Trehalose 6,6′-Dimycolate (Cord Factor) Enhances Neovascularization through Vascular Endothelial Growth Factor Production by Neutrophils and Macrophages

Abstract

Trehalose 6,6-dimycolate (TDM) plays important roles in the development of granulomatous inflammation during infection with Mycobacterium spp., Rhodococcus spp., etc. To reveal the augmenting effect of TDM on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue air pouches induced by Rhodococcus sp. strain 4306 TDM dissolved in Freund's incomplete adjuvant (FIA), comparing them to pouches treated with FIA alone. Histologically, granulomatous tissue and new vessel formation, which reached a maximum at day 7, was greatly enhanced by treatment with TDM. At day 1, VEGF-positive neutrophils accumulated in the pouch wall with frequency of 95% of total infiltrating cells, adhering to TDM-containing micelles. By day 3, granulomatous tissue and new vessels started to develop, and VEGF-positive macrophages appeared in a small number and gradually increased in number thereafter. The pouch contents of VEGF, interleukin-1␤, tumor necrosis factor alpha, and transforming growth factor ␤ were significantly elevated in TDM-treated pouches, with peaks at days 1, 0.5, 1, and 3, respectively, compared to those of control pouches, while that of basic fibroblast growth factor showed no significant increase. Treatment with anti-VEGF antibody inhibited TDM-induced granulomatous tissue formation and neovascularization, and administration of recombinant VEGF into pouches treated with FIA alone induced neovascularization comparable to that in the TDM-treated pouches. Incubation of neutrophils and macrophages on TDM-coated plastic dishes increased the VEGF release. The present results indicate that TDM augments VEGF production by neutrophils and macrophages and induces neovascularization in the granulomatous tissue.

In vitro cytokine stimulation assay for glycolipid biosurfactant from Rhodococcus ruber: role of monocyte adhesion

“…It was also found that a GLB-covering film partly prevents monocyte adhesion to plastic. However, our findings are in controversy with the published results (Sakaguchi et al 2000;Ozeki et al 2006) showing increased TNF-a release by murine neutrophils and macrophages after the incubation in TDM-coated microplates, and with data of Lima et al (2001) showing high cytokine (TNF-a, IL-6, 10, 12, IFN-c) release by murine macrophages incubated in TDM-covered glass coverslips. One possible explanation for the difference between our and other authors' results lies in the fact that cytokine secretion by monocytes is a complex process requiring both cell adhesion and exposure to a chemical inducer such as bacterial antigen or endotoxin (Petit-Bertron et al 2003).…”

“…GLBcoated plates were prepared as described by Sakaguchi et al (2000). Namely, the GLB was dissolved in isopropanol to the concentrations of 1, 10, 100 and 1,000 lg/mL (similar to those used for emulsion preparation), and 100-lL aliquots were placed into wells.…”

“…Surface-active trehalose di-and monomycolates of pathogenic (M. tuberculosis, C. diphtheriae) and opportunistic (M. avium-M. intracellulare group, N. asteroids, C. matruchotii, C. xerosis) mycolata play a key role in the pathogenesis of infections caused by these actinobacteria and are characterized by pronounced immunomodulating effects (Ryll et al 2001). Biological activity of these glycolipid biosurfactants (GLBs) is significant, but apparent or potential pathogenicity of the producer strains and high toxicity of produced glycolipids hinder their biomedical applications (Sakaguchi et al 2000;Franzetti et al 2010). Therefore, the search for GLB producers among nonpathogenic actinobacteria is of importance.…”

“…In some new in vitro protocols, to study the cytokine-stimulating activity of TDM, murine neutrophils and macrophages were incubated in TDMcoated microplates (Sakaguchi et al 2000;Okamoto et al 2006;Ozeki et al 2006) and onto TDM-coated coverslips (Lima et al 2001). This method avoids the emulsification procedure and allows preparation of stock TDM-coated microplates or coverslips, which could be stored and used in mass immunological tests.…”

Granulomatous Tissue Formation of Shikon and Shikonin by Air Pouch Method.

“…10) In this study, it can be certified that SK and shikonin enhance neovascularization in granulomatous tissue formation in the mice air pouch through VEGF produced by infiltrated neutrophils.…”