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(Investigative Ophthalmology and Visual Science. 2005;46:1010-1016.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0788
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Retinal Disease in Mice Lacking Hypoxia-Inducible Transcription Factor-2{alpha}

Kan Ding,1 Marzia Scortegagna,1 Robyn Seaman,2 David G. Birch,2,3 and Joseph A. Garcia1

1From the Departments of Internal Medicine and 3Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas; and the 2Retina Foundation of the Southwest, Dallas, Texas.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To characterize ocular disease in HIF-2{alpha}-null mice.

METHODS. Histologic, electroretinographic (ERG), and molecular studies were performed on samples obtained from age- and gender-matched HIF-2{alpha}-null (HIF-2{alpha}-KO), HIF-2{alpha}-heterozygous (HIF-2{alpha}-HET), and wild-type (WT) littermate mice.

RESULTS. HIF-2{alpha}-KO mice exhibited marked thinning of the retina and abnormal retinal vasculature. The pathologic changes in HIF-2{alpha}-KO mice were associated with a virtual absence of postreceptor function. The expression of a surrogate marker for HIF-2{alpha} mRNA localized to vascular endothelial, amacrine, and retinal pigment epithelial (RPE) cells. Several HIF-2{alpha} target genes involved in angiogenesis, retinal protection, and stress responses have altered expression patterns in HIF-2{alpha}-KO retinas.

CONCLUSIONS. HIF-2{alpha}-KO mice exhibit marked retinopathy consistent with complete loss of vision by 1 month of age. Impaired HIF-2{alpha} signaling in HIF-2{alpha}-KO mice likely produces functional deficits in cell types in which HIF-2{alpha} normally is expressed, ultimately resulting in retinopathy. Future studies will address whether the molecular abnormalities described in this study are directly responsible for the retinal disease in HIF-2{alpha}-KO mice.

The retina is among the most metabolically active tissues in the body, in part due to rods, which constantly shed their outer segment tips, and to the retinal pigment epithelium (RPE), which phagocytoses the shed tips. As a consequence of shedding and the active metabolic state associated with light exposure,1 the RPE is exposed to increased oxidative stress from reactive oxygen intermediates (ROIs) generated in vitro2 3 or in vivo.4 Unopposed oxidative stress in the RPE leads to altered mitochondrial biophysical properties,5 mitochondrial DNA damage,6 and/or apoptosis. Mitochondria are not only targets for but are themselves a major source of ROIs within the cell.7 8 Enhanced mitochondrial ROI generation is a causative factor in some mitochondrial disease states. Retinopathy is associated with several mitochondrial disease syndromes.9 Morphologic changes in the retina and the RPE of rodents also occur with aging, another condition associated with mitochondrial dysfunction.10

To alleviate increased oxidative stress, the RPE has nonenzymatic5 11 and enzymatic antioxidant defense mechanisms. The latter include the antioxidant enzymes (AOE) catalase, superoxide dismutases (SODs), and glutathione peroxidases,12 whose function is to eliminate ROI or their derivatives. Expression of several AOE genes is induced with oxidative stress, notably that of SOD2 encoding the mitochondria-localized manganese superoxide dismutase (MnSOD) antioxidant enzyme.13 The induction of AOE gene expression is mediated in part by ROI-responsive signaling pathway(s). Lower levels of AOE gene expression result in oxidative stress-induced pathologic changes. These changes may occur with or without overt mitochondrial dysfunction.14 15 Hence, retinal disease attributable to oxidative stress may involve perturbations in mitochondrial physiology, RPE function, or ROI homeostasis.

Members of the hypoxia inducible factor (HIF) family are activated by hypoxia and increased oxidative stress. Target genes for HIF-1{alpha}, the founding member of the HIF family, include key regulators of glucose and glycolytic metabolism as well as cellular factors involved in angiogenesis.16 17 HIF-2{alpha}, also known as endothelial PAS domain protein 1 (EPAS1), shows many similarities with HIF-1{alpha}.18 However, several molecular, biochemical, and physiologic observations suggest that HIF-1{alpha} and HIF-2{alpha} are not entirely redundant. Data from in vitro studies have implicated HIF-2{alpha} in the control of specific cellular factors involved in angiogenesis. A recent in vivo study demonstrated oxidative stress-induced injury in HIF-2{alpha}-KO mice, suggesting a role for HIF-2{alpha} in antioxidant defense mechanisms.19

