Role of nitric oxide in apoptosis of human peritoneal and adhesion fibroblasts after hypoxia.
OBJECTIVE
To study the modulation of the inducible nitric oxide synthase/nitric oxide (iNOS/NO) expression system in fibroblasts isolated from human peritoneum and adhesion tissues by hypoxia.


METHODS
Prospective experimental study.


METHODS
University medical center.


METHODS
Cultures of fibroblasts from both peritoneum and adhesion tissues of five patients.


METHODS
Hypoxia treatment of the primary cultured fibroblasts.


METHODS
We used Western and Northern blots to determine whether iNOS mRNA and its protein were present in peritoneal and adhesion fibroblasts and whether this expression is modulated by hypoxia. Multiplex reverse transcription polymerase chain reaction (RT-PCR) technique was used to quantify type I collagen mRNA in response to NG-nitro-L-arginine methyl ester (L-NAME). A terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) assay was used to quantify apoptosis in response to NO donor S-nitro-N-acetyl-penicillamine (SNAP) treatment. A Griess assay was used to measure NO levels.


RESULTS
Peritoneal fibroblasts have significantly higher NO levels than adhesion fibroblasts. Hypoxia decreased NO in peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition, hypoxia increased both mRNA and protein levels of the iNOS gene in peritoneal and adhesion fibroblasts. Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts. In contrast, SNAP had no effect on apoptosis of peritoneal fibroblasts. Inhibition of NO by L-NAME treatment increased type I collagen mRNA levels in peritoneal fibroblasts.


CONCLUSIONS
Our findings confirm that adhesion fibroblasts produce less NO than normal peritoneal fibroblasts; NO may be the mechanism responsible for the creation and persistence of the adhesion phenotype.
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, The C.S. Mott Center for Human Growth and Development, Detroit, Michigan 48201, USA. g.saed@wayne.edu
Ghassan M
GM
Saed
Husam M
HM
Abu-Soud
Michael P
MP
Diamond
D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010532:Peritoneal Diseases / Q000378:metabolism / Q000503:physiopathology*; D010537:Peritoneum / Q000503:physiopathology*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D000267:Tissue Adhesions / Q000378:metabolism / Q000503:physiopathology
D024042:Collagen Type I; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D019331:NG-Nitroarginine Methyl Ester