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Hyperthermia-induced upregulation of vascular endothelial growth factor in retinal pigment epithelial cells is regulated by mitogen-activated protein kinases

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Abstract

Purpose

Localized application of hyperthermia is a potential treatment for retinal diseases. Vascular endothelial growth factor (VEGF) derived from the retinal pigment epithelium (RPE) is implicated in a variety of retinal pathologies. As it has been recently shown that hyperthermia may induce VEGF in the RPE, the aim of this study was to investigate hyperthermia-induced VEGF secretion and the pathways of hyperthermal VEGF upregulation in the RPE.

Material and methods

The human RPE cell line (Arpe-19) was exposed to 40°, 42°, 45° and 50 °C for one, five and 15 min. Cell viability was evaluated using a trypan blue exclusion assay, VEGF secretion was evaluated by an enzyme-linked immunosorbent assay ELISA) and VEGF expression was investigated using a Western blot. Involvement of mitogen-activated protein kinase (MAPK) pathways (ERK1/2, JNK, p38) and transient receptor potential vanilloid (TRPV) channels on VEGF induction was investigated using commercially available inhibitors (U0126, SB203580, SP600125, ruthenium red). Expression and phosphorylation of MAPKs was investigated using a Western blot.

Results

Hyperthermia induces time- and temperature-dependent cell death in human RPE cells. VEGF expression and secretion is induced by hyperthermia in a time- and temperature-dependent manner mediated by p38 and to a lesser degree by JNK. TRPV channels seem to play a minor role in regulation of hyperthermia-induced VEGF secretion.

Conclusions

Hyperthermia induces temperature-dependent secretion of VEGF in the RPE, which is mediated by p38 and, to a lesser extent, JNK. This may lead to undesired effects from hyperthermal treatment of retinal diseases.

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References

  1. Strauss O (2005) The retinal pigment epithelium in visual function. Physiol Rev 85:845–881

    Article  CAS  PubMed  Google Scholar 

  2. Ambati J, Fowler BJ (2012) Mechanisms of age-related macular degeneration. Neuron 75:26–39

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Chen H, Liu B, Lukas TJ, Neufeld AH (2008) The aged retinal pigment epithelium/choroid: a potential substratum for the pathogenesis of age-related macular degeneration. PLoS ONE 3:e2339

    Article  PubMed Central  PubMed  Google Scholar 

  4. McLeod DS, Grebe R, Bhutto I, Merges C, Baba T, Lutty GA (2009) Relationship between RPE and choriocapillaris in age-related macular degeneration. Invest Ophthalmol Vis Sci 50:4982–4991

    Article  PubMed  Google Scholar 

  5. Bhutto I, Lutty G (2012) Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch's membrane/choriocapillaris complex. Mol Asp Med 33:295–317

    Article  CAS  Google Scholar 

  6. Klettner A (2013) Physiological Functions of VEGF in the Retina and Its Possible Implications of Prolonged Anti-VEGF Therapy. In: Parker ML(edt) Vascular Endothelial Growth Factor: Biology, Regulation and Clinical Significance. Nova Biomedical pp117-136.

  7. Kurihara T, Westenskow PD, Bravo S, Aguilar E, Friedlander M (2012) Targeted deletion of Vegfa in adult mice induces vision loss. J Clin Invest 122:4213–4217

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Grunwald JE, Daniel E, Huang J, Ying GS, Maguire MG, Toth CA, Jaffe GJ, Fine SL, Blodi B6, Klein ML, Martin AA, Hagstrom SA, Martin DF; CATT Research Group (2014) Risk of geographic atrophy in the comparison of age-related macular degeneration treatments trials. Ophthalmology 121:150–156

    Article  Google Scholar 

  9. Witmer AN, Vrensen GF, Van Noorden CJ (2003) Schlingemann RO (2003) Vascular endothelial growth factors and angiogenesis in eye disease. Prog Retin Eye Res 22:1–29

    Article  CAS  PubMed  Google Scholar 

  10. Blaauwegeers HGT, Holtkamp GM, Rutten H, Witmer AN, Koolwijk P, Partanen TA, Alitalo K, Kroon ME, Kijlstra A, van Hinsbergh VW, Schlingemann RO (1999) 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

