- Research article
- Open Access
Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection
BMC Genomics volume 9, Article number: 73 (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
In previous experiments we observed an almost complete protection of photoreceptors against light induced degeneration when exposed at 4 hours after hypoxic preconditioning. Protection, however, was lost after prolonged reoxygenation of 16 hours suggesting a rather short-lived neuroprotective effect of hypoxic preconditioning . To analyze the time frame of hypoxia-induced neuroprotection in more detail, we allowed preconditioned mice to reoxygenate for 4 h, 8 h, 12 h and 16 h, respectively, before they were exposed to high levels of white light. As expected, retinal morphology (Fig. 1A) was almost completely preserved in mice illuminated after a reoxygenation period of 4 h. Only slight disturbances and vesiculations in the rod outer segments but no apoptotic nuclei with condensed chromatin were observed.
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.
After normalization of the data by GCRMA algorithm to minimize the theoretical appearance of false positive signals we detected 431 differentially regulated genes immediately after hypoxia, 227 of which had a fold change above 2 (0 h, Fig. 2). The number of regulated genes decreased gradually until 16 h after hypoxia when just 3 genes showed altered expression levels (Fig. 2). The application of our statistical filter for a maximal acceptable FDR suggested that there are (statistically estimated) 25, 23, 6 and 1 false positive genes in the analyzed list of differentially regulated genes at 0 h, 2 h, 4 h and 16 h, respectively.
Prominently regulated genes
The genes with the strongest regulation after hypoxic exposure are listed in Table 1 [for complete gene lists see additional file 2]. The most significantly upregulated gene (>118-fold) was found to be Obox6, a homeobox-containing, putative transcriptional activator of unknown function mainly expressed in oocytes . The gene with the strongest downregulation was the cold-inducible RNA binding motif protein 3 (Rbm3) with a 19-fold reduced expression immediately after hypoxia (Table 1).
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
The expression of a total of 32 genes identified by the microarrays was tested by realtime PCR. Differential expression in response to hypoxia was verified for 21 (66%) genes (Fig. 3 and additional file 3). Quality control analysis showed that the integrities of both retinal RNA and chip surfaces were high [QC analysis, see additional file 1] and are thus unlikely to be responsible for the somewhat low confirmation frequency.
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
The bioinformatics resource DAVID was used to study the impact of differentially expressed genes on known biological processes . We obtained 39 functional groups for upregulated genes and 8 groups for downregulated genes immediately after hypoxia (H0). Significant functional groups included 'apoptosis', 'cell cycle' or 'negative regulation of transcription' (note that some genes appear in more than one group) were found in the list of upregulated genes (Table 2). Lists of all functional related groups can be found in additional file 4. Gene signalling networks affected by hypoxic preconditioning were identified using Ingenuity Pathway analysis and the complete lists of regulated genes (Fig 4 and see additional file 5). The centre of the strongest affected pathway was occupied by p21, one of the most strongly upregulated genes by hypoxia (Fig. 4). All genes belonging to this pathway were differentially regulated. 70% of the genes were induced indicating that the pathway was activated rather than repressed. All genes of this pathway, which showed at least a two-fold differential regulation on the chip, were tested by real-time PCR. Ten of the 16 tested genes were confirmed to be regulated by hypoxia (Table 3). This suggested that the p21-pathway was indeed strongly affected by the hypoxic preconditioning protocol. Since it is known that p21 not only inhibits cell cycle but can also repress apoptosis , it was considered as a strong candidate for the involvement in neuroprotection by hypoxic preconditioning.
The influence of p21 on retinal neuroprotection in the model of light induced degeneration
The hypothesis that p21 is important for neuroprotection in the retina after hypoxic preconditioning was directly tested using p21 knockout animals. Both, normoxic and hypoxic preconditioned p21-/- mice were exposed to high intensity visible light for 2 hours and retinal morphology was analyzed 10 days thereafter (Fig. 5). As expected, normoxic control p21-/- mice showed strong damage after light exposure with the loss of all photoreceptors in the central retina. If p21 was involved in neuroprotection after hypoxic exposure, preconditioned p21-/- mice should show an increased susceptibility to light damage as compared to wild type mice. However, photoreceptors of the p21 knockout mice were completely protected after preconditioning (Fig. 5). The quantification of cell death by biochemical assays (data not shown) supported our conclusion that p21 does not contribute significantly to the neuroprotective effect observed after hypoxic preconditioning. Furthermore, most genes identified by Ingenuity Pathway analysis as being part of the p21 signalling network were similarly regulated in the presence or absence of functional p21. The only exception was Semaphorin 3c (Sema3c), which showed no hypoxic upregulation in the absence of p21 (data not shown).