HIF-2{alpha}-KO mice closely resemble SOD2-KO mice, a mouse model of oxidative-stress induced mitochondrial disease. Both HIF-2{alpha}-KO and SOD2-KO mice have impaired mitochondrial-dependent metabolism, elevated oxidative stress, and similar multiple organ disease.20 21 22 Molecular profiling of HIF-2{alpha}-KO mice revealed decreased Sod2 gene expression, an abnormal finding given Sod2 expression normally increases in response to increased oxidative stress. HIF-2{alpha} efficiently transactivates the SOD2 and other AOE promoters, establishing a putative role for HIF-2{alpha} in the in vivo response to oxidative stress.19 We reasoned that the absence of HIF-2{alpha} would have a profound effect on retinal structure and function. In this study, we examine the histologic, functional, and molecular consequences of the HIF-2{alpha}-null state on the retina.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice
All data reported in this study were obtained from F1 progeny originating from crosses of heterozygous HIF-2{alpha} mice maintained on a C57/BL6J (male) or 129S6/SvEvTac (female) background. Mice were housed in an institutional animal care facility, maintained on a 12-hour light–dark cycle, and fed standard chow with 11% fat content. All procedures performed in this study were approved by the University of Texas Southwest Medical Center (UTSWMC) institutional animal care and use committee and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Behavior
Open-field activity measurements of four pairs of 1-month-old HIF-2{alpha}-KO (KO) or wild-type (WT) control mice were acquired immediately after introduction into an infrared beam-break activity detector (ENV-510; Med Associates Inc., St. Albans, VT). Mice were acclimated for 2 hours before testing and then were examined under standard light conditions (fluorescent room lighting) for a single 12-minute period beginning 2 to 3 hours into the light cycle.

Histology
Eyes of five independent pairs of 1-month-old mice were examined for histologic studies. For hematoxylin-eosin (H&E) staining, the eyes were enucleated, fixed at 4°C overnight in 4% paraformaldehyde and 1x phosphate-buffered saline (PBS), paraffin-embedded, and sectioned. H&E staining of all samples was performed as WT and HIF-2{alpha}-KO pairs, to minimize variability in staining techniques.

Data from retinal morphometric analysis, acquired in a blinded manner from digital images from five pairs of HIF-2{alpha}-KO and WT mice, was analyzed by t-test.22 The mean retinal thickness was calculated from measurements of six consecutive retinal sections for each eye sample taken in two locations: central retina (within one optic nerve diameter from the optic nerve margin) and peripheral retina (within one 40x field from the anterior margin ora serrata). The parameters measured were thickness of: combined nerve fiber and ganglion layers (NFL/GCL); the inner plexiform (IPL), inner nuclear (INL), outer plexiform (OPL), outer nuclear (ONL), photoreceptor (PR) layers; and the retinal pigment epithelium (RPE).

Retinal vasculature was examined by a modification of the lead sulfide technique for adenosine triphosphatase (ADPase) activity staining.23 For lectin staining, paraffin sections from methyl Carnoy-fixed eyes were stained with biotinylated Griffonia simplicifolia lectin B4 (Vector Laboratories, Burlingame, CA) and streptavidin-horseradish peroxidase (HRP; Vector Laboratories). For ß-galactosidase staining, eye samples from HIF-2{alpha}-HET mice were lightly fixed in 4% paraformaldehyde and 1x PBS, incubated with staining buffer at room temperature for 24 hours, rinsed with PBS, and either flatmounted or paraffin embedded, sectioned, and counterstained for retinal cross sections.

