July 2011
Volume 52, Issue 8
Free
Retinal Cell Biology  |   July 2011
Normoxic Activation of Hypoxia-Inducible Factors in Photoreceptors Provides Transient Protection against Light-Induced Retinal Degeneration
Author Affiliations & Notes
  • Christina Lange
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Severin R. Heynen
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Naoyuki Tanimoto
    Institute of Ophthalmic Research, Center of Ophthalmology, Division of Ocular Neurodegeneration, Tubingen, Germany;
  • Markus Thiersch
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Yun-Zheng Le
    Department of Cell Biology and Dean A. McGee Eye Institute, Oklahoma City, Oklahoma; and
  • Isabelle Meneau
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Mathias W. Seeliger
    Institute of Ophthalmic Research, Center of Ophthalmology, Division of Ocular Neurodegeneration, Tubingen, Germany;
  • Marijana Samardzija
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Christian Caprara
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
  • Christian Grimm
    From the Lab for Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Zurich, Switzerland;
    Zurich Center for Integrative Human Physiology (ZIHP), Zurich, Switzerland.
  • Corresponding author: Christian Grimm, Lab for Retinal Cell Biology, Department Ophthalmology, USZ, University of Zurich, Wagistrasse 14, CH 8952 Schlieren, Switzerland; cgrimm@opht.uzh.ch
Investigative Ophthalmology & Visual Science July 2011, Vol.52, 5872-5880. doi:10.1167/iovs.11-7204
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Christina Lange, Severin R. Heynen, Naoyuki Tanimoto, Markus Thiersch, Yun-Zheng Le, Isabelle Meneau, Mathias W. Seeliger, Marijana Samardzija, Christian Caprara, Christian Grimm; Normoxic Activation of Hypoxia-Inducible Factors in Photoreceptors Provides Transient Protection against Light-Induced Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(8):5872-5880. doi: 10.1167/iovs.11-7204.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose.: Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection.

Methods.: Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG.

Results.: The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls.

Conclusions.: Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.

Retinitis pigmentosa (RP) is characterized by the progressive loss of photoreceptors causing severe visual disturbances and blindness in humans. To investigate molecular mechanisms of photoreceptor degeneration, different mouse models have been developed. The model of light-induced photoreceptor cell death is widely used to analyze immediate changes in gene expression during degeneration or to test the efficacy of neuroprotective strategies 1 such as hypoxic preconditioning. It has been shown that a short pre-exposure of mice to reduced levels of oxygen completely protects photoreceptors from light-induced degeneration. 2  
To cope with the stress imposed by reduced oxygen availability during hypoxia, cells regulate expression of genes for survival, growth, or metabolism. 3 This regulation is achieved mainly by special transcription factors called hypoxia inducible factors (HIFs). HIFs consist of two subunits, α and β. The HIF β-subunit (aryl-hydrocarbon receptor nuclear translocator [ARNT]) is constitutively expressed and located in the nucleus. HIF1A, HIF2A, and HIF3A, the three known isoforms of the HIF α-subunit, are expressed but quickly degraded in conditions of normal oxygen availability (normoxia). Hydroxylation of HIF α-subunits by prolyl hydroxylases (egl nine [EGLN] family) leads to their recognition by the von Hippel-Lindau (VHL) protein. VHL forms a multimeric protein complex together with Cullin-2, RBX1, and the elongins B and C, 4 6 and acts as a E3-ubiquitinase targeting HIF α proteins to proteasomal degradation. 7 Another hydroxylase, the factor inhibiting HIF (FIH), hydroxylates HIF-α at an asparagine residue, inhibiting p300 coactivator recruitment and thus transcriptional activity. 8 Hydroxylation of HIF-α proteins by EGLNs and FIH depends on the availability of oxygen. 8,9 Hence, hydroxylation is strongly reduced during hypoxia and HIF α-subunits are not recognized by VHL. Thus HIF α proteins are stabilized, enter the nucleus, bind HIF-β and p300, and participate in the regulation of gene expression. 
It has been shown that during hypoxic preconditioning, HIF1A and HIF2A are stabilized and that HIF target genes are differentially regulated in the retina. 2,10,11 Erythropoietin (Epo) was shown to be strongly induced after hypoxic exposure, and injection of recombinant EPO was protective against light-induced retinal degeneration. 2 Using a genetic approach, we recently showed that HIF1A is not essential in rods for protection by hypoxic preconditioning. 10 However, this does not exclude that rod-specific activation of HIF1A contributes to neuroprotection after hypoxic preconditioning. In addition, HIF2A and/or other factors that are expressed in rods and regulated by hypoxia might be involved in neuroprotection by hypoxic preconditioning. Alternatively, hypoxia might regulate factors in other cell types in the retina. Such factors might act in trans to protect rods. To address these questions, we investigated whether a hypoxia-like response in rod photoreceptors is sufficient to prevent retinal degeneration in normoxia. To achieve normoxic induction of a hypoxia-like response, we generated photoreceptor-specific Vhl knockdown mice using the Cre-lox system. Because the absence of VHL leads to increased stability of HIF α-subunits in normoxia, Vhl knockdowns may mimic the hypoxic response at normal oxygen levels. In case of protection, VHL protein may provide a target for tissue-specific pharmacological interventions in the future. 
Materials and Methods
Mice and Genotyping
Mice 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. All mice were maintained as breeding colonies at the University of Zurich in a 12-hour/12-hour light/dark cycle (60 lux). 
Wild-type BALB/c mice were from a breeding colony at the University Hospital Zurich. 129S-Vhlhtm1jae/J-mice (hereafter Vhlflox/flox mice), which have loxP sites flanking exon 1 and part of the promoter of the Vhl gene, 12 were purchased from Jackson Laboratory (Bar Harbor, ME). To generate rod photoreceptor-specific Vhl knockdowns, Vhlflox/flox mice were crossed with mice expressing Cre recombinase under the control of the opsin promoter (LMOPC1, hereafter opsin-Cre mice). In these mice, Cre expression in rods starts at approximately postnatal day 7 and increases up to 6 weeks of age. 13 The breeding colonies were kept on the light-sensitive Rpe65450Leu background. 14 A red fluorescence protein (RFP) reporter strain (ROSA-flox-RFP; kindly provided by Wolf-Dietrich Hardt, ETH, Zurich, Switzerland) was crossed with opsin-Cre mice to assay the expression pattern of Cre recombinase. 
The following primers were used to detect wild-type (wt) and Vhl-flox alleles: forward (forw) (5′-TGAGTATGGGATAACGGGTTGAAC-3′) and reverse (rev) (5′-AGAACTGACTGACTTCCACTGATGC-3′). The wt allele was identified as a 125-basepair (bp) PCR fragment and the Vhl-flox as a 317-bp fragment after gel electrophoresis. Presence of the opsin-cre transgene was tested by PCR using the following primer pair: forw (5′-AGGTGTAGAGAAGGCACTTAGC-3′) and rev (5′-CTAATCGCCATCTTCCAGCAGG-3′). In the presence of the transgene, the amplification reaction resulted in the production of a 411-bp fragment. To detect excision of floxed sequences in the Vhl gene, genomic DNA was isolated from retinal tissue and tested by PCR using the following primers: forw_un-excised (5′-CTGGTACCCACGAAACTGTC-3′), forw_excised (5′-CTAGGCACCGAGCTTAGAGGTTTGCG-3′) and rev_both (5′-CTGACTTCCACTGATGCTTGTCACAG-3′). The excised allele was identified as a 260-bp and the unexcised allele as a 460-bp fragment. Presence or absence of the RFP transgene was tested using the ROSA-tg forward primer: 5′-GCGAAGAGTTTGTCCTCAACC-3′ or the ROSA-wt forward primer: 5′-GGAGCGGGAGAAATGGATATG-3′, respectively, together with the ROSA-common reverse primer: 5′-AAAGTCGCTCTGAGTTGTTAT-3′. An amplification product of 300 bp or of 500 bp indicated the presence or absence, respectively, of the RFP locus. 
Laser Capture Microdissection
After mice were killed, their eyes were enucleated, immediately embedded in tissue freezing medium (Leica Microsystems Nussloch GmbH, Nussloch, Germany), and frozen in a 2-methylbutane bath cooled with liquid nitrogen. Retinal sections (20 μm) were fixed (5 minutes in acetone), air dried (5 minutes), and dehydrated (30 seconds in 100% ethanol, 5 minutes in xylene). Microdissection was performed using an Arcturus XT Laser capture device (Molecular Devices, Sunnyvale, CA). RNA was isolated using the Arcturus kit for RNA isolation (Molecular Devices) according to the manufacturer's directions including a DNase treatment to digest residual genomic DNA. Equal amounts of RNA were used for reverse transcription using oligo(dT) and M-MLV reverse transcriptase (Promega, Madison, WI). 
Semiquantitative Real-Time PCR
Retinas were removed through a slit in the cornea and immediately frozen in liquid nitrogen. Total RNA was prepared using a kit (RNeasy RNA isolation kit; Qiagen, Hilden, Germany) according to the manufacturer's directions including a DNase treatment to digest residual genomic DNA. Equal amounts of RNA were used for reverse transcription using oligo(dT) and M-MLV reverse transcriptase (Promega). Relative quantification of cDNA was conducted via real-time PCR using a kit (LightCycler 480 Sybr Green I Master kit; Roche Diagnostics, Basel, Switzerland), a LightCycler 480 instrument (Roche Diagnostics) and specific primer pairs (Table 1). Expression was normalized to Actb (β-actin) and relative quantification was calculated using the comparative threshold method (ΔΔCT) and commercial software (Light Cycler 480 software; Roche). 
Table 1.
 
