January 2015
Volume 56, Issue 1
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Retina  |   January 2015
Regulation of Fibroblast Growth Factor 2 Expression in Oxygen-Induced Retinopathy
Author Affiliations & Notes
  • Li Fang
    Department of Pediatrics, Penn State Hershey Eye Center, Penn State Hershey College of Medicine, Hershey, Pennsylvania, United States
  • Alistair J. Barber
    Department of Ophthalmology, Penn State Hershey Eye Center, Penn State Hershey College of Medicine, Hershey, Pennsylvania, United States
  • Jeffrey S. Shenberger
    Department of Pediatrics, Penn State Hershey Eye Center, Penn State Hershey College of Medicine, Hershey, Pennsylvania, United States
    Department of Pediatrics, Baystate Medical Center, Springfield, Massachusetts, United States
Investigative Ophthalmology & Visual Science January 2015, Vol.56, 207-215. doi:10.1167/iovs.14-15616
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      Li Fang, Alistair J. Barber, Jeffrey S. Shenberger; Regulation of Fibroblast Growth Factor 2 Expression in Oxygen-Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(1):207-215. doi: 10.1167/iovs.14-15616.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Fibroblast growth factor (FGF) 2 is a potent endothelial cell mitogen and survival factor that is postulated to participate in the pathogenesis of retinopathy of prematurity (ROP). The purpose of the current study was to determine the transcriptional and translational regulation of FGF2 expression in oxygen-induced retinopathy (OIR), the animal model of ROP.

Methods.: We examined FGF2 protein and mRNA expression and optokinetic visual responses in transgenic mice possessing a dual-luciferase bicistronic transgene containing a 5′-internal ribosome entry site (IRES) of FGF2.

Results.: We found that retinal FGF2 protein isoform expression varies with age but not in response to OIR. Analysis of luciferase, protein, and mRNA data indicate that FGF2 protein expression is translationally repressed during the vaso-obliterative phase of OIR, possibly by inhibiting elongation. At the transition from vaso-obliteration to neovascularization, heightened FGF2 protein expression corresponds to maintenance of IRES activity and diminished cap-dependent translational activity. During neovascularization, FGF2 expression is primarily regulated by transcription. Mice recovering from OIR display alterations in visual optokinetic responses and increased FGF2 protein expression at 6 weeks of age.

Conclusions.: In total, these findings illustrate the complexity of translational and transcriptional regulation of FGF2 protein expression in OIR. The augmentation of FGF2 expression and reduced optokinetic responses during the resolution of surface vasculopathy may indicate a role for FGF2 in the maintenance of neuroretinal function in OIR/ROP.

