The Axon Guidance Molecule Netrin-4 is Expressed by Müller Cells and Contributes to Angiogenesis in the Retina

Retinal glial (Müller) cells are involved in a wide range of developmental mechanisms, including axon guidance and angiogenesis. This study was undertaken to explore whether Netrin-4, an axonal guidance molecule, is expressed by Müller cells and promotes angiogenesis-related activities. Netrin-4 was found through all retinal layers, and its expression was demonstrated in Müller cells, retinal pigment epithelium cells and bovine retinal endothelial cells (BRECs). Co-localization of Netrin-4 with Müller cell-specific molecules [cellular retinaldehyde-binding protein (cRALBP), vimentin] was observed in the ganglion cell layer, nerve fiber layer, and at the outer limiting membrane. Under hypoxic conditions, the release of Netrin-4 from Müller cells was increased, with mRNA levels upregulated in a hypoxia-inducible factor-1-dependent manner and dependent on the concomitantly induced release of vascular endothelial growth factor. These findings were consistent with an intensified immunofluorescence of Netrin-4 labeling in the postischemic retinas after ischemia-reperfusion. Netrin-4 stimulated BRECs to increase phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK)-1/-2, and p38, in a dose-dependent manner. Synthetic inhibitors of the MAP kinases were able to suppress Netrin-4-induced migration and proliferation of BRECs suggesting that both MAP kinases are differentially involved in Netrin-4-induced angiogenesis. Two receptors for Netrins, i.e., deleted in colorectal cancer (DCC) and uncoordinated-5-homolog 1 (Unc5H1), were detected in BRECs. DCC is at least partially required for Netrin-4-induced activation of ERK-1/-2. These data suggest that Müller glial cells contribute to, and may modulate, retinal Netrin-4 levels. This may be a novel pathway of Müller cell-mediated control of retinal angiogenesis, particularly under hypoxic/ischemic conditions when the cells upregulate Netrin-4 expression.