    Article  Google Scholar 

  11. Klettner A, Roider J (2012) Mechanisms of Pathological VEGF Production in the Retina and Modification with VEGF-Antagonists. In: Stratton R, Hauswirth W, Gardner T (eds) Oxidative Stress in Applied Basic Research and Clinical Practise: Studies on Retinal and Choroidal Disorders. Humana Press, New York, pp 277–307

  12. Klettner A, Koinzer S, Meyer T, Roider J (2013) Toll-like receptor 3 activation in retinal pigment epithelium cells- Mitogen-activated protein kinase pathways of cell death and vascular endothelial growth factor secretion. Acta Ophthalmol 91:e211–e218

    Article  CAS  PubMed  Google Scholar 

  13. Kannan R, Zhang N, Sreekumar P, Spee C, Rodriguez A, Barron E, Hinton DR (2006) Stimulation of apical and basolateral vascular endothelial growth factor-A and vascular endothelial growth factor-C secretion by oxidative stress in polarized retinal pigment epithelial cells. Mol Vis 12:1649–1659

    CAS  PubMed  Google Scholar 

  14. Coassin M, Duncan KG, Bailey KR, Singh A, Schwartz DM (2010) Hypothermia reduces secretion of vascular endothelial growth factor by cultured retinal pigment epithelial cells. Br J Ophthalmol 94:1678–1683

    Article  PubMed  Google Scholar 

  15. Cordeiro S, Seyler S, Stindl J, Milenkovic VM, Strauss O (2010) Heat-sensitive TRPV channels in retinal pigment epithelial cells: regulation of VEGF-A secretion. Invest Ophthalmol Vis Sci 51:6001–6008

    Article  PubMed  Google Scholar 

  16. Parver LM, Auker C, Carpenter DO (1980) Choroidal blood flow as a heat dissipating mechanism in the macula. Am J Ophthalmol 89:641–646

    Article  CAS  PubMed  Google Scholar 

  17. Nickla DL, Wallman J (2010) The multifunctional choroid. Prog Retin Eye Res 29:144–168

    Article  PubMed Central  PubMed  Google Scholar 

  18. Metelitsina TI, Grunwald JE, DuPont JC, Ying GS, Brucker JA, Dunaief JL (2008) Foveolar choroidal circulation and choroidal neovascularization in age-related macular degeneration. Invest Ophthalmol Vis Sci 49:358–363

    Article  PubMed Central  PubMed  Google Scholar 

  19. Boltz A, Luksch A, Wimpissinger B, Maar N, Weigert G, Frantal S, Brannath W, Garhöfer G, Ergun E, Stur M, Schmetterer L (2010) Choriodal blood flow and progression of age-related macular degeneration in the fellow eye in patients with unilateral choroidal neovascularization. Invest Ophthalmol Vis Sci 51:4220–4225

    Article  PubMed  Google Scholar 

  20. Journee-de Korver JG, Oosterhuis JA, van Best JA, Fakkel J (1991) Xenon arch photocoagulator used for transpupillary hyperthermia. Doc Ophthalmol 78:183–187

    Article  CAS  PubMed  Google Scholar 

  21. Oosterhuis JA, Journee-de Korver HG, Kakebeeke-Kemme HM, Bleeker JC (1995) Transpupillary thermotherapy in choroidal melanomas. Arch Ophthalmol 113:315–321

    Article  CAS  PubMed  Google Scholar 

  22. Nowak MS, Jurowski P, Grzybowski A, Gos R, Pastuszka M, Kapica A, Smigielski J (2012) A prospective study on different methods for the treatment of choroidal neovascularization. The efficacy of verteporfin photodynamic therapy, intravitreal bevacizumab and transpupillary thermotherapy in patients with neovascular age-related macular degeneration. Med Sci Monit 18:CR374–CR380

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Söderberg AC, Algvere PV, Hengstler JC, Söderberg P, Seregard S, Kvanta A (2012) Combination therapy with low-dose transpupillary thermotherapy and intravitreal ranibizumab for neovascular age-related macular degeneration: a 24-month prospective randomised clinical study. Br J Ophthalmol 96:714–718

    Article  PubMed  Google Scholar 

  24. Francis JH, Abramson DH, Brodie SE, Marr BP (2013) Indocyanine green enhanced transpupillary thermotherapy in combination with ophthalmic artery chemosurgery for retinoblastoma. Br J Ophthalmol 97:164–168