Retinopathy of prematurity (ROP) is a major cause of potentially preventable blindness in developed countries.1 The pathogenesis of ROP entails two distinct phases: the primary cessation and regression of retinal vascular elements induced by the abrupt increase in O2 tension upon preterm delivery, followed by a secondary vasoproliferative phase stimulated by a surge in growth factor expression within the hypoxic, yet highly metabolic and undervascularized, neuroretina. It is the development of neovascularization (NV) during the second phase of ROP that has the potential to induce fibrous scar formation and retinal detachment in the most advanced cases, leading to irreversible loss of vision.2 Although O2-mediated changes in VEGF expression are known to be integral to development of both phases of ROP, inhibition of VEGF expression fails to completely eliminate NV, suggesting that additional factors may potentiate or stabilize the neovascular elements.3,4 
Fibroblast growth factor (FGF) 2, a potent smooth muscle, endothelial, and neuronal progenitor cell mitogen, is expressed throughout the retina and central nervous system.5,6 Upregulation of FGF2 expression has been documented in the vitreous and neovascular retina of patients with diabetic retinopathy, in the vitreous of infants with operative ROP, and in the retina during the neovascular phase of oxygen-induced retinopathy (OIR) in mice.79 The OIR mouse model recapitulates the biphasic retinal vascular response seen in infants with ROP.10 In the OIR model, exposure of newborn mouse pups to hyperoxia initially produces central retinal vaso-obliteration—an approximation of the rapid increase in oxygen tension experienced by premature infants. Return of pups to room air generates hypoxia in the avascular retina, inducing the expression of vascular growth factors that stimulate aberrant vascular growth, a process analogous to the neovascular phase in developing ROP. Hence, OIR represents an opportunity to link growth factor expression with vascular pathology and neural retinal function. 
The regulation of FGF2 expression is complex, mediated by transcriptional, posttranscriptional, and translational processes. Although the native FGF2 transcript yields several mRNAs in humans differing in the length of the 3′-untranslated region (3′-UTR), only the longest transcript is utilized in nontransformed cells.11 This solitary mRNA generates up to five protein products, depending on the start codon selected within the 5′-UTR. The length and GC-rich nature of the 5′-UTR leader sequence imparts significant inhibition to ribosomal scanning, limiting the efficiency of cap-dependent translation of FGF2 mRNAs.12 Like many growth factor transcripts with complex secondary structure, the 5′-UTR leader of FGF2 contains an internal ribosome entry site (IRES), a sequence motif capable of supporting cap-independent translation.13 During periods of hypoxia/ischemia, transgenic mice expressing a FGF2 reporter transgene display enhanced IRES activity, leading to the hypothesis that hypoxia may also drive FGF2 IRES activity in ROP/OIR.14 In this regard, the goal of this project was to define the temporal expression pattern of retinal FGF2 protein during the course of OIR. Furthermore, given that recent human studies indicate that infants with ROP have reduced contrast sensitivity months after the spontaneous regression of NV, we sought to correlate alterations in OIR-induced optokinetic responses with FGF2 expression weeks following the induction of OIR.15 
Methods
OIR Model and Animals
All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Pennsylvania State University College of Medicine in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Genetically engineered C57BL/6 x CBA mice containing the human FGF2 IRES transgene located between an upstream, cap-dependent Renilla luciferase (RL) reporter and a downstream cap-independent firefly luciferase (FL) reporter were backcrossed with wild-type C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) to more closely approximate a standard wild-type C57BL/6 background commonly used in models of OIR.16 The expression of the transgene during development and tissue specificity of the reporter has been previously reported.16,17 Retention of reporter gene expression within the retina and other tissues of the hybrid mice was confirmed by RT-PCR and luciferase analysis. The OIR model used was identical to that previously described by Smith et al.10 Briefly, mice dam/pup pairings, culled to a litter size of 7 to 10, were placed into Plexiglas exposure chambers overnight beginning on postnatal day-of-life 7 (P7). In the OIR protocol, mice were exposed to 75% O2 for 5 days (through P12), after which they were returned to room air. Administration of O2 was continually adjusted and monitored using a computerized system (BioSpherix Oxycycler; Reming Bioinstruments, Redfield, NY, USA). Control animals were exposed to ambient room air in identical chambers for durations identical to those for OIR mice. In both chambers, CO2 concentrations were adjusted by the degree of chamber leak and kept at <0.5%. Dams in both groups were supplied with standard mouse chow and water ad libitum, which was replenished at P12 and as needed thereafter. Routine day/light cycles of 12 hours were used and temperature and humidity maintained at 26°C and 75%–80%, respectively. 
Retinal Flatmounts
Mouse pups were euthanized using pentobarbital and the eyes enucleated. The eyes were fixed in 4% paraformaldehyde for 60 minutes at room temperature then washed three times in PBS and stored at 4°C. Retinas were separated from the sclera, retinal pigmented epithelium, lens, and cornea and marked for orientation. Thereafter, retinas were incubated with 500 μL of Alexa Fluor 594 conjugated isolectin (B4-594; Life Technologies Corp., Carlsbad, CA, USA) solution overnight at 4°C in the dark. Following rinsing, retinas were flat-mounted ganglion side up on glass slides and coverslipped in aqueous mounting medium containing antifade reagent. Digital images of each retinal quadrant were taken with a fluorescence microscope at 4× and the quadrants merged together in Photoshop CS4 (Adobe Systems, Inc., San Jose, CA, USA) to produce an image of the entire retina for further analysis. Flat-mounted retinas from control and OIR reporter and C57BL/6 mice (two to three mice per condition) were examined for vascular regression at P12 and for NV at P17. 
Retinal Lysate Preparation and Immunoblotting