Electrophysiology
Five independent pairs of age-matched (4–5 weeks old, designated as 1 month old), gender-matched WT and HIF-2{alpha}-KO mice were examined for ERG analysis.24 Subsequently, seven HIF-2{alpha}-HET mice (6–8 weeks old, designated as 2 months old) were tested, along with six additional WT mice (also 6–8 weeks old, designated as 2 months old). Mice were dark-adapted overnight before testing. Eyes were dilated with scopolamine hydrobromide (Hyoscine) the day before examination. For ERG analyses, mice were anesthetized with tribromoethanol (Avertin; Sandofi Winthrop Laboratories, New York, NY), a topical corneal anesthetic (1% proparacaine hydrochloride) was administered, and eyes were moistened with 1% carboxymethylcellulose sodium. A gold-wire loop placed on the cornea served as the active electrode and was referenced to a gold-wire loop placed in the mouth. A needle electrode placed in the tail served as the ground electrode. Body temperature was maintained at 37.5°C, using a heating pad and continuously monitoring with a rectal probe. Signals were amplified (AM502 differential amplifier; x10,000; 3 dB down at 2 and 10,000 Hz; Tektronix, Beaverton, OR) before analog–digital conversion. PC-based custom software was used for stimulus control and timing, data acquisition, averaging, and analysis.

Full-field ERGs were obtained in a Ganzfeld dome. Responses to flashes from near-threshold (–3.0 log scotopic td-s) to 1.3 log scotopic td-s were elicited by a photostimulator unit (Grass-Telefactor, West Warwick, RI). A xenon gas tube driven by a 1600-W/s power pack was used to produce achromatic flashes ranging from 1.8 to 3.7 log scotopic td-s. Calibrated apertures of various diameters were used to control the energy of the flash. A series of cone b-waves were elicited by stimuli ranging from 0.6 to 1.8 log scotopic td-s (–0.6 to +0.6 log cd/m2) in the presence of a 40-cd/m2 background.

Molecular Biology
Quantitative real-time RT-PCR identified changes in gene expression of candidate genes on a sequence detection system and software (7000 Prism; Applied Biosystems, Inc. [ABI], Foster City, CA). Gene-specific primers were used to examine known HIF-1 target genes, angiogenesis related genes, erythropoietin signaling genes, primary AOE genes, and a housekeeping gene for the internal standard (Actb). Expression was compared using the threshold cycle method normalized to ß-actin. The data are the overall mean of three to five set means, each generated from triplicate real-time RT-PCR data points. Each independent set consists of two different RNA pools, WT and HIF-2{alpha}-KO, with each pool generated from the tissues of three 1-month-old mice matched for genotype, gender, age and litter.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
While assessing overall locomotion levels of HIF-2{alpha}-KO mice, we noticed unusual activity patterns. In the open-field test, HIF-2{alpha}-KO mice avoided crossing the center portion of the open-field apparatus and instead almost exclusively limited their activity to the periphery (Fig. 1) . Although open-field activity assays are not routinely used in evaluating visually impaired mice, we reasoned impaired visual acuity might be a cause for such behavior in HIF-2{alpha}-KO mice, given the coexistence of retinopathy in these mice.19 Histologic studies were performed to gain a better determination of the retinopathy in HIF-2{alpha}-KO mice.



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  		FIGURE 1. Open-field activity measurements of WT or HIF-2{alpha}-KO mice performed over a 12-minute period under standard light conditions. Note that the HIF-2{alpha}-KO mouse avoided crossing the center of the field, whereas the WT mouse crossed this region multiple times. This pattern of center avoidance was typical of HIF-2{alpha}-KO mice, even of HIF-2{alpha}-KO mice with activity levels closer to those of WT mice.

 
Examination of HIF-2{alpha}-KO retinal cross sections revealed dramatically reduced thickness, particularly in the periphery (Figs. 2A 2B) . Higher magnification demonstrated marked abnormalities in multiple cell layers (Figs. 2C 2D) including decreased PR, OPL, and IPL widths (Fig. 3) , with the extent of the decrease dependent on retinal location and layer. For the NFL/GCL, IPL, INL, and OPL, the reduction in retinal thickness was comparable in the center and periphery. For the ONL and PR layer, however, there was a much greater reduction in the periphery than in the center. The RPE appeared of comparable thickness in HIF-2{alpha}-KO and WT mice at all locations.



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  		FIGURE 2. (A, B) Low- and (C, D) high-magnification views of H&E stained, paraffin-embedded retinal samples from (A, C) WT control or (B, D) HIF-2{alpha}-KO mice. The sections include the lens (L) and optic nerve (ON) for orientation. Note the marked diminution of the IPL located between the GCL and INL as well as diminution of the OPL located between the INL and ONL. The intravitreous vessels in the HIF-2{alpha}-KO sections were segments from a persistent hyaloid artery.

