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Courtney L. Meier Jewett, Hsin Chien, Richard D. Dix; Murine Cytomegalovirus Infection of Cultured Macrophage Cell Lines Results in Upregulation of VEGF Production. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2337.
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Cousins, Espinosa-Heidmann, and our laboratory have shown previously that C57BL/6 mice infected systemically with murine cytomegalovirus (MCMV) for 12 weeks exhibit increased size and severity of experimental choroidal neovascularization (CNV). Additional studies suggested that enhanced CNV is due to a significant increase in production of VEGF by circulating macrophages possibly activated by MCMV infection. If true, macrophage cell lines grown in culture should also exhibit increased production of VEGF following MCMV infection.
Monolayers of IC-21 and RAW 264.7 macrophage cell lines were inoculated with either MCMV (moi = 2.5), UV-inactivated MCMV, maintenance medium only (negative control), or maintenance medium containing either lipopolysaccharide (100 ng/ml) or lipoteichoic acid (80 ug/ml) (positive controls). All cells were harvested at 24 or 48 hours post infection and subjected to real time RT-PCR assay for quantification of VEGF and TNF-α mRNAs. Monolayers of MCMV-infected IC-21 macrophages were also analyzed for production of VEGF protein by immunostaining.
Whereas MCMV-infected IC-21 macrophages resulted in significant upregulation of both VEGF and TNF-α mRNA levels (5-fold and 8-fold, respectively), MCMV-infected RAW 264.7 macrophages also showed significant increase of VEGF mRNA levels (13-fold), but not of TNF-α mRNA levels. No significant change in mRNA levels was observed in either IC-21 or RAW 264.7 macrophages inoculated with UV-inactivated MCMV. Immunostaining confirmed VEGF production by MCMV-infected IC-21 macrophages.
Two macrophage cell lines demonstrated upregulation of VEGF mRNA production in response to MCMV infection, one also showing a concomitant upregulation of VEGF protein. IC-21 macrophages, but not RAW 264.7 macrophages, also demonstrated MCMV-induced upregulation of TNF-α mRNA, a marker for macrophage activation. The IC-21 macrophage cell line will be used for future studies designed to identify the virus gene(s) responsible for VEGF upregulation.
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