    Article  PubMed  Google Scholar 

  25. Caminal JM, Mejia-Castillo KA, Arias L, Catala J, Rubio M, Garcia P, Pujol O, Arruga J (2013) Subthreshold transpupillary thermotherapy in management of foveal subretinal fluid in small pigmented choroidal lesions. Retina 33:194–199

    Article  PubMed  Google Scholar 

  26. Sharma T, Krishnan T, Gopal L, Nagpal A, Khetan V, Rishi P (2011) Transpupillary thermotherapy for circumscribed choroidal hemangioma: clinical profile and treatment outcome. Ophthalmic Surg Lasers Imaging 42:360–368

    Article  PubMed  Google Scholar 

  27. Rougier MB, Francois L, Fourmaux E, Isber R, Colin J, Korobelnik JF (2005) Complications and lack of benefit after transpupillary thermotherapy for occult choroidal neovascularization: 1-year results. Retina 25:784–788

    Article  PubMed  Google Scholar 

  28. Yarovoy AA, Magaramov DA, Bulgakova ES (2010) Which choroidal melanoma should be treated with primary transpupillary thermotherapy? Our experience from 78 patients. Eur J Ophthalmol 20:186–193

    PubMed  Google Scholar 

  29. Parrozzani R, Boccassini B, De Belvis V, Radin PR, Midena E (2009) Long term outcome of transpupillary thermotherapy as primary treatment of selected choroidal melanoma. Acta Ophthalmol 87:789–792

    Article  PubMed  Google Scholar 

  30. Finger PT, Lipka AC, Lipkowitz J, Jofe M, McCormick SA (2000) Failure of transpupillary thermotherapy (TTT) for choroidal melanoma: two cases with histopathological correlation. Br J Ophthalmol 84:1075–1082

    Article  CAS  PubMed  Google Scholar 

  31. Klettner A, Westhues D, Lassen J, Bartsch S, Roider J (2013) Regulation of constitutive vascular endothelial growth factor secretion in retinal pigment epithelium/choroid organ cultures: p38, nuclear factor kappaB, and the vascular endothelial growth factor receptor-2/phosphatidylinositol 3 kinase pathway. Mol Vis 19:281–291

  32. Berra E, Pagès G, Pouysségur J (2000) MAP kinases and hypoxia in the control of VEGF expression. Cancer Metastasis Rev 19:139–145

    Article  CAS  PubMed  Google Scholar 

  33. Milanini-Mongiat J, Pouysségur J (2002) Identification of two p42/p44 mitogen activated kinases phosphorylation sites on Sp1: Their implication on vascular endothelial growth factor gene transcription. J Biol Chem. doi:10.1074/jbc.M201753200

    PubMed  Google Scholar 

  34. Pagès G, Berra E, Milanini J, Levy AP, Pouysségur J (2000) Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem 275:26484–26491

    Article  PubMed  Google Scholar 

  35. Kawano Y, Utsunomiya-Kai Y, Kai K, Miyakawa I, Ohshiro T, Narahara H (2012) The production of VEGF involving MAP kinase activation by low level laser therapy in human granulosa cells. Laser Ther 21:269–274

    Article  PubMed Central  PubMed  Google Scholar 

  36. Raidl M, Sibbing B, Strauch J, Müller K, Nemat A, Schneider PM, Hag H, Erdmann E, Koch A (2007) Impaired TNFalpha-induced VEGF expression in human airway smooth muscle cells from smokers with COPD: role of MAPkinases and histone acetylation–effect of dexamethasone. Cell Biochem Biophys 49:98–110

    Article  CAS  PubMed  Google Scholar 

  37. Tanaka T, Kanai H, Sekiguchi K, Aihara Y, Yokoyama T, Arai M, Kanda T, Nagai R, Kurabayashi M (2000) Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J Mol Cell Cardiol 32:1955–1967

    Article  CAS  PubMed  Google Scholar 

  38. Bian ZM, Elner SG, Elner VM (2007) Thrombin-induced VEGF expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 48:2738–2746

    Article  PubMed Central  PubMed  Google Scholar 

  39. Weng CY, Kothary PC, Verkade AJ, Reed DM, Del Monte MA (2009) MAP kinase pathway is involved in IGF-1-stimulated proliferation of human retinal pigment epithelial cells (hRPE). Curr Eye Res 34:867–876

    Article  CAS  PubMed  Google Scholar 

  40. Klettner A, Roider J (2009) Constitutive and oxidative-stress-induced expression of VEGF in the RPE are differently regulated by different Mitogen-activated protein kinases. Graefes Arch Clin Exp Ophthalmol 247:1487–1492

