Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

Zhao,Jianfeng; Geng,Yu; Hua,Hairong; Cun,Biyun; Chen,Qianbo; Xi,Xiaoting; Yang,Liushu; Li,Yan

doi:10.3892/etm.2015.2697

Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

Authors:
- Jianfeng Zhao
- Yu Geng
- Hairong Hua
- Biyun Cun
- Qianbo Chen
- Xiaoting Xi
- Liushu Yang
- Yan Li
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Affiliations: Department of Ophthalmology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650031, P.R. China
Published online on: August 21, 2015 https://doi.org/10.3892/etm.2015.2697
Pages: 1404-1412
Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to examine the mechanisms through which fenofibrate inhibits the ability of human retinal pigment epithelial cells (RPE cells) exposed to hypoxia to stimulate the proliferation and migration of human umbilical vein endothelial cells (HUVECs). For this purpose, RPE cells and HUVECs were divided into the following groups: RPE-normoxia, RPE + fenofibrate, RPE-hypoxia, RPE hypoxia + fenofibrate; HUVECs normal culture and HUVECs + RPE-hypoxia culture supernatant. RPE cell hypoxia was induced by cobalt(II) chloride (CoCl2). A superoxide anion probe was used to measure the production of superoxide anion, which is indicative of hypoxic conditions. Cell proliferation was assessed by MTT assay, and the expression of vascular endothelial growth factor C (VEGFC) and vascular endothelial growth factor receptor-3 (VEGFR-3) in the RPE cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The migration ability of the HUVECs was determined by scratch-wound assay, and the angiogenic ability of the HUVECs was examined by measuring cell lumen formation. The mRNA and protein expression levels of VEGFC and VEGFR-3 in the RPE cells were measured by RT-qPCR and western blot analysis, respectively. Our results revealed that fenofibrate inhibited the increase in the expression and release of VEGFC and VEGFR-3 into the RPE cell culture supernatant induced by exposure to hypoxia. The culture of HUVECs in medium supernatant of RPE cells epxosed to hypoxia enhanced the viability and migration ability of the HUVECs and promoted lumen formation; these effects were inhibited by fenofibrate. In conclusion, our data demonstrated that the exposure of RPE cells to hypoxia induced the expression and release of VEGFC and VEGFR-3 into the cell culture supernatant. The culture of HUVECs in conditioned medium from RPE cells exposed to hypoxia increased VEGFC and VEGFR-3 expression, and promoted the proliferation and migration of the HUVECs, as well as capillary tube formation, suggesting that RPE cells play an important role in the formation of choroidal neovascularization resulting from hypoxia. Fenofibrate inhibited the upregulation of VEGFC and VEGFR-3 in the RPE cells exposed to hypoxia, and thus reduced the ability of HUVECs to form new blood vessels.