Primers Used for Real-Time PCR
Table 1.
 
Primers Used for Real-Time PCR
Gene Upstream (5′–3′) Downstream (5′–3′) Annealing Temperature (°C) Product (bp)
Adm ttcgcagttccgaaagaagt ggtagctgctggatgcttgta 62 77
Actb cgacatggagaagatctggc caacggctccggcatgtgc 62 153
Bnip3 cctgtcgcagttgggttc gaagtgcagttctacccaggag 60 93
Egln1 cattgttggcagaaggtgtg caaaggactacagggtctcca 62 70
Epo gccctgctagccaattcc gccctgctagccaattcc 60 128
Gnat1 gaggatgctgagaaggatgc tgaatgttgagcgtggtcat 58 209
Gnat2 gcatcagtgctgaggacaaa ctaggcactcttcgggtgag 58 192
Mt1 gaatggaccccaactgctc gcagcagctcttcttgcag 62 104
Mt2 tgtacttcctgcaagaaaagctg acttgtcggaagcctctttg 62 94
Sag ttacaagccttccaacctctgac accagcacaacaccatctacag 64 189
Scl2a1 cagtgtatcctgttgcccttctg gccgaccctcttctttcatctc 62 151
Rho cttcacctggatcatggcgtt ttcgttgttgacctcaggcttg 62 130
Vegfa acttgtgttgggaggaggatgtc aatgggtttgtcgtgtttctgg 60 171
Vhl gagggacccgttccaataat ttggcaaaaataggctgtcc 60 364
Fgf2 tgtgtctatcaagggagtgtgtgc accaactggagtatttccgtgaccg 62 158
Edn2 agacctcctccgaaagctg ctggctgtagctggcaaag 60 64
Stat3 caaaaccctcaagagccaagg tcactcacaatgcttctccgc 62 133
Gfap ccaccaaactggctgatgtctac ttctctccaaatccacacgagc 62 240
Socs3 atttcgcttcgggactagc aacttgctgtgggtgaccat 58 126
Lif aatgccacctgtgccatacg caacttggtcttctctgtcccg 60 216
Cntf ctctgtagccgctctatctg ggtacaccatccactgagtc 58 125
Western Blotting Analysis
Retinas were homogenized in 0.1 M Tris–HCl (pH 8) at 4°C and protein content was determined using Bradford reagent. Standard SDS-PAGE and Western blotting were performed. For immunodetection, the following antibodies were used: anti-HIF1A (no. Nb100–479; Novus Biologicals, Cambridge, UK, 1:1000), anti-HIF2A (no. Nb100–122, Novus Biologicals; 1:1000), anti-pSTAT3 (no. 9131; Cell Signaling Technology, Danvers, MA; 1:500) and anti-ACTB (no. A5441; Sigma-Aldrich, St. Louis, MO; 1:5000). Blots were incubated overnight at 4°C with primary antibodies followed by a 1-hour incubation with HRP-conjugated secondary antibodies. Immunoreactivity was visualized using the Renaissance-Western blot detection kit (PerkinElmer Life Sciences, Emeryville, CA). 
Electroretinography
ERGs were recorded according to previously described procedures. 15,16 The ERG equipment consisted of a Ganzfeld bowl, a direct current amplifier, and a PC-based control and recording unit (Multiliner Vision; VIASYS Health Care GmbH, Hoechberg, Germany). Mice were dark adapted overnight and anesthetized with ketamine (66.7 mg/kg body weight) and xylazine (11.7 mg/kg body weight). Pupils were dilated and single-flash ERG responses were obtained under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was accomplished with a background illumination of 30 cd/m2 starting 10 minutes before recording. Single white-flash stimulus intensity ranged from –4 to 1.5 log cd · s/m2 under scotopic and from –2 to 1.5 log cd · s/m2 under photopic conditions, divided into 10 and 8 steps, respectively. Ten responses were averaged with an interstimulus interval (ISI) of either 5 seconds (for –4, –3, –2, –1.5, –1, and –0.5 log cd · s/m2) or 17 seconds (for 0, 0.5, 1, and 1.5 log cd · s/m2). 
Light Exposure, Cell Death Detection, and Morphology
Mice were dark adapted overnight and pupils dilated with 1% ophthalmic solution (Cyclogyl; Alcon, Cham, Switzerland) and 5% phenylephrine (Ciba Vision, Niederwangen, Switzerland) 1 hour before exposure to white fluorescent light (13′000 lux) for 1 hour. 
After light exposure, mice were placed in darkness overnight. Thereafter, mice were kept in normal cyclic light until they were killed. To quantify apoptosis, mice were killed 36 hours after light exposure. Nucleosomal release was determined using a cell death detection (CDD) kit (Roche Diagnostics) according to the manufacturer's recommendations. 
For light microscopy, eyes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C overnight. For each eye, the superior and the inferior retinas were prepared, washed in cacodylate buffer, incubated in osmium tetroxide for 1 hour, dehydrated, and embedded in resin Epon 812. Sections (0.5 μm) were prepared from the lower central retina and counterstained with methylene blue. 
Hypoxic Preconditioning
Adult (8–10 weeks of age) BALB/c mice were exposed to reduced oxygen concentrations in a hypoxic chamber with food and water ad libitum. O2 levels were reduced in steps of 2% by changing the O2/N2 ratio to reach a final O2 concentration of 7% within 90 minutes. After 6 hours of hypoxia, mice were killed immediately to isolate retinal tissue for gene expression analysis. 
Rhodopsin Measurements
Mice were dark adapted overnight. Retinas were removed through a slit in the cornea under dim red light and placed in 1 mL of distilled H2O for 1 minute. After 3 minutes of centrifugation at 15,000g, the supernatant was discarded and 700 μL of 1% hexadecyltrimethylammonium bromide (Fluka Chemie, Buchs, Switzerland) in H2O was added to the pellet. Retinas were mechanically homogenized with a polytron (20 seconds, 3000 rpm), centrifuged for 3 minutes at 15,000g, and the supernatant was collected. The absorption at 500 nm was measured before and after exposure to bright white light (20,000 lux for 1 minute). The amount of rhodopsin present per retina was calculated using the following formula derived from the Lambert-Beer equation: ρ = vol × c = vol × Δabs500/(E500 × l × n). ρ is the amount of rhodopsin per retina (in moles); vol is the volume of the sample (in liters); c is the concentration of rhodopsin per retina (M); Δabs500 is the difference between absorption of the sample at 500 nm before and after bleaching; E500 is the extinction coefficient of rhodopsin at 500 nm (4.2 × 104 cm × M); l is the path length of the cuvette (in cm); and n is the number of retinas. 
Results
Successful Knockdown of Vhl in Photoreceptors
Expression of Cre recombinase in our line of LMOPC1 mice was specific to the photoreceptor layer but showed a patchy pattern (Figs. 1A, 1B). Analysis of genomic DNA from retinal tissue of 10-week-old Vhlflox/flox and Vhlflox/flox ;opsin-cre mice showed excision of floxed DNA sequences in mice expressing CRE recombinase but not in control mice (Fig. 1C). Because excision is specific for rod photoreceptors, 13 whole retinal samples contain both the floxed (unexcised; from retinal cells without CRE expression) and the excised allele. Semiquantitative real-time PCR on samples obtained by laser capture microdissection revealed lower expression levels of Vhl mRNA in the ONL of Vhlflox/flox ;opsin-cre mice (CRE+) compared to their control (Vhlflox/flox ;CRE−) littermates (Fig. 1D). The reason for the upregulation of Vhl expression in the other cell layers is unclear, but might involve compensatory effects. 
Figure 1.
 
