Jiahui Wu

TLR9 Agonist Regulates Angiogenesis and Inhibits Corneal Neovascularization

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.

Interleukin-33 regulates tissue remodelling and inhibits angiogenesis in the eye

Age‐related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Loss of retinal pigment epithelium (RPE) is a major pathological hallmark in AMD with or without pathological neovascularization. Although activation of the immune system is implicated in disease progression, pathological pathways remain diverse and unclear. Here, we report an unexpected protective role of a pro‐inflammatory cytokine, interleukin‐33 (IL‐33), in ocular angiogenesis. IL‐33 and its receptor (ST2) are expressed constitutively in human and murine retina and choroid. When RPE was activated, IL‐33 expression was markedly elevated in vitro. We found that IL‐33 regulated tissue remodelling by attenuating wound‐healing responses, including reduction in the migration of choroidal fibroblasts and retinal microvascular endothelial cells, and inhibition of collagen gel contraction. In vivo, local administration of recombinant IL‐33 inhibited murine choroidal neovascularization (CNV) formation, a surrogate of human neovascular AMD, and this effect was ST2‐dependent. Collectively, these data demonstrate IL‐33 as a potential immunotherapy and distinguishes pathways for subverting AMD pathology.

Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways

There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease.

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.

Role of interleukin 33 in neovascular age-related macular degeneration

Abstract Background Age-related macular degeneration is the leading cause of irreversible blindness in the developed world, and in its most acute form, secondary to angiogenesis in neovascular age-related macular degeneration. Inflammation has a key role in its pathogenesis, particularly in angiogenesis. We have shown that retinal pigment epithelium (RPE) produces interleukin 33 (IL33), which regulates cellular responses in the underlying choroidal stroma. To further our understanding we assessed the role of IL33 in ocular angiogenesis. Methods Human retinal microvascular endothelial cells and RPE-choroid explants were treated with various doses of IL33, and assayed by real-time PCR, western blot, and ELISA. Laser-induced choroidal neovascularisation was used as a model of neovascular age-related macular degeneration. Wild-type (WT), ST2–/–, and IL33–/– mice were administered four laser burns in each eye before receiving IL33 as an intravitreal injection at a range of doses. The development of neovascular lesions was assessed by optical coherence tomography and immunofluorescence 7 days after injection. Findings IL33 treatment of human retinal microvascular endothelial cells impaired their ability to migrate, whereas IL33–ST2 signalling regulated vascular sprouting in RPE-choroidal explants. Compellingly, intravitreal IL33 significantly attenuated choroidal neovascularisation in WT mice, resulting in a 50% reduction in the volume of choroidal neovascular lesions (p=0·007). IL33 did not have an effect in angiogenesis in mice lacking ST2, the IL33 receptor. Furthermore, IL33–/– mice developed larger choroidal neovascular lesions, suggesting that choroidal neovascularisation was also regulated by endogenous IL33. Interpretation We found that the alarmin proinflammatory cytokine IL33 can inhibit the formation of choroidal neovascularisation in mice and is ST2 dependent. IL33 could represent an immunotherapeutic-based strategy for the treatment of age-related macular degeneration. Funding National Institute for Health Research, National Eye Research Centre.