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1	Nowak JZ: Age-related macular degeneration (AMD): Pathogenesis and therapy. Pharmacol Rep. 58:353–363. 2006.PubMed/NCBI
2	Das A and McGuire PG: Retinal and choroidal angiogenesis: Pathophysiology and strategies for inhibition. Prog Retin Eye Res. 22:721–748. 2003. View Article : Google Scholar : PubMed/NCBI
3	Campochiaro PA, Soloway P, Ryan SJ and Miller JW: The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration. Mol Vis. 5:341999.PubMed/NCBI
4	Ambati J, Ambati BK, Yoo SH, Ianchulev S and Adamis AP: Age-related macular degeneration: Etiology, pathogenesis, and therapeutic strategies. Surv Ophthalmol. 48:257–293. 2003. View Article : Google Scholar : PubMed/NCBI
5	Blaauwgeers HG, Holtkamp GM, Rutten H, Witmer AN, Koolwijk P, Partanen TA, Alitalo K, Kroon ME, Kijlstra A, van Hinsbergh VW, et al: Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol. 155:421–428. 1999. View Article : Google Scholar : PubMed/NCBI
6	Ozturk BT, Bozkurt B, Kerimoglu H, Okka M, Kamis U and Gunduz K: Effect of serum cytokines and VEGF levels on diabetic retinopathy and macular thickness. Mol Vis. 15:1906–1914. 2009.PubMed/NCBI
7	Zhang P, Wang Y, Hui Y, Hu D, Wang H, Zhou J and Du H: Inhibition of VEGF expression by targeting HIF-1 alpha with small interference RNA in human RPE cells. Ophthalmologica. 221:411–417. 2007. View Article : Google Scholar : PubMed/NCBI
8	Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, et al: VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 161:1163–1177. 2003. View Article : Google Scholar : PubMed/NCBI
9	Gaengel K and Betsholtz C: Endocytosis regulates VEGF signalling during angiogenesis. Nat Cell Biol. 15:233–235. 2013. View Article : Google Scholar : PubMed/NCBI
10	Carvalho B, Freitas-Costa P, Pinheiro-Costa J, Falcêo M, Carneiro Â and Falcão-Reis F: Evaluation of antiangiogenic treatment results in choroidal neovascularization related to pathological myopia. Acta Med Port. 27:49–58. 2014.(In Portuguese). PubMed/NCBI
11	Casanovas O, Hicklin DJ, Bergers G, Hanahan D, Carneiro Â and Falcão-Reis F: Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell. 8:299–309. 2005. View Article : Google Scholar : PubMed/NCBI
12	Jain RK: Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science. 307:58–62. 2005. View Article : Google Scholar : PubMed/NCBI
13	Jones D, Xu Z, Zhang H, He Y, Kluger MS, Chen H and Min W: Functional analyses of the bone marrow kinase in the X chromosome in vascular endothelial growth factor-induced lymphangiogenesis. Arterioscler Thromb Vasc Biol. 30:2553–2561. 2010. View Article : Google Scholar : PubMed/NCBI
14	Tammela T, Zarkada G, Wallgard E, Murtomäki A, Suchting S, Wirzenius M, Waltari M, Hellström M, Schomber T, Peltonen R, et al: Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature. 454:656–660. 2008. View Article : Google Scholar : PubMed/NCBI
15	Clarijs R, Schalkwijk L, Hofmann UB, Ruiter DJ and de Waal RM: Induction of vascular endothelial growth factor receptor-3 expression on tumor microvasculature as a new progression marker in human cutaneous melanoma. Cancer Res. 62:7059–7065. 2002.PubMed/NCBI
16	Paavonen K, Puolakkainen P, Jussila L, Jahkola T and Alitalo K: Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am J Pathol. 156:1499–1504. 2000. View Article : Google Scholar : PubMed/NCBI
17	Yuasa T, Takahashi S, Hatake K, Yonese J and Fukui I: Biomarkers to predict response to sunitinib therapy and prognosis in metastatic renal cell cancer. Cancer Sci. 102:1949–1957. 2011. View Article : Google Scholar : PubMed/NCBI
18	Chen Y, Hu Y, Lin M, Jenkins AJ, Keech AC, Mott R, Lyons TJ and Ma JX: Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models. Diabetes. 62:261–272. 2013. View Article : Google Scholar : PubMed/NCBI
19	Trudeau K and Roy S, Guo W, Hernández C, Villarroel M, Simó R and Roy S: Fenofibric acid reduces fibronectin and collagen type IV overexpression in human retinal pigment epithelial cells grown in conditions mimicking the diabetic milieu: Functional implications in retinal permeability. Invest Ophthalmol Vis Sci. 52:6348–6354. 2011. View Article : Google Scholar : PubMed/NCBI
20	Walker AE, Kaplon RE, Lucking SM, Russell-Nowlan MJ, Eckel RH and Seals DR: Fenofibrate improves vascular endothelial function by reducing oxidative stress while increasing endothelial nitric oxide synthase in healthy normolipidemic older adults. Hypertension. 60:1517–1523. 2012. View Article : Google Scholar : PubMed/NCBI
21	Sharma N, Ooi JL, Ong J and Newman D: The use of fenofibrate in the management of patients with diabetic retinopathy: an evidence-based review. Aust Fam Physician. 44:367–370. 2015.PubMed/NCBI
22	Fagan XJ and Chong EW: Fenofibrate and diabetic retinopathy. Clin Experiment Ophthalmol. 43:297–299. 2015. View Article : Google Scholar : PubMed/NCBI
23	Simó R, Simó-Servat O and Hernández C: Is fenofibrate a reasonable treatment for diabetic microvascular disease? Curr Diab Rep. 15:242015. View Article : Google Scholar : PubMed/NCBI
