title: Reactive nitrogen species augment fibroblast-mediated collagen gel contraction, mediator production, and chemotaxis.
abstract: Reactive nitrogen species (RNS) such as peroxynitrite cause cellular injury and tissue inflammation. Excessive production of nitrotyrosine, which is a footprint of RNS, has been observed in the airways of patients with asthma and chronic obstructive pulmonary disease, disorders characterized by tissue remodeling. The aim of this study was to evaluate whether RNS can affect tissue remodeling through direct effects on fibroblasts, and to determine if these effects depend on production of transforming growth factor-beta (TGF-beta). To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess fibroblast-mediated contraction of floating gels and chemotaxis toward fibronectin. In addition, the ability of fibroblasts to release TGF-beta1, fibronectin, and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay. Authentic peroxynitrite significantly augmented gel contraction (P < 0.01) and chemotaxis (P < 0.01) compared with control in a concentration-dependent manner. Similarly, the peroxynitrite donor 3-morpholynosidenonimine hydrochloride (SIN-1) also augmented gel contraction (P < 0.01). RNS also significantly increased TGF-beta1 (P < 0.01), fibronectin (P < 0.01), and VEGF (P < 0.01) release into the media in both 3D gel and monolayer culture. Anti-TGF-beta antibody reversed RNS-augmented gel contraction (P < 0.01) and mediator production (P < 0.01). Anti-TGF-beta antibody also partially, but significantly, reversed RNS-augmented chemotaxis toward fibronectin (P < 0.01). Finally, peroxynitrite enhanced expression of alpha5beta1 integrin, which is a receptor for fibronectin (P < 0.01), and neutralizing anti-TGF-beta antibody suppressed peroxynitrite-augmented alpha5beta1 expression (P < 0.01). These results suggest that RNS can affect the tissue repair process by modulating TGF-beta1.
authors.affiliation: University of Nebraska Medical Center, 985885 Nebraska Medical Center, Omaha, NE 68198-5885, USA. srennard@unmc.edu
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Sugiura
authors.forename: Xiangde
authors.initials: X
authors.lastname: Liu
authors.forename: Tetsu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Togo
authors.forename: Ronald F
authors.initials: RF
authors.lastname: Ertl
authors.forename: Shin
authors.initials: S
authors.lastname: Kawasaki
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kamio
authors.forename: Xing Qi
authors.initials: XQ
authors.lastname: Wang
authors.forename: Lijun
authors.initials: L
authors.lastname: Mao
authors.forename: Lei
authors.initials: L
authors.lastname: Shen
authors.forename: Cory M
authors.initials: CM
authors.lastname: Hogaboam
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Rennard
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D003094:Collagen / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D005782:Gels; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D039081:Integrin alpha5beta1 / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D051381:Rats; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D000906:Antibodies; D005353:Fibronectins; D005782:Gels; D039081:Integrin alpha5beta1; D026361:Reactive Nitrogen Species; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D030421:Peroxynitrous Acid; D003094:Collagen; D006861:Hydrogen Peroxide