Protein analysis was assessed in control and OIR mouse retinal extracts (four mice per condition) at P8, 12, 13, 15, 17, and 21 and 6 weeks of age. Flash-frozen retinas were homogenized in 80 μL of CHAPS buffer (40 mM HEPES, pH 7.5; 120 mM NaCl; 1 mM EDTA; 10 mM pyrophosphate; 10 mM β-glycerophosphate; 40 mM NaF; 1.5 mM sodium orthovanadate; 0.1 mM PMSF; 1 mM benzamidine; 1 mM dithiothreitol; 0.3% CHAPS) supplemented with protease and phosphatase inhibitors (Complete Mini, EDTA-free, and PhosSTOP; Roche, Branford, CT, USA) using a Bullet Blender and 30 mg of 0.5-mm stainless beads (speed 5.5 for 3 minutes; Next Advance, New York, NY, USA). Lysates were cleared by centrifugation and the protein concentration of the supernatant determined using the BCA assay (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Protein (20–30 μg) was separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF), blocked with 5% milk in Tris-buffered saline with Tween (TBST), and blotted with FGF2 (1:1000; EMD Millipore, Temecula, CA, USA) and β-actin (1:10,000; Sigma-Aldrich Corp., St. Louis, MO, USA) antibodies. A control lane of whole mouse brain lysate was included on each gel. Blots were developed by chemiluminescence and specific band densities quantified using the GeneGnome imaging system (SynGene USA, Frederick, MD, USA) as previously described.18 Loading was controlled by normalization to β-actin within each gel, and development was standardized across gels by normalization to the expression of FGF2 in whole mouse brain. 
Quantification by RT-PCR
Relative amounts of FGF2 and VEGF-A isoform mRNA were quantified by RT-PCR (ABI 7900; Life Technologies Corp.). RNA from four retinas from each time point and condition were extracted using RNeasy Mini and QIA shredder kits (Qiagen, Inc., Valencia, CA, USA). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corp.), with each reaction containing 4 mM deoxyribonucleotide triphosphate, 1X RT random primers, and 125 U of MultiScript reverse transcriptase using a profile of 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and a 4°C hold. The cDNA was amplified in triplicate using SYBR Green (Qiagen, Inc.) and 0.3-μM forward and reverse primers using the following profile: 50°C for 2 minutes, followed by 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds and 72°C for 30 seconds. The following mouse primer sequences were utilized: FGF2 forward: 5′-CACCAGGCCACTTCAAGGA-3′; FGF2 reverse: 5′-GATGGATGCGCAGGAAGAA-3′; VEGF-120 and VEGF-164 forward: 5′-GGAGAGATGAGCTTCCTACAGCA-3′; VEGF-120 reverse: 5′-CTGAACAAGGCTCACAGTGATTTT-3′, VEGF-164 reverse: 5′-CCTTGGCTTGTCACATTTTTCT-3′; GAPDH forward: 5′-TGCACCACCAACTGCTTAG-3′; GAPDH reverse: 5′-GGATGCAGGGATGATGTTC-3′. Standard curves were generated for each primer pair using serial dilutions, after which the relative amount of mRNA was calculated based on the average threshold cycle (Ct) value after removal of outliers with a coefficient of variation >17%. Values of FGF2 and VEGF-A isoform mRNA were normalized to GAPDH and expressed in arbitrary units. Transcript levels for FGF were measured in four control and four OIR mice at P8, 12, 13, 15, and 17 and for VEGF at P12, 15, and 17. 
Luciferase Assay
Frozen retinas from 5 to 10 control and OIR mice at P8, 12, 13, 15, and 17 were homogenized in passive lysis buffer (Promega Corp., Madison, WI, USA) using the Bullet Blender and were cleared by centrifugation. Duplicate firefly and Renilla luciferase activities in the supernatants were measured using the Dual Luciferase Kit (Promega, Corp.) in a single tube luminometer (Femtomaster FB12; Zylux Corp., Huntsville, AL, USA) and corrected to protein concentration. 
Immunohistochemistry
In order to describe FGF2 distribution in the retina, paraffin-embedded eyes from a control and OIR mouse at P17 were cut into 5-μm cross-sections and mounted onto glass slides. Tissue sections were dehydrated in xylene, rehydrated through graded ethanol, and subjected to microwave antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 2 minutes. Sections were then deparaffinized, endogenous peroxides quenched with 3% H2O2, sections were rehydrated, and blocked with 10% normal goat serum for 1 hour. Retinal cross-sections were then incubated with anti-mouse FGF2 antibody (1:100; EMD Millipore) at 4°C overnight. After thorough washing in PBS, sections were incubated with biotin-labeled anti-mouse antibody for 30 minutes and developed using VECTASTAIN Elite ABC reagent (Vector Laboratories, Inc., Burlingame, CA, USA). 
Optokinetics
Optokinetic responses were studied in 6-week-old control and OIR mice (four to eight per group) using a computer-controlled rotating virtual cylinder of vertical white/black grating projected onto four LCD panels (OptoMotry; Cerebral Mechanics, Inc., Lethbridge, AB, Canada).19 Animals were placed on a central platform and allowed to acclimate to the chamber prior to measurements. During studies, head position was monitored with an overhead digital camera that superimposed crosshairs on the animal's snout to continuously track head position. The axial coordinates of the crosshair relative to the LCD panel dictated the hub of the virtual cylinder, thereby enabling the cylinder wall to be maintained at a constant distance from the animal's vantage point. Positive optokinetic responses were judged by denoting horizontal head and neck movements in concert with the direction of the rotation. Spatial frequency threshold (SFT) in cycles/degree (c/d) represented the grating width at which tracking response could no longer be elicited at a constant contrast of 100%. Percent of contrast sensitivity (CS) was determined at constant grating frequencies of 0.092 and 0.64 c/d, which represented the contrast at which tracking can no longer be elicited at the specified frequency. Each measurement was repeated three times per animal and averaged. 
Statistical Analysis
At each time point, 9 to 16 animals were utilized per condition, with the exception of P21, in which only four animals per condition were studied. Protein, mRNA, luciferase activity, and optokinetic responses were compared with one-way ANOVA or two-way ANOVA with Tukey multiple comparisons testing. Individual comparisons at specific time points were made via t-tests or Mann-Whitney rank sum tests when appropriate for nonnormally distributed data (SigmaPlot v12; Systat Software, Inc., San Jose, CA, USA). For simplicity, all data, including that analyzed by nonparametric tests, are listed as mean ± SEM, and the statistical significance is set at P < 0.05. 
Results
Characterization of Retinopathy in Reporter Mice
To fully analyze the translational regulation of FGF2 during OIR, we utilized transgenic mice containing the FGF2 IRES sequence positioned between Renilla and firefly luciferase reporters—a construct capable of delineating relative cap-dependent and -independent efficiencies.16 Retinal vascular development, judged in flat-mounted retinas, was similar between reporter and C57BL/6 mice, with full vascularization occurring by P12 (Fig. 1). Exposure of reporter mice to the OIR protocol produced significant centripedal vascular regression at P12 and marked NV at P17 analogous, but not necessarily equivalent, to that observed in C57BL/6 mice.10 The presence of NV was confirmed on retinal cross-sections from reporter mice (and C57BL/6 mice [not shown]) as the presence of cells anterior to the internal limiting membrane on hematoxylin-stained sections. These cells were absent in control retinas from both strains. Immunoreactive FGF2 in retinal cross-sections from reporter mice was localized within the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) at P17 (Fig. 2). 
Figure 1
 
