Toll-like Receptor 4 Regulation of Corneal Angiogenesis

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.