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).
Doonan F, Donovan M, Cotter TG: Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration. J Neurosci. 2003, 23 (13): 5723-5731.
Dalke C, Graw J: Mouse mutants as models for congenital retinal disorders. Exp Eye Res. 2005, 81 (5): 503-512. 10.1016/j.exer.2005.06.004.
Wenzel A, Grimm C, Samardzija M, Reme CE: Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration. Prog Retin Eye Res. 2005, 24 (2): 275-306. 10.1016/j.preteyeres.2004.08.002.
Emerson MR, Nelson SR, Samson FE, Pazdernik TL: A global hypoxia preconditioning model: neuroprotection against seizure-induced specific gravity changes (edema) and brain damage in rats. Brain Res Brain Res Protoc. 1999, 4 (3): 360-366. 10.1016/S1385-299X(99)00041-0.
Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, Kamada T: 'Ischemic tolerance' phenomenon found in the brain. Brain Res. 1990, 528 (1): 21-24. 10.1016/0006-8993(90)90189-I.
Cai Z, Manalo DJ, Wei G, Rodriguez ER, Fox-Talbot K, Lu H, Zweier JL, Semenza GL: Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation. 2003, 108 (1): 79-85. 10.1161/01.CIR.0000078635.89229.8A.
Dong JW, Zhu HF, Zhu WZ, Ding HL, Ma TM, Zhou ZN: Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res. 2003, 13 (5): 385-391. 10.1038/sj.cr.7290184.
Gidday JM, Fitzgibbons JC, Shah AR, Park TS: Neuroprotection from ischemic brain injury by hypoxic preconditioning in the neonatal rat. Neurosci Lett. 1994, 168 (1–2): 221-224. 10.1016/0304-3940(94)90455-3.
Grimm C, Wenzel A, Groszer M, Mayser H, Seeliger M, Samardzija M, Bauer C, Gassmann M, Reme CE: HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med. 2002, 8 (7): 718-724. 10.1038/nm723.
Roth S, Li B, Rosenbaum PS, Gupta H, Goldstein IM, Maxwell KM, Gidday JM: Preconditioning provides complete protection against retinal ischemic injury in rats. Invest Ophthalmol Vis Sci. 1998, 39 (5): 777-785.
Zhang C, Rosenbaum DM, Shaikh AR, Li Q, Rosenbaum PS, Pelham DJ, Roth S: Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Invest Ophthalmol Vis Sci. 2002, 43 (9): 3059-3066.
Zhu Y, Zhang Y, Ojwang BA, Brantley MA, Gidday JM: Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1. Invest Ophthalmol Vis Sci. 2007, 48 (4): 1735-1743. 10.1167/iovs.06-1037.
Michiels C: Physiological and pathological responses to hypoxia. Am J Pathol. 2004, 164 (6): 1875-1882.
Fisher SA, Burggren WW: Role of Hypoxia in the Evolution and Development of the Cardiovascular System. Antioxid Redox Signal. 2007
Cringle SJ, Yu DY, Yu PK, Su EN: Intraretinal oxygen consumption in the rat in vivo. Invest Ophthalmol Vis Sci. 2002, 43 (6): 1922-1927.
Semenza GL: HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol. 2000, 88 (4): 1474-1480.
Fruehauf JP, Meyskens FL: Reactive oxygen species: a breath of life or death?. Clin Cancer Res. 2007, 13 (3): 789-794. 10.1158/1078-0432.CCR-06-2082.
Bardos JI, Ashcroft M: Negative and positive regulation of HIF-1: a complex network. Biochim Biophys Acta. 2005, 1755 (2): 107-120.
Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S, Rosenbaum PS, Cerami A, Brines M, Rosenbaum DM: Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2002, 99 (16): 10659-10664. 10.1073/pnas.152321399.
Grimm C, Wenzel A, Stanescu D, Samardzija M, Hotop S, Groszer M, Naash M, Gassmann M, Reme C: Constitutive overexpression of human erythropoietin protects the mouse retina against induced but not inherited retinal degeneration. J Neurosci. 2004, 24 (25): 5651-5658. 10.1523/JNEUROSCI.1288-04.2004.