Vasculopathy of OIR in FGF2 reporter mice. Photomicrographs of representative flat-mounted retinas from C57BL/6 and FGF2 reporter mice are depicted. In room air (RA) at P12, both mice are fully vascularized. In mice exposed to 75% O2 (Ox) from P7 to P12, both variants undergo similar degrees of vascular regression. Likewise, after 5 days in room air, P17 animals show significant NV.
Figure 1
 
Vasculopathy of OIR in FGF2 reporter mice. Photomicrographs of representative flat-mounted retinas from C57BL/6 and FGF2 reporter mice are depicted. In room air (RA) at P12, both mice are fully vascularized. In mice exposed to 75% O2 (Ox) from P7 to P12, both variants undergo similar degrees of vascular regression. Likewise, after 5 days in room air, P17 animals show significant NV.
Figure 2
 
FGF2 immunoreactivity at P17. Photographs (40×) of retinal cross-sections taken from control and OIR animals at P17 prepared as described in the Methods section. Arrows denote clusters of immunoreactive neovascular cells anterior to the internal limiting membrane. FGF2 immunoreactivity was noted within select cells in the GCL and in general throughout the ONL. Images were obtained approximately one-third of the distance from the optic disc to periphery.
Figure 2
 
FGF2 immunoreactivity at P17. Photographs (40×) of retinal cross-sections taken from control and OIR animals at P17 prepared as described in the Methods section. Arrows denote clusters of immunoreactive neovascular cells anterior to the internal limiting membrane. FGF2 immunoreactivity was noted within select cells in the GCL and in general throughout the ONL. Images were obtained approximately one-third of the distance from the optic disc to periphery.
FGF2 Expression During OIR
Analysis of retinal FGF2 mRNA levels revealed time-dependent increases in FGF2 mRNA in both controls and OIR animals from P8 through P17 (Fig. 3). Expression of FGF2 mRNA was greater in OIR than controls at P8, P13, P15, and P17, with nearly a 4-fold increase in expression at the latter time points. In comparison to FGF2 mRNA, the expression of total VEGF, VEGF164, and VEGF120 isoform mRNAs were decreased in OIR animals relative to controls at P12, a trend that was reversed at P15 and P17 (Fig. 4), illustrating the unique transcriptional activation patterns of the two retinal growth factors. 
Figure 3
 
Retinal FGF2 mRNA expression during OIR. Retinal FGF2 mRNA levels from whole mouse retinas were determined by RT-PCR and normalized to GAPDH. FGF2 expression increased over time in both controls (dark columns) and OIR (light columns) mice. FGF2 expression was increased in OIR animals relative to controls at P8, P13, P15, and P17. Columns represent mean of four animals, and bars indicate SEM. *P < 0.05.
Figure 3
 
Retinal FGF2 mRNA expression during OIR. Retinal FGF2 mRNA levels from whole mouse retinas were determined by RT-PCR and normalized to GAPDH. FGF2 expression increased over time in both controls (dark columns) and OIR (light columns) mice. FGF2 expression was increased in OIR animals relative to controls at P8, P13, P15, and P17. Columns represent mean of four animals, and bars indicate SEM. *P < 0.05.
Figure 4
 
Impact of OIR on retinal VEGF mRNA. Mouse retinal VEGF-A mRNA levels determined by RT-PCR were normalized to GAPDH (n = 4 each time and condition). Total VEGF, VEGF120, and VEGF164 mRNA expression was decreased in OIR (light columns) mouse pups relative to controls (dark columns) at P12 (†P < 0.05). At both P15 and P17, however, OIR mice had greater expression of total, VEGF120, and VEGF164 mRNA relative to control mice (*P < 0.05). Bars indicate SEM.
Figure 4
 
Impact of OIR on retinal VEGF mRNA. Mouse retinal VEGF-A mRNA levels determined by RT-PCR were normalized to GAPDH (n = 4 each time and condition). Total VEGF, VEGF120, and VEGF164 mRNA expression was decreased in OIR (light columns) mouse pups relative to controls (dark columns) at P12 (†P < 0.05). At both P15 and P17, however, OIR mice had greater expression of total, VEGF120, and VEGF164 mRNA relative to control mice (*P < 0.05). Bars indicate SEM.
The FGF2 mRNA encodes distinct protein isoforms expressed by utilization of several alternative CUG start codons in addition to the classical AUG start codon.20 In mice, alternative start site selection generates two high molecular weight (HMW) species of 21 and 22 kDa, while activation of the classic AUG produces a single, low molecular weight (LMW) 17.5-kDa isoform.20 Immunoblotting identified all three isoforms at each age, but the distribution of the HMW and LMW proteins varied with age (Fig. 5A). In control pups, total FGF2 varied little, but significantly, over the 2-week examination period, with decrements noted at P12 and P13. Age produced a shift in isoform distribution favoring HMW proteins (ANOVA, P < 0.05). From P8 to P17, the ratio of HMW:LMW increased more than 10-fold in both control and OIR groups (P8: Con [control] = 0.21 ± 0.02; OIR = 0.14 ± 0.06; P17: Con = 2.47 ± 0.21; OIR = 2.60 ± 0.21; P < 0.01). Although the OIR protocol failed to alter the ratio of HMW-to-LMW FGF2, exposure to 75% O2 decreased LMW, HMW, and total FGF2 protein expression relative to controls (P12; Fig. 5B). Upon return to room air at P13, total, LMW, and HMW FGF2 protein expression increased in a time-dependent manner. Relative to controls, total FGF2 protein and both isoforms were greater at P13, P17, and P21. Of note, the expression trends for total FGF2 protein in C57BL/6 animals was similar to that of hybrids at P12, P15, and P17 in the hybrid mice (not shown). 
Figure 5
 
