TLR9 Agonist Regulates Angiogenesis and Inhibits Corneal Neovascularization

Abstract

Myeloid cells are highly adaptable and may positively or negatively regulate angiogenesis dependent on the cognate and soluble signals they receive. Toll-like receptors (TLRs) initiate immune responses, orchestrate adaptive immune responses, and regulate vascular endothelial growth factor (VEGF)-mediated angiogenesis during wound healing. We investigated the possible role of TLR ligands in attenuation of new vessel growth via regulation of expression of VEGF or soluble fms-like tyrosine kinase-1 (sFlt-1) in both an aortic ring assay and a model of suture-induced corneal angiogenesis. The TLR3 ligand [poly(I:C)] markedly suppressed VEGF secretion and stimulated sFlt-1 release from macrophages. The aortic ring assay demonstrated that new vessels were promoted by the TLR2 ligand (heat killed Listeria monocytogenes) and the TLR4 ligand (lipopolysaccharide), concomitant with increased VEGF and matrix metalloproteinase 9 secretion. In contrast, the TLR9 ligand [oligodeoxynucleotide (ODN)1826] stimulated sFlt-1 secretion from macrophages and reduced the number of aortic ring vessel sprouts. ODN1826 also significantly reduced the length and volume of both hemangiogenesis and lymphangiogenesis in the suture-induced corneal angiogenesis model. Furthermore, 53 angiogenic factors were examined via protein array and compared between ODN1826- and water-treated corneas to interrogate the pathway of ODN1826 inhibition, demonstrating an up-regulation of Serpin E1 signal. Further clinical and IHC analyses of the aortic ring assay indicated that TLR9 suppressed tip cell migration and recruitment of mural cells and adventitial macrophages.