Knockdown of Vhl in photoreceptors of 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Expression pattern of Cre recombinase in ROSA-flox-RFP;opsin-Cre mice. Red, red fluorescent protein, expressed only in cells where Cre was active. Scale bar, 100 μm. (B) higher magnification of the section shown in (A). (C) PCR amplification of genomic DNA isolated from total retinal tissue of three different Vhlflox/flox (CRE–) and three Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. The amplified fragment from the floxed gene (floxed) has a length of 460 bp. CRE-mediated excision of the floxed sequence (excised) resulted in a 260-bp fragment. (D) Semiquantitative real-time PCR after laser capture microdissection showing relative Vhl mRNA levels in the different retinal layers (as indicated) of Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice. Values were normalized to Actb and expressed relatively to the respective values for each layer in Vhlflox/flox mice, which were set to 1. Shown are mean values ± SD of three different mice amplified in duplicates. ONH, optic nerve head; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 1.
 
Knockdown of Vhl in photoreceptors of 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Expression pattern of Cre recombinase in ROSA-flox-RFP;opsin-Cre mice. Red, red fluorescent protein, expressed only in cells where Cre was active. Scale bar, 100 μm. (B) higher magnification of the section shown in (A). (C) PCR amplification of genomic DNA isolated from total retinal tissue of three different Vhlflox/flox (CRE–) and three Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. The amplified fragment from the floxed gene (floxed) has a length of 460 bp. CRE-mediated excision of the floxed sequence (excised) resulted in a 260-bp fragment. (D) Semiquantitative real-time PCR after laser capture microdissection showing relative Vhl mRNA levels in the different retinal layers (as indicated) of Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice. Values were normalized to Actb and expressed relatively to the respective values for each layer in Vhlflox/flox mice, which were set to 1. Shown are mean values ± SD of three different mice amplified in duplicates. ONH, optic nerve head; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Stabilization of HIF-α Proteins Leads to Increased Expression of HIF Target Genes in 10-Week-Old Vhlflox/flox ;opsin-cre Mice
Downregulation of Vhl in photoreceptors led to a stabilization of HIF-α proteins in normoxic retinas. Increased levels of both HIF1A and HIF2A were detected in retinas of 10-week-old Vhlflox/flox ;opsin-cre mice (CRE+; Fig. 2A). In addition to HIF-α stabilization, we noticed strong phosphorylation of STAT3. Expression levels of total STAT3 protein was also slightly elevated in Vhl knockdown retinas. Although STAT3 is reported to be part of the HIF-pathway, 17 it is unclear whether the increased phosphorylation of STAT3 is a consequence of the stabilized HIF-α proteins or whether it is due to other mechanisms in the Vhl knockdown retinas. Importantly, however, stabilization of HIF-α proteins and phosphorylation of STAT3 are features also found in the hypoxic retina. Therefore, the molecular response in the Vhl knockdowns may resemble the response in retinas of mice preconditioned by hypoxia. To determine whether the stabilized HIFs are transcriptionally active in normoxia, we analyzed expression of several hypoxia-responsive genes 11 in retinas of 10-week-old Vhlflox/flox ;opsin-cre mice (Fig. 2B) and in wild-type mice after hypoxic preconditioning (Fig. 2C). Expression of adrenomedullin (Adm), prolyl hydroxylase 2 (Egln1), solute carrier family 2 (facilitated glucose transporter) member 1 (Slc2a1, also called Glut1), and vascular endothelial growth factor (Vegf) was induced to a similar extent in normoxic Vhlflox/flox ;opsin-cre mice and in hypoxic wild-type mice (after exposure to 7% oxygen for 6 hours). Regulation of these hypoxia response genes is largely attributed to HIF1. 18 20 Expression of BCL2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3), metallothionein 1 (Mt1), and metallothionein 2 (Mt2) was also upregulated in Vhlflox/flox ;opsin-cre mice but not to the same extent as in hypoxic wild-type mice. Expression of erythropoietin Epo, which is reported to be activated by HIF2, 21 was induced in hypoxic but not in Vhlflox/flox ;opsin-cre mice. This suggests that the stabilized HIF2 was not transcriptionally active, that induction of Epo during hypoxia is regulated by cells other than rod photoreceptors, or that Epo expression in photoreceptors is not controlled by HIF2. 
Figure 2.
 
Stabilization and activation of transcription factors in 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Western blot analysis showing protein expression of HIF1A, HIF2A, phosphorylated STAT3 (p-STAT3), and STAT3 in four different Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. ACTB served as a control for equal loading. (B) Total retinal RNA was prepared from 10-week-old Vhlflox/flox ;opsin-cre mice and expression levels of individual genes (as indicated) were determined by semiquantitative real-time PCR. Levels were normalized to Actb and expressed relatively to the expression in Vhlflox/flox control mice (set to 1). (C) Total retinal RNA was prepared from wild-type mice after hypoxic preconditioning. Expression levels were normalized to Actb and expressed relatively to expression in normoxic control mice (set to 1). Shown are mean values ± SD of four retinas amplified in duplicates.
Figure 2.
 
