Role of CpG oligodeoxynucleotide in angiogenesis

Abstract Background Angiogenesis involves multiple cell types, including close association with immune cells and vascular growth factors. We have previously shown that the toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (ODN) inhibits corneal angiogenesis. We aimed to investigate how CpG ODNs regulate vascular endothelial cells, pericytes, and macrophages and whether macrophages are required for the anti-angiogenic process. Methods Regulation of angiogenesis by CpG ODN was determined in various in-vivo models (corneal neovascularisation and laser-induced choroidal neovascularisation). Human umbilical vein endothelial cells (HUVEC) were stimulated with CpG ODN to determine tube formation and cell migration. Migration of either pericytes or bone-marrow-derived macrophages was determined by co-culture with HUVEC medium conditioned with CpG ODN. Before CpG ODN treatment, macrophages were depleted by clodronate, or macrophage subpopulations were selectively depleted with antibodies to chemokine receptor type 2 (anti-CCR2). Findings CpG ODN suppressed suture-induced corneal neovascularisation and laser-induced choroidal neovascularisation. Conditioned HUVEC medium enhanced macrophage migration but limited pericyte mobilisation. After macrophage depletion by clodronate or anti-CCR2, a reduction in suture-induced corneal neovascularisation was noted; however, treatment with CpG ODN did not augment this response in the absence of macrophages. CpG ODN promoted macrophage M1 polarisation, and macrophages lacking CCR2 attenuated HUVEC tube formation. Interpretation We have shown that the regulation of angiogenesis by CpG ODN is ubiquitous through a variety of pathways. CpG ODN maintains endothelial cells in stalk status and reduces pericyte migration to support vessel formation. Macrophage depletion subverts any additive anti-angiogenic regulation of CpG ODN treatment. CpG ODN promotes macrophage mobilisation with mainly M1 phenotype of which probably the CCR2 population regulates endothelial cells as a result of anti-angiogenesis. Funding Rosetrees Trust, Academy of Medical Sciences, National Institute for Health Research, National Eye Research Centre, Fight for Sight PhD studentship.