OBJECTIVETo investigate the impact of human peritoneal mesothelial cells (HPMC) on angiogenesis factor expression and secretion of ovarian carcinoma cell line SKOV3.

METHODSThe conditioned medium with HPMC was tested by ELISA for tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-1beta). Millicell was used to co-culture HPMC and ovarian carcinoma cell line SKOV3 in the presence or absence of neutralizing antibody against TNF-alpha or IL-1beta. RT-PCR was used to detect vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene expression in SKOV3 cells. VEGF and bFGF protein levels in the SKOV3 conditioned medium were assessed by ELISA.

RESULTSConditioned medium with HPMC contained both TNF-alpha and IL-1beta. SKOV3 co-cultured with HPMC expressed higher levels of VEGF and bFGF mRNA and secreted at increased levels of both VEGF and bFGF, in comparison with those in SKOV3 cells cultured alone (P < 0.01). Addition of neutralizing antibody against TNF-alpha or IL-1beta during co-cultures resulted in decrease in mRNA expression and secretion of VEGF and bFGF in SKOV3 cells. When both antibodies were administered during co-culture, additive decrease was observed.

CONCLUSIONHPMC can act in a paracrine fashion to stimulate ovarian tumor cells to produce and secret at increased levels of VEGF and bFGF through TNF-alpha and IL-1beta, and contribute to angiogenesis and peritoneal metastasis of ovarian cancer.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Culture Media, Conditioned ; metabolism ; Cystadenocarcinoma, Serous ; genetics ; metabolism ; pathology ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; secretion ; Female ; Fibroblast Growth Factor 2 ; genetics ; secretion ; Gene Expression ; drug effects ; Humans ; Interleukin-1beta ; immunology ; secretion ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Peritoneal Cavity ; cytology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; immunology ; secretion ; Vascular Endothelial Growth Factor A ; genetics ; secretion

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Antibodies, Neutralizing/immunology ; Cathepsin L/genetics/*metabolism ; *Cell Movement ; Cells, Cultured ; Comet Assay ; Dependovirus/genetics ; Endothelial Cells/cytology/*metabolism ; Fibroblast Growth Factor 2/genetics/immunology/*metabolism ; Gene Transfer Techniques ; Humans ; Immunoblotting ; JNK Mitogen-Activated Protein Kinases ; Lac Operon/genetics ; Mass Spectrometry ; Matrix Metalloproteinase 1/biosynthesis/genetics ; Muscle, Skeletal/*metabolism ; Neovascularization, Physiologic ; Plasminogen Activator Inhibitor 1/biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-gamma in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-beta, is important in wound healing. We investigated the role of FGF2 in IFN-gamma-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-gamma-induced emphysema, lung targeted IFN-gamma transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 microg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-gamma in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-gamma but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-gamma-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.
Animals ; Asthma/drug therapy/*prevention & control ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Emphysema/drug therapy/*prevention & control ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2/deficiency/*metabolism/*therapeutic use ; Flow Cytometry ; Inflammation/immunology ; Interferon-gamma/*biosynthesis/genetics ; Interleukin-13 ; Lipopolysaccharides/administration & dosage/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Pulmonary Eosinophilia ; Recombinant Proteins/administration & dosage/therapeutic use