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

Jiahui Wu,Andrew D Dick,Lei Liu,Michael B Powner,David A Copland,Jian Liu,Wenru Su,Paolo Madeddu,Marcus Fruttiger

doi:10.1038/srep31873

Abstract

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.

Highlights

Mouse aortic rings were treated with three classes of murine CpG-ODN and their non-CpG-ODN controls
We demonstrate the direct anti-angiogenic effect of CpG-ODNs on vascular endothelial cells independent of TLR9 signalling
TLR9 is indispensable for macrophage regulation of angiogenesis, as the effect is lost in the absence of CpG motif or in TLR9 deficient mice

Summary

Introduction

All three classes of CpG-ODNs (class A: ODN1585; class B: ODN1826; class C: ODN2395) suppressed aortic ring sprouts compared to medium control at a dose-dependent manner. As the natural ODN backbone phosphodiester (PD) is degraded by nucleases[14], most of the current synthetic CpG-ODNs used in tumour clinical trials are phosphorothioate (PS)-modified to enhance stability[14,15,16]. CpG-ODN activation of TLR9 induces anti-tumour immunity and suppresses angiogenesis in tumours[12,18,19,20,21,22]. We show that suppression of endothelial cell activity by CpG-ODNs is dependent upon PS-modified backbone and is not TLR9-dependent. TLR9-dependent macrophage activation by CpG-ODNs was required to promote M1 phenotype and modulate angiogenesis through regulation of endothelial cell function

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