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C. Rask-Madsen, R. Abramov, M. Poillucci, A. Clermont, L. P. Aiello, G. L. King; PKC Regulates Retinal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4960.
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© ARVO (1962-2015); The Authors (2016-present)
Activation of microglia in the retina and macrophages in other tissues is critical for angiogenesis. Because the ε isoform of protein kinase C (PKC) contributes to macrophage activation and innate immunity, we aimed to determine whether PKCε is involved in retinal angiogenesis.
Oxygen-induced retinopathy was produced in PKCε-/- mice and controls by placing pups with their lactating mother in 75% oxygen from postnatal day 7 (P7) to P12. Adenoviral vectors were used to express wild-type or mutant PKCε in cell culture. Real-time PCR was used to measure mRNA, and protein expression was analyzed by Western blotting.
Pre-retinal neovascularization in PKC-/- mice was only 14±1% of the wild-type response (n=18 per group, p=0.00002). Specifically, the average number of preretinal nuclei per 6 µm ocular section, counted by an observer unware of genotype, was 47.6±8.4, 18.8±4.4, and 6.7±1.3 in PKCε+/+, PKCε+/-, and PKCε-/- mice, respectively. mRNA expression of vascular endothelial growth factor (VEGF) and mRNA and protein expression of its receptor KDR were not different in retinas of PKCε+/+, PKCε+/-, or PKCε-/- mice (p>0.3). Furthermore, in bovine retinal endothelial cells overexpressing wild-type or kinase-dead PKCε, and in lung endothelial cells isolated from PKCε-/- mice, no difference was observed in expression of KDR or in VEGF signaling compared to control conditions. However, phosphorylation of IΚBα at Ser32/36, an index of NFΚB activation, was more than 5-fold lower in retinas from PKCε-/- mice than PKCε+/+ mice at P17. Expression of pro-inflammatory factors peaked at different times during retinopathy: compared to mRNA levels at P12, tumor necrosis factor-α (Tnf) increased 86-fold at P13, interleukin-1β (Il1b) increased 28-fold at P14, and inducible nitric oxide (Nos2) increased 10-fold at P16, suggesting that some of these factors may precede and possibly facilitate angiogenesis, while others may be a result of the angiogenic response. At P12.5, just 12 hours after initiation of retinal ischemia, expression of monocyte chemoattractant protein-1 (MCP-1/Ccl2), which is central to macrophage recruitment, increased 18-fold in PKCε+/+ mice compared to PKCε+/+ mice not exposed to oxygen, but less than 6-fold in PKCε-/- mice (p=0.002).
Knockout of PKCε dramatically reduces pre-retinal neovascularization in oxygen-induced retinopathy. PKCε may promote angiogenesis by regulating macrophage recruitment or microglia activation. Therefore, PKCε could be a novel target for prevention of proliferative retinopathy.
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