Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection
© Thiersch et al; 2008
Received: 22 October 2007
Accepted: 08 February 2008
Published: 08 February 2008
Retinal degeneration is a main cause of blindness in humans. Neuroprotective therapies may be used to rescue retinal cells and preserve vision. Hypoxic preconditioning stabilizes the transcription factor HIF-1α in the retina and strongly protects photoreceptors in an animal model of light-induced retinal degeneration. To address the molecular mechanisms of the protection, we analyzed the transcriptome of the hypoxic retina using microarrays and real-time PCR.
Hypoxic exposure induced a marked alteration in the retinal transcriptome with significantly different expression levels of 431 genes immediately after hypoxic exposure. The normal expression profile was restored within 16 hours of reoxygenation. Among the differentially regulated genes, several candidates for neuroprotection were identified like metallothionein-1 and -2, the HIF-1 target gene adrenomedullin and the gene encoding the antioxidative and cytoprotective enzyme paraoxonase 1 which was previously not known to be a hypoxia responsive gene in the retina. The strongly upregulated cyclin dependent kinase inhibitor p21 was excluded from being essential for neuroprotection.
Our data suggest that neuroprotection after hypoxic preconditioning is the result of the differential expression of a multitude of genes which may act in concert to protect visual cells against a toxic insult.
Retinal blinding diseases like retinitis pigmentosa (RP) and age related macular degeneration (AMD) are characterized by a progressive retinal degeneration which involves the apoptotic loss of photoreceptor cells. Although significant progress in the understanding of the molecular mechanisms leading to AMD and RP has been made in recent years, efficient treatments to successfully prevent loss of vision are still not available.
Neuroprotection is a strategy to preserve retinal function. It aims at the interference with regulatory mechanisms of cell death to protect photoreceptor cells. To successfully target these mechanisms it is necessary to understand the molecular signalling networks in the degenerating retina. Since neither an extrinsic (activation of caspases via death receptors) nor an intrinsic death pathway (release of cytochrome c from mitochondria) seems to be activated during retinal degeneration , mechanisms of photoreceptor cell death are still poorly understood. Several models of inherited  and induced  retinal degeneration are used to study the molecular events of photoreceptor apoptosis. Inherited models mostly show a slow progression of retinal degeneration resulting in constant but low levels of apoptosis. Models of induced retinal degeneration, like the light damage model , are easy to handle and the synchronized response to the apoptotic stimulus may raise apoptotic factors above detection threshold allowing their detailed investigation.
Various preconditioning protocols are used as a strategy to protect tissues from degenerative processes. Especially ischemic and hypoxic preconditioning successfully reduced the severity of induced or inherited degeneration in tissues like brain [4, 5] heart [6, 7] and the retina [8–12]. Hypoxia describes a state of low oxygen. It appears pathologically during several diseases like cancer, stroke or heart infarction  but also physiologically during development in many tissues . In the adult retina, increased oxygen consumption during night time leads to borderline hypoxic conditions . To cope with the reduced oxygen availability cells differentially regulate genes including factors involved in an anti-apoptotic response [16, 17]. A key regulator of the tissue response to hypoxia is the transcription factor hypoxia inducible factor 1 (HIF-1), a heterodimeric protein consisting of the constitutively and stably expressed hypoxia inducible factor 1β (HIF-1β) and oxygen regulated subunit hypoxia inducible factor 1α (HIF-1α). During hypoxia HIF-1α is stabilized, enters the nucleus, recruits HIF-1β and regulates the expression of target genes involved in different pathways like apoptosis, metabolism or angiogenesis .
Hypoxic preconditioning was shown to stabilize HIF-1α in the retina [9, 12]. Stabilization of this transcription factor induces the expression of target genes with neuroprotective properties like vascular endothelial growth factor (Vegf) and erythropoietin (Epo) suggesting a link between HIF-1 driven gene expression and neuroprotection [9, 12]. Exogenous application of Epo not only protects retinal ganglion cells in a model of ischemia-reperfusion injury  but also photoreceptors in the model of light induced retinal degeneration [20, 21]. However, protection of visual cells from light damage was weaker than after hypoxic preconditioning suggesting that factors in addition to Epo contribute to retinal protection by hypoxia. The identification of these factors is essential for the development of efficient neuroprotective strategies focused on the prevention of retinal degeneration.
