V.J. Dudley

Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network.

Müller cells have recently been found to produce select angiogenic substances. In choosing a more comprehensive approach, we wanted to study the genomic response of Müller cells to hypoxia to identify novel angiogenic genes. An established Müller cell line (rMC-1) was exposed to standard or hypoxic conditions. We analyzed gene expression with three independent microarrays and determined differential expression levels compared to normoxia. Selected genes were confirmed by real-time PCR (RTPCR). Subcellular localization of proteins was examined by immunocytochemistry. A network-based pathway analysis was performed to investigate how those genes may contribute to angiogenesis. We found 19,004 of 28,000 known rat genes expressed in Müller cells. 211 genes were upregulated by hypoxia 1.5 to 14.9-fold (p<0.001, FDR<or=5%) and 220 genes were downregulated 1.5-4.6-fold (p<0.001, FDR<or=5%). Unexpectedly, expression patterns of cell proliferation, differentiation and organogenesis were increased besides predictable declines in cell function. Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization and macular degeneration (VLDLR) and in ocular mesodermal development and differentiation (TRIB3), respectively. VLDLR was upregulated 3.1-fold (p=0.001, RTPCR 3.0-fold) and TRIB3 2.8-fold (p=0.025, RTPCR 5.1-fold). VEGF was increased 3.1-fold (p=0.003, RTPCR 8.3-fold) and apelin, a novel factor of retinal angiogenesis, 5.6-fold (p=0.006, RTPCR 8.7-fold). A network of interacting angiogenic genes was identified in silico that included VLDLR as a surface receptor. VLDLR protein localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. We conclude that hypoxia triggers an angiogenic network response in Müller cells with VLDLR as a novel node and gene expression patterns of proliferation, differentiation and organogenesis.

Characterization of Green Fluorescent Protein-Expressing Retinal Cells in CD 44-Transgenic Mice

Sensory information in the retina is transferred from rod and cone photoreceptors to higher visual centers via numerous parallel circuits that sample the photoreceptor mosaic independently. Each circuit consists of a unique combination of ganglion cell, bipolar and amacrine cell types. The morphology and physiological responses of many amacrine cells have been characterized. However, the synaptic connections and retinal circuits in which they participate are only rarely understood. A major problem that has prevented fuller characterization of retinal circuitry is the need for specific cellular markers for the more than 50 inner retinal cell types. One potential strategy for labeling cells is to use transgenic expression of a reporter gene in a specific cell type. In a recent study of cluster of differentiation 44 (CD44)-enhanced green fluorescent protein (EGFP) transgenic mice, we observed that the green fluorescent protein (GFP) was expressed in a population of amacrine and ganglion cells in the inner nuclear layer (INL) and the GCL. To characterize the morphology of the GFP-labeled cells, whole mount preparations of the retina were used for targeted iontophoretic injections of Lucifer Yellow and Neurobiotin. Furthermore, immunocytochemistry was used to characterize the antigenic properties of the cells. We found that many GFP-expressing cells were GABAergic and also expressed calretinin. In addition to the somatic staining, there was a strong GFP(+)-band located about 50-60% depth in the inner plexiform layer (IPL). Double labeling with an antibody to choline acetyltransferase (ChAT) revealed that the GFP-band was located at strata 3 inner retina. The best-labeled GFP-expressing cell type in the INL was a wide-field amacrine cell that ramified in stratum 3. The GFP-expressing cells in the GCL resemble the type B1, or possibly A2 ganglion cells. The CD44-EGFP mice should provide a valuable resource for electrophysiological and connectivity studies of amacrine cells in the mouse retina.