Stabilization and activation of transcription factors in 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Western blot analysis showing protein expression of HIF1A, HIF2A, phosphorylated STAT3 (p-STAT3), and STAT3 in four different Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. ACTB served as a control for equal loading. (B) Total retinal RNA was prepared from 10-week-old Vhlflox/flox ;opsin-cre mice and expression levels of individual genes (as indicated) were determined by semiquantitative real-time PCR. Levels were normalized to Actb and expressed relatively to the expression in Vhlflox/flox control mice (set to 1). (C) Total retinal RNA was prepared from wild-type mice after hypoxic preconditioning. Expression levels were normalized to Actb and expressed relatively to expression in normoxic control mice (set to 1). Shown are mean values ± SD of four retinas amplified in duplicates.
Knockdown of Vhl in Rod Photoreceptors Does Not Affect Morphology and Function in Young Mice but Leads to Retinal Degeneration in Old Mice
To assess a potential spontaneous degeneration of photoreceptor cells in Vhl knockdown mice, we analyzed ERG responses (Figs. 3A–D) in young Vhlflox/flox ;opsin-cre and Vhlflox/flox littermates. ERG traces and scotopic and photopic b-wave amplitudes in 17-week-old Vhl knockdown mice did not differ from those in control Vhlflox/flox littermates and were similar to those in wild-type mice. 16 Normal retinal structure in young Vhl knockdown animals was further supported by comparable retinal morphologies (see below) and by similar expression levels of the photoreceptor marker genes rod transducin (Gnat1), cone transducin (Gnat2), S-antigen (Sag), and rod opsin (Rho) in 10-week old Vhlflox/flox ;opsin-cre and Vhlflox/flox littermates (Fig. 3E). 
Figure 3.
 
Normal retinal function in 17-week-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 17-week-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the time point of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Semiquantitative real-time PCR for photoreceptor-specific genes (as indicated) in 10-week-old Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre (gray bars). Values were normalized to Actb and expressed relatively to the value of Vhlflox/flox mice, which was set to 1. Shown are mean values ± SD of four animals amplified in duplicates.
Figure 3.
 
Normal retinal function in 17-week-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 17-week-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the time point of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Semiquantitative real-time PCR for photoreceptor-specific genes (as indicated) in 10-week-old Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre (gray bars). Values were normalized to Actb and expressed relatively to the value of Vhlflox/flox mice, which was set to 1. Shown are mean values ± SD of four animals amplified in duplicates.
ERG recordings from 1-year-old Vhlflox/flox ;opsin-cre mice, however, revealed a reduction of a- and b-wave amplitudes under both scotopic and photopic conditions (Figs. 4A–D). The strong reduction in the a-wave amplitude suggested that photoreceptors might be injured or lost. This was confirmed by the morphologic analysis of 1-year-old retinas (Fig. 4E). Compared with the ONL of age-matched Vhlflox/flox and opsin-cre control mice, the ONL of Vhlflox/flox ;opsin-cre mice was thinned, with only three to four rows of photoreceptor nuclei remaining. In addition, the structure of inner and outer segments appeared severely disintegrated. Thus, knockdown of Vhl did not affect retinal function and morphology in mice up to 17 weeks of age but caused retinal degeneration in old mice. 
Figure 4.
 
Retinal degeneration in 1-year-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 1-year-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the timing of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Representative morphologic sections from the lower central retina of 1-year-old Vhlflox/flox , of opsin-cre and of Vhlflox/flox ;opsin-cre mice. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.
Figure 4.
 
Retinal degeneration in 1-year-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 1-year-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the timing of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Representative morphologic sections from the lower central retina of 1-year-old Vhlflox/flox , of opsin-cre and of Vhlflox/flox ;opsin-cre mice. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.
Knockdown of Vhl in Rod Photoreceptors Delays Light-Induced Photoreceptor Degeneration in Young Mice
To test whether the activation of the hypoxia-like response in photoreceptors during normoxic conditions is similarly neuroprotective to light exposure as hypoxic preconditioning, we used 10-week-old Vhlflox/flox ;opsin-cre mice. At this age, retinal morphology (Fig. 5) and expression of photoreceptor-specific genes (Fig. 3) were still similar to controls. In addition, scotopic and photopic b-wave amplitudes in 17-week-old knockdowns were indistinguishable from controls, suggesting that also at 10 weeks, retinal function was comparable. Mice were exposed to 13′000 lux of white light for 1 hour and retinal morphology was analyzed and cell death quantified at 36 hours thereafter (Figs. 5A, 5B). At this time point, photoreceptor outer segments were better preserved (Fig. 5A, middle panels, black arrows) and fewer pyknotic nuclei (Fig. 5A, white arrows) were found in the ONL of Vhlflox/flox ;opsin-cre mice. Reduced cell death was confirmed by the ELISA-based quantification of free nucleosomes generated by internuceleosomal DNA cleavage during apoptotic cell death. Although variable within groups, light-exposed Vhlflox/flox ;opsin-cre mice showed significantly less cell death compared to their control littermates and to mice expressing solely opsin-cre (Fig. 5B). 
Figure 5.
 
Transient photoreceptor protection in Vhlflox/flox;opsin-cre mice. (A) Morphologic sections of dark controls (DC), and at 36 hours (+36h) and 10 days (+10d) after light exposure from retinas of 10-week-old Vhlflox/flox and Vhlflox/flox ;opsin-cre mice. Shown are representative sections of the lower central retina, the most affected region in our light damage setup. Arrows indicate the layer of photoreceptor segments; arrowheads show examples of pyknotic nuclei. (B) Quantification of retinal cell death at 36 hours after light exposure of opsin-cre (black bars), Vhlflox/flox (white bars) and Vhlflox/flox;opsin-cre mice (gray bars). Values are presented as box blots representing the median and the lower and upper quartiles. Whiskers show the complete range of all data points. Values were calculated relative to the value of a positive control (provided by the kit), which was set to 100%. n = 1 (DC, opsin-cre); n = 7 (DC, Vhlflox/flox ); n = 5 (DC, Vhlflox/flox ;opsin-cre); n = 4 (+36h, opsin-cre); n = 9 (+36h, Vhlflox/flox ); n = 6 (+36h, Vhlflox/flox ;opsin-cre). (C) Relative rhodopsin levels of retinas at 10 days after light exposure (LE) in Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre mice (gray bars). Values are expressed relative to the corresponding dark controls (DC), which were set to 100%. Shown are mean values ± SD of six retinas. (D) Basal rhodopsin levels in retinas of dark-adapted and not light-exposed opsin-cre (black bar), Vhlflox/flox (white bar) and Vhlflox/flox ;opsin-cre (gray bar) mice. Values are expressed relative to the values of Vhlflox/flox mice, which were set to 100%. Shown are mean values ± SD of six retinas. *P < 0.05; **P < 0.01. Significance was calculated using an unpaired t-test. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 50 μm.
Figure 5.
 
