Abstract
Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of biological and pathological effects in wet age-related macular degeneration and proliferative diabetic retinopathy. However, its specific mechanism is still not fully understood. Here, we examined the effects of VEGF on choroid-retinal endothelial cells (RF/6A) proliferation and tube formation, and the underlying signal pathways responsible in this process. RF/6A cells were pretreated with MEK inhibitor or PI3K inhibitor, and then incubated in a hypoxia chamber. Real-time PCR and Western blot analysis were carried out to explore VEGF expression on mRNA and protein levels. Hypoxia inducible factor-1α (HIF-1α) and VEGFR2 expression levels were also investigated in the presence and absence of hypoxic conditions. CCK-8 analysis and tube formation assay were tested under hypoxia, exogenous recombinant VEGF, and different signal pathway inhibitors, respectively. Mean while, the PI3K/Akt and MEK/ERK pathways in this process were also investigated. Our results showed that VEGF, HIF-1α, VEGFR2, p-ERK, and p-Akt were up-regulated in RF/6A cells under hypoxic conditions. MEK inhibitor (PD98059) and PI3K inhibitor (LY294002) decreased ERK and Akt activity, respectively, and reduced VEGF expression. VEGF-induced RF/6A proliferation and tube formation requires MEK/ERK and PI3K/Akt signaling, and both of the two pathways were needed in regulating VEGF expression. These suggest that VEGF plays an important role in RF/6A proliferation and tube formation, and MEK/ERK and PI3K/Akt pathway may be responsible for this process.
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Friedman DS, O’Colmain BJ, Munoz B, Tomany SC, McCarty C, de Jong PT, Nemesure B, Mitchell P, Kempen J (2004) Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 122:564–572
Calcutt NA, Cooper ME, Kern TS, Schmidt AM (2009) Therapies for hyperglycaemia-induced diabetic complications: from animal models to clinical trials. Nat Rev Drug Discov 8:417–429
Skeie JM, Mullins RF (2008) Elastin-mediated choroidal endothelial cell migration: possible role in age-related macular degeneration. Invest Ophthalmol Vis Sci 49:5574–5580
Dong X, Wang YS, Dou GR, Hou HY, Shi YY, Zhang R, Ma K, Wu L, Yao LB, Cai Y, Zhang J (2011) Influence of Dll4 via HIF-1alpha-VEGF signaling on the angiogenesis of choroidal neovascularization under hypoxic conditions. PLoS One 6:e18481
Yang XM, Wang YS, Zhang J, Li Y, Xu JF, Zhu J, Zhao W, Chu DK, Wiedemann P (2009) Role of PI3K/Akt and MEK/ERK in mediating hypoxia-induced expression of HIF-1alpha and VEGF in laser-induced rat choroidal neovascularization. Invest Ophthalmol Vis Sci 50:1873–1879
Ruiz DAC, Lambrechts D, Mazzone M, Carmeliet P (2009) Role and therapeutic potential of VEGF in the nervous system. Physiol Rev 89:607–648
Cobb MH (1999) MAP kinase pathways. Prog Biophys Mol Biol 71:479–500
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331
Kandel ES, Hay N (1999) The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res 253:210–229
Grossniklaus HE, Green WR (2004) Choroidal neovascularization. Am J Ophthalmol 137:496–503
Arjamaa O, Nikinmaa M, Salminen A, Kaarniranta K (2009) Regulatory role of HIF-1alpha in the pathogenesis of age-related macular degeneration (AMD). Ageing Res Rev 8:349–358
Zhang P, Zhang X, Hao X, Wang Y, Hui Y, Wang H, Hu D, Zhou J (2009) Rac1 activates HIF-1 in retinal pigment epithelium cells under hypoxia. Graefes Arch Clin Exp Ophthalmol 247:633–639
Zhang P, Wang Y, Hui Y, Hu D, Wang H, Zhou J, Du H (2007) Inhibition of VEGF expression by targeting HIF-1 alpha with small interference RNA in human RPE cells. Ophthalmologica 221:411–417
Lou DA, Hu FN (1987) Specific antigen and organelle expression of a long-term rhesus endothelial cell line. In Vitro Cell Dev Biol 23:75–85
Shao H, Tan Y, Eton D, Yang Z, Uberti MG, Li S, Schulick A, Yu H (2008) Statin and stromal cell-derived factor-1 additively promote angiogenesis by enhancement of progenitor cells incorporation into new vessels. Stem Cells 26:1376–1384
Stone EM (2006) A very effective treatment for neovascular macular degeneration. N Engl J Med 355:1493–1495
Frampton JE (2013) Ranibizumab: a review of its use in the treatment of neovascular age-related macular degeneration. Drugs Aging 30:331–358
Wang FZ, Fei HR, Li XQ, Shi R, Wang DC (2012) Perifosine as potential anti-cancer agent inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells. Mol Cell Biochem 368:1–8
Dai C, Zheng CQ, Meng FJ, Zhou Z, Sang LX, Jiang M (2013) VSL#3 probiotics exerts the anti-inflammatory activity via PI3K/Akt and NF-kappaB pathway in rat model of DSS-induced colitis. Mol Cell Biochem 374:1–11
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We would like to thank Wen-cheng Zhao for his help on the manuscript and Dr. Yabo Gao for providing some reagents.
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Jin, J., Yuan, F., Shen, Mq. et al. Vascular endothelial growth factor regulates primate choroid-retinal endothelial cell proliferation and tube formation through PI3K/Akt and MEK/ERK dependent signaling. Mol Cell Biochem 381, 267–272 (2013). https://doi.org/10.1007/s11010-013-1710-y
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DOI: https://doi.org/10.1007/s11010-013-1710-y