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S. A. McClellan, Y. Zhang, M. Wu, L. D. Hazlett; TLR4 Regulates HIF-1 and Inflammatory Angiogenesis in Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):414.
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Previous work has shown that after P. aeruginosa corneal infection, TLR4 LPS deficient (TLR4lps-d) BALB/c mice had worsened disease outcome when compared with TLR4 sufficient mice (Huang, Du et al. 2006). Deficiency also resulted in less ocular (limbal and peripheral cornea) vascularity, suggesting that the absence of functional TLR4 decreases vascularity and worsens disease. Therefore this study tests whether TLR4 regulates inflammatory angiogenesis after P. aeruginosa corneal infection.
: When tested, TLR4lps-d mice had reduced TLR2 mRNA levels, therefore, to specifically knockdown only TLR4, wildtype (wt) BALB/c mice received subconjunctival and topical siRNA TLR4 treatment. Controls were similarly treated with scrambled siRNA. Disease was graded after infection by clinical score, photography, real-time RT-PCR, ELISA, and confocal microscopy.
: TLR4 knockdown resulted in more severe corneal disease and reduced ocular vascularity. Decreased mRNA levels for VEGF-A and VEGF-R1 and increased levels of HIF-1α, but no difference in VEGF-R2, were observed in TLR4 knockdown vs control treated mice. ELISA analysis revealed enhanced protein levels for VEGF-A, VEGF-R1 and HIF-1α (active). Confocal microscopy revealed greater VEGF-A and VEGF-R1 (co-localized with F4/80 positive cells) and HIF-1α staining in the TLR4 knockdown vs control treated mice.
TLR4 appears to modulate inflammatory angiogenesis by regulating expression of HIF-1α, which in turn regulates VEGF-A and its receptor VEGF-R1. After TLR4 knockdown, all three molecules are elevated leading to decreased vascularity.
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