Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes.
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.
Buhimschi
I A
IA
Division of Perinatal Research, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore 21201-1559, USA.
Kramer
W B
WB
Buhimschi
C S
CS
Thompson
L P
LP
Weiner
C P
CP
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
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