We used whole genome microarrays and real-time PCR to detect expression of differentially regulated genes after hypoxic preconditioning in adult mouse retinas. The analysis of the hypoxic transcriptome characterized the response of the retina to low oxygen levels. Cyclin-dependent kinase inhibitor 1a (p21) was among the most strongly induced genes and occupied a central position in a differentially regulated gene network affecting cellular growth and proliferation. Using p21 gene knockout animals, we analyzed the impact of this gene on retinal neuroprotection in the model of light induced retinal degeneration.
Time frame of neuroprotection after hypoxic preconditioning
Exposure after 8 h of reoxygenation resulted in the appearance of some apoptotic photoreceptor nuclei. 12 h of reoxygenation further reduced the protection against light damage as evidenced by the appearance of many nuclei with condensed chromatin and an almost complete disintegration of rod inner (RIS) and rod outer segments (ROS). Retinas of mice illuminated 16 hours after hypoxic preconditioning were as susceptible to light damage as retinas of normoxic control mice (Fig. 1A).
The retinal response to hypoxic preconditioning
It is well known that hypoxia alters the gene expression profile in a given tissue  in an attempt to cope with the unfavourable condition. One of the major factors regulating this response is the transcription factor HIF-1, which is activated in the hypoxic retina (Fig. 1B) . Similarly, the pro-survival transcription factor Stat3 , which has been reported to be induced in several hypoxic tissues , was phosphorylated and thus activated in the hypoxic retina (Fig. 1B). The different levels of activation (shown are examples of two mice) point to a certain variability in the response to hypoxia between individual mice. Nevertheless, the activation of these transcription factors suggests a differential regulation of a multitude of potentially neuroprotective genes in the retina by hypoxic preconditioning. Based on the time frame of neuroprotection (Fig. 1A), we analyzed the gene expression pattern in the retina at 0 h, 2 h, 4 h, and at 16 h after hypoxia (see Methods).
Hierarchical clustering of gene chip data showed strong similarities of the three replica-chips of a respective time point after hypoxia [see additional file 1]. Such clustering was not observed in normoxic samples suggesting that hypoxia induced a strong and specific response in the retina. This hypoxic response quickly vanished and at 4 h after hypoxic preconditioning the retinal transcriptome was similar to normoxic controls.
Prominently regulated genes
Top 50 differentially regulated genes immediately after hypoxic preconditioning
oocyte specific homeobox 6
RIKEN cDNA 8430408G22 gene
cyclin-dependent kinase inhibitor 1A (p21)
RIKEN cDNA 4833408G04 gene
retinal G protein coupled receptor
apolipoprotein L domain containing 1
histone 2, H2aa2
phosphatidylinositol transfer protein, membrane-associated 2
retinol dehydrogenase 5
X transporter protein 3 similar 1 gene
sterile alpha motif domain containing 7
DNA segment, Chr 7, Brigham & Women's Genetics 0826 expressed
arrestin domain containing 2
RIKEN cDNA A330049M08 gene
solute carrier family 19 (thiamine transporter), member 2
H3 histone, family 3B
solute carrier family 7 (cationic amino acid transporter, y+ system), member 1
RIKEN cDNA 6820408C15 gene (6820408C15Rik), mRNA
expressed sequence C80120
RNA binding motif protein 3
similar to retinoic acid, EGF, and NGF upregulated
budding uninhibited by benzimidazoles 1 homolog, beta (S. cerevisiae)
budding uninhibited by benzimidazoles 1 homolog, beta (S. cerevisiae)
RNA binding motif protein 12B
transformed mouse 3T3 cell double minute 1
WD repeat domain 39
Proline rich 14
IBR domain containing 2 (Ibrdc2), mRNA
histone 2, H3c1
death inducer-obliterator 1
zinc finger protein 354B
myocyte enhancer factor 2C
Nudcd1 NudC domain containing 1 3.19 3.84E-04
RNA binding motif protein 4
RIKEN cDNA 1700129I04 gene
IWS1 homolog (S. cerevisiae)
Most interestingly, expression of several genes with a potential capacity to protect photoreceptors against light-induced cell death was upregulated (Table 1). To this group belong p21 and Bcl2l10. In addition, genes belonging to oxidative stress response pathways or lipid metabolism like metallothioneins (Mt1 and Mt2), transthyretin (Ttr) and paraoxonase1 (Pon1) were induced as was the expression of adrenomedullin (Adm) which was previously shown to respond to hypoxic conditions  and to have neuroprotective properties .
