1 2 OSTEOPONTIN INHIBITS OSMOTIC SWELLING OF RETINAL GLIAL 3 (MU ¨ LLER) CELLS BY INDUCING RELEASE OF VEGF 4 V. WAHL, a Q1 S. VOGLER, a A. GROSCHE, a T. PANNICKE, a 5 M. UEFFING, b,c P. WIEDEMANN, d A. REICHENBACH, a 6 S. HAUCK b AND A. BRINGMANN d * 7 a Paul Flechsig Institute of Brain Research, University of Leipzig, 8 Leipzig, Germany 9 b Research Unit Protein Science, Helmholtz Zentrum Mu ¨ nchen, 10 German Research Center for Environmental Health, Neuherberg, 11 Germany 12 c Center of Ophthalmology, Institute for Ophthalmic Research, 13 University of Tu ¨ bingen, Tu ¨ bingen, Germany 14 d Department of Ophthalmology and Eye Hospital, University of 15 Leipzig, Leipzig, Germany 16 Abstract—Osmotic swelling of retinal neurons and glial cells is an important pathogenic factor of retinal edema for- mation. Here, we show that the neuroprotective factor oste- opontin (OPN), which is released from retinal glial (Mu¨ ller) cells after stimulation of the cells with glial cell line-derived neurotrophic factor (Del Rı´o et al., 2011, Glia 59:821–832), inhibits the swelling of rat Mu¨ller cells induced by hypoos- motic exposure of retinal slices in the presence of barium ions and H 2 O 2 , respectively, and in slices of postischemic retinas. OPN did not inhibit the hypoosmotic swelling of bipolar cells in slices of control and postischemic retinas. The inhibitory eﬀect of OPN on Mu¨ller cell swelling was dose-dependent, with a half-maximal eﬀect at 0.6 ng/ml. The eﬀect of OPN was abrogated in the presence of pharma- cological blockers of vascular endothelial growth factor (VEGF) receptor-2, metabotropic glutamate receptors, and purinergic receptors (P2Y 1 , adenosine A 1 receptors), as well as of a neutralizing anti-VEGF antibody. The data suggest that OPN induces the release of VEGF, glutamate, ATP, and adenosine from Mu¨ ller cells. The eﬀect of OPN was also prevented by blockers of voltage-gated sodium channels (tetrodotoxin), T-type voltage-gated calcium channels (kur- toxin), potassium channels (cloﬁlium), and chloride chan- nels 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The swelling-inhibitory eﬀect of OPN was dependent on intracellular calcium signaling, activation of phospholipase C and protein kinase C, and vesicular exocytosis of gluta- mate. In retinal slices, Mu¨ ller glial cells display immunoreac- tivity of OPN. The data suggest that Mu¨ ller cell-derived OPN has (in addition to the eﬀects on photoreceptors and retinal neurons) autocrine eﬀects. The neuroprotective eﬀects of OPN may be in part mediated by the prevention of cytotoxic Mu¨ ller cell swelling and the release of VEGF and adenosine from Mu¨ller cells. Ó 2013 Published by Elsevier Ltd. on behalf of IBRO. Key words: osteopontin, VEGF, osmotic swelling, ischemia, glia, retina. 17 18 INTRODUCTION 19 Retinal diseases such as age-related macular 20 degeneration, retinitis pigmentosa, and glaucoma are 21 characterized by a degeneration of photoreceptors and/ 22 or inner retinal neurons. Various neurotrophic and 23 growth factors, or combinations of the factors, promote 24 the survival of photoreceptors and retinal neurons. Brain- 25 derived neurotrophic factor (BDNF), ciliary neurotrophic 26 factor, basic ﬁbroblast growth factor (bFGF), and other 27 factors rescue photoreceptors and retinal neurons such 28 as retinal ganglion and bipolar cells from apoptosis 29 under distinct conditions (reviewed in Bringmann et al., 30 2009). However, the mechanisms of the neuroprotective 31 action of the factors are not fully understood. It has been 32 proposed that part of the neurotrophic rescue of 33 photoreceptor and retinal neurons is indirect, mediated 34 by the interaction of neurotrophic factors with retinal glial 35 (Mu¨ller) cells that in turn release trophic factors that act 36 directly on photoreceptor and neuronal cells (Wen et al., 37 1995; Wexler et al., 1998; Harada et al., 2000; Wahlin 38 et al., 2000; Garcia and Vecino, 2003). One factor which 39 is proposed to protect photoreceptors indirectly via the 40 activation of Mu¨ller cells is the glial cell line-derived 41 neurotrophic factor (GDNF) (Hauck et al., 2006). GDNF 42 protects retinal neurons from excitotoxicity by inducing 43 upregulation of glutamate transporters in retinal glial 44 cells (Koeberle and Ba¨hr, 2008). GDNF induces the 45 expression of BDNF and bFGF in Mu¨ller cells (Harada 46 et al., 2003). GDNF was also shown to induce 47 expression and secretion of osteopontin (OPN) from 48 murine Mu¨ller cells (Del Rı´o et al., 2011). OPN itself 49 exerts a pro-survival eﬀect in cultured photoreceptor 0306-4522/13 $36.00 Ó 2013 Published by Elsevier Ltd. on behalf of IBRO. http://dx.doi.org/10.1016/j.neuroscience.2013.04.045 * Corresponding author. Address: Department of Ophthalmology and Eye Hospital, University of Leipzig, Liebigstrasse 10-14, D-04103 Leipzig, Germany. Tel: +49-0-341-9721557; fax: +49-0-341- 9721659. E-mail address: bria@medizin.uni-leipzig.de (A. Bringmann). Abbreviations: BAPTA-AM, bis-(o-aminophenoxy)ethane-N,N,N 0 ,N 0 - tetra-acetic acid acetoxymethyl ester; BDNF, brain-derived neurotrophic factor; bFGF, basic ﬁbroblast growth factor; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; GDNF, glial cell line-derived neurotrophic factor; Go¨6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro- 13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; HEPES, hydroxyethyl piperazineethanesulfonic acid; LY341495, (2S)-2-amino- 2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; MRS2179, N 6 -methyl-2 0 -deoxyadenosine-3 0 ,5 0 -bisphosphate; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; OPN, osteopontin; SNAP, S-nitroso-N-acetyl-penicillamine; SU1498, (E)-3-(3,5-diisopro- pyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino-carbonyl]acrylnitrile; U73122, 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione; VEGF, vascular endothelial growth factor. Neuroscience xxx (2013) xxx–xxx Please cite this article in press as: Wahl V et al. Osteopontin inhibits osmotic swelling of retinal glial (Mu¨ller) cells by inducing release of VEGF. Neu- roscience (2013), http://dx.doi.org/10.1016/j.neuroscience.2013.04.045 1 NSC 14575 No. of Pages 14, Model 5G 13 May 2013