Abstract
TLR4 is important in P. aeruginosa keratitis, yet whether it regulates angiogenesis, contributing to disparate disease outcome in murine models, remains unknown. When tested in susceptible (C57BL/6, B6) and resistant (BALB/c) mice, TLR4 mRNA levels were increased in B6 over BALB/c mice after infection. Protein levels of VEGF-A, and VEGFR1 also were increased, while VEGF-R2 was unchanged. To test the significance of higher corneal levels of VEGF-R1 in B6 mice, blood vessel ingrowth from the limbus was documented and quantitated after infection, and showed decreased ingrowth in B6 vs BALB/c mice. To further determine whether changes in TLR4 levels modulated angiogenesis, TLR4 was knocked down in B6 mice. VEGF-R1, VEGF-A and VEGFR-2 protein levels were slightly reduced, (significant only for VEGF-R1). In contrast, similar knockdown in BALB/c mice increased VEGF-A and VEGF-R1, with no difference in VEGFR-2 between groups. Immunohistochemistry using dual staining for VEGF-R1 and macrophages from TLR4 knockdown or TLR4 LPS deficient (TLR4lps-d) BALB/c mice showed increased VEGF-R1 staining in both groups over controls. ELISA confirmed that TLR4lps-d infected BALB/c mice also had increased corneal protein levels of VEGF-R1 and VEGF-A, with no difference in VEGF-R2. The studies provide evidence that TLR4 disparately regulates angiogenic molecules in the infected cornea of susceptible and resistant mice and that higher levels of VEGF-A and VEGF-R1 correlate with decreased vascularity and poor outcome.
Keywords: TLR4, angiogenesis, bacteria, cornea, inflammation
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