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M. Wu, Y. Zhang, S. A. McClellan, R. P. Barrett, L. D. Hazlett; Analysis of Angiogenic Molecules in Cornea: In vivo and in vitro Studies. Invest. Ophthalmol. Vis. Sci. 2010;51(13):413.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work has shown that BALB/c (resistant) vs C57BL/6 (B6, susceptible) mice have increased conjunctival vascularity after P. aeruginosa infection which we hypothesize correlates with resistance. This study examines angiogenic molecule expression in the cornea of these inbred mice and whether there may be correlation with TLR4 levels which are higher after infection in B6 than BALB/c mice.
Real time RT-PCR, cell culture assays, cell transfection, and confocal microscopy were used.
mRNA expression of VEGF-A was higher in B6 mice (whole cornea at 1, 3, and 5 days p.i.), in the epithelium at 1 and 5 days p.i., and in the stroma at 5 days p.i. mRNA levels of VEGF-A receptors, R1 and R2 and HIF-1α also were tested in epithelium vs stroma of the two mouse strains. R1 and R2 levels were elevated in B6 stroma at 1 and 5 days p.i. HIF-1α was elevated at 5 days p.i. in both the epithelium and stroma of B6 mice. Mouse derived epithelial cells (A61) stimulated with endotoxin (LPS) (1µg/ml for 18 h) showed increased mRNA levels of TLR4, VEGF-A, VEGFR1 and HIF-1α, but not VEGFR2 over controls. Transformed human corneal epithelial cells transfected with TLR4/MD2, when stimulated similarly with LPS showed increased immunostaining for HIF-1α, but a disparate pattern of angiogenenic molecule upregulation when compared with control plasmid transfected or A61 cells.
Higher TLR4 levels in B6 vs BALB/c mice after infection appears to correlate with decreased vascularity due to increased HIF-1α, VEGF-A and R1 levels which may downregulate blood vessel formation. Both the epithelium and stroma appear to have a role in regulating these molecules which are tightly regulated by TLR4 levels.
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