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Ana J Chucair-Elliott, Katie M Hudson, Min Zheng, Daniel J Carr; HSV-1 Corneal Infection Drives Robust Lymphangiogenesis Beyond Virus Resolution. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4059.
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Herpes simplex virus type I (HSV-1) is a leading cause of infectious corneal blindness and herpetic stromal keratitis (HSK). A hallmark feature of HSK is neovascularization of the cornea. Our goal is to identify the growth factors and cells that contribute to the maintenance and further development of lymphatic vessels in the cornea following resolution of HSV-1 infection.
C57BL/6J mice were infected with 1,000 plaque forming units (PFU) of HSV-1. Corneas were harvested at 0, 6, 10, 14, 21, and 30 days post-infection (pi) and assessed for CD31high blood vessels and CD31lo/LYVE-1+ lymphatic vessels via confocal microscopy. Expression levels for 20 genes associated with neovascularization and inflammation were determined in the cornea using Affymetrix quantigene assays. The expression of IL-6, VEGF A, VEGF C, IL-1β, and MMP-9 were determined at the protein level by suspension array and ELISA assays. Infiltrating T cells and myeloid cells were quantified by flow cytometry.
Lymphatic vessel area in the cornea proper significantly increases following viral clearance and continues to increase up to day 30 pi. At similar time points, gene expression of IL-6, IL-1β, MMP9, LTβ, CCL21A, VEGFC, MMP2, HGF, PDGFB, ANGPT1, and VEGF A were elevated compared to uninfected controls. The expression of IL-6 and MMP9 proteins was significantly elevated at day 14 pi. A modest increase in VEGF A and IL-1β proteins was observed at this time point. The CD4+ and CD8 T+ cell infiltration peaked at day 14 pi but was still evident at day 30 pi. Neutrophils were significantly elevated by day 6 pi and remained elevated up to day 30 pi.
Previous studies have indicated HSV-1 induces corneal lymphangiogenesis during acute infection via VEGF A production by infected epithelial cells. Our results reveal corneal lymphangiogenesis is maintained after viral clearance (day 10 pi) and continues to develop at time points consistent with the development of HSK (day 14 pi) and establishment of viral latency (day 30 pi). Increases in corneal lymphangiogenesis were associated with an influx of T cells and myeloid cells, mostly neutrophils. These cells may be a source for the significant rise in pro-angiogenic growth factors and inflammatory cytokines observed after viral clearance. It is anticipated results from this study will identify novel targets for the treatment of corneal neovascularization following HSV-1 infection.
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