Quantification of pigment epithelium-derived factor (PEDF) in an ex vivo coculture of retinal pigment epithelium cells and neuroretina

Salvatore Di Lauro1,2, Maria Teresa Garcia-Gutierrez1, Ivan Fernandez-Bueno1,3,4

1. Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Valladolid, Spain
2. Departamento de Oftalmología, Hospital Clinico Universitario de Valladolid, Valladolid, Spain
3. Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leon, Valladolid, Spain
4. Red Temática de Investigación Cooperativa en Salud (RETICS), Oftared, Instituto de Salud Carlos III, Valladolid, Spain


Purpose: To quantify the pigment epithelium-derived factor (PEDF) levels in a coculture model of physically separated neuroretina and retinal pigment epithelium (RPE) cells, and to point out its potential role in neuroretinal maintenance. Methods: RPE cells and neuroretina explants were isolated from porcine eyes. RPE cells were expanded and seeded on the bottom of Transwell® culture inserts. Neuroretina explants were cultured alone (controls) on Transwell® culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane, during 9 days. PEDF concentration in the culture medium at 3, 5, 7 and 9 days of culture was determined by enzyme-linked immunosorbent assays (ELISA) specific for porcine samples. Mean statistical analysis were performed with Pair-wise Student’s-tests. Results: Culture medium collected from neuroretina cultures without and with RPE cells contained detectable levels of PEDF in both conditions and at all evaluated time-points. At 3, 5, and 7 days and through the whole time of culture PEDF concentration was significantly higher in cocultures with RPE cells (p<0.05). Conclusions: RPE cells neuroprotective role may be linked to the beneficial effects of neurotrophic factors, such as PEDF, secreted or induced by RPE cells during co-culture.
Keywords: Ex vivo model; Retinal pigment epithelium (RPE); Neuroretina; Pigment Epithelium-Derived Factor (PEDF); Neuroprotection
Alt, A., Hilgers, R.-D., Tura, A., Nassar, K., Schneider, T., Hueber, A., Januschowski, K., Grisanti, S., Lüke, J., Lüke, M., 2013. The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina. Cell. Physiol. Biochem. 32, 218–34. https://doi.org/10.1159/000350138

Barnstable, C.J., Tombran-Tink, J., 2004. Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular targets and therapeutic potential. Prog. Retin. Eye Res. 23, 561–577. https://doi.org/10.1016/J.PRETEYERES.2004.05.002

Di Lauro, S., Rodriguez-Crespo, D., Gayoso, M.J.M.J., Garcia-Gutierrez, M.T.M.T., Pastor, J.C.C., Srivastava, G.K.G.K., Fernandez-Bueno, I., 2016. A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells. Mol. Vis. 22, 243–53.

Fernandez-Bueno, I., Garcia-Gutierrez, M.T., Srivastava, G.K., Gayoso, M.J., Gonzalo-Orden, J.M., Pastor, J.C., 2013. Adalimumab (Tumor necrosis factor-blocker) reduces the expression of glial fibrillary acidic protein immunoreactivity increased by exogenous tumor necrosis factor alpha in an organotypic culture of porcine neuroretina. Mol. Vis. 19.

Fernandez-Bueno, I., Pastor, J.C.J.C., Gayoso, M.J.M.J., Alcalde, I., Garcia, M.T.M.T., 2008. Müller and macrophage-like cell interactions in an organotypic culture of porcine neuroretina. Mol. Vis. 14, 2148–56.

Fisher, S.K., Lewis, G.P., Linberg, K.A., Barawid, E., Verardo, M.R., 1995. Cellular Remodeling in Mammalian Retina Induced by Retinal Detachment, Webvision: The Organization of the Retina and Visual System.

Ghosh, F., Taylor, L., Arnér, K., 2012. Exogenous glutamate modulates porcine retinal development in vitro. Dev. Neurosci. 34, 428–39. https://doi.org/10.1159/000343721

Jablonski, M.M., Tombran-Tink, J., Mrazek, D.A., Iannaccone, A., 2000. Pigment epithelium-derived factor supports normal development of photoreceptor neurons and opsin expression after retinal pigment epithelium removal. J. Neurosci. 20, 7149–57.