Some pro-apoptotic genes were downregulated after hypoxic preconditioning (Table 1) like Mef2c, a transcription factor involved in neuronal loss in Parkinson's disease  and in the regulation of apoptosis in macrophages . The genes belonging to the Rbm family of genes are also of high interest since some members of this family are known to have an impact on apoptosis regulation .
Verification of Affymetrix microarray data
Since many of the genes which were not confirmed by real-time PCR to be differentially regulated had a low fold-change on the microarrays, we used only genes with a minimal fold change of 2 for the investigations of functionally related groups (see below). Figure 3 shows the real-time PCR data of 4 selected genes with potential neuroprotective properties (p21, Pon1, Ttr, Adm). Consistent with the microarray results, these genes were strongly upregulated immediately after hypoxia and returned quickly to normal expression levels during reoxygenation.
Biological functional groups and pathway analysis
Differentially regulated genes with possible impact on cell survival and neuroprotection; detected and functionally clustered by DAVID.
apoptosis (p ≥ 0.032)
cyclin-dependent kinase inhibitor 1a (p21)
zinc finger and btb domain containing 16
vascular endothelial growth factor a
growth arrest and dna-damage-inducible 45 gamma
calcium/calmodulin-dependent protein kinase id
nerve growth factor receptor (TNFR superfamily, member 16)
cell cycle (p ≥ 0.013)
cyclin-dependent kinase inhibitor 1A (P21)
nucleolar and spindle associated protein 1
tousled-like kinase 2 (Arabidopsis)
B-cell translocation gene 3
retroviral integration site 2
protein tyrosine phosphatase 4a1
growth arrest and DNA-damage-inducible 45 gamma
vascular endothelial growth factor A
gene model 877 (NCBI)
synaptonemal complex protein 3
negative regulation of transcription (p ≥ 0.031)
zinc finger and btb domain containing 16
Fatty acid binding protein 4, adipocyte (Fabp4), mRNA
inhibitor of DNA binding 1
nuclear transcription factor, X-box binding 1
Max interacting protein 1
regulation of transcription (p ≥ 0.09)
ribosomal protein S6 kinase, polypeptide 5
signal transducer and activator of transcription 1
Trf (TATA binding protein-related factor)-proximal protein homolog (Drosophila)
WD repeat domain 39
zinc finger protein 354B
myocyte enhancer factor 2C
death inducer-obliterator 1
thyroid hormone receptor alpha
small nuclear RNA activating complex, polypeptide 3
Proline rich 14
Real-Time PCR results of 16 genes belonging to the p21 pathway compared to the fold change (FC) detected by Affymetrix microarrays.
FC Affymetrix Chip
The influence of p21 on retinal neuroprotection in the model of light induced degeneration
Hypoxic preconditioning is strongly neuroprotective and prevents photoreceptor apoptosis after exposure to high levels of visible light . The transcription factors HIF-1 and Stat3 are activated. This suggests differential regulation of the expression of various target genes which was confirmed by the detection of 431 differentially regulated genes immediately after hypoxia. More than 50% of these genes showed at least a 2-fold difference in the expression level as compared to normoxic retinas. Among those were also Rgr and Lrat, two genes highly expressed in the retinal pigment epithelium. Genes normally not or only barely expressed in the neuronal retina may easily reach a high-fold induction when the tissue is contaminated with neighbouring cells expressing the respective gene at high levels. Low levels of oxygen during hypoxic preconditioning may have altered the physical interaction properties between neuronal retina and retinal pigment epithelium (RPE) leading to an increased contamination rate of the retina by cells of the RPE during tissue isolation. Thus, fold inductions have to be interpreted cautiously.