Transient photoreceptor protection in Vhlflox/flox;opsin-cre mice. (A) Morphologic sections of dark controls (DC), and at 36 hours (+36h) and 10 days (+10d) after light exposure from retinas of 10-week-old Vhlflox/flox and Vhlflox/flox ;opsin-cre mice. Shown are representative sections of the lower central retina, the most affected region in our light damage setup. Arrows indicate the layer of photoreceptor segments; arrowheads show examples of pyknotic nuclei. (B) Quantification of retinal cell death at 36 hours after light exposure of opsin-cre (black bars), Vhlflox/flox (white bars) and Vhlflox/flox;opsin-cre mice (gray bars). Values are presented as box blots representing the median and the lower and upper quartiles. Whiskers show the complete range of all data points. Values were calculated relative to the value of a positive control (provided by the kit), which was set to 100%. n = 1 (DC, opsin-cre); n = 7 (DC, Vhlflox/flox ); n = 5 (DC, Vhlflox/flox ;opsin-cre); n = 4 (+36h, opsin-cre); n = 9 (+36h, Vhlflox/flox ); n = 6 (+36h, Vhlflox/flox ;opsin-cre). (C) Relative rhodopsin levels of retinas at 10 days after light exposure (LE) in Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre mice (gray bars). Values are expressed relative to the corresponding dark controls (DC), which were set to 100%. Shown are mean values ± SD of six retinas. (D) Basal rhodopsin levels in retinas of dark-adapted and not light-exposed opsin-cre (black bar), Vhlflox/flox (white bar) and Vhlflox/flox ;opsin-cre (gray bar) mice. Values are expressed relative to the values of Vhlflox/flox mice, which were set to 100%. Shown are mean values ± SD of six retinas. *P < 0.05; **P < 0.01. Significance was calculated using an unpaired t-test. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 50 μm.
At 10 days after light exposure, however, photoreceptors in Vhlflox/flox;opsin-cre mice had degenerated to a similar extent as in Vhlflox/flox controls (Fig. 5A, bottom panels). Accordingly, both genotypes showed a comparable reduction in rhodopsin levels (as a measure for surviving photoreceptors) to 58% (Vhlflox/flox ) and 52% (Vhlflox/flox ;opsin-cre) of their respective dark control levels, which were set to 100% (Fig. 5C). This suggests that the knockdown of Vhl delayed but did not prevent photoreceptor degeneration after light exposure. We noted that basal levels of dark-adapted rhodopsin levels (before light exposure) were reduced in untreated Vhl knockdown animals to 76% of the levels found in control Vhlflox/flox or opsin-cre mice (Fig. 5D). Rhodopsin regeneration, however, was comparable to that in wild-type and reached 8 pmol/min (data not shown). This value is relevant for light damage susceptibility and close to data published for pigmented wild-type animals expressing the RPE65-450Leu variant. 14 Similarly, expression of Rho RNA was not affected (Fig. 3E), and intracellular localization of rod opsin was normal (determined by immunofluorescence according to 22 ; data not shown) in the knockdown. Thus, the reason for the reduced basal levels of rhodopsin in Vhl knockdown retinas is unclear. 
Because protection was only transient and not permanent, as observed after hypoxic preconditioning, mechanisms of protection might be different in Vhl knockdowns and the hypoxic wild-type retina. To address potential mechanisms for the transient protection against degeneration, we analyzed expression levels of several genes known to be involved in the retinal response to injury or stress. Of particular interest were genes implicated in the protective pathway controlled by leukemia inhibitory factor (LIF) after light exposure. 23,24 Basal expression of several genes downstream of LIF (Fgf2, Edn2, Stat3, Socs3, and Gfap) was higher in knockdown animals (Fig. 6, triangles) than in controls (Fig. 6, squares). Basal expression of Cntf and Lif itself was not affected. After light exposure, however, expression levels of all these genes converged in the two strains and already at 12 hours after exposure, levels were no longer significantly different. The expression pattern of the individual genes after light exposure in wild-type mice is in accordance with data published previously. 24,25  
Figure 6.
 
Gene expression after light exposure. Relative gene expression levels in retinas of 10-week-old Vhlflox/flox (squares, blue) and Vhlflox/flox ;opsin-cre (triangles, red) mice before (dark) or at 12, 24, 36, and 48 hours after exposure to 13′000 lux of white light for 1 hour. Values are expressed relative to values of dark-adapted Vhlflox/flox mice which were set to 1. Shown are mean ± SD values of three mice. Expression in the two strains were compared at individual time points using a Student's t-test. *P < 0.05; **P < 0.01.
Figure 6.
 