    Article  PubMed  Google Scholar 

  41. Kunchithapautham K, Rohrer B (2011) Sublytic membrane-attack-complex (MAC) activation alters regulated rather than constitutive vascular endothelial growth factor (VEGF) secretion in retinal pigment epithelium monolayers. J Biol Chem 286:23717–23724

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Klettner A, Koinzer S, Waetzig V, Herdegen T, Roider J (2010) Deferoxamine mesylate is toxic for retinal pigment epithelium cells, and its toxicity is mediated by p38. Cutan Ocul Toxicol 29:122–129

    Article  CAS  PubMed  Google Scholar 

  43. Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–169

    Article  CAS  PubMed  Google Scholar 

  44. Zeng F, Zhang M, Xu Y, Xu H (2013) ARMS2 interference leads to decrease of proinflammatory mediators. Graefes Arch Clin Exp Ophthalmol 251:2539–2544

    Article  CAS  PubMed  Google Scholar 

  45. Yokoyama K, Kimoto K, Itoh Y, Nakatsuka K, Matsuo N, Yoshioka H, Kubota T (2012) The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol 250:15–23

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Ahmado A, Carr AJ, Vugler AA, Semo M, Gias C, Lawrence JM, Chen LL, Chen FK, Turowski P, da Cruz L, Coffey PJ (2011) Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19. Invest Ophthalmol Vis Sci 52:7148–7159

    Article  CAS  PubMed  Google Scholar 

  47. Alge CS, Hauck SM, Priglinger SG, Kampik A, Ueffing M (2006) Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival. J Proteome Res 5:862–878

    Article  CAS  PubMed  Google Scholar 

  48. Cai H, Del Priore LV (2006) Gene expression profile of cultured adult compared to immortalized human RPE. Mol Vis 12:1–14

    CAS  PubMed  Google Scholar 

  49. Tian J, Ishibashi K, Honda S, Boylan SA, Hjelmeland LM, Handa JT (2005) The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions. Br J Ophthalmol 89:1510–1517

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Ford KM, Saint-Geniez M, Walshe T, Zahr A, D'Amore PA (2011) Expression and role of VEGF in the adult retinal pigment epithelium. Invest Ophthalmol Vis Sci 52:9478–9487

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Bergmann M, Holz F, Kopitz J (2011) Lysosomal stress and lipid peroxidation products induce VEGF-121 and VEGF-165 expression in ARPE-19 cells. Graefes Arch Clin Exp Ophthalmol 249:1477–1483

    Article  CAS  PubMed  Google Scholar 

  52. Bai L, Zhu X, Ma T, Wang J, Wang F, Zhang S (2013) The p38 MAPK NF-κB pathway, not the ERK pathway, is involved in exogenous HIV-1 Tat-induced apoptotic cell death in retinal pigment epithelial cells. Int J Biochem Cell Biol 45:1794–1801

    Article  CAS  PubMed  Google Scholar 

  53. Watanabe K, Zhang XY, Kitagawa K, Yunoki T, Hayashi A (2009) The effect of clonidine on VEGF expression in human retinal pigment epithelial cells (ARPE-19). Graefes Arch Clin Exp Ophthalmol 247:207–213

    Article  CAS  PubMed  Google Scholar 

  54. Chen Y, Kijlstra A, Chen Y, Yang P (2011) IL-17A stimulates the production of inflammatory mediators via Erk1/2, p38 MAPK, PI3K/Akt, and NF-κB pathways in ARPE-19 cells. Mol Vis 17:3072–3077

    CAS  PubMed Central  PubMed  Google Scholar 

  55. Sekiyama E, Saint-Geniez M, Yoneda K, Hisatomi T, Nakao S, Walshe TE, Maruyama K, Hafezi-Moghadam A, Miller JW, Kinoshita S, D'Amore PA (2011) Heat treatment of retinal pigment epithelium induces production of elastic lamina components and antiangiogenic activity. FASEB J 26:567–575

    Article  PubMed  Google Scholar 

  56. Yin L, Wu X, Gong Y, Shi Y, Qiu Y, Zhang H, Liu X, Gu Q (2011) OX-LDL up-regulates the vascular endothelial growth factor-to-pigment epithelium-derived factor ratio in human retinal pigment epithelial cells. Curr Eye Res 36:379–385