Ku B, Liang C, Jung JU, Oh BH. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. 2011. Cell Research. 21:627–641.

Labrador-Velandia, S., Alonso-Alonso, M.L., Di Lauro, S., García-Gutierrez, M.T., Srivastava, G., Pastor, J.C., Fernandez-Bueno, I., 2019. Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures. Exp. Eye Res. 185, 107671. https://doi.org/10.1016/j.exer.2019.05.011

Lange, J., Yafai, Y., Reichenbach, A., Wiedemann, P., Eichler, W., 2008. Regulation of Pigment Epithelium–Derived Factor Production and Release by Retinal Glial (Müller) Cells under Hypoxia. Investig. Opthalmology Vis. Sci. 49, 5161. https://doi.org/10.1167/iovs.08-2201

Martínez-Fernández de la Cámara, C., Sequedo, M.D., Gómez-Pinedo, U., Jaijo, T., Aller, E., García-Tárraga, P., García-Verdugo, J.M., Millán, J.M., Rodrigo, R., 2013. Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina. Exp. Eye Res. 111, 122–33. https://doi.org/10.1016/j.exer.2013.03.015

Murakami Y, Ikeda Y, Yonemitsu Y, Onimaru M, Nakagawa K, Kohno R, Miyazaki M, Hisatomi T, Nakamura M, Yabe T, Hasegawa M, Ishibashi T, Sueishi K., 2008 Inhibition of nuclear translocation of apoptosis-inducing factor is an essential mechanism of the neuroprotective activity of pigment epithelium-derived factor in a rat model of retinal degeneration. Am J Pathol. Nov; 173(5):1326-38.

Pang, I.-H., Zeng, H., Fleenor, D.L., Clark, A.F., 2007. Pigment epithelium-derived factor protects retinal ganglion cells. BMC Neurosci. 8, 11. https://doi.org/10.1186/1471-2202-8-11

Pastor, J.C., Coco, R.M., Fernandez-Bueno, I., Alonso-Alonso, M.L., Medina, J., Sanz-Arranz, A., Rull, F., Gayoso, M.J., Dueñas, A., Garcia-Gutierrez, M.T., Gonzalez-Buendia, L., Delgado-Tirado, S., Abecia, E., Ruiz-Miguel, M., Serrano, M.A., Ruiz-Moreno, J.M., Srivastava, G.K., 2017. Acute retinal damage after using a toxic perfluoro-octane for vitreo-retinal surgery. Retina 37. https://doi.org/10.1097/IAE.0000000000001680

Pastor, J.C., de la Rúa, E.R., Martín, F., 2002. Proliferative vitreoretinopathy: risk factors and pathobiology. Prog. Retin. Eye Res. 21, 127–44.

Rodriguez-Crespo, D., Di Lauro, S., Singh, A.K.A.K., Garcia-Gutierrez, M.T.M.T., Garrosa, M., Pastor, J.C.C., Fernandez-Bueno, I., Srivastava, G.K.G.K., 2014. Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs. Cell Tissue Res. 358, 705–716. https://doi.org/10.1007/s00441-014-1987-5

Strauss, O., 2005. The retinal pigment epithelium in visual function. Physiol. Rev. 85, 845–81. https://doi.org/10.1152/physrev.00021.2004

Unterlauft, J.D., Claudepierre, T., Schmidt, M., Müller, K., Yafai, Y., Wiedemann, P., Reichenbach, A., Eichler, W., 2014. Enhanced survival of retinal ganglion cells is mediated by Müller glial cell-derived PEDF. Exp. Eye Res. 127, 206–14. https://doi.org/10.1016/j.exer.2014.08.004

Ms. Ref. No.: A0201008     RA    
Received: 12/09/2019  
Accepted: 06/11/2019   
Published: 15/04/2020 

How to cite this paper