Gene expression after light exposure. Relative gene expression levels in retinas of 10-week-old Vhlflox/flox (squares, blue) and Vhlflox/flox ;opsin-cre (triangles, red) mice before (dark) or at 12, 24, 36, and 48 hours after exposure to 13′000 lux of white light for 1 hour. Values are expressed relative to values of dark-adapted Vhlflox/flox mice which were set to 1. Shown are mean ± SD values of three mice. Expression in the two strains were compared at individual time points using a Student's t-test. *P < 0.05; **P < 0.01.
Discussion
Hypoxic preconditioning protects photoreceptors against light-induced degeneration. 2 The exact mechanisms are not known but may include differential regulation of neuroprotective genes. Hypoxia-inducible transcription factors such as HIF1 and HIF2 are activated during hypoxic preconditioning in the whole retina 10 and the product of at least one of their target genes, EPO, has been shown to protect photoreceptors. 2,26 Here we show that lack of VHL induced a sustained activation of HIF transcription factors in normoxic photoreceptors that led to a hypoxia-like response and provided protection against light-induced cell death. However, this protection was only transient and did not reach the level observed after hypoxic preconditioning. 
Knockdown of Vhl in rod photoreceptors stabilized HIF1A and HIF2A and induced phosphorylation of STAT3, leading to the increased expression of a set of target genes that were also activated in the wild-type retina after exposure to hypoxia (Fig. 2). Because the knockdown of Vhl was specific to rod photoreceptors, our data suggest that expression of these genes was regulated in rods by HIFs and/or pSTAT3. Epo was the only tested gene that was not induced in Vhlflox/flox ;opsin-cre mice. Because lack of VHL is sufficient to induce Epo expression in the retina, as shown in Vhlflox/flox ;α-cre mice, 22 our results suggest that upregulation of Epo in hypoxic wild-type mice may not occur in rods but in other cell types of the retina. Alternative explanations include the possibility that Epo expression in rods is not regulated by one of the factors activated here. 
Although retinas of Vhlflox/flox ;opsin-cre mice had a normal retinal morphology and function, levels of rhodopsin were reduced. This cannot be explained by a reduced number of photoreceptor cells because expression of rod and cone marker genes were normal in 10-week-old Vhlflox/flox ;opsin-cre mice. Rhodopsin regeneration was also not affected and reached similar kinetics as in wild-type control mice. In addition, we did not detect mislocalization of rod opsin in retinas of Vhlflox/flox ;opsin-cre mice (data not shown). Thus, the reason for the reduced rhodopsin levels remains unclear and warrants further investigation. 
Transient Protection against Light Damage
Knockdown of Vhl in rods resulted in a transient protection (or delayed degeneration) of photoreceptor cells against light damage. Although light damage depends on light absorption by the visual pigment, 27 29 light damage susceptibility is determined by rhodopsin regeneration kinetics rather than by the absolute levels of dark-adapted rhodopsin. 14,30 Because regeneration rates were identical in both wild-type and Vhl knockdown mice, protection was thus not mediated by differences in photon absorption. 
The delayed degeneration suggests that rods lacking VHL have activated some mechanisms that can postpone but not prevent the death of injured photoreceptors. A potential factor involved in this process may be FGF2, which was shown previously to be neuroprotective for photoreceptors. 31,32 The increased basal expression of Fgf2 in Vhlflox/flox ;opsin-cre mice before light exposure (Fig. 6) may support survival of photoreceptors initially. After light exposure, however, expression levels were similar to levels in light-exposed control mice. Because photoreceptors in control mice degenerate, such levels are obviously not sufficient for a long-term rescue of injured photoreceptors. Interestingly, Lif knockout and wild- type mice have similar Fgf2 expression levels before light exposure, but whereas wild-type mice upregulate Fgf2 expression in response to photoreceptor injury, Lif knockouts do not. 25 As a consequence, Lif knockouts show a light damage severity similar to that of wild-type mice early (unpublished data) but a stronger degeneration late (10 days) after exposure. 25 Therefore, the expression profile of Fgf2 correlates with degeneration severity early or late after photoreceptor injury. 
The mechanism leading to increased expression of Fgf2 in the absence of VHL needs to be determined. However, Vhl knockdown mice also have increased basal levels of Edn2 (Fig. 6), and we recently showed that activation of the receptor for Edn2 (EDNRB) induces Fgf2 expression. 23 Thus, it might be that the increased levels of HIF1 in the Vhl knockdown retinas upregulate expression of Edn2 in photoreceptors, as shown in other systems. 33 EDN2 may then cause induction of Fgf2 via activation of EDNRB. Thus, increased expression of Fgf2 may be a consequence of the increased Edn2 expression through HIF1A stabilization in rods of the Vhl knockdown mice. Alternatively, lack of VHL might also directly regulate Edn2, as suggested for Edn1. 34  
The restriction of the hypoxia-like response to rod photoreceptors might be another reason for the lack of full protection in the Vhlflox/flox ;opsin-cre mice. In contrast to the knockdown mice, hypoxic preconditioning induces a systemic response and may thus involve factors induced in other cell types of the retina. Epo, for example, is strongly upregulated in the hypoxic retina and can protect photoreceptors against light damage 2 and in the rds mouse. 26 Epo, however, was not upregulated in the Vhlflox/flox ;opsin-cre knockdown mice. Thus, at least one neuroprotective factor contributing to protection after hypoxic preconditioning was missing. The complete protection of photoreceptors in Vhl knockdown mice after hypoxic exposure (data not shown) suggests that retinas lacking VHL in rods were still able to react to acute hypoxia and further supports the conclusion that mechanisms in addition to rod-specific activation of HIF and STAT3 transcription factors are required for full protection. 
Thus, the rod-specific knockdown of Vhl mimics the response of the retina to hypoxic preconditioning only partially. Whether or not a more complete retinal knockdown of Vhl would result in increased protection cannot be tested because generation of such mice would require an inactivation of Vhl in all cells of the adult retina. Inactivation of Vhl already during retinal development (e.g., by using the α-cre mouse) results in severe abnormalities of the retinal vasculature and strong retinal degeneration. 22,35 Because long-term lack of VHL and/or activation of HIF transcription factors leads to retinal degeneration (Fig. 4), the Vhl knockdown may not only trigger a protective reaction but simultaneously may also alter the physiology of rods and/or of the cellular environment, leading to a retinal stress counteracting the protection. Hence, only a short hypoxic period may be tolerated by the retina and be protective. Long-term hypoxia 36 or a long-term hypoxia-like response (this work) may lead to degeneration. 
In summary, we show that a photoreceptor-specific Vhl knockdown induced a hypoxia-like response that protected photoreceptors against light-induced cell death in 10-week-old mice. However, protection was only transient and did not reach the level detected after hypoxic preconditioning. We hypothesize that the full and sustained protection of photoreceptors after hypoxic exposure requires regulation of factors in addition to the ones observed here, and/or that hypoxia-mediated protection of photoreceptors is controlled mainly by other retinal cells in a paracrine fashion. 
Footnotes
 Supported by Swiss National Science Foundation (Grant 3100A0-117760), Deutsche Forschungsgemeinschaft (DFG, Grants Se837/5-2, Se837/6-1, Se837/7-1), German Ministry of Education and Research (BMBF, Grant 0314106), Fritz Tobler Foundation, and H. Messerli Foundation.
Footnotes
 Disclosure: C. Lange, None; S.R. Heynen, None; N. Tanimoto, None; M. Thiersch, None; Y.-Z. Le, None; I. Meneau, None; M.W. Seeliger, None; M. Samardzija, None; C. Caprara, None; C. Grimm, None
The authors thank Coni Imsand and Hedwig Wariwoda for excellent technical assistance. 
References
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. [CrossRef] [PubMed]
Grimm C Wenzel A Groszer M . HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med. 2002;8(7):718–724. [CrossRef] [PubMed]
Webb JD Coleman ML Pugh CW . Hypoxia, hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing. Cell Mol Life Sci. 2009;66(22):3539–3554. [CrossRef] [PubMed]
Iwai K Yamanaka K Kamura T . Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci USA. 1999;96(22):12436–12441. [CrossRef] [PubMed]
Kamura T Koepp DM Conrad MN . Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science. 1999;284(5414):657–661. [CrossRef] [PubMed]
Kibel A Iliopoulos O DeCaprio JA Kaelin WGJr . Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science. 1995;269(5229):1444–1446. [CrossRef] [PubMed]
Ohh M Park CW Ivan M . Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol. 2000;2(7):423–427. [CrossRef] [PubMed]
Mahon PC Hirota K Semenza GL . FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001;15(20):2675–2686. [CrossRef] [PubMed]
Jaakkola P Mole DR Tian YM . Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–472. [CrossRef] [PubMed]
Thiersch M Lange C Joly S Heynen S Le YZ Samardzija M Grimm C . Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors. Eur J Neurosci. 2009;29(12):2291–2302. [CrossRef] [PubMed]
Thiersch M Raffelsberger W Frigg R Samardzija M Wenzel A Poch O Grimm C . Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection. BMC Genomics. 2008;9:73. [CrossRef] [PubMed]
Haase VH Glickman JN Socolovsky M Jaenisch R . Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor. Proc Natl Acad Sci USA. 2001;98(4):1583–1588. [CrossRef] [PubMed]
Le YZ Zheng L Zheng W Ash JD Agbaga MP Zhu M Anderson RE . Mouse opsin promoter-directed Cre recombinase expression in transgenic mice. Mol Vis. 2006;12:389–398. [PubMed]
Wenzel A Reme CE Williams TP Hafezi F Grimm C . The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J Neurosci. 2001;21(1):53–58. [PubMed]
Seeliger MW Grimm C Stahlberg F . New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis. Nat Genet. 2001;29(1):70–74. [CrossRef] [PubMed]
Tanimoto N Muehlfriedel RL Fischer MD Fahl E Humphries P Biel M Seeliger MW . Vision tests in the mouse: functional phenotyping with electroretinography. Front Biosci. 2009;14:2730–2737. [CrossRef]
Jung JE Lee HG Cho IH . STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells. FASEB J. 2005;19(10):1296–1298. [PubMed]
Garayoa M Martinez A Lee S . Hypoxia-inducible 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. [CrossRef] [PubMed]
Warnecke C Weidemann A Volke M . The specific contribution of hypoxia-inducible factor-2alpha to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors. Exp Cell Res. 2008;314(10):2016–2027. [CrossRef] [PubMed]
Zhang H Bosch-Marce M Shimoda LA . Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem. 2008;283(16):10892–10903. [CrossRef] [PubMed]
Haase VH . Hypoxic regulation of erythropoiesis and iron metabolism. Am J Physiol Renal Physiol. 2010;299(1):F1–13. [CrossRef] [PubMed]
Lange C Caprara C Tanimoto N . Retina-specific activation of a sustained hypoxia-like response leads to severe retinal degeneration and loss of vision. Neurobiol Dis. 2011;41(1):119–130. [CrossRef] [PubMed]
Joly S Lange C Thiersch M Samardzija M Grimm C . Leukemia inhibitory factor extends the lifespan of injured photoreceptors in vivo. J Neurosci. 2008;28(51):13765–13774. [CrossRef] [PubMed]
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. [CrossRef] [PubMed]
Burgi S Samardzija M Grimm C . Endogenous leukemia inhibitory factor protects photoreceptor cells against light-induced degeneration. Mol Vis. 2009;15:1631–1637. [PubMed]
Rex TS Wong Y Kodali K Merry S . Neuroprotection of photoreceptors by direct delivery of erythropoietin to the retina of the retinal degeneration slow mouse. Exp Eye Res. 2009;89(5):735–740. [CrossRef] [PubMed]
Grimm C Wenzel A Hafezi F Yu S Redmond TM Reme CE . Protection of Rpe65-deficient mice identifies rhodopsin as a mediator of light-induced retinal degeneration. Nat Genet. 2000;25(1):63–66. [CrossRef] [PubMed]
Noell WK Albrecht R . Irreversible effects on visible light on the retina: role of vitamin A. Science. 1971;172(978):76–79. [CrossRef] [PubMed]
Williams TP Howell WL . Action spectrum of retinal light-damage in albino rats. Invest Ophthalmol Vis Sci. 1983;24(3):285–287. [PubMed]
Keller C Grimm C Wenzel A Hafezi F Reme C . Protective effect of halothane anesthesia on retinal light damage: inhibition of metabolic rhodopsin regeneration. Invest Ophthalmol Vis Sci. 2001;42(2):476–480. [PubMed]
O'Driscoll C O'Connor J O'Brien CJ Cotter TG . Basic fibroblast growth factor-induced protection from light damage in the mouse retina in vivo. J Neurochem. 2008;105(2):524–536. [CrossRef] [PubMed]
Faktorovich EG Steinberg RH Yasumura D Matthes MT LaVail MM . Basic fibroblast growth factor and local injury protect photoreceptors from light damage in the rat. J Neurosci. 1992;12(9):3554–3567. [PubMed]
Na G Bridges PJ Koo Y Ko C . Role of hypoxia in the regulation of periovulatory EDN2 expression in the mouse. Can J Physiol Pharmacol. 2008;86(6):310–319. [CrossRef] [PubMed]
Wykoff CC Pugh CW Maxwell PH Harris AL Ratcliffe PJ . Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling. Oncogene. 2000;19(54):6297–6305. [CrossRef] [PubMed]
Kurihara T Kubota Y Ozawa Y . von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina. Development. 2010;137(9):1563–1571. [CrossRef] [PubMed]
Neubauer JA . Invited review: physiological and pathophysiological responses to intermittent hypoxia. J Appl Physiol. 2001;90(4):1593–1599. [PubMed]
Figure 1.
 
