title: Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes.
abstract: OBJECTIVE
  The mechanisms underlying membrane rupture at term and preterm are obscure. Collagenolytic activity of matrix metalloproteinases in amniochorionic membranes increases during spontaneous term and preterm labor associated with intra-amniotic infection. We sought to test the hypothesis that reduction-oxidation homeostasis, which is altered in inflammatory states, directly regulates amniochorionic matrix metalloproteinases.
  
  
  METHODS
  Membranes were collected from 7 patients undergoing elective cesarean delivery at term, rinsed thoroughly, and immediately incubated in phosphate-buffered sodium chloride solution at 37 degrees C for 24 hours. Matrix metalloproteinase activity in the culture medium was assayed by substrate-gel electrophoresis and normalized against the dry weight of the tissue incubated. Superoxide anions were generated in the presence of membranes by a xanthine (2 mmol/L) and xanthine oxidase (20 mU/mL) mixture and monitored by reduction of ferri-cytochrome c to ferro-cytochrome c. Incubations were performed in the presence of xanthine alone, a xanthine-xanthine oxidase mixture, superoxide dismutase (500 U/mL), a xanthine-xanthine oxidase-superoxide dismutase mixture, nitro-L-arginine (a nitric oxide synthase inhibitor, 1 mmol/L), xanthine-xanthine oxidase-nitro-L-arginine, S-nitroso-N -acetylpenicillamine (a nitric oxide donor, 10 mmol/L), xanthine-xanthine oxidase-S-nitroso-N -acetylpenicillamine, N -acetylcysteine (a thiol-containing antioxidant, 0.1, 1, or 10 mmol/L), lipopolysaccharide (100 ng/mL), or lipopolysaccharide-N -acetylcysteine. Intracellular generation of superoxide anions was monitored by the reduction of nitroblue tetrazolium to formazan.
  
  
  RESULTS
  Basal matrix metalloproteinase 9 and matrix metalloproteinase 2 levels were detected in all samples. Superoxide anions significantly increased matrix metalloproteinase 9 activity but did not increase matrix metalloproteinase 2 activity, which effect was reversed by the addition of superoxide dismutase. N-acetylcysteine reduced basal activity of both matrix metalloproteinase 9 and matrix metalloproteinase 2 to 20%. Importantly, N-acetylcysteine completely inhibited intracellular formazan formation in cultured membranes both in the absence and in the presence of lipopolysaccharide. Neither nitric oxide synthase inhibition nor the nitric oxide donor S-nitroso-N -acetylpenicillamine had any effect on fetal membrane matrix metalloproteinase activity.
  
  
  CONCLUSIONS
  Matrix metalloproteinase activity in human fetal membranes is reduction-oxidation (redox)-regulated. Matrix metalloproteinase 9 activity in human fetal membranes is directly increased by superoxide anion, a byproduct of macrophages and neutrophils. Neither nitric oxide donors nor nitric oxide synthase inhibitors significantly affect matrix metalloproteinase activity in human fetal membranes. The glutathione precursor N-acetylcysteine dramatically inhibits amniochorionic matrix metalloproteinase activity in addition to inhibiting intrinsic superoxide generation within the tissue. Thus thiol-reducing agents, such as N-acetylcysteine, may be beneficial in preventing preterm premature rupture of the membranes.
authors.affiliation: Division of Perinatal Research, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore 21201-1559, USA.
authors.forename: I A
authors.initials: IA
authors.lastname: Buhimschi
authors.forename: W B
authors.initials: WB
authors.lastname: Kramer
authors.forename: C S
authors.initials: CS
authors.lastname: Buhimschi
authors.forename: L P
authors.initials: LP
authors.lastname: Thompson
authors.forename: C P
authors.initials: CP
authors.lastname: Weiner
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry; D002585:Cesarean Section; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D005321:Extraembryonic Membranes / Q000201:enzymology*; D005260:Female; D016166:Free Radical Scavengers / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008070:Lipopolysaccharides / Q000737:chemistry; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000502:physiology*; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000737:chemistry; D019335:Nitroarginine / Q000737:chemistry; D010084:Oxidation-Reduction; D010396:Penicillamine / Q000031:analogs & derivatives / Q000737:chemistry; D011247:Pregnancy; D017382:Reactive Oxygen Species / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine; D013482:Superoxide Dismutase / Q000737:chemistry; D019820:Xanthine / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry
chemical_list: D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D019820:Xanthine; D019335:Nitroarginine; D026423:S-Nitroso-N-Acetylpenicillamine; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D010396:Penicillamine; D000111:Acetylcysteine