Retinal FGF2 protein expression during OIR. Whole retinas from mice exposed to room air (control) or the OIR paradigm were homogenized and lysates separated by SDS-PAGE. They were transferred to PVDF membranes and immunoblotted for FGF2. Quantitation was performed by densitometry and normalized to the expression of β-actin. (A) Representative immunoblots from control (dark columns) and OIR (light columns) mice at P8 to P21 showing high and low molecular weight (MW) FGF2 isoforms. (B) Histograms depicting mean expression of total, high MW, and low MW FGF2 normalized to β-actin (n = 4 per time and condition). The dagger symbol (†) indicates OIR values significantly less and the asterisk (*) OIR values significantly greater than control at a given time point. For all, P < 0.05, and bars indicate SEM.
Figure 5
 
Retinal FGF2 protein expression during OIR. Whole retinas from mice exposed to room air (control) or the OIR paradigm were homogenized and lysates separated by SDS-PAGE. They were transferred to PVDF membranes and immunoblotted for FGF2. Quantitation was performed by densitometry and normalized to the expression of β-actin. (A) Representative immunoblots from control (dark columns) and OIR (light columns) mice at P8 to P21 showing high and low molecular weight (MW) FGF2 isoforms. (B) Histograms depicting mean expression of total, high MW, and low MW FGF2 normalized to β-actin (n = 4 per time and condition). The dagger symbol (†) indicates OIR values significantly less and the asterisk (*) OIR values significantly greater than control at a given time point. For all, P < 0.05, and bars indicate SEM.
Translational Regulation of FGF2 Expression During OIR
To assess the contribution of cap-dependent and -independent translation of FGF2 mRNA during OIR, whole retinal lysates from reporter mice were analyzed for RL and FF luciferase activities, respectively, at P8 through P17 (Figs. 6A, 6B, respectively). The RL activity was significantly higher than FF activity at all time points regardless of conditions, indicating the predominance of cap-dependent mRNA translation. At P8, however, the FF/RL ratio was significantly greater in control and OIR animals than at all other times (Fig. 6C), reflecting the lower RL activity at this age. Exposure to hyperoxia (P8 and P12) had little effect on FF or RL activities. Return to room air at P13, on the other hand, suppressed RL activity, leading to a significant increase in the FF/RL ratio (Fig. 6C). From P15 onward, both RL and FF activity increased in OIR mice, though increases in RL were larger, leading to a reduction in the FF/RL ratio. These findings imply that unique regulatory events modulate cap-dependent and cap-independent mRNA translation during the early and latter stages of OIR that correlate with the development of NV. 
Figure 6
 
Translational regulation of FGF2 expression. Mice containing a bicistronic, dual-luciferase transgene containing the FGF2 IRES were exposed to room air (control, dark columns) or the OIR (light columns) paradigm. Whole retinas were homogenized and lysates analyzed for Renilla and firefly luciferase activity representing IRES-mediated mRNA translation and cap-dependent mRNA translation, respectively, as described in the Methods section. Figure illustrates the effect of OIR on (A) Renilla luciferase (RR); (B) firefly luciferase (FL); and (C) the RR/FL ratio. Columns represent mean of 5 to 10 animals, and bars indicate SEM. The dagger symbol (†) indicates OIR values less than and the asterisk (*) OIR values greater than control at a given time point. For all, P < 0.05.
Figure 6
 
Translational regulation of FGF2 expression. Mice containing a bicistronic, dual-luciferase transgene containing the FGF2 IRES were exposed to room air (control, dark columns) or the OIR (light columns) paradigm. Whole retinas were homogenized and lysates analyzed for Renilla and firefly luciferase activity representing IRES-mediated mRNA translation and cap-dependent mRNA translation, respectively, as described in the Methods section. Figure illustrates the effect of OIR on (A) Renilla luciferase (RR); (B) firefly luciferase (FL); and (C) the RR/FL ratio. Columns represent mean of 5 to 10 animals, and bars indicate SEM. The dagger symbol (†) indicates OIR values less than and the asterisk (*) OIR values greater than control at a given time point. For all, P < 0.05.
Long-Term Effect of OIR on FGF2 Expression and Visual Function
Previous studies in C57BL/6 mice demonstrate that resolution of OIR-induced NV occurs within 5 weeks.10 To assess FGF2 expression and visual function in resolving OIR, we performed immunoblotting of retinal extracts and optokinetic measurements in 6-week-old control and OIR animals. At 6 weeks of age, total and LMW FGF2 protein expression remained elevated in OIR animals relative to controls (LMW: Con = 0.9 ± 0.1; OIR = 2.9 ± 1.1, and total: Con = 1.2 ± 0.2; OIR = 5.0 ± 1.7, arbitrary units, P < 0.05; Fig. 7A). Meanwhile, spatial frequency threshold, which tests the ability to discriminate grating size at maximal contrast, was significantly reduced in OIR animals (Fig. 7B). Contrast sensitivity, a measure of the ability to track different grating widths at varying contrasts, was similar at 0.064 c/d, but poorer in OIR mice at 0.092 c/d. 
Figure 7
 
