Targeting Hif1a rescues cone degeneration and prevents subretinal neovascularization in a model of chronic hypoxia

All experimental procedures were performed according to ‘The Association for Research in Vision and Ophthalmology’ statement on animal use in ophthalmic and vision research and the regulation of the veterinary authorities of Kanton Zurich, Switzerland. R91W;Nrl ^-/- mice were generated by crossing Rpe65 ^R91W (R91W) [33] to Nrl ^-/- mice [34], and were described recently [32]. BPCre;R91W;Nrl ^-/- ;Vhl ^f/f (=cone ^ΔVhl ) mice were generated by breeding R91W;Nrl ^-/- mice to Vhl ^f/f mice [35] and mice expressing the Cre recombinase under the transcriptional control of the blue cone opsin (BP) promoter [36]. To generate BPCre;R91W;Nrl ^-/- ;Vhl ^f/f ;Hif1a ^f/f (=cone ^ΔVhlHif1a ) mice, BPCre;R91W;Nrl ^-/- ;Vhl ^f/f were bred to Hif1a ^f/f mice [37]. R91W;Nrl ^-/- ;Vhl ^f/f and R91W;Nrl ^-/- ;Vhl ^f/f ;Hif1a ^f/f littermates without Cre recombinase served as respective control mice (=ctrl). Genotyping was performed by PCR using DNA isolated from ear clips and primer pairs as described previously [38, 39]. Presence of BPCre was tested using the following primer pair: forward (5’-GGACATGTTCAGGGATCGCCAGGCG-3’) and reverse (5’-GCATAACCAGTGAAACAGCATTGCTG-3’). The amplification reaction resulted in a 268 bp fragment in the presence of the transgene. To test for deletion of floxed sequences, genomic DNA was isolated from retinal tissues and tested by PCR using appropriate primer pairs as described [38, 39]. 129S6 (Taconic, Ejby, Denmark) mice were used as wild-type controls. To test expression of the Cre recombinase BPCre;R91W;Nrl ^-/- mice were bred to a ZsGreen reporter line (Ai6 mice, Gt(ROSA)26Sor ^{tm6(CAG-ZsGreen1)Hze} , [40]). Mice of both sexes were used for experiments and were housed at the animal facility of the University Zurich under a 14 h : 10 h light/dark cycle with lights on at 6 am and lights off at 8 pm. Food and water were provided ad libitum.

To evaluate retinal morphology, eyes were enucleated and fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 7.2, 0.1 M), according to the previously described procedure [41]. By cutting through the optic nerve head, nasal and temporal halves of the eyecups were separated and embedded in epon plastic. Semi-thin cross sections (0.5 μm) were counterstained with toluidine blue and analyzed by light microscopy (Axioplan; Zeiss, Jena, Germany). Thickness of the outer nuclear layer was measured at indicated distances from the optic nerve head using the Adobe Photoshop CS6 ruler tool (Adobe Systems, Inc., San Jose, CA, USA) on reconstructed retinal panorama images.

After euthanasia, eyes were marked nasally, enucleated and fixed in 4% paraformaldehyde (PFA) in phosphate buffer for 1 h at 4°C. Cornea and lens were removed and the dissected eyecups postfixed for 2 h in 4% PFA. After immersion in 30% sucrose (in PBS 0.1 M) the eyecups were embedded in tissue freezing medium (O.C.T., Leica Biosystems Nussloch GmbH, Nussloch, Germany), frozen in a 2-methylbutane bath cooled by liquid nitrogen and stored at -80°C. Cryosections (12 μm) were blocked (3% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA), 0.3% Triton X-100 (Sigma) in PBS) and incubated with the following primary antibodies overnight at 4°C: Isolectin GS-IB₄-Alexa594 from Griffonia simplicifolia (1:300, I21413; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-allograft inflammatory factor 1 (alias IBA1, 1:1000, 019-19741; Wako, Neuss, Germany), rabbit anti-albumin (ALB, 1:500, RARaAlb; Nordic Immunology, Tilburg, Netherlands). Sections were washed with PBS, incubated with secondary antibodies (Cy3-labeled, Jackson ImmunoResearch Laboratories, Westgrove, PA, USA) for 1 h at room temperature (RT), counterstained with DAPI (4',6-Diamidine-2'-phenylindole dihydrochloride, Roche, Basel, Switzerland) and analyzed by fluorescence microscopy (Axioplan; Zeiss).

Eyes were isolated and incubated for 5 to 10 minutes in 2% PFA in PBS as described recently [42]. After removal of cornea and lens, the retina was dissected and flat-mounted in PBS. After postfixation in 4% PFA for 1 h at RT, flat-mounts were blocked (3% normal goat serum, 