Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces corneal angiogenesis in rats

Abstract

Neovascularization or angiogenesis is required for the progression of chronic inflammation. The mechanism of inflammatory neovascularization in tuberculosis remains unknown. Trehalose 6, 6'-dimycolate (TDM) purified from Mycobacterium tuberculosis was injected into rat corneas. TDM challenge provoked a local granulomatous response in association with neovascularization. Neovascularization was seen within a few days after the challenge, with the extent of neovascularization being dose dependent, although granulomatous lesions developed 14 days after the challenge. Cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), IL-1beta, and vascular endothelial growth factor (VEGF), were found in lesions at the early stage (within a few days after the challenge) and were detectable until day 21. Neovascularization was inhibited substantially by neutralizing antibodies to VEGF and IL-8 but not IL-1beta. Treatment with anti-TNF-alpha antibodies resulted in partial inhibition. TDM possesses pleiotropic activities, and the cytokine network plays an important role in the process of neovascularization.

FIG. 1

Induction of corneal neovascularization in rats by TDM challenge. Corneas challenged with vehicle alone did not develop neovascularization. TDM challenge induced corneal neovascularization by day 3 that persisted up to day 21 in a dose-dependent fashion. Arrowheads indicate new blood vessels in the cornea.

FIG. 2

Time kinetic study of corneal neovascularization induced by TDM challenge. New vessel growth is expressed as the maximal lengths and widths of new vessels. Data are means ± SD (n = 10/group). The asterisks indicate statistical significance relative to results with control (CTRL) rats challenged with vehicle alone (P < 0.02).

FIG. 3

Histopathology of rat corneas challenged with TDM. Histologic sections (4 μm thick) of formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and eosin. Polymorphonuclear leukocytes infiltrated primarily at the site of TDM challenge until day 3. Focal accumulation of macrophages, including epithelioid macrophages, was evident by day 14, suggesting the development of granulomas. Neovascularization was found around granulomas but not in the center of the lesion (indicated by arrows). In the immunohistochemical analysis, the new blood vessels were positive for factor VIII-related antigen, a specific marker for endothelial cells (arrows).

FIG. 4

Antigenic cytokine levels in corneas challenged with TDM. Local cytokine levels were measured with commercially available EIA kits. Data are means ± SD (n = 10/group). The asterisks indicate statistical significance relative to values for control rats challenged with vehicle alone (∗, P < 0.0001; ∗∗, P < 0.001; ∗∗∗, P < 0.05; ∗∗∗∗, P < 0.001).

FIG. 5

Inhibition of TDM-induced corneal neovascularization by in vivo treatment with neutralizing anti-cytokine antibodies (Ab) to IL-1β, TNF-α, IL-8, and VEGF. Administration of antibodies to IL-8 and VEGF substantially inhibited neovascularization, although antibodies to IL-1β and TNF-α did not induce significant inhibition. Antibodies were injected into the sites indicated by arrowheads. The pictures represent lesions 6 days after the treatment.

FIG. 6

Morphometric analyses of the inhibition of TDM-induced corneal neovascularization by in vivo treatment with neutralizing anti-cytokine antibody (Ab). Data are means ± SD (n = 10/group). The asterisks indicate statistical significance relative to values for control (CTRL) rats challenged with vehicle alone (∗, P < 0.01; ∗∗, P < 0.05).