Reoxygenation caused the rapid return to a normal gene expression pattern. This is in line with a model of hypoxic preconditioning in brain where it was shown that differential gene regulation was low between 12 and 18 hours of reoxygenation . In models of ischemic preconditioning (IPC), however, differential gene expression is observed immediately after the stimulus until up to 7 days after preconditioning [34–36]. This goes together with a long lasting neuroprotective effect of IPC observed in the retina  and in brain  suggesting that mechanisms of IPC may differ from those of acute hypoxic preconditioning. An extended neuroprotection by hypoxic preconditioning may be achieved by the repetitive exposure to low oxygen .
Similarities of hypoxic preconditioning and IPC
Although mechanisms of hypoxic preconditioning and IPC may differ, few genes were found to be differentially regulated in both types of preconditioning protocol. Among those are metallothionein 2 (Mt2), C/ebpd (an apoptosis-related transcription factor) and p21 [35, 38]. The identification of these genes makes them strong candidates for playing a role in general retinal neuroprotection.
An involvement of p21 was directly tested using the respective knockout animal. As a HIF-1 target gene , p21 was not only very strongly regulated but was also at the center of a highly regulated gene network (Fig. 4). Although p21 can be pro-apoptotic  and can trigger non-apoptotic cell death , it is also known to have antiapoptotic properties . However, the test of p21 knockout mice in the model of light induced degeneration revealed no significant impact of p21 on neuroprotection against light damage. Despite the lack of p21, all other genes of the p21 pathway (except for Sema3c) showed the same response to hypoxic preconditioning as in wild type mice. This raises the possibility that other genes of the p21 network might influence retinal neuroprotection. Specific candidates are Timp3 which has been reported to be a promoter of apoptosis through the inhibition of metalloproteinases  and Egf which has proven anti-apoptotic properties .
Mt2, as a gene also detected in both preconditioning schemes, may play an important role as a scavenger of free radicals . It is interesting to note that metallothioneins are also induced after ischemic preconditioning of the rat spinal cord  and that they have been reported to be neuro- and cardioprotective, respectively, in various degenerative models [46, 47]. In addition, metallothioneins have been shown to be induced in light-damaged retinas  and to protect retinas from oxidative stress caused by the glutamate analogue NMDA . Further experiments are clearly needed to evaluate the impact of this protein in retinal neuroprotection.
The low similarity of the transcriptome after IPC and hypoxic preconditioning may be surprising but might be based on the different nature of the preconditioning protocols. Whereas IPC normally uses a very short (minutes) ischemic stimulus followed by reperfusion, our protocol of hypoxic preconditioning uses exposure to 6 hours of low oxygen concentrations followed by the immediate analysis. The different length of exposure to low oxygen and the interrupted supply of nutrients in one (IPC) but not the other protocol might essentially explain the differences in the gene expression patterns.
Strong candidate genes for neuroprotection: Adm, Pon1
Adrenomedullin (Adm) is a multifunctional protein involved in angiogenesis, cancer promotion, host defence and neuroprotection . Elevated levels of Adm were found in plasma of patients suffering from retinitis pigmentosa . Previous reports identified Adm as a target gene of HIF-1α [52, 53] linking it to a possible HIF – mediated protection mechanism.
Interestingly, some genes which so far were not described in the context of hypoxia, like paraoxonase 1 (Pon1), were also highly induced. Pon1 is a high-density lipoprotein (HDL) associated enzyme which plays a major role in the prevention of lipid peroxidation [54, 55]. Since retinal degeneration involves oxidative stress and inhibition of lipid peroxidation protects against light damage  Pon1 may have an important role in retinal protection after hypoxic preconditioning. Recently, Pon1 levels were found to be reduced in serum of AMD patients whereas a marker for oxidative stress was elevated . This may suggest that elevated levels of Pon1 in our model might reduce oxidative stress and prevent photoreceptor degeneration. Interestingly, C57Bl/6 mice which have a reduced sensitivity to light damage show a higher basal expression of Pon1 than light sensitive strains (data not shown). If the anti-oxidative enzyme Paraoxonase 1 was involved in the protection of the retina against oxidative damage, the different basal expression levels of Pon1 might contribute to the different light damage susceptibilities of various mouse strains.