24	Lazzeri S, Orlandi P, Figus M, Fioravanti A, Cascio E, Di Desidero T, Agosta E, Canu B, Sartini MS, Danesi R, et al: The rs2071559 AA VEGFR-2 genotype frequency is significantly lower in neovascular age-related macular degeneration patients. ScientificWorldJournal. 2012:4201902015.
25	Izuta H, Chikaraishi Y, Shimazawa M, Mishima S and Hara H: 10-Hydroxy-2-decenoic acid, a major fatty acid from royal jelly, inhibits VEGF-induced angiogenesis in human umbilical vein endothelial cells. Evid Based Complement Alternat Med. 6:489–494. 2009. View Article : Google Scholar : PubMed/NCBI
26	Pourgholami MH, Khachigian LM, Fahmy RG, Badar S, Wang L, Chu SW and Morris DL: Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis. Biochem Biophys Res Commun. 397:729–734. 2010. View Article : Google Scholar : PubMed/NCBI
27	Liu HA, Liu YL, Ma ZZ, Wang JC and Zhang Q: A lipid nanoparticle system improves siRNA efficacy in RPE cells and a laser-induced murine CNV model. Invest Ophthalmol Vis Sci. 52:4789–4794. 2011. View Article : Google Scholar : PubMed/NCBI
28	Cao J, Zhao L, Li Y, Liu Y, Xiao W, Song Y, Luo L, Huang D, Yancopoulos GD, Wiegand SJ, et al: A subretinal matrigel rat choroidal neovascularization (CNV) model and inhibition of CNV and associated inflammation and fibrosis by VEGF trap. Invest Ophthalmol Vis Sci. 51:6009–6017. 2010. View Article : Google Scholar : PubMed/NCBI
29	Querques G, Thirkill CE, Hagege H, Soubrane G and Souied EH: Choroidal neovascularization associated with cancer-associated retinopathy. Acta Ophthalmol. 88:571–575. 2010. View Article : Google Scholar : PubMed/NCBI
30	Liu X, Du J and Xi Q: HIF-1α ODD polypeptides increased the expression of HIF1 and VEGF in hypoxic rat cortical neuron. Neurol Sci. 32:1029–1033. 2011. View Article : Google Scholar : PubMed/NCBI
31	Miyasaka A, Oda K, Ikeda Y, Sone K, Fukuda T, Inaba K, Makii C, Enomoto A, Hosoya N, Tanikawa M, et al: PI3K/mTOR pathway inhibition overcomes radioresistance via suppression of the HIF1-α/VEGF pathway in endometrial cancer. Gynecol Oncol. 138:174–180. 2015. View Article : Google Scholar : PubMed/NCBI
32	Nakajima T, Anayama T, Koike T, Shingyoji M, Castle L, Kimura H, Yoshino I and Yasufuku K: Endobronchial ultrasound doppler image features correlate with mRNA expression of HIF1-α and VEGF-C in patients with non-small-cell lung cancer. J Thorac Oncol. 7:1661–1667. 2012. View Article : Google Scholar : PubMed/NCBI
33	Wang H, Geisen P, Wittchen ES, King B, Burridge K, D'Amore PA and Hartnett ME: The role of RPE cell-associated VEGF₁₈₉ in choroidal endothelial cell transmigration across the RPE. Invest Ophthalmol Vis Sci. 52:570–578. 2011. View Article : Google Scholar : PubMed/NCBI
34	Padera TP and Jain RK: VEGFR3: A new target for antiangiogenesis therapy? Dev Cell. 15:178–179. 2008. View Article : Google Scholar : PubMed/NCBI
35	Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K, Breitman M and Alitalo K: Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science. 282:946–949. 1998. View Article : Google Scholar : PubMed/NCBI
36	Wittig C, Scheuer C, Parakenings J, Menger MD and Laschke MW: Geraniol suppresses angiogenesis by downregulating vascular endothelial growth factor (VEGF)/VEGFR-2 signaling. PLoS One. 10:e01319462015. View Article : Google Scholar : PubMed/NCBI
37	Yi X, Mai LC, Uyama M and Yew DT: Time-course expression of vascular endothelial growth factor as related to the development of the retinochoroidal vasculature in rats. Exp Brain Res. 118:155–160. 1998. View Article : Google Scholar : PubMed/NCBI
38	Le YZ, Bai Y, Zhu M and Zheng L: Temporal requirement of RPE-derived VEGF in the development of choroidal vasculature. J Neurochem. 112:1584–1592. 2010. View Article : Google Scholar : PubMed/NCBI
39	Milosavljevic D, Griglio S, Le Naour G and Chapman MJ: Preferential reduction of very low density lipoprotein-1 particle number by fenofibrate in type IIB hyperlipidemia: Consequences for lipid accumulation in human monocyte-derived macrophages. Atherosclerosis. 155:251–260. 2001. View Article : Google Scholar : PubMed/NCBI
40	Panigrahy D, Kaipainen A, Huang S, Butterfield CE, Barnés CM, Fannon M, Laforme AM, Chaponis DM, Folkman J and Kieran MW: PPARalpha agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition. Proc Natl Acad Sci USA. 105:985–990. 2008. View Article : Google Scholar : PubMed/NCBI
41	Meissner M, Stein M, Urbich C, Reisinger K, Suske G, Staels B, Kaufmann R and Gille J: PPARalpha activators inhibit vascular endothelial growth factor receptor-2 expression by repressing Sp1-dependent DNA binding and transactivation. Circ Res. 94:324–332. 2004. View Article : Google Scholar : PubMed/NCBI
42	Noonan JE, Jenkins AJ, Ma JX, Keech AC, Wang JJ and Lamoureux EL: An update on the molecular actions of fenofibrate and its clinical effects on diabetic retinopathy and other microvascular end points in patients with diabetes. Diabetes. 62:3968–3975. 2013. View Article : Google Scholar : PubMed/NCBI
43	Ge Y, Liu J, Yang X, Zhu H, Yang B, Zhao K, Wu Z, Cheng G, Wang F, Ni F, et al: Fenofibrate enhances radiosensitivity of esophageal squamous cell carcinoma by suppressing hypoxia-inducible factor-1α expression. Tumour Biol. 35:10765–10771. 2014. View Article : Google Scholar : PubMed/NCBI