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Chang He, Zijing Huang, Jing Wang, Tian Zhou, Xiaowei Sun, Xialin Liu; TLR4-mediated Necroptosis of Microglia Contributes to Ischemia-induced Retinal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3663.
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© ARVO (1962-2015); The Authors (2016-present)
Ischemic vascular disease in the retina is sight-threatening, and its pathogenetic mechanism is still not well elucidated. This study aimed to explore a novel role of microglia in ischemia-induced retinal angiogenesis through TLR4-mediated necroptosis, a programmed necrosis, using an oxygen-induced retinopathy (OIR) murine model.
OIR was established in C57BL/6 wild type (WT) and TLR4 deficient mice. At the peak of angiogenesis at P17, retinae were extracted for detecting necroptosis markers by Western blot and immunofluorescent staining on retinal whole mounts or cryosections. Some WT-OIR mice were received 1 mM/1μL necrostatin-1 (a specific inhibitor of necroptosis) by intravitreal injection at P12 and the neovascular areas were analyzed at P17. In vitro, BV-2 murine microglia cells were transfected with TLR4-siRNA and stimulated under hypoxia for necroptosis analysis.
OIR was successfully generated in WT mice with the peak time of angiogenesis at P17, when the necroptosis markers, receptor interacting protein (RIP) kinases 1, RIP3, and downstream molecule mixed lineage kinase domain-like (MLKL), were significantly stimulated compared with non-OIR mice. RIP1 and RIP3 signals were distributed mainly around the vascular tufts in OIR, and co-localized with Iba1 positive microglia but not other retinal cells (RGC, dipolar, neuron or photoreceptor). Interestingly, necrostatin-1 treatment largely rescued the retina from RIP1/3-mediated necroptosis, suppressed pro-inflammatory and pro-angiogenic cytokines (IL-6, VEGF, FGF2 and CCL2), and inhibited pathological angiogenesis. Furthermore, TLR4 activation was found to be required for microglia necroptosis during angiogenesis, as evidenced by TLR4-deficient OIR mice, as well as in vitro cultured microglia with TLR4 knockdown by siRNA under hypoxic condition.
Our findings uncover a novel mechanism that TLR4-mediated microglia necroptosis plays a pivotal role in the control of inflammation and angiogenesis in ischemic retinopathy. This provides insight knowledge to pathological angiogenesis and new potential therapeutic approaches on the basis of modulation of necroptosis in microglia.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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