Knockdown of Vhl in photoreceptors of 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Expression pattern of Cre recombinase in ROSA-flox-RFP;opsin-Cre mice. Red, red fluorescent protein, expressed only in cells where Cre was active. Scale bar, 100 μm. (B) higher magnification of the section shown in (A). (C) PCR amplification of genomic DNA isolated from total retinal tissue of three different Vhlflox/flox (CRE–) and three Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. The amplified fragment from the floxed gene (floxed) has a length of 460 bp. CRE-mediated excision of the floxed sequence (excised) resulted in a 260-bp fragment. (D) Semiquantitative real-time PCR after laser capture microdissection showing relative Vhl mRNA levels in the different retinal layers (as indicated) of Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice. Values were normalized to Actb and expressed relatively to the respective values for each layer in Vhlflox/flox mice, which were set to 1. Shown are mean values ± SD of three different mice amplified in duplicates. ONH, optic nerve head; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 1.
 
Knockdown of Vhl in photoreceptors of 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Expression pattern of Cre recombinase in ROSA-flox-RFP;opsin-Cre mice. Red, red fluorescent protein, expressed only in cells where Cre was active. Scale bar, 100 μm. (B) higher magnification of the section shown in (A). (C) PCR amplification of genomic DNA isolated from total retinal tissue of three different Vhlflox/flox (CRE–) and three Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. The amplified fragment from the floxed gene (floxed) has a length of 460 bp. CRE-mediated excision of the floxed sequence (excised) resulted in a 260-bp fragment. (D) Semiquantitative real-time PCR after laser capture microdissection showing relative Vhl mRNA levels in the different retinal layers (as indicated) of Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice. Values were normalized to Actb and expressed relatively to the respective values for each layer in Vhlflox/flox mice, which were set to 1. Shown are mean values ± SD of three different mice amplified in duplicates. ONH, optic nerve head; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 2.
 
Stabilization and activation of transcription factors in 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Western blot analysis showing protein expression of HIF1A, HIF2A, phosphorylated STAT3 (p-STAT3), and STAT3 in four different Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. ACTB served as a control for equal loading. (B) Total retinal RNA was prepared from 10-week-old Vhlflox/flox ;opsin-cre mice and expression levels of individual genes (as indicated) were determined by semiquantitative real-time PCR. Levels were normalized to Actb and expressed relatively to the expression in Vhlflox/flox control mice (set to 1). (C) Total retinal RNA was prepared from wild-type mice after hypoxic preconditioning. Expression levels were normalized to Actb and expressed relatively to expression in normoxic control mice (set to 1). Shown are mean values ± SD of four retinas amplified in duplicates.
Figure 2.
 
Stabilization and activation of transcription factors in 10-week-old Vhlflox/flox ;opsin-cre mice. (A) Western blot analysis showing protein expression of HIF1A, HIF2A, phosphorylated STAT3 (p-STAT3), and STAT3 in four different Vhlflox/flox (CRE–) and Vhlflox/flox ;opsin-cre (CRE+) mice at 10 weeks of age. ACTB served as a control for equal loading. (B) Total retinal RNA was prepared from 10-week-old Vhlflox/flox ;opsin-cre mice and expression levels of individual genes (as indicated) were determined by semiquantitative real-time PCR. Levels were normalized to Actb and expressed relatively to the expression in Vhlflox/flox control mice (set to 1). (C) Total retinal RNA was prepared from wild-type mice after hypoxic preconditioning. Expression levels were normalized to Actb and expressed relatively to expression in normoxic control mice (set to 1). Shown are mean values ± SD of four retinas amplified in duplicates.
Figure 3.
 
Normal retinal function in 17-week-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 17-week-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the time point of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Semiquantitative real-time PCR for photoreceptor-specific genes (as indicated) in 10-week-old Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre (gray bars). Values were normalized to Actb and expressed relatively to the value of Vhlflox/flox mice, which was set to 1. Shown are mean values ± SD of four animals amplified in duplicates.
Figure 3.
 
Normal retinal function in 17-week-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 17-week-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the time point of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Semiquantitative real-time PCR for photoreceptor-specific genes (as indicated) in 10-week-old Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre (gray bars). Values were normalized to Actb and expressed relatively to the value of Vhlflox/flox mice, which was set to 1. Shown are mean values ± SD of four animals amplified in duplicates.
Figure 4.
 
Retinal degeneration in 1-year-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 1-year-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the timing of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Representative morphologic sections from the lower central retina of 1-year-old Vhlflox/flox , of opsin-cre and of Vhlflox/flox ;opsin-cre mice. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.
Figure 4.
 
