Department of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development, Division of Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. gsaed@med.wayne.edu
Hypoxia induces the adhesion phenotype, characterized by enhanced extracellular matrix molecule and cytokine expression. Additionally, hypoxia reduces myeloperoxidase (MPO) activity in normal peritoneal fibroblasts to basal levels of adhesion fibroblasts indicating the importance of this enzyme in the development of the adhesion phenotype and also in tissue fibrosis. Immunohistochemistry was used to detect and localize MPO and inducible nitric oxide synthase (iNOS) in fibroblasts. Silencing of these genes was performed using siRNA technology. Levels of iNOS, MPO, type I collagen, and transforming growth factor were detected using real-time reverse transcription-polymerase chain reaction (RT-PCR), while HPLC was used to measure nitrate/nitrite levels. Our results show a unique interaction between MPO and iNOS, which are colocalized in both cell lines. Silencing iNOS reduced MPO and nitric oxide levels while silencing MPO had similar results, but to a lesser extent in both cell types. Additionally, silencing iNOS reduced type I collagen and transforming growth factor-beta in adhesion fibroblasts, but to a lesser extent in peritoneal fibroblasts. These studies identify MPO and iNOS as key enzymes in the cellular response to hypoxia and consequent development of tissue fibrosis.