FGF2 protein expression and optokinetic responses in OIR mice. Mice were exposed to room air (control) or the OIR paradigm and allowed to further recover in room air until 6 weeks of life. (A) Western blots of whole retina lysates from 6-week-old mice separated by SDS-PAGE and immunoblotted for FGF2. (B) Histograms depicting mean spatial frequency threshold at maximal contrast and contrast sensitivity at fixed gratings in control (dark columns) and OIR (light columns) mice at 6 weeks of age. Columns represent mean of four to eight mice, bars indicate SEM, and an asterisk (*) indicates differences between control and OIR (P < 0.05).
Figure 7
 
FGF2 protein expression and optokinetic responses in OIR mice. Mice were exposed to room air (control) or the OIR paradigm and allowed to further recover in room air until 6 weeks of life. (A) Western blots of whole retina lysates from 6-week-old mice separated by SDS-PAGE and immunoblotted for FGF2. (B) Histograms depicting mean spatial frequency threshold at maximal contrast and contrast sensitivity at fixed gratings in control (dark columns) and OIR (light columns) mice at 6 weeks of age. Columns represent mean of four to eight mice, bars indicate SEM, and an asterisk (*) indicates differences between control and OIR (P < 0.05).
Discussion
Angiogenesis in the newborn retina is a dynamic process influenced by age, growth factor expression, and fluctuations in tissue oxygen levels. Utilizing genetically modified mice containing the human FGF2 IRES reporter enabled us to delineate temporal patterns of retinal FGF2 expression in the newborn period and throughout the progression of OIR, thereby indicating distinct roles for cap-dependent and -independent regulation. Over the first 3 weeks of life, retinal FGF2 protein expression declines modestly and transiently despite a doubling of mRNA levels. Advancing age also influences FGF2 protein isoform expression, revealing an increase in HMW forms and a reduction in LMW protein. Developmentally, the decline in LMW FGF2 protein beyond P8 correlated with attainment of full retinal vascularization. Unlike VEGF, administration of hyperoxia augments retinal FGF2 mRNA expression during the vaso-obliterative phase of OIR. This finding is consistent with investigations reporting that hyperoxia stimulates FGF2 promoter activity via both Egr-1 (early growth response-1) and Sp1 (stimulating protein-1) transcription factors in lung cells and increases FGF2 mRNA expression in adult mouse lungs.2123 Nevertheless, despite transcriptional upregulation, both HMW and LMW isoform contents are reduced in the presence of hyperoxia. Although we previously found that higher levels of oxygen inhibit cap-dependent translation in the lungs of newborn rats, exposure to 75% O2 failed to elicit alterations in luciferase activities of either the cap-dependent or -independent FGF2 reporters.24 Taken together, these findings suggest that chronic hyperoxia is more likely to inhibit elongation rather than initiation, as previously shown in endothelial cells.25 
Local environmental stressors such as hypoxia are known to inhibit initiation through a reduction in mammalian target of rapamycin (mTOR)–dependent phosphorylation of 4E-binding protein.26 Ischemia, for example, reduces global cap-dependent translation, yet the presence of the VEGF-A IRES permits continued efficient translation.14,27 Under similar circumstances at the onset of phase II of OIR (P13), the relative hypoxia generated by the abrupt switch from 75% O2 to room air increases total FGF2 protein and induces a shift in translational mechanisms favoring the IRES. Close examination of luciferase activities of the FGF2 constructs reveals the change in translational priority can be attributed to reduced cap-dependent (decreased RL) translation rather than enhanced cap-independent translation. During this period, FGF2 mRNA expression increases marginally, indicating that IRES activity is capable of sustaining translation during the low O2 state. In late phase 2, transcriptional activation appears to be the primary driver for the FGF2 protein expression owing to the greatly enhanced cap-dependent and -independent translational activities. In C67BL/6 mice, resolution of NV in OIR (beyond P17) is accompanied by a decline in retinal VEGF protein and mRNA.7,28 The expression of FGF2 protein, in contrast, remains elevated in OIR reporter mice at P21 and at 6 weeks of age. Unfortunately we did not analyze mRNA or luciferase activities at these latter times and therefore cannot determine the regulatory processes involved. 
Age-related alterations in growth factor expression and vascularity during the development and resolution of OIR/ROP are pathophysiologically meaningful, but by themselves say little regarding vision. In humans, spontaneously regressed ROP results in delayed luminance and chromatic contrast sensitivity years after the initial injury.15 In the current study, OIR was associated with reduced spatial frequency threshold at 6 weeks of age, suggesting a deficit in outer retina function. Previous work in the rat model of OIR revealed that fluctuating hypoxia/hyperoxia produced similar reductions in the optokinetic responses.29 In former premature infants, resolved mild stage 1 to 2 ROP produces structural retinal changes, including the development of a shallow and wider foveal pit and the presence of intraretinal vessels overlying the fovea, all of which may influence visual acuity.30 Mild forms of ROP that fully regress are also associated with long-term deficits in rod, cone, and postphotoreceptor neural function in electroretinogram studies.3133 These laboratory and clinical findings indicate that the term “resolved ROP or OIR” is accurate only in reference to the surface vascular elements and not necessarily to the structural and functional integrity of the retina as a whole. 
The persistence of augmented FGF2 expression weeks after induction of OIR raises broader speculation as to the role of FGF2 in the progression of retinopathy. Mice deficient in total FGF2 possess normal-appearing retinal vasculature and develop NV during OIR that is indistinguishable from wild-type counterparts.34 Likewise, retinas from mice overexpressing FGF2 in photoreceptors are normally vascularized and retain the OIR vasculopathy, indicating that FGF2 is neither sufficient nor necessary for the development of NV.34 While the influence of FGF2 on the resolution of NV is currently unknown, the abnormalities in behavioral visual responses in OIR coupled with the heightened FGF2 protein expression point to a potential role in neuroprotection. In retinal degeneration, reports document temporal increases in FGF2 expression that protect the retina by delaying or diminishing photoreceptor cell death.35,36 Indeed, forced overexpression of FGF2 decreases rod cell death in the hyperoxia model of macular degeneration.36 Conceivably, hyperoxic injury may induce early response genes such as Egr-1 during phase 1, which in turn upregulate FGF2 expression to preserve photoreceptor integrity. The subsequent hypoxia incurred in phase II may further promote FGF2 expression through HIF-1α stabilization.37 Alternatively, FGF2 may aid in maintaining vascular-associated glia/microglia to facilitate revascularization following hypoxia.38 
Despite generating functional theories regarding the regulation of FGF2 expression during OIR, the present study has some limitations. Although the luciferase data signifies phase-specific differences in the translational regulation of FGF2, total retinal protein synthesis was not measured, rendering universal application of such changes speculative. Since the newborn retina continues to grow throughout the course of OIR, it would appear that only select transcripts, rather than the entire transcriptome, will be poorly translated. Finally, we did not assess optokinetic responses at a point of confirmed OIR resolution, thus we cannot determine if the observed alterations are permanent or will resolve over time. 
In summary, these data illustrate that retinal FGF2 protein expression varies according to the phase of OIR and involves complex interactions of translational and transcriptional regulatory processes. Alterations in FGF2 protein isoform expression, on the other hand, are dependent upon age rather than OIR, which may be influenced by FGF2 ingested during nursing.39 Finally, a role for FGF2 in neuroretinal function is suggested by the persistent increase in expression that accompanies impaired spatial frequency threshold and contrast sensitivity during the resolution of surface vasculopathy, a result that may have future therapeutic implications. 
Acknowledgments
Disclosure: L. Fang, None; A.J. Barber, None; J.S. Shenberger, None 
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Figure 1
 
