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Jiahui Wu, Andrew D Dick, Lei Liu; Three classes of Cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) regulate angiogenesis via distinct pathways.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3240.
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Cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODNs) have been classified depending on the characteristics of elicited immune response. This is a result of variance in backbone, sequences, numbers and locations of CpG. Here we aim to interrogate whether and how type A, B and C CpG ODNs regulate angiogenesis.
Mouse bone marrow derived macrophages were stimulated with type A or B CpG ODNs or their controls for 24h. Chloroquine, an endosome/lysosome acidification inhibitor, was used to elucidate whether CpG ODNs regulate VEGF and sFlt1 independent of TLR9 activation. For angiogenesis assay, the number of aortic sprouting neovascular outgrowths was measured after treatment in a dose response with three types of CpG ODNs over 7d. VEGF and sFlt1 expression in both macrophages and aortic ring cultures were examined by ELISA and qPCR. The angiogenic regulation of type C CpG ODN was further determined in HUVEC tube formation assay. Finally, type A or B CpG ODN was administered subconjunctivally to assess effect on BALB/c mouse suture-induced corneal angiogenesis model. Volumes of both blood and lymphatic corneal vessels were quantified after 7d.
In macrophages, VEGF expression was suppressed by type A ODN and sFlt1 expression was increased by type B ODN, both responses abolished by chloroquine. All 3 types of ODN suppressed aortic angiogenesis in a dose dependent manner. Their regulation of VEGF and sFlt expression was distinct. Type C ODN suppressed HUVEC tube formation, in a dose dependent manner. In vivo, Type B CpG ODN suppressed both corneal hem- and lymph-angiogenesis as demonstrated by shorter lengths of vessel outgrowth as well as overall vessel volume compared to vehicle control. There was no difference between type A CpG ODN and vehicle groups in this assay.
ODNs regulate VEGF and sFlt in macrophages dependent on TLR9 activation. Although type A, B and C CpG ODNs were all able to suppress aortic sprouts, only type B restrained corneal hem- and lymph-angiogenesis in vivo. The data infers that different classes of CpG ODNs regulated angiogenesis via different pathways which predicates future targets for therapeutic suppression of angiogenesis.
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