Rex TS, Allocca M, Domenici L, Surace EM, Maguire AM, Lyubarsky A, Cellerino A, Bennett J, Auricchio A: Systemic but not intraocular Epo gene transfer protects the retina from light-and genetic-induced degeneration. Mol Ther. 2004, 10 (5): 855-861. 10.1016/j.ymthe.2004.07.027.
Shih SC, Claffey KP: Hypoxia-mediated regulation of gene expression in mammalian cells. Int J Exp Pathol. 1998, 79 (6): 347-357. 10.1046/j.1365-2613.1998.00088.x.
Stephanou A: Role of STAT-1 and STAT-3 in ischaemia/reperfusion injury. J Cell Mol Med. 2004, 8 (4): 519-525. 10.1111/j.1582-4934.2004.tb00476.x.
Jung JE, Lee HG, Cho IH, Chung DH, Yoon SH, Yang YM, Lee JW, Choi S, Park JW, Ye SK, Chung MH: STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells. Faseb J. 2005, 19 (10): 1296-1298.
Rajkovic A, Yan C, Yan W, Klysik M, Matzuk MM: Obox, a family of homeobox genes preferentially expressed in germ cells. Genomics. 2002, 79 (5): 711-717. 10.1006/geno.2002.6759.
Udono T, Takahashi K, Nakayama M, Yoshinoya A, Totsune K, Murakami O, Durlu YK, Tamai M, Shibahara S: Induction of adrenomedullin by hypoxia in cultured retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2001, 42 (5): 1080-1086.
Xia CF, Yin H, Borlongan CV, Chao J, Chao L: Adrenomedullin gene delivery protects against cerebral ischemic injury by promoting astrocyte migration and survival. Hum Gene Ther. 2004, 15 (12): 1243-1254. 10.1089/hum.2004.15.1243.
Smith PD, Mount MP, Shree R, Callaghan S, Slack RS, Anisman H, Vincent I, Wang X, Mao Z, Park DS: Calpain-regulated p35/cdk5 plays a central role in dopaminergic neuron death through modulation of the transcription factor myocyte enhancer factor 2. J Neurosci. 2006, 26 (2): 440-447. 10.1523/JNEUROSCI.2875-05.2006.
Fu W, Wei J, Gu J: MEF2C mediates the activation induced cell death (AICD) of macrophages. Cell Res. 2006, 16 (6): 559-565. 10.1038/sj.cr.7310073.
Sutherland LC, Rintala-Maki ND, White RD, Morin CD: RNA binding motif (RBM) proteins: a novel family of apoptosis modulators?. J Cell Biochem. 2005, 94 (1): 5-24. 10.1002/jcb.20204.
Dennis G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA: DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003, 4 (5): P3-10.1186/gb-2003-4-5-p3.
Mahyar-Roemer M, Roemer K: p21 Waf1/Cip1 can protect human colon carcinoma cells against p53-dependent and p53-independent apoptosis induced by natural chemopreventive and therapeutic agents. Oncogene. 2001, 20 (26): 3387-3398. 10.1038/sj.onc.1204440.
Tang Y, Pacary E, Freret T, Divoux D, Petit E, Schumann-Bard P, Bernaudin M: Effect of hypoxic preconditioning on brain genomic response before and following ischemia in the adult mouse: identification of potential neuroprotective candidates for stroke. Neurobiol Dis. 2006, 21 (1): 18-28. 10.1016/j.nbd.2005.06.002.
Carmel JB, Kakinohana O, Mestril R, Young W, Marsala M, Hart RP: Mediators of ischemic preconditioning identified by microarray analysis of rat spinal cord. Exp Neurol. 2004, 185 (1): 81-96. 10.1016/j.expneurol.2003.09.007.
Kamphuis W, Dijk F, van Soest S, Bergen AA: Global gene expression profiling of ischemic preconditioning in the rat retina. Mol Vis. 2007, 13: 1020-1030.
Kawahara N, Wang Y, Mukasa A, Furuya K, Shimizu T, Hamakubo T, Aburatani H, Kodama T, Kirino T: Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats. J Cereb Blood Flow Metab. 2004, 24 (2): 212-223. 10.1097/01.WCB.0000106012.33322.A2.
Barone FC, White RF, Spera PA, Ellison J, Currie RW, Wang X, Feuerstein GZ: Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke. 1998, 29 (9): 1937-1950. discussion 1950-1931
Kamphuis W, Dijk F, van Soest S, Bergen AA: Ischemic preconditioning alters the pattern of gene expression changes in response to full retinal ischemia. Mol Vis. 2007, 13: 1892-1901.
Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, Huang LE: HIF-1alpha induces cell cycle arrest by functionally counteracting Myc. Embo J. 2004, 23 (9): 1949-1956. 10.1038/sj.emboj.7600196.
Chen T, Turner J, McCarthy S, Scaltriti M, Bettuzzi S, Yeatman TJ: Clusterin-mediated apoptosis is regulated by adenomatous polyposis coli and is p21 dependent but p53 independent. Cancer Res. 2004, 64 (20): 7412-7419. 10.1158/0008-5472.CAN-04-2077.
Kwon YH, Jovanovic A, Serfas MS, Tyner AL: The Cdk inhibitor p21 is required for necrosis, but it inhibits apoptosis following toxin-induced liver injury. J Biol Chem. 2003, 278 (32): 30348-30355. 10.1074/jbc.M300996200.
Sharma RR, Ravikumar TS, Raimo D, Yang WL: Induction of p21WAF1 expression protects HT29 colon cancer cells from apoptosis induced by cryoinjury. Ann Surg Oncol. 2005, 12 (9): 743-752. 10.1245/ASO.2005.11.021.
Wetzel M, Rosenberg GA, Cunningham LA: Tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal sensitivity to doxorubicin-induced apoptosis. Eur J Neurosci. 2003, 18 (5): 1050-1060. 10.1046/j.1460-9568.2003.02838.x.
Hicks D, Heidinger V, Mohand-Said S, Sahel J, Dreyfus H: Growth factors and gangliosides as neuroprotective agents in excitotoxicity and ischemia. Gen Pharmacol. 1998, 30 (3): 265-273. 10.1016/S0306-3623(97)00356-X.
Andrews GK, Lee DK, Ravindra R, Lichtlen P, Sirito M, Sawadogo M, Schaffner W: The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development. Embo J. 2001, 20 (5): 1114-1122. 10.1093/emboj/20.5.1114.
Ebadi M, Brown-Borg H, El Refaey H, Singh BB, Garrett S, Shavali S, Sharma SK: Metallothionein-mediated neuroprotection in genetically engineered mouse models of Parkinson's disease. Brain Res Mol Brain Res. 2005, 134 (1): 67-75. 10.1016/j.molbrainres.2004.09.011.
Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN: Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest. 1997, 100 (6): 1501-1506. 10.1172/JCI119672.
Chen L, Wu W, Dentchev T, Wong R, Dunaief JL: Increased metallothionein in light damaged mouse retinas. Exp Eye Res. 2004, 79 (2): 287-293. 10.1016/j.exer.2004.05.004.
Suemori S, Shimazawa M, Kawase K, Satoh M, Nagase H, Yamamoto T, Hara H: Metallothionein, an endogenous antioxidant, protects against retinal neuron damage in mice. Invest Ophthalmol Vis Sci. 2006, 47 (9): 3975-3982. 10.1167/iovs.06-0275.
Miyashita K, Itoh H, Arai H, Suganami T, Sawada N, Fukunaga Y, Sone M, Yamahara K, Yurugi-Kobayashi T, Park K, Oyamada N, Sawada N, Taura D, Tsujimoto H, Chao TH, Tamura N, Mukoyama M, Nakao K: The neuroprotective and vasculo-neuro-regenerative roles of adrenomedullin in ischemic brain and its therapeutic potential. Endocrinology. 2006, 147 (4): 1642-1653. 10.1210/en.2005-1038.
Vingolo EM, Lupo S, Domanico D, Cotesta D, Petramala L, Grenga R, Letizia C: Adrenomedullin plasma concentrations in patients with retinitis pigmentosa. Clin Biochem. 2005, 38 (8): 735-738. 10.1016/j.clinbiochem.2005.04.007.
Garayoa M, Martinez A, Lee S, Pio R, An WG, Neckers L, Trepel J, Montuenga LM, Ryan H, Johnson R, Gassmann M, Cuttitta F: Hypoxiainducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis. Mol Endocrinol. 2000, 14 (6): 848-862. 10.1210/me.14.6.848.
Leonard MO, Cottell DC, Godson C, Brady HR, Taylor CT: The role of HIF-1 alpha in transcriptional regulation of the proximal tubular epithelial cell response to hypoxia. J Biol Chem. 2003, 278 (41): 40296-40304. 10.1074/jbc.M302560200.
