Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells
Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization.
Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24 h or 48 h) or hypoxic conditions (1% O2, 24 h). Part of the cells were re-oxygenated (24 h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1β, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24 h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24 h.
Hypoxia induced significant (P < 0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1β or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4 h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS.
Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.
KeywordsHypoxia Inflammation VEGF Interleukins Human RPE cells
We warmly thank Dr. Ewen MacDonald for the language revision and Res. Dir. Emeritus Antero Salminen for the valuable collaboration, discussions, and critical review of the manuscript. Part of the results was published as an abstract at EVER meeting (Acta Ophthalmol. 2015, http://dx.doi.org/10.1111/j.1755-3768.2015.0426).
OA, VA, KK, and AK: designing, supervision, and reporting of the study. NP, VA, AK, TC, and GP: collection, analysis, and interpretation of the data as well as critical revision of the manuscript.
Compliance with ethical standards
All procedures performed in the experiments involving human material were in accordance with the local institutional ethical review board and with the Helsinki Declaration. For this type of a study, a formal consent was not required.
The research was financially supported by the Academy of Finland (Health Research Council project 297,267), the Finnish Cultural Foundation (Central and North-Savo Foundations), the Alfred Kordelin Foundation, the Emil Aaltonen Foundation, the Finnish Eye Foundation, and the Kuopio University Hospital (VTR funding), GINOP- 2.3.2–15–2016-00006 (Hungary), co-financed by the EU and the European Regional Development Fund.
Conflict of interest
All authors certify that they have no involvement in any organization with any financial interest in the subject matter or the materials discussed in the this manuscript.
- 16.Canataroglu H, Varinli I, Ozcan AA, Canataroglu A, Doran F, Varinli S (2005) Interleukin (IL)-6, interleukin (IL)-8 levels and cellular composition of the vitreous humor in proliferative diabetic retinopathy, proliferative vitreoretinopathy, and traumatic proliferative vitreoretinopathy. Ocul Immunol Inflamm 13:375–381CrossRefPubMedGoogle Scholar
- 22.Szatmári-Tóth M, Kristóf E, Veréb Z, Akhtar S, Facskó A, Fésüs L, Kauppinen A, Kaarniranta K, Petrovski G (2016) Clearance of autophagy-associated dying retinal pigment epithelial cells — a possible source for inflammation in age-related macular degeneration. Cell Death Dis 7(9):e2367. doi: 10.1038/cddis.2016.133 CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Lally DR, Shah CP, Heier JS (2016) Vascular endothelial growth factor and diabetic macular edema. Surv Ophthalmol. doi: 10.1016/j.survophthal.2016.03.010