Vasculopathy of OIR in FGF2 reporter mice. Photomicrographs of representative flat-mounted retinas from C57BL/6 and FGF2 reporter mice are depicted. In room air (RA) at P12, both mice are fully vascularized. In mice exposed to 75% O2 (Ox) from P7 to P12, both variants undergo similar degrees of vascular regression. Likewise, after 5 days in room air, P17 animals show significant NV.
Figure 1
 
Vasculopathy of OIR in FGF2 reporter mice. Photomicrographs of representative flat-mounted retinas from C57BL/6 and FGF2 reporter mice are depicted. In room air (RA) at P12, both mice are fully vascularized. In mice exposed to 75% O2 (Ox) from P7 to P12, both variants undergo similar degrees of vascular regression. Likewise, after 5 days in room air, P17 animals show significant NV.
Figure 2
 
FGF2 immunoreactivity at P17. Photographs (40×) of retinal cross-sections taken from control and OIR animals at P17 prepared as described in the Methods section. Arrows denote clusters of immunoreactive neovascular cells anterior to the internal limiting membrane. FGF2 immunoreactivity was noted within select cells in the GCL and in general throughout the ONL. Images were obtained approximately one-third of the distance from the optic disc to periphery.
Figure 2
 
FGF2 immunoreactivity at P17. Photographs (40×) of retinal cross-sections taken from control and OIR animals at P17 prepared as described in the Methods section. Arrows denote clusters of immunoreactive neovascular cells anterior to the internal limiting membrane. FGF2 immunoreactivity was noted within select cells in the GCL and in general throughout the ONL. Images were obtained approximately one-third of the distance from the optic disc to periphery.
Figure 3
 
Retinal FGF2 mRNA expression during OIR. Retinal FGF2 mRNA levels from whole mouse retinas were determined by RT-PCR and normalized to GAPDH. FGF2 expression increased over time in both controls (dark columns) and OIR (light columns) mice. FGF2 expression was increased in OIR animals relative to controls at P8, P13, P15, and P17. Columns represent mean of four animals, and bars indicate SEM. *P < 0.05.
Figure 3
 
Retinal FGF2 mRNA expression during OIR. Retinal FGF2 mRNA levels from whole mouse retinas were determined by RT-PCR and normalized to GAPDH. FGF2 expression increased over time in both controls (dark columns) and OIR (light columns) mice. FGF2 expression was increased in OIR animals relative to controls at P8, P13, P15, and P17. Columns represent mean of four animals, and bars indicate SEM. *P < 0.05.
Figure 4
 