 


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  		FIGURE 3. Retinal thickness parameters of WT or HIF-2{alpha}-KO mice for the retinal layers, as shown. Mean thicknesses are presented along with error bars for SEM. WC, central retinal thickness in WT mice; KC, central retinal thickness in HIF-2{alpha}-KO mice; WP, peripheral retinal thickness in WT mice; KP, peripheral retinal thickness in HIF-2{alpha}-KO mice.

 
Gross examination of eyes from HIF-2{alpha}-KO mice revealed intraocular vessels consistent with a persistent hyaloid artery (Figs. 4A 4B) . Fluorescein angiography indicated gross attenuation and tortuosity of the major retinal vessels with dropout of the capillary bed (data not shown). ADPase staining of retinal flatmounts demonstrated tortuous central retinal vasculature and diminished vasculature in the periphery (Figs. 4C 4D) . A persistent hyaloid artery and related structures characterized higher resolution, lectin-stained preparations (Figs. 5A 5B 5C 5D 5E 5F) . The surrogate marker for HIF-2{alpha} gene expression in this knockout strain, a nuclear-localized form of ß-galactosidase,25 was prominently expressed in the vasculature of HIF-2{alpha}-HET retinas (Fig. 6A) . Retinal flatmount cross sections revealed localization of ß-galactosidase activity in vascular endothelial cells as well as cells within the GCL and the amacrine cell and RPE layers (Fig. 6B) .



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  		FIGURE 4. Gross examinations of eyes (A, B) and adenosine diphosphatase (ADPase) staining of retinal flatmounts (C, D) from (A, C) WT or (B, D) HIF-2{alpha}-KO mice. For the ADPase stained samples, the persistent hyaloid artery in the HIF-2{alpha}-KO retinas was carefully removed by microdissection to visualize the retinal vessels. The ADPase staining, visible as intense black, revealed a markedly abnormal HIF-2{alpha}-KO retinal vascular bed.

 


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  		FIGURE 5. Low- (A, B) and high- (C, F) magnification of lectin-stained eye sections from a (A, C, E) WT or (B, D, F) HIF-2{alpha}-KO mouse. In addition to the reduced thickness of the retina (R), the hyaloid artery (arrowheads) originating from the optic nerve (ON) of the HIF-2{alpha}-KO eye (B, D) was prominent in comparison with that in the WT eye (A, C). Hyaloid artery remnants (arrowheads) in the vitreous (V) remained attached to the posterior aspect of the lens in the HIF-2{alpha}-KO eye (F), whereas similar findings were not evident in the WT eye (E). (F, inset) A high-magnification view of a typical hyaloid artery remnant observed in a HIF-2{alpha}-KO eye.

 


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  		FIGURE 6. (A) Low- and high- (inset) magnification of ß-galactosidase–stained flatmount retina from a HIF-2{alpha}-HET mouse. The nuclear-localized ß-galactosidase activity appears as intense blue staining and is evident in vascular endothelial cells. (B) ß-Galactosidase staining of retinal sections from HIF-2{alpha}-HET mice. Nuclear-localized ß-galactosidase activity localized to cells residing in the GCL, INL, and RPE (arrows) in addition to the retinal vascular endothelial cells (unmarked).

 
ERG studies in HIF-2{alpha}-KO mice were compared with those in WT mice. The initial 50 ms of the responses to high-intensity stimuli were examined (Figs. 7A 7B) , along with computer-generated fits (dashed curves) of the predominant phototransduction model26 to the leading edges of the a-wave ensemble.27 Maximum rod a-wave amplitude in the HIF-2{alpha}-KO mouse was approximately one fourth that of the WT mouse (note difference in scale). The sensitivity parameter (log S) derived from the leading edge in this HIF-2{alpha}-KO mouse was 1.6 s–2 · td-s–1 compared with 1.8 s–2 · td-s–1 in the WT mouse.



