title: Trichloroethylene decreases heat shock protein 90 interactions with endothelial nitric oxide synthase: implications for endothelial cell proliferation.
abstract: Trichloroetheylene (TRI) is an environmental pollutant that has been linked to congenital heart defects (CHD). Endothelial nitric oxide synthase (eNOS) generation of nitric oxide (NO) plays an important role in endothelial cell proliferation, which is considered essential for normal blood vessel growth and development. We hypothesized that TRI alters the balance of NO and superoxide anion (O2-) to impair endothelial cell proliferation. Proliferating endothelial cells were pretreated with TRI (5 microM) and then stimulated with the calcium ionophore, A23187 (5 microM), to determine changes in endothelial cell and eNOS function with respect to NO and O2- generation. Immunoblots of eNOS, phospho-eNOS at serine 1179 (S1179), and the levels of associated heat shock protein 90 (hsp90) were used to define the activation state of eNOS. The effects of TRI (0.05-100 microM) on vascular endothelial growth factor (VEGF, 0.58 nM) induced endothelial cell proliferation were determined from cell counts. TRI decreased A23187-stimulated nitrite + nitrate production from 1.99 +/- 0.90 to 0.89 +/- 0.51 pmol/mg protein (p < 0.05; n = 6). In controls, Lomega-nitroargininemethylester (L-NAME) increased A23187-stimulated O2- production from 0.130 +/- 0.089 to 0.214 +/- 0.071 nmol/min/mg protein (p < 0.05; n = 5). In TRI-treated cultures, however, L-NAME decreased A23187-stimulated O2- production from 0.399 +/- 0.121 to 0.199 +/- 0.055 nmol/min/mg protein (p < 0.05; n = 5). TRI decreased hsp90 associated with eNOS by 46.7% and inhibited VEGF-stimulated endothelial cell proliferation by 12 to 35%. These data show that TRI alters hsp90 interactions with eNOS and induces eNOS to shift from NO to O2- generation. Our findings provide new insight into how TRI alters endothelial and eNOS function to impair VEGF-stimulated endothelial proliferation. Such changes in endothelial function may play an important role in the development of congenital heart defects.
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, Cardiovascular Center, Milwaukee, Wisconsin 53226, USA. JOU@mcw.edu
authors.forename: Jingsong
authors.initials: J
authors.lastname: Ou
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Ou
authors.forename: D Gail
authors.initials: DG
authors.lastname: McCarver
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Hines
authors.forename: Keith T
authors.initials: KT
authors.lastname: Oldham
authors.forename: Allan W
authors.initials: AW
authors.lastname: Ackerman
authors.forename: Kirkwood A
authors.initials: KA
authors.lastname: Pritchard
mesh_terms: D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011233:Precipitin Tests; D013481:Superoxides / Q000378:metabolism; D014241:Trichloroethylene / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D007476:Ionophores; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D014241:Trichloroethylene; D009569:Nitric Oxide; D000001:Calcimycin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester