ABSTRACT
OBJECTIVE: The ability of erythropoietin (EPO) to elicit a pro-angiogenic effect on human mesenchymal stem cells (hMSC) was tested. hMSC are currently under study as therapeutic delivery agents that target tumor vessels. Hypoxia favors the differentiation of hMSC towards a pro-angiogenic program. However, the classical angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, are not fully capable of restoring this effect. The hypoxia-regulated factor, EPO, induces angiogenesis in endothelial cells. Here, EPO's pro-angiogenic effect on hMSC was analyzed. METHODS: hMSC were tested for EPO receptor expression by western blot, immunofluorescence, and flow cytometry assays. Downstream receptor signaling components JAK and STAT were measured by standard assays. Pro-angiogenesis effects mediated by EPO treatment of hMSC were measured by proliferation, cytokine, or pro-angiogenesis factor secretion, metalloprotease activation, migration, invasion, wound healing, and tubule formation assays. RESULTS: hMSC express the cognate EPO receptor and are capable of promoting angiogenesis following EPO treatment in all the angiogenesis assays tested. EPO-treated hMSC proliferate and secrete pro-angiogenesis factors more readily than untreated hMSC. EPO leads to increased hMSC chemotaxis, migration, and activation of matrix metalloprotease-2. This treatment causes greater recruitment of vessels as measured in an in vivo angiogenesis assay. CONCLUSION: EPO is capable of eliciting a pro-angiogenesis program in hMSC that instigates secretion of angiogenic factors and the subsequent recruitment of endothelium. This study defines a novel mechanism for tumor cell recruitment of blood vessels that is important to consider in the design of stem cell-based therapies.
Subject(s)
Erythropoietin/pharmacology , Mesenchymal Stem Cells/drug effects , Neovascularization, Physiologic , Blotting, Western , Cells, Cultured , Flow Cytometry , Fluorescent Antibody Technique , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Receptors, Erythropoietin/metabolismABSTRACT
Based on cDNA microarray results, integrin-linked kinase (ILK) emerged as an interesting candidate in hypoxia-mediated survival mechanisms employed by cancer cells. This notion was confirmed here by the following observations: the 5' promoter region of the ilk gene contains hypoxia responsive elements (HRE) that bind hypoxia-inducible factor (HIF) transcription factor complexes and drive HRE-luciferase gene expression in reporter assays; ILK protein and kinase activity are induced following hypoxia; downstream targets of ILK signaling are induced following hypoxia treatment; inhibition of ILK leads to increased apoptosis; and HIF and ILK are co-localized within human cancer tissues. The identification of ILK as a player in hypoxia survival signaling employed by cancer cells further validates ILK as a unique target for cancer therapy.
Subject(s)
Hypoxia/enzymology , Protein Serine-Threonine Kinases/genetics , Apoptosis , Breast Neoplasms/enzymology , Carcinoma, Hepatocellular , Cell Line, Tumor , DNA, Neoplasm/genetics , Female , Genes, Reporter , Humans , Immunohistochemistry , Kidney Neoplasms/enzymology , Liver Neoplasms , Oligonucleotide Array Sequence Analysis , Ovarian Neoplasms/enzymology , Protein Serine-Threonine Kinases/biosynthesis , TransfectionABSTRACT
BACKGROUND/AIMS: We developed a single-chain antibody fragment (scFv) to the non-structural 3 protein (NS3) of hepatitis C virus (HCV) and tested its ability to interfere with the HCV replication cycle in infected hepatocytes. METHODS: The variable regions of the human monoclonal antibody CM3.B6 that recognizes a conformational epitope within the helicase domain of NS3 were introduced into adenoviral vectors for expression in mammalian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellular expression of the scFv on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse transcription-polymerase chain reaction. RESULTS: Transduction of HepG2 cells by the recombinant adenoviruses resulted in stable, efficient expression of scFv in the cytoplasm that was non-toxic to the cells. The scFv specifically bound to its cognate antigen. Significantly, intracellular expression of scFv resulted in a decrease in HCV genomic RNA in HCV infected hepatocytes. CONCLUSIONS: These results indicate that specific binding of a scFv to NS3 may inhibit one or more functions of this essential viral protein thus interfering with the HCV replication cycle.