Ferre N, Marsillach J, Camps J, Mackness B, Mackness M, Riu F, Coll B, Tous M, Joven J: Paraoxonase-1 is associated with oxidative stress, fibrosis and FAS expression in chronic liver diseases. J Hepatol. 2006, 45 (1): 51-59. 10.1016/j.jhep.2005.12.018.
Mackness B, Hine D, Liu Y, Mastorikou M, Mackness M: Paraoxonase-1 inhibits oxidised LDL-induced MCP-1 production by endothelial cells. Biochem Biophys Res Commun. 2004, 318 (3): 680-683. 10.1016/j.bbrc.2004.04.056.
Tanito M, Li F, Elliott MH, Dittmar M, Anderson RE: Protective effect of TEMPOL derivatives against light-induced retinal damage in rats. Invest Ophthalmol Vis Sci. 2007, 48 (4): 1900-1905. 10.1167/iovs.06-1057.
Baskol G, Karakucuk S, Oner AO, Baskol M, Kocer D, Mirza E, Saraymen R, Ustdal M: Serum paraoxonase 1 activity and lipid peroxidation levels in patients with age-related macular degeneration. Ophthalmologica. 2006, 220 (1): 12-16. 10.1159/000089269.
Song Q, Kuang Y, Dixit VM, Vincenz C: Boo, a novel negative regulator of cell death, interacts with Apaf-1. Embo J. 1999, 18 (1): 167-178. 10.1093/emboj/18.1.167.
Zhang H, Holzgreve W, De Geyter C: Bcl2-L-10, a novel anti-apoptotic member of the Bcl-2 family, blocks apoptosis in the mitochondria death pathway but not in the death receptor pathway. Hum Mol Genet. 2001, 10 (21): 2329-2339. 10.1093/hmg/10.21.2329.
Donovan M, Cotter TG: Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development. Cell Death Differ. 2002, 9 (11): 1220-1231. 10.1038/sj.cdd.4401105.
Argraves WS, Larue AC, Fleming PA, Drake CJ: VEGF signaling is required for the assembly but not the maintenance of embryonic blood vessels. Dev Dyn. 2002, 225 (3): 298-304. 10.1002/dvdy.10162.
Nishijima K, Ng YS, Zhong L, Bradley J, Schubert W, Jo N, Akita J, Samuelsson SJ, Robinson GS, Adamis AP, Shima DT: Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am J Pathol. 2007, 171 (1): 53-67. 10.2353/ajpath.2007.061237.
Ferrari G, Pintucci G, Seghezzi G, Hyman K, Galloway AC, Mignatti P: VEGF, a prosurvival factor, acts in concert with TGF-beta1 to induce endothelial cell apoptosis. Proc Natl Acad Sci USA. 2006, 103 (46): 17260-17265. 10.1073/pnas.0605556103.
Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM: A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature. 2000, 405 (6782): 85-90. 10.1038/35011084.
Hong JR, Lin GH, Lin CJ, Wang WP, Lee CC, Lin TL, Wu JL: Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish. Development. 2004, 131 (21): 5417-5427. 10.1242/dev.01409.
Hisatomi T, Sakamoto T, Sonoda KH, Tsutsumi C, Qiao H, Enaida H, Yamanaka I, Kubota T, Ishibashi T, Kura S, Susin SA, Kroemer G: Clearance of apoptotic photoreceptors: elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin alphavbeta3. Am J Pathol. 2003, 162 (6): 1869-1879.
Gordon WC, Casey DM, Lukiw WJ, Bazan NG: DNA damage and repair in light-induced photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2002, 43 (11): 3511-3521.
Hoppeler T, Hendrickson P, Dietrich C, Reme C: Morphology and timecourse of defined photochemical lesions in the rabbit retina. Curr Eye Res. 1988, 7 (9): 849-860. 10.3109/02713688808997242.
Baghdoyan S, Dubreuil P, Eberle F, Gomez S: Capture of cytokineresponsive genes (NACA and RBM3) using a gene trap approach. Blood. 2000, 95 (12): 3750-3757.
Baldi A, Battista T, De Luca A, Santini D, Rossiello L, Baldi F, Natali PG, Lombardi D, Picardo M, Felsani A, Paggi MG: Identification of genes down-regulated during melanoma progression: a cDNA array study. Exp Dermatol. 2003, 12 (2): 213-218. 10.1034/j.1600-0625.2003.00026.x.