Retinal degeneration in 1-year-old Vhlflox/flox ;opsin-cre mice. (A) Representative scotopic (dark-adapted) and (B) photopic (light-adapted) single-flash ERGs with increasing light intensities recorded from 1-year-old Vhlflox/flox control and Vhlflox/flox ;opsin-cre mice as indicated. The vertical line crossing each trace shows the timing of the light flash. (C) Scotopic and (D) photopic b-wave amplitudes from control (black, n = 3) and Vhlflox/flox ;opsin-cre mice (red, n = 3) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quartile range, whiskers indicate the 5% and 95% quantiles, and the asterisks indicate the median of the data. (E) Representative morphologic sections from the lower central retina of 1-year-old Vhlflox/flox , of opsin-cre and of Vhlflox/flox ;opsin-cre mice. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.
Figure 5.
 
Transient photoreceptor protection in Vhlflox/flox;opsin-cre mice. (A) Morphologic sections of dark controls (DC), and at 36 hours (+36h) and 10 days (+10d) after light exposure from retinas of 10-week-old Vhlflox/flox and Vhlflox/flox ;opsin-cre mice. Shown are representative sections of the lower central retina, the most affected region in our light damage setup. Arrows indicate the layer of photoreceptor segments; arrowheads show examples of pyknotic nuclei. (B) Quantification of retinal cell death at 36 hours after light exposure of opsin-cre (black bars), Vhlflox/flox (white bars) and Vhlflox/flox;opsin-cre mice (gray bars). Values are presented as box blots representing the median and the lower and upper quartiles. Whiskers show the complete range of all data points. Values were calculated relative to the value of a positive control (provided by the kit), which was set to 100%. n = 1 (DC, opsin-cre); n = 7 (DC, Vhlflox/flox ); n = 5 (DC, Vhlflox/flox ;opsin-cre); n = 4 (+36h, opsin-cre); n = 9 (+36h, Vhlflox/flox ); n = 6 (+36h, Vhlflox/flox ;opsin-cre). (C) Relative rhodopsin levels of retinas at 10 days after light exposure (LE) in Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre mice (gray bars). Values are expressed relative to the corresponding dark controls (DC), which were set to 100%. Shown are mean values ± SD of six retinas. (D) Basal rhodopsin levels in retinas of dark-adapted and not light-exposed opsin-cre (black bar), Vhlflox/flox (white bar) and Vhlflox/flox ;opsin-cre (gray bar) mice. Values are expressed relative to the values of Vhlflox/flox mice, which were set to 100%. Shown are mean values ± SD of six retinas. *P < 0.05; **P < 0.01. Significance was calculated using an unpaired t-test. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 50 μm.
Figure 5.
 
Transient photoreceptor protection in Vhlflox/flox;opsin-cre mice. (A) Morphologic sections of dark controls (DC), and at 36 hours (+36h) and 10 days (+10d) after light exposure from retinas of 10-week-old Vhlflox/flox and Vhlflox/flox ;opsin-cre mice. Shown are representative sections of the lower central retina, the most affected region in our light damage setup. Arrows indicate the layer of photoreceptor segments; arrowheads show examples of pyknotic nuclei. (B) Quantification of retinal cell death at 36 hours after light exposure of opsin-cre (black bars), Vhlflox/flox (white bars) and Vhlflox/flox;opsin-cre mice (gray bars). Values are presented as box blots representing the median and the lower and upper quartiles. Whiskers show the complete range of all data points. Values were calculated relative to the value of a positive control (provided by the kit), which was set to 100%. n = 1 (DC, opsin-cre); n = 7 (DC, Vhlflox/flox ); n = 5 (DC, Vhlflox/flox ;opsin-cre); n = 4 (+36h, opsin-cre); n = 9 (+36h, Vhlflox/flox ); n = 6 (+36h, Vhlflox/flox ;opsin-cre). (C) Relative rhodopsin levels of retinas at 10 days after light exposure (LE) in Vhlflox/flox (white bars) and Vhlflox/flox ;opsin-cre mice (gray bars). Values are expressed relative to the corresponding dark controls (DC), which were set to 100%. Shown are mean values ± SD of six retinas. (D) Basal rhodopsin levels in retinas of dark-adapted and not light-exposed opsin-cre (black bar), Vhlflox/flox (white bar) and Vhlflox/flox ;opsin-cre (gray bar) mice. Values are expressed relative to the values of Vhlflox/flox mice, which were set to 100%. Shown are mean values ± SD of six retinas. *P < 0.05; **P < 0.01. Significance was calculated using an unpaired t-test. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 50 μm.
Figure 6.
 
Gene expression after light exposure. Relative gene expression levels in retinas of 10-week-old Vhlflox/flox (squares, blue) and Vhlflox/flox ;opsin-cre (triangles, red) mice before (dark) or at 12, 24, 36, and 48 hours after exposure to 13′000 lux of white light for 1 hour. Values are expressed relative to values of dark-adapted Vhlflox/flox mice which were set to 1. Shown are mean ± SD values of three mice. Expression in the two strains were compared at individual time points using a Student's t-test. *P < 0.05; **P < 0.01.
Figure 6.
 
Gene expression after light exposure. Relative gene expression levels in retinas of 10-week-old Vhlflox/flox (squares, blue) and Vhlflox/flox ;opsin-cre (triangles, red) mice before (dark) or at 12, 24, 36, and 48 hours after exposure to 13′000 lux of white light for 1 hour. Values are expressed relative to values of dark-adapted Vhlflox/flox mice which were set to 1. Shown are mean ± SD values of three mice. Expression in the two strains were compared at individual time points using a Student's t-test. *P < 0.05; **P < 0.01.
Table 1.
 
Primers Used for Real-Time PCR
Table 1.
 
Primers Used for Real-Time PCR
Gene Upstream (5′–3′) Downstream (5′–3′) Annealing Temperature (°C) Product (bp)
Adm ttcgcagttccgaaagaagt ggtagctgctggatgcttgta 62 77
Actb cgacatggagaagatctggc caacggctccggcatgtgc 62 153
Bnip3 cctgtcgcagttgggttc gaagtgcagttctacccaggag 60 93
Egln1 cattgttggcagaaggtgtg caaaggactacagggtctcca 62 70
Epo gccctgctagccaattcc gccctgctagccaattcc 60 128
Gnat1 gaggatgctgagaaggatgc tgaatgttgagcgtggtcat 58 209
Gnat2 gcatcagtgctgaggacaaa ctaggcactcttcgggtgag 58 192
Mt1 gaatggaccccaactgctc gcagcagctcttcttgcag 62 104
Mt2 tgtacttcctgcaagaaaagctg acttgtcggaagcctctttg 62 94
Sag ttacaagccttccaacctctgac accagcacaacaccatctacag 64 189
Scl2a1 cagtgtatcctgttgcccttctg gccgaccctcttctttcatctc 62 151
Rho cttcacctggatcatggcgtt ttcgttgttgacctcaggcttg 62 130
Vegfa acttgtgttgggaggaggatgtc aatgggtttgtcgtgtttctgg 60 171
Vhl gagggacccgttccaataat ttggcaaaaataggctgtcc 60 364
Fgf2 tgtgtctatcaagggagtgtgtgc accaactggagtatttccgtgaccg 62 158
Edn2 agacctcctccgaaagctg ctggctgtagctggcaaag 60 64
Stat3 caaaaccctcaagagccaagg tcactcacaatgcttctccgc 62 133
Gfap ccaccaaactggctgatgtctac ttctctccaaatccacacgagc 62 240
Socs3 atttcgcttcgggactagc aacttgctgtgggtgaccat 58 126
Lif aatgccacctgtgccatacg caacttggtcttctctgtcccg 60 216
Cntf ctctgtagccgctctatctg ggtacaccatccactgagtc 58 125
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×