistic, suggesting independent pathways.
authors.affiliation: Division of Maternal and Fetal Medicine, Department of Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520-8063, USA. mert.bahtiyar@yale.edu
authors.forename: Mert Ozan
authors.initials: MO
authors.lastname: Bahtiyar
authors.forename: Catalin
authors.initials: C
authors.lastname: Buhimschi
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Ravishankar
authors.forename: Joshua
authors.initials: J
authors.lastname: Copel
authors.forename: Errol
authors.initials: E
authors.lastname: Norwitz
authors.forename: Svena
authors.initials: S
authors.lastname: Julien
authors.forename: Seth
authors.initials: S
authors.lastname: Guller
authors.forename: Irina A
authors.initials: IA
authors.lastname: Buhimschi
mesh_terms: D000818:Animals; D002908:Chronic Disease; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005317:Fetal Growth Retardation / Q000097:blood / Q000201:enzymology* / Q000209:etiology*; D000860:Hypoxia / Q000150:complications*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D000072483:Placenta Growth Factor; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000097:blood*; D051381:Rats; D017207:Rats, Sprague-Dawley; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000097:blood*; D042442:Vascular Endothelial Growth Factors / Q000097:blood*
chemical_list: D004791:Enzyme Inhibitors; C000608038:Pgf protein, rat; D011257:Pregnancy Proteins; D042442:Vascular Endothelial Growth Factors; D000072483:Placenta Growth Factor; D019001:Nitric Oxide Synthase; C501164:Flt1 protein, rat; D040281:Vascular Endothelial Growth Factor Receptor-1; D019331:NG-Nitroarginine Methyl Ester