Gene expression patterns in hypoxic and post-hypoxic adult rat retina with special reference to the NMDA receptor and its interactome

Abstract

Purpose: A gene expression analysis of hypoxic rat retina was undertaken to gain a deeper understanding of the possible molecular mechanisms that underlie hypoxia-induced retinal pathologies and identify possible therapeutic targets.

Methods: Rats were made severely hypoxic (6%-7% O(2)) for 3 h. Some rats were sacrificed at this time, and others were allowed to recover for 24 h under normoxic conditions. A focused oligonucleotide microarray of 1,178 genes, qRT-PCR of selected transcripts, and western analysis of hypoxia inducible factor-1alpha (HIF-1alpha) were used to compare retinas from the hypoxic and recovery groups to control animals that were not made hypoxic. SAM analysis was used to identify statistically significant changes in microarray data, and the bioinformatics programs GoMiner, Gene Set Enrichment Analysis (GSEA), and HiMAP were used to identify significant ontological categories and analyze the N-methyl-D-aspartate (NMDA) receptor interactome.

Results: HIF-1alpha protein, but not mRNA, was elevated up to 15-fold during hypoxia, beginning at 0.5 h, the shortest duration examined. Of the total of 1,178 genes examined by microarray, 119 were significantly upregulated following hypoxia. Of these, 86 were still significantly upregulated following recovery. However, 24 genes were significantly downregulated following hypoxia, with 12 still significantly downregulated after recovery. Of the 1035 genes that did not change with hypoxia, the expression of 36 genes was significantly changed after recovery. Ontological analyses showed significant upregulation of a large number of genes in the glutamate receptor family, including 3 of the 5 NMDA subunits. qRT-PCR analysis further corroborated these findings. Genes known to directly interact specifically with the NR1 subunit of the NMDA receptor were identified using HiMAP databases. GSEA analysis revealed that these genes were not affected by either hypoxia or altered after recovery.

Conclusions: The identification of gene expression alterations as a function of hypoxia and recovery from hypoxia is important to understand the molecular mechanisms underlying retinal dysfunction associated with a variety of diseases. Gene changes were identified in hypoxic retina that could be linked to specific networks. Retinas recovering from hypoxia also showed distinct patterns of gene expression that were different from both normoxic control retinas and hypoxic retinas, indicating that hypoxia initiates a complex pattern of gene expression. Diseases of which hypoxia is a component may exhibit the several changes found here. Several potential therapeutic targets have been identified by our approach, including modulation of NMDA receptor expression and signaling, which until now have only been shown to play a role in responding to ischemia.

Figure 1

HIF-1α mRNA and protein expression in rat retina during hypoxia. Rats were exposed to 6%–7% O₂ (hypoxia) for varying durations up to 6 h. A: HIF-1α mRNA abundance, normalized to acidic ribosomal protein P0 mRNA, was calculated by qRT–PCR. Data represent mean (±SD) of 3 independent experiments. No significant differences (n/s; p>0.05) were observed between control and hypoxic HIF-1α mRNA expression levels at any time point. B: Quantitation of HIF-1α protein expression in nuclear protein extracts by western analysis. Data represent mean (±SD) of at least 3 independent experiments and were normalized to β-actin expression levels. HIF-1α protein levels were higher in all hypoxic samples relative to controls (ANOVA followed by post-hoc test; p<0.05) but were not different between any two hypoxic time points. The inset depicts the western result from a representative time course experiment, as well as the return of HIF-1α protein levels to control levels 24 h after exposure to 3 h hypoxia.

Figure 2

qRT–PCR analysis of known hypoxia-associated mRNAs. For each mRNA, transcript abundance, normalized to acidic ribosomal protein P0, was calculated by qRT–PCR. Values were then further normalized to the control level of each transcript. Data represent mean (±SD) of 5 independent experiments. A: The expression of VEGF and VEGF receptors Flk-1 and Flt-1 mRNA was measured in the rat retina immediately following 3 h of hypoxia and after 24 h of recovery in air. VEGF and Flk-1 were significantly higher in hypoxic samples as compared to control. Flt-1 tended to increase during hypoxia, but the difference from control was not significant. B: The expression of erythropoietin (EPO) and erythropoietin receptor (EPOR) mRNA was measured in the rat retina immediately following 3 h of hypoxia and after 24 h of recovery in air. EPO mRNA levels were significantly higher in hypoxic samples as compared to control. A significant difference was also observed for the EPOR control and hypoxia mRNA levels between hypoxic and recovery samples. The asterisks indicate significance levels assessed via ANOVA followed by post-hoc tests: * - p<0.05; ** - p<0.01; *** - p<0.001.

Figure 3

Summary of microarray data. Shown is an examination of the microarray databased on the number of genes upregulated, downregulated, or not changed. These gene changes were categorized into major functional groups. Upward arrows indicate numbers of genes that were significantly upregulated; down arrows indicate those that were downregulated; and horizontal bars represent those that were not altered. The recovery column shows how the genes in each category were affected during recovery. Ellipses indicate that these were not the only categories affected.

Figure 4

NMDA receptor subunit mRNA expression in the rat retina following hypoxia and 24-h recovery in air. For each mRNA, transcript abundance, normalized to acidic ribosomal protein P0, was measured by qRT–PCR. Values were then further normalized to the control level of each transcript. Data represent mean (±SD) of 5 independent experiments. NMDAR1 mRNA levels were higher in hypoxic samples compared to control and in recovery samples compared to control. NMDAR2C mRNA levels were higher in hypoxia samples compared to the control samples. NMDAR2D mRNA levels were higher in hypoxia samples compared to the control samples. The asterisks indicate significance levels assessed via ANOVA followed by post-hoc tests: * - p<0.05; ** - p<0.01; *** - p<0.001.

Figure 5

NMDAR1 Interactome. A: The molecular network of direct physical, transcriptional, and enzymatic interactions with NMDAR1 (GRIN1), referred to as the NMDAR1 “interactome,” was derived from the HiMAP database. Using evidence from literature-confirmed interactions within the Human Protein Reference Database and predicted interactions generated by Bayesian Analysis, greater than >40,000 molecular relationships were queried. B: Subsets of the NMDA Interactome were created based on ligand specificity. In these subsets, the 25 genes in the NMDA interactome (above) were organized into progressively smaller subsets and further analyzed by GSEA.