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  		FIGURE 7. Full-field ERGs from a representative WT (A, C, E, G) or HIF-2{alpha}-KO (B, D, F, H) mouse. Note increased gain (x5) in WT relative to HIF-2{alpha}-KO graphs. The y-axis represents amplitude (microvolts) and the x-axis represents time (milliseconds). (A, B) Rod a-waves to flashes ranging from 1.8 to 3.7 log scotopic td-s. Dashed curves: best fits of the rod phototransduction model. (C, D) Rod b-waves to series of flashes from threshold to 1.3 log scotopic td-s (0.3 log-steps). (E, F) Oscillatory potentials digitally isolated with a band-pass filter 6 dB down at 54 and 1037 Hz. (G, H) Light-adapted cone b-waves to flashes ranging from 0.6 to 1.8 log scotopic td-s obtained in the presence of a rod-saturating background (40 cd/m2).

 
Full–field ERGs to stimuli ranging from –3.0 to 1.3 log scotopic td-s were recorded for mice from each group. In WT mice (Fig. 7C) , responses to these stimuli contain three components: the a-wave, the b-wave, and the oscillatory potentials (OPs), the latter being substantial in the mouse. In contrast, responses from HIF-2{alpha}-KO mice were primarily electro-negative (Fig. 7D) . OPs (Figs. 7E 7F) were virtually extinguished in HIF-2{alpha}-KO mice. Representative cone responses to achromatic stimuli presented in the presence of a background were recorded (Figs. 7G 7H) . There were virtually no cone b-waves in HIF-2{alpha}-KO mice. A slight negativity was evident in most responses, presumably originating from the cone PRs.

Summary values for ERG parameters derived from response functions were compared among WT, HIF-2{alpha}-HET, and HIF-2{alpha}-KO mice (Fig. 8) . HIF-2{alpha}-KO mice were significantly different from both WT and HIF-2{alpha}-HET mice in rod a-wave maximum amplitude (F2,18 = 11.44, P = 0.0006), rod b-wave maximum amplitude (F2,18 = 40.31, P = 0.000001), and cone b-wave maximum amplitude (F2,18 = 4.81, P = 0.02). Log S, the sensitivity parameter of the a-wave that relates to transduction gain, was not significantly different among groups (F2,18 = 2.1, P = 0.15).



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  		FIGURE 8. Summary of ERG parameters for WT, HIF-2{alpha}-HET, or HIF-2{alpha}-KO mice. Mean ± SEM rod a-wave, rod b-wave, and cone b-wave amplitude are presented. Comparisons were made by ANOVA. No significant differences were found between WT and HIF-2{alpha}-HET mice. Maximum amplitudes were significantly lower in HIF-2{alpha}-KO mice compared with either WT or HIF-2{alpha}-HET mice for rod a-wave (F2,18 = 11.44, P = 0.0006), rod b-wave (F2,18 = 40.31, P = 0.000001), and cone b-wave (F2,18 = 4.81, P = 0.02).

 
Gene expression patterns for relevant molecular signaling pathways in HIF-2{alpha}-KO retinal samples was determined (Table 1) . HIF-1 target genes (HO-1, eNos, Vegf-A, and Flk1) were minimally elevated in HIF-2{alpha}-KO retinas, although not to statistically significant levels. Other genes previously implicated as HIF-2{alpha}-specific (Tie2) were minimally depressed, although again not to statistically significant levels. The antiangiogenic factors thrombospondin 1 and 2 (Tsp1 and -2) were upregulated in HIF-2{alpha}-KO retinas, whereas the antiangiogenic factor pigment epithelium-derived factor (PEDF) was unchanged. The HIF-2{alpha}-regulated gene erythropoietin,28 a cytokine with retinal protective effects, was decreased in HIF-2{alpha}-KO retinas, and erythropoietin receptor levels trended lower in HIF-2{alpha}-KO retinas.