Liu J, Narasimhan P, Lee YS, Song YS, Endo H, Yu F, Chan PH: Mild hypoxia promotes survival and proliferation of SOD2-deficient astrocytes via c-Myc activation. J Neurosci. 2006, 26 (16): 4329-4337. 10.1523/JNEUROSCI.0382-06.2006.
Liu T, Laurell C, Selivanova G, Lundeberg J, Nilsson P, Wiman KG: Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis. Cell Death Differ. 2007, 14 (3): 411-421. 10.1038/sj.cdd.4402022.
Wenzel A, Grimm C, Samardzija M, Reme CE: The genetic modifier Rpe65Leu(450): effect on light damage susceptibility in c-Fos-deficient mice. Invest Ophthalmol Vis Sci. 2003, 44 (6): 2798-2802. 10.1167/iovs.02-1134.
Samardzija M, Wenzel A, Aufenberg S, Thiersch M, Reme C, Grimm C: Differential role of Jak-STAT signaling in retinal degenerations. Faseb J. 2006, 20 (13): 2411-2413. 10.1096/fj.06-5895fje.
Raffelsberger W, Krause Y: RReportGenerator: Automatic reports from routine statistical analysis using R. Bioinformatics. 2008, 24 (2): 276-278. 10.1093/bioinformatics/btm556.
Wu Z, Rafael A, Irizarry R, Gentleman R, Martinez-Murillo F, Spencer F: A model-based background adjustment for oligonucleotide expression arrays. J Am Stat Assoc. 2004, 99: 909-917. 10.1198/016214504000000683.
Ploner A, Calza S, Gusnanto A, Pawitan Y: Multidimensional local false discovery rate for microarray studies. Bioinformatics. 2006, 22 (5): 556-565. 10.1093/bioinformatics/btk013.
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5 (10): R80-10.1186/gb-2004-5-10-r80.
Database for Annotation, Visualization and Integrated Discovery (DAVID). [http://david.abcc.ncifcrf.gov/home.jsp]
Ingenuity Systems, CA, USA. [http://www.ingenuity.com]
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.
MT, RF, MS and CG performed experiments. MT, WR and OP analyzed microarray data. Study was designed by MT, RF, AW and CG. Manuscript was written by MT and CG.
Electronic supplementary material
Additional file 2: Table 1, 2, 3 Gene Lists of differentially regulated genes. Table 1 List of differentially regulated genes immediately after hypoxia (H0). Table 2 List of differentially regulated genes 2 h after hypoxia (H2). Table 3 List of differentially regulated genes 4 h after hypoxia (H4). File contains all genes found to be differentially regulated immediately after hypoxia (Table 5 H0), 2 h after hypoxia (Table 6, H2) and 4 h after hypoxia (Table 7, H4). (XLS 132 KB)
Additional file 3: Table 4 Real time PCR results. Table 4 Real-Time PCR results of the expression of genes detected by Affymetrix microarrays as differentially regulated by hypoxic preconditioning. File contains all genes revealed by microarray analyses, which were tested by real time PCR. (DOC 138 KB)
Additional file 4: Table 5,6 List of genes functionally clustered by DAVID. Table 5 Differentially up-regulated genes functionally clustered by DAVID. Table 6 Differentially down-regulated genes functionally clustered by DAVID. File contains functional clusters of genes found to be differentially regulated immediately after hypoxia (H0 data). (XLS 92 KB)
Additional file 5: Fig 2, 3 Hypoxic preconditioning gene networks discovered by Ingenuity Pathway analysis. Fig 2 affected gene network discovered by Ingenuity Pathway Analysis and classified as: DNA replication, recombination, cell cycle and cancer. Fig. 3 affected gene network discovered by Ingenuity Pathway Analysis and classified as: Cell death, cellular development, hematological system development & function. File contains schemes of pathways, which are affected by hypoxic preconditioning. Some genes connected to cMyc (Fig. 7) and to TGF-b (Fig. 8) are differentially regulated. (DOC 586 KB)
Authors’ original submitted files for images
About this article
Cite this article
Thiersch, M., Raffelsberger, W., Frigg, R. et al. Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection. BMC Genomics 9, 73 (2008). https://doi.org/10.1186/1471-2164-9-73
- Retinitis Pigmentosa
- Ingenuity Pathway Analysis
- Hypoxic Exposure
- Retinal Degeneration