Impact of OIR on retinal VEGF mRNA. Mouse retinal VEGF-A mRNA levels determined by RT-PCR were normalized to GAPDH (n = 4 each time and condition). Total VEGF, VEGF120, and VEGF164 mRNA expression was decreased in OIR (light columns) mouse pups relative to controls (dark columns) at P12 (†P < 0.05). At both P15 and P17, however, OIR mice had greater expression of total, VEGF120, and VEGF164 mRNA relative to control mice (*P < 0.05). Bars indicate SEM.
Figure 4
 
Impact of OIR on retinal VEGF mRNA. Mouse retinal VEGF-A mRNA levels determined by RT-PCR were normalized to GAPDH (n = 4 each time and condition). Total VEGF, VEGF120, and VEGF164 mRNA expression was decreased in OIR (light columns) mouse pups relative to controls (dark columns) at P12 (†P < 0.05). At both P15 and P17, however, OIR mice had greater expression of total, VEGF120, and VEGF164 mRNA relative to control mice (*P < 0.05). Bars indicate SEM.
Figure 5
 
Retinal FGF2 protein expression during OIR. Whole retinas from mice exposed to room air (control) or the OIR paradigm were homogenized and lysates separated by SDS-PAGE. They were transferred to PVDF membranes and immunoblotted for FGF2. Quantitation was performed by densitometry and normalized to the expression of β-actin. (A) Representative immunoblots from control (dark columns) and OIR (light columns) mice at P8 to P21 showing high and low molecular weight (MW) FGF2 isoforms. (B) Histograms depicting mean expression of total, high MW, and low MW FGF2 normalized to β-actin (n = 4 per time and condition). The dagger symbol (†) indicates OIR values significantly less and the asterisk (*) OIR values significantly greater than control at a given time point. For all, P < 0.05, and bars indicate SEM.
Figure 5
 
Retinal FGF2 protein expression during OIR. Whole retinas from mice exposed to room air (control) or the OIR paradigm were homogenized and lysates separated by SDS-PAGE. They were transferred to PVDF membranes and immunoblotted for FGF2. Quantitation was performed by densitometry and normalized to the expression of β-actin. (A) Representative immunoblots from control (dark columns) and OIR (light columns) mice at P8 to P21 showing high and low molecular weight (MW) FGF2 isoforms. (B) Histograms depicting mean expression of total, high MW, and low MW FGF2 normalized to β-actin (n = 4 per time and condition). The dagger symbol (†) indicates OIR values significantly less and the asterisk (*) OIR values significantly greater than control at a given time point. For all, P < 0.05, and bars indicate SEM.
Figure 6
 
Translational regulation of FGF2 expression. Mice containing a bicistronic, dual-luciferase transgene containing the FGF2 IRES were exposed to room air (control, dark columns) or the OIR (light columns) paradigm. Whole retinas were homogenized and lysates analyzed for Renilla and firefly luciferase activity representing IRES-mediated mRNA translation and cap-dependent mRNA translation, respectively, as described in the Methods section. Figure illustrates the effect of OIR on (A) Renilla luciferase (RR); (B) firefly luciferase (FL); and (C) the RR/FL ratio. Columns represent mean of 5 to 10 animals, and bars indicate SEM. The dagger symbol (†) indicates OIR values less than and the asterisk (*) OIR values greater than control at a given time point. For all, P < 0.05.
Figure 6
 
Translational regulation of FGF2 expression. Mice containing a bicistronic, dual-luciferase transgene containing the FGF2 IRES were exposed to room air (control, dark columns) or the OIR (light columns) paradigm. Whole retinas were homogenized and lysates analyzed for Renilla and firefly luciferase activity representing IRES-mediated mRNA translation and cap-dependent mRNA translation, respectively, as described in the Methods section. Figure illustrates the effect of OIR on (A) Renilla luciferase (RR); (B) firefly luciferase (FL); and (C) the RR/FL ratio. Columns represent mean of 5 to 10 animals, and bars indicate SEM. The dagger symbol (†) indicates OIR values less than and the asterisk (*) OIR values greater than control at a given time point. For all, P < 0.05.
Figure 7
 
FGF2 protein expression and optokinetic responses in OIR mice. Mice were exposed to room air (control) or the OIR paradigm and allowed to further recover in room air until 6 weeks of life. (A) Western blots of whole retina lysates from 6-week-old mice separated by SDS-PAGE and immunoblotted for FGF2. (B) Histograms depicting mean spatial frequency threshold at maximal contrast and contrast sensitivity at fixed gratings in control (dark columns) and OIR (light columns) mice at 6 weeks of age. Columns represent mean of four to eight mice, bars indicate SEM, and an asterisk (*) indicates differences between control and OIR (P < 0.05).
Figure 7
 
FGF2 protein expression and optokinetic responses in OIR mice. Mice were exposed to room air (control) or the OIR paradigm and allowed to further recover in room air until 6 weeks of life. (A) Western blots of whole retina lysates from 6-week-old mice separated by SDS-PAGE and immunoblotted for FGF2. (B) Histograms depicting mean spatial frequency threshold at maximal contrast and contrast sensitivity at fixed gratings in control (dark columns) and OIR (light columns) mice at 6 weeks of age. Columns represent mean of four to eight mice, bars indicate SEM, and an asterisk (*) indicates differences between control and OIR (P < 0.05).
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