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  		TABLE 1. Molecular Expression Profiles for HIF-2{alpha}-null Retinas

 
Expression of HIF-2{alpha}-regulated and other AOE genes in HIF-2{alpha}-KO retinas was measured.19 The glutathione peroxidase–encoding genes Gpx1 and -3 levels were unchanged. Sod2 gene expression trended toward lower levels. Levels of the copper/zinc superoxide dismutase-encoding gene Sod1 mRNA as well as the catalase-encoding gene Cas1 mRNA were decreased. The oxidative stress–induced factor metallothionein-2 (Mt2) trended toward increased expression levels, whereas expression of the gene encoding Chop-10 (Ddit3) did not differ significantly. The molecular findings, in summary, reveal increased thrombospondin, depressed erythropoietin signaling, and dysregulated AOE gene expression in a pathophysiological state characterized by ischemic retinopathy.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The HIF-2{alpha}-null state has profound effects on retinal function as assessed by ERG. The reduction in maximum rod a-wave amplitude, a direct reflection of rod PR function, is consistent with the extensive retinopathy. The S parameter of the rod a-wave was near normal, suggesting a preserved functional status of the remaining rods. The rod b-wave was dramatically reduced in amplitude consistent with the presence of extensive inner retinal abnormalities. The cone a-wave is extremely small in the mouse29 ; nevertheless, there is the suggestion of a remnant cone a-wave despite the almost complete loss of the cone b-wave in HIF-2{alpha}-KO mice. These ERG findings are reminiscent of those observed in rodents raised from birth in a hyperoxygenated environment30 31 or in premature human infants or kittens treated with hyperoxygen therapy,32 33 conditions associated with increased oxidative stress.

The marked retinal thinning in HIF-2{alpha}-KO mice is similar to other mouse models of oxidative stress-induced retinal disease including {alpha}-TTF and SOD2-KO mice.22 34 SOD2-KO mice typically do not survive past 1 to 2 weeks of age and have multiple organ diseases including retinopathy. The retinopathy has not been extensively characterized due to the neonatal death of SOD2-KO mice. Treatment of SOD2-KO mice with an antioxidant enzyme mimetic compound (MnTBAP) increases their lifespan and corrects much of the multiple organ disease otherwise seen in these mice. However, MnTBAP-treated SOD2-KO mice have neurologic and retinal disease by 1 month of age, presumably due to an inability of MnTBAP to cross the brain–blood and retinal–blood barrier.22 34

In MnTBAP-treated SOD2-KO mice, the retinopathy is characterized by central thinning of the NFL/GCL/IPL and INL, no significant thinning of the ONL, decreased thickness of the PR layer, no overt change in the RPE, and a lack of lipofuscin deposition at 3 weeks of age.22 34 In another mouse model of SOD2 deficiency, hammerhead ribozymes delivered by adenoassociated viral vectors (Rz-SOD2) to 2-month-old mice were used to reduce endogenous Sod2 gene expression in the eye.35 These reductions resulted in a substantial decrease in Sod2 gene expression in vitro and in the development of retinopathy in vivo that is evident by 4 months after injection.

The similarity of the HIF-2{alpha}-KO to the SOD2-KO phenotype is probably due in part to an ineffective induction of SOD2 and to reductions of other major AOE genes in HIF-2{alpha}-KO mice. The altered AOE gene expression patterns in HIF-2{alpha}-KO retinas are consistent with the proposed role for HIF-2{alpha} in the in vivo regulation of the AOE genes.19 The physiological consequence of altered AOE gene expression is an inadequate oxidative stress defense. Additional experiments will address whether specific alterations in AOE gene expression patterns result in the pathologic features of HIF-2{alpha}-KO mice.

The depressed AOE gene expression patterns may be a direct consequence of the HIF-2{alpha}-null state. Alternatively, a reduced hypoxic drive as a consequence of PR dropout may result in less induction of HIF-1{alpha}–dependent genes. Diminished induction of HIF-1{alpha} target genes would probably have adverse effects on vascular development and retinal survival. Expression of erythropoietin, a factor induced by HIF-1{alpha}36 and -2{alpha}28 with retina-protective effects in vitro and in vivo,37 38 39 was reduced in HIF-2{alpha}-KO retinas. However, the inability of exogenous erythropoietin to correct the retinopathy in HIF-2{alpha}-knockdown mice28 or HIF-2{alpha}-KO mice (data not shown) suggests that erythropoietin signaling may be affected in a more complex manner. In this respect, the lower trends in erythropoietin receptor gene expression may also contribute to impaired erythropoietin signaling.