Additional genes with potential neuroprotective function
Bcl2-like 10 (Bcl2l10) is a anti-apoptotic member of the Bcl2 family  acting to suppress cell death by preventing cytochrome c release, casp-3 activation and mitochondrial membrane collapse . However, retinal degeneration induced by acute light exposure may not depend on cytochrome c release or caspase activation . Therefore, upregulation of Bcl2l10 might not be responsible for photoreceptor protection by hypoxic preconditioning.
Induction of the HIF-1 target gene Vegfa is an attempt to increase tissue oxygen levels by improving blood circulation through the formation of new vessels . In the retina Vegfa is also recognized as a pro-survival factor protecting retinal neurons against ischemic injury . However, Vegfa is discussed to have also pro-apoptotic properties  and its potential role in the preconditioning scheme is unclear. Ptdsr encodes a posphatidylserine receptor involved in the clearance of apoptotic cells  and it has been shown that lack of Ptdsr activity can increase tissue damage through the stimulation of apoptosis in cells neighbouring apoptotic cells . Ptdsr is also involved in the elimination of apoptotic debris of dying photoreceptors by macrophage-mediated phagocytosis which is important for the maintenance of retinal tissue integrity .
Downregulated genes with a possible impact on cell death included Mef2c and genes of the Rbm family of protein. Mef2c triggers apoptosis in macrophages  and may be involved in dopaminergic neuron death in Parkinson's disease . Because macrophages seem to play an important role in light induced apoptosis [67, 68] a potential influence on neuroprotection may be possible but needs further investigation. This is also true for the identified members of the Rbm family. Although these proteins have been implicated in the modulation of apoptosis , and downregulation of Rbm3 has been specifically connected to the regulation of cell cycle progression  and the inhibition of apoptosis , their role is still controversial.
Since hypoxia can either lead to adaptation and protection  or to apoptosis  it may not be surprising that we identified several genes which may rather be involved in promoting apoptosis than in its inhibition. Neuroprotection by hypoxic preconditioning may thus depend on a balance between numerous anti- and proapoptotic factors. The loss of individual proteins like p21 may not be sufficient to shift the balance towards apoptosis. Likewise, it might require several different antiapoptotic factors to fully protect the retina. Full neuroprotection may only be achieved by controlling the central regulators of the hypoxic response like the transcription factors HIF and/or STAT3.
Animals, hypoxic preconditioning and light damage
Animals were treated in accordance with the regulations of the Veterinary Authority of Zurich and with the statement of 'The Association for Research in Vision and Ophthalmology' for the use of animals in research. BALB/c mice were purchased from Harlan (The Netherlands) and p21-/- mice on a mixed Bl/6;129S2 background were obtained from Jackson Laboratory (Bar Harbor, USA). All mice were homozygous for the light sensitive Rpe65450Leu variant . Hypoxic preconditioning (6% O2 for 6 hours) was performed as described previously . Reoxygenation was allowed in darkness for 4 h, 8 h, 12 h and 16 h in normal room air. After reoxygenation BALB/c mice were exposed to 5'000 lux of white fluorescent light for 1 h and analyzed at time points as indicated.
Pupils of p21-/- animals (pigmented) were dilated in dim red light using 1% Cyclogyl (Alcon, Cham, Switzerland) and 5% Phenylephrine (Ciba Vision, Niederwangen, Switzerland) 45 minutes prior to illumination. Light dose (13'000 lux) and exposure duration (2 h) was adjusted according to the decreased light damage susceptibility of this mouse strain. After light exposure animals remained in darkness until analyzed or at the most for 36 h.
For morphology mice were sacrificed 36 h or 10 days after light exposure and eyes were enucleated and processed as previously described .