    Article  CAS  PubMed  Google Scholar 

  57. Vay L, Gu C, McNaughton PA (2012) The thermo-TRP ion channel family: properties and therapeutic implications. Br J Pharmacol 165:787–801

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Saint-Geniez M, Maharaj ASR, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D'Amore PA (2008) Endogenous VEGF is required for visual function: Evidence for a survival role on Müller cells and photoreceptors. PLoS One 3:e3554

    Article  PubMed Central  PubMed  Google Scholar 

  59. Saint-Geniez M, Kurihara T, Sekiyama E, Maldonado A, D'Amore P (2009) An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris. Proc Natl Acad Sci U S A 106:18751–18756

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  60. Byeon SH, Lee SC, Choi SH, Lee HK, Lee JH, Chu YK, Kwon OW (2010) Vascular endothelial growth factor as an autocrine survival factor for retinal pigment epithelial cells under oxidative stress via the VEGFR-2/PI3K/Akt pathway. Invest Ophthalmol Vis Sci 51:1190–1197

  61. El-Remessy AB, Bartoli M, Platt DH, Fulton D, Caldwell RB (2005) Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration. J Cell Sci 118:243–252

    Article  CAS  PubMed  Google Scholar 

  62. Kilic Ü, Kilic E, Järve A, Guo Z, Spudich A, Bieber K, Barzena U, Bassetti CL, Marti HH, Hermann DM (2006) Human vascular endothelial growth factor protects axotomized retinal ganglion cells in vivo by activating ERK1/2 and Akt pathways. J Neurosci 26:12439–12446

    Article  CAS  PubMed  Google Scholar 

  63. Miller DW, Joussen AM, Holz FG (2003) Die molekularen Mechanismen der neovaskulären AMD. Ophthalmologe 100:92–96

    Article  CAS  PubMed  Google Scholar 

  64. Scholl S, Kirchhof J, Augustin AJ (2010) Pathophysiology of macular edema. Ophthalmologica 224:8–15

    Article  CAS  PubMed  Google Scholar 

  65. Tolentino MJ (2009) Current molecular understanding and future treatment strategies for pathological ocular neovascularization. Curr Mol Med 9:973–981

    Article  CAS  PubMed  Google Scholar 

  66. Yang H, Jager MJ, Grossniklaus HE (2010) Bevacizumab suppression of establishment of micrometastases in experimental ocular melanoma. Invest Ophthalmol Vis Sci 51:2835–2842

    Article  PubMed Central  PubMed  Google Scholar 

  67. Stoffelns BM (2002) Transpupillary thermotherapy (TTT) for malignant choroidal melanoma. Acta Ophthalmol 80:25–31

    Article  CAS  Google Scholar 

  68. Stoffelns BM (2006) Morphologische und funktionelle Ergebnisse nach transpupillarer Thermotherapie (TTT) maligner Aderhautmelanome. Klin Monatsbl Augenheilkd 223:74–80

    Article  CAS  PubMed  Google Scholar 

  69. Schöpfer K, Stoffelns B (2010) Development of choroidal neovascularisation following transpupillary thermotherapy (TTT) for a small malignant melanoma of the choroid at the posterior pole – a case report. Med Laser Appl 25:223–228

    Article  Google Scholar 

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Acknowledgements

The authors thank Ms. Serap Luick for her excellent technical assistance. This study was presented at the Tagung der Vereinigung Norddeutscher Augenärzte 2012, the Meeting of the German Society of Ophthalmology 2012, and at the EVER Meeting 2012.

Declaration of interest statement

No conflict of interest exists regarding this study. Independent of this study, AK has been consultant for and has received honoraria for lectures by Novartis Pharma.

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Authors and Affiliations

  1. Department of Ophthalmology, University Medical Center, University of Kiel, Arnold-Heller-Str. 3, 24105, Kiel, Germany

    Hendrik Faby, Jost Hillenkamp, Johann Roider & Alexa Klettner

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  1. Hendrik Faby
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  2. Jost Hillenkamp
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Correspondence to Alexa Klettner.

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Faby, H., Hillenkamp, J., Roider, J. et al. Hyperthermia-induced upregulation of vascular endothelial growth factor in retinal pigment epithelial cells is regulated by mitogen-activated protein kinases. Graefes Arch Clin Exp Ophthalmol 252, 1737–1745 (2014). https://doi.org/10.1007/s00417-014-2750-z

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