The vascular pattern in the eyes from HIF-2{alpha}-KO mice is characterized by an avascular periphery, an atrophic central retinal vascular pattern without prominent radial sprouting, and a persistent hyaloid artery. The minimally reduced expression of Tie2, a proangiogenic HIF target gene encoding a receptor tyrosine kinase, may be of relevance to the HIF-2{alpha}-KO retinal phenotype. The absence of Tie2 has profound effects on vascular development.40 Elimination of the Tie2 ligand, angiopoietin-2, results in an avascular peripheral retinopathy and a persistent hyaloid artery,41 similar to that observed in HIF-2{alpha}-KO mice. Inhibition of Tie2 signaling leads to increased expression of the antiangiogenic factor thrombospondin 1 (Tsp1),42 a finding also noted in HIF-2{alpha}-KO retinas. Whether decreased Tie2 expression or increased Tsp1 expression is responsible for the HIF-2{alpha}-KO vascular phenotype may be amenable to testing in other genetic models.

We have not identified what aspects of the HIF-2{alpha}-KO phenotype—abnormal retinal vasculature, retinopathy, and/or impaired oxidative stress response—are primary versus secondary features. Retinal vascular abnormalities may be a consequence of abnormal vascular development,43 PR loss (leading to a reduced hypoxic drive for angiogenesis or subsequent capillary atrophy),44 45 or an ineffective oxidative stress defense.46 Retinopathy may develop as a consequence of PR dropout,47 inadequate vascular development (and therefore impaired oxygen delivery),43 a reduced capacity for oxidative metabolism,48 or oxidative stress-induced injury.22 An increased oxidative stress state may result from less efficient induction of HIF-2{alpha}-dependent AOE genes, PR loss (leading to a reduced hypoxic drive for HIF-1{alpha}-dependent stress response genes), or ineffectual RPE.49 The interrelationship of these processes make it difficult to identify the major inciting factor in HIF-2{alpha}-KO retinopathy. Future studies using primary cells from HIF-2{alpha}-KO mice may better define the causative pathophysiological processes.

It is not evident when the pathologic processes observed in 1-month-old HIF-2{alpha}-KO retinas are initiated. Neonatal rodents are particularly sensitive to exogenous oxidative stress, probably as a result of the postnatal AOE gene expression profile.50 51 The features apparent at 1 month of age may also be due to developmental abnormalities rather than to processes begun at a postnatal stage. Likewise, we cannot ascertain which of the retinal cell types that express HIF-2{alpha} are most affected by the HIF-2{alpha}-null state. More sophisticated mouse models and primary cell analyses are needed to address these questions.

HIF target genes serve a protective function against environmental stressors. An impaired induction of HIF-2{alpha} target genes, as with HIF-2{alpha}-KO mice, is probably an important contributing factor in the development of certain retinopathies. We speculate that perturbations induced by impaired HIF-2{alpha} gene expression, directly or indirectly lead to reductions in AOE gene expression, increased thrombospondin gene expression, and altered erythropoietin signaling. The combined effects of these altered gene expression patterns predispose HIF-2{alpha}-KO mice to ischemic retinopathy. Future studies detailing the molecular, biochemical, cellular, and physiological effects of the HIF-2{alpha}-null state on eye function will have important implications for treatment of human eye disease states.


   Acknowledgements
 
The authors thank SandiJo Estill and Carol Dudley for assistance with behavioral assays and the UTSWMC Molecular Pathology Core for use of their facilities.


   Footnotes
 
Supported in part by National Heart, Lung, and Blood Institute Grant K08-HL67154; American Heart Association/Texas Affiliate Grant 0160073Y (JAG); American Cancer Society Grant IRG0219601 (JAG); National Institute of Mental Health Grant 1P50MH66172 (JAG); National Eye Institute Grant EY05235 (DGB); the Donald W. Reynolds Foundation (JAG); and the Foundation Fighting Blindness (DGB).

Submitted for publication July 6, 2004; revised October 25, 2004; accepted November 13, 2004.

Disclosure: K. Ding, None; M. Scortegagna, None; R. Seaman, None; D.G. Birch, None; J.A. Garcia, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Joseph A. Garcia, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573; joseph.garcia{at}utsouthwestern.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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