RNA isolation and Affymetrix microarrays
Retinas were isolated immediately, 2 h, 4 h and 16 h after hypoxic preconditioning, frozen in liquid nitrogen and stored at -70°C. Normoxic controls were treated in parallel and collected at the same time points. For Affymetrix microarrays 3 retinas of 3 different mice were pooled. This procedure was repeated 3 times to generate independent biological triplicates. RNA was extracted using the RNeasy isolation kit (Qiagen, Hilden, Germany), including a DNase treatment to digest residual genomic DNA. RNA was processed according to standard procedures and hybridized to Affymetrix GeneChip® Mouse Genome 430 2.0 microarrays. The 3 experimental replicates were hybridized independently resulting in three microarray replicates per condition. In total, 24 Affymetrix gene chips were hybridized with RNA from 72 retinas of 72 mice.
Quality control (QC) and Affymetrix microarray analysis
To analyze the quality of the results after gene chip hybridization we employed RReporterGenerator  combining Affymetrix-style QC, RMA and residual QC. The complete report is available in additional file 1.
Affymetrix raw gene expression data were summarized and normalized using the GCRMA procedure . The data were filtered in order to remove probe-sets with constant low-level expression. Probe-sets were removed which showed replicate means for a given time-point for both treatments below the threshold separating the two peaks of the bimodal distribution of signal-intensity values. This procedure was performed independently for each of the differential testing procedures. The filtered data-sets were subsequently subjected to t-tests with multiple testing correction and control of the false discovery rate (FDR) using OCplus package  available under Bioconductor . By comparing the plotted number of differentially expressed genes at various FDR levels, corresponding threshold values for the maximum acceptable FDR were chosen with the aim of keeping homogenous groups with similar FDR together.
To group differentially regulated genes according to their biological function the Affymetrix IDs were imported into the Database for Annotation, Visualization and Integrated Discovery (DAVID) from the National Institute of Allergy and Infectious Diseases (NIAID), NIH [31, 79] and into Ingenuity Pathway Analyses from Ingenuity Systems .
cDNA was prepared from equal amounts of total retinal RNA, using oligo(dT) primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). 10 ng of cDNA was amplified in a LightCycler 480 instrument (Roche Diagnostics AG, Rotkreuz, Switzerland) using LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics AG) and appropriate primer pairs [see additional file 6]. mRNA levels were normalized to β-actin and relative values were calculated using a respective calibrator.
Retinas were homogenized in 0.1 M Tris/HCl (pH 8.0) by sonification at 4°C. The protein content was determined using a Bradford assay (Bio-Rad, Munich, Germany). Protein extracts were mixed with SDS sample buffer and incubated for 10 min at 90°C. Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. After blocking with 5% non-fat dry milk (Bio-Rad, Munich, Germany) in TBST (Tris/HCl 10 mM, pH 8; 150 mM NaCl; 0.05% Tween-20) membranes were incubated with primary antibodies at 4°C over night. Primary antibodies used were: rabbit anti-HIF-1α (Novus Biologicals NB 100–479; 1:1000), rabbit anti-phospho-STAT3 (Cell Signalling; 1:500), rabbit anti-STAT3 (Cell Signalling 1:1000) and goat anti-β-actin (Santa Cruz; 1:1000). After incubation with horseradish peroxidase labelled secondary antibodies for 1 h at room temperature the protein bands were visualized by the application of a chemiluminescent substrate (PerkinElmer, Boston, USA) and exposure to a Super RX film (Fujifilm, Dielsdorf, Switzerland).
The authors thank Coni Imsand, Gaby Hoegger, Hedwig Wariwoda, Philipp Huber and the Plate-Forme BioInformatique de Strasbourg for excellent technical assistance. This work was supported by the Swiss National Science Foundation (SNF, grant 3100A0-105793), the Fritz-Tobler-Foundation, the Centre of Integrative Human Physiology (CIHP) of the University of Zurich and the European Union (Evi-GenoRet, LSHG-CT-512036). WR was supported by the European Retinal Research Training Network 'RETNET' (MRTN-CT-2003-504003), CNRS, INSERM and Université Louis Pasteur de Strasbourg. Prof. Klara Landau is acknowledged for constant support.
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