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Figure Lengend Snippet: Mice were infected with HSV-1 and corneas were harvested at the indicated times pi to for gene and protein expression analysis. Gene expression is categorized as (A) high relative expression, (B) medium relative expression, and (C) low relative expression in the corneas at times pi. (D) Heatmap clustering/analysis of pro-angiogenic gene expression at times pi. (E) Protein levels of interleukin-6 (IL-6), (F) angiopoietin-2 (Ang-2), (G) fibroblast growth factor-2 (FGF-2), (H) hepatocyte growth factor (HGF), (I) VEGF-A, and (J) matrix metalloproteinase-9 (MMP-9) in time-course experiments. Data represent summary of mean ± SEM of 2-3 independent experiments with n = 5–9 mice per time point. * p<0.1, ** p<0.01, *** p<0.001, ****p<0.0001 comparing infected mice to uninfected (UI) control animals as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: For mast cell and FGF-2 co-staining, rabbit anti-mouse FGF-2 (Abcam) was used as primary antibody followed by an overnight incubation in a cocktail of donkey anti-rabbit TRITC (Jackson Immunoresearch) and FITC-conjugated Avidin (Biolegend).

Techniques: Infection, Expressing

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Figure Lengend Snippet: Mice were inoculated with HSV-1, and neutralizing antibody against FGF-2 or isotype control antibody (5μg/10μl PBS) were injected subjconjunctivally at days 8, 10, and 12 pi. (A) Representative Z-stacked corneal images depicting lymphatic (green) and blood (red) vessels at day 14 pi comparing isotype control antibody (IgG) to anti-FGF-2 antibody-treated mice. Metamorph quantification of corneal area containing (B) LYVE-1 + lymphatic vessels, and (C) CD31 + blood vessels per 100X field of view. (D) Protein expression of pro-angiogenic factors at day 14 pi comparing isotypic control to anti-FGF-2 antibody-treated mice. (E) Representative Z-stacked corneal images depicting lymphatic (green) and blood (red) vessels at day 21 pi comparing isotypic control to anti-FGF-2 antibody-treated mice. Metamorph quantification of corneal area containing (F) LYVE-1 + lymphatic vessels, and (G) CD31 + blood vessels per 100X field of view. (H) Protein expression of pro-angiogenic factors at day 21 pi comparing isotypic control to anti-FGF-2 antibody-treated mice. (I) Leukocyte content in the cornea at day 21 pi indicating (from left to right) neutrophils, macrophages, inflammatory monocytes, CD4 + T cells, CD8 + T cells, and NK cells. Data represent summary of mean ± SEM of two independent experiments (A–C and E–G) with n = 10 corneas, three independent experiments (D and H) with n = 6–12 mice, three independent experiments (H and I) with n = 10–11 mice. ** p<0.01, **p<0.001, ****p<0.0001 (A–C, E–G, and I) as determined by Student’s t test. * p<0.1, **p<0.01, ***p<0.001, ****p<0.0001 (D and H) as determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Bars for each imaged frame are 200 μm.

Article Snippet: For mast cell and FGF-2 co-staining, rabbit anti-mouse FGF-2 (Abcam) was used as primary antibody followed by an overnight incubation in a cocktail of donkey anti-rabbit TRITC (Jackson Immunoresearch) and FITC-conjugated Avidin (Biolegend).

Techniques: Injection, Expressing

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Figure Lengend Snippet: Mice were inoculated with HSV-1 or left uninfected. At days 8, 10, and 12 pi, anti-FGF-2 antibody or isotype-matched control antibody (5μg/10μl PBS) were administered to infected mice by subconjunctival injection. (A) Functional vision of the mice was measured using opto-kinetic response tracking in optomotry at the indicated days pi. (B) Corneal sensation of mice was measured using a Cochet-Bonnet ethesiometer at the indicated days pi. Data represent the summary of mean ± SEM of two independent experiments, n = 7–8 mice/group. * p<0.1, **p<0.01, ****p<0.0001 as determined by two-way ANOVA followed by Dunnet’s multiple comparisons test (comparing IgG and Anti-FGF-2 treated animals to UI control group).

Article Snippet: For mast cell and FGF-2 co-staining, rabbit anti-mouse FGF-2 (Abcam) was used as primary antibody followed by an overnight incubation in a cocktail of donkey anti-rabbit TRITC (Jackson Immunoresearch) and FITC-conjugated Avidin (Biolegend).

Techniques: Infection, Injection, Functional Assay

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Figure Lengend Snippet: (A) Naive (uninfected) cornea exhibits basal level of VEGF-A which is normally neutralized by soluble form of VEGFR-1 present within the cornea. Resident APCs (such as macrophages) decorate the posterior half of the corneal stroma. Limbal vasculature including blood and lymphatic vessels do not penetrate the center cornea under normal conditions. (B) Following HSV-1 infection, VEGF-A is predominantly produced by the infected corneal epithelial cells that drive corneal neovascularization. When the virus is still present in the corneal epithelium, immune cells including PMN, inflammatory monocytes, macrophages, T cells, and NK cells infiltrate the cornea. Various cytokines and growth factors such as IL-6, MMP-9, FGF-2, Ang-2, and HGF are also produced within the cornea leading to further inflammation. (C) When the virus is cleared in the cornea, the immune cells continue to infiltrate, pro-angiogenic factors continue to be produced, and neovascularization prolongs/sustains. However, immune cells do not have major contribution to corneal neovascularization. MMP-9 is dispensable, IL-6 is necessary but not sufficient, and FGF-2 is crucial for HSV-1-induced corneal neovascularization. Importantly, FGF-2 controls the expression of VEGF-A, IL-6, Ang-2, HGF, and MMP-9. The primary source of FGF-2 may not be the leukocytes. Instead, resident non-hematopoietic cells of the cornea including fibroblast (keratocytes), epithelium, endothelium, blood endothelial cells (BEC), and lymphatic endothelial cells (LEC) might play important roles in such process.

Article Snippet: For mast cell and FGF-2 co-staining, rabbit anti-mouse FGF-2 (Abcam) was used as primary antibody followed by an overnight incubation in a cocktail of donkey anti-rabbit TRITC (Jackson Immunoresearch) and FITC-conjugated Avidin (Biolegend).

Techniques: Infection, Produced, Expressing