High-mobility group box-1 mediates toll-like receptor 4-dependent angiogenesis

Abstract

Objective: Inflammation is closely linked to angiogenesis, and Toll-like receptors (TLRs) are the key mediators of inflammatory responses. However, the impact of TLRs on angiogenesis is incompletely understood. In this study, we determined the involvement of TLRs in angiogenesis.

Methods and results: In a mouse model of alkali-induced corneal neovascularization (CNV), we found that CNV was attenuated in TLR4-/- but not TLR2-/- mice. Further study revealed that the absence of TLR4 led to decreased production of proangiogenic factors in association with reduced accumulation of macrophages at the site of wounds, which was associated with reduced expression of high-mobility group box-1 (HMGB1) protein, an endogenous ligand for TLR4. Topical application of HMGB1 to the injured cornea promoted CNV with increased macrophage accumulation in wild-type mice but not in TLR4-/- mice. HMGB1 treatment in vitro also promoted the production of proangiogenic factors by mouse macrophages in a TLR4-dependent manner. Furthermore, antagonists of HMGB1 and TLR4 reduced CNV and macrophage recruitment in the injured cornea of wild-type mice.

Conclusions: Our results suggest that the release of HMGB1 in the wounds initiates TLR4-dependent responses that contribute to neovascularization. Thus, targeting HMGB1-TLR4 signaling cascade may constitute a novel therapeutic approach to angiogenesis-related diseases.

Figure 1.

Alkali injury-induced CNV in WT and TLR4-deficient mice. A, Macroscopic appearance of WT (left panels) and TLR4-deficient (right panels) mouse eyes 7 days (upper panels) and 14 days (lower panels) after alkali injury. Images were taken with a slit lamp to show the frontal and lateral view of each eye. B, Corneal tissues were immunostained with anti-CD31 Abs. Magnification ×400. C and D, Quantitative analysis of data presented in B. CNV numbers per mm² in hot spots (C) or percentage of CNV areas in hot spots (D) were determined on corneas. Data represent means±SEM (n=6 animals). *P<0.05 vs WT mice.

Figure 2.

Macrophage accumulation and angiogenic factor expression in injured corneas. A, Corneal tissues from WT mice (left panels) or TLR4^−/− (right panels) mice were stained with fluorescein isothiocyanate–conjugated anti-F4/80 monoclonal Abs. Magnification ×400. B, The numbers of infiltrating F4/80-positive macrophages were determined, and the means±SEM are shown (n=6). *P<0.05 vs WT mice. C to E, Angiogenic factor expression in injured corneas. The mRNA expression of VEGF (C), b-FGF (D), and TGFβ1 (E) in wound sites was determined by quantitative reverse transcription–PCR. Results are expressed as mean±SEM of fold increase over control. *P<0.05 vs WT mice.

Figure 3.

The expression of TLR4, HMGB1, and RAGE in corneas after alkali injury. A, Quantitative reverse transcription–PCR to assess TLR4 gene expression in corneas of WT mice. Data represent means±SEM (n=6). *P<0.05 vs normal corneas. B, Immunofluorescence analysis of TLR4 protein expression in intact or wounded cornea from WT mice. Magnification ×400. C, Real-time PCR analysis of HMGB1 expression in wounded corneas at the indicated time points. Results were expressed as mean±SEM (n=6) of fold increase over control. *P<0.05 vs WT mice. D, HMGB1 protein expression in injured corneas. Whole eyes were obtained and processed for immunohistochemical analysis using an anti-HMGBI Ab. Magnification ×400. E, Real-time PCR analysis of RAGE expression in wounded corneas. All values represent mean±SEM (n=6). *P<0.05 vs intact corneas.

Figure 4.

The effects of topical HMGB1 application on CNV. A and B, Macroscopic appearances of WT (A) and TLR4^−/− (B) mice topically applied with HMGB1 2 weeks after alkali injury are shown. LPS treatment was used as a positive control. Images were taken with a slit lamp (left panels), and the corresponding cryosections from treated corneal tissues were immunostained with anti-CD31 Ab (right panels). Magnification ×400. C and D, Quantitative analysis of data presented in A and B. The CNV numbers per mm² in hot spots (C) and percentage of CNV areas in hot spots (D) were determined. Data represent means±SEM (n=6). *P<0.05 vs PBS-treated groups. E, Corneal tissues removed 4 days after injury from WT (left panel) and TLR4^−/− (right panel) mice were stained with anti-F4/80 monoclonal Ab. Magnification ×400. F, The numbers of F4/80-positive macrophages were determined, and the mean±SEM are shown (n=6). *P<0.05 vs PBS-treated groups.

Figure 5.

The effects of HMGB1 on angiogenic factor expression by murine peritoneal macrophages. A, Peritoneal macrophages from wT and TLR4^−/− mice were incubated with 1.0 μg/mL recombinant HMGB1 for 12 hours. The levels of VEGF, b-FGF, and TGFβ1 mRNA were determined by quantitative reverse transcription–PCR, and data represent means±SEM. *P<0.05 vs WT mice. B, Peritoneal macrophages from WT mice were stimulated in the absence (upper panels) or presence of HMGB1 (lower panels) for 24 hours. The cells were stained with anti-VEGF Ab and then Cy3-conjugated secondary Ab and Hoechst 33342. Original magnification, ×400.

Figure 6.

Antiangiogenic effects of Box A and LPS-RS. A, Macroscopic observations of CNV in WT mice instilled with the antagonists 2 weeks after alkali injury are shown. Images were taken with a slit lamp (left panels), and the corresponding cryosections from corneal tissues were immunostained with anti-CD31 Ab (right panels). Magnification ×400. B and C, Quantitative analysis of data presented in A. The CNV numbers per mm² in hot spots (B) and percentage of CNV areas in hot spots (C) were determined. Each value represents the mean±SEM (n=6). *P<0.05 vs PBS-treated groups. D, Corneal tissues removed 4 days after the injury were stained with anti-F4/80 monoclonal Ab. Original magnification, ×400. E, The numbers of F4/80-positive macrophages were determined, and the mean±SEM are shown (n=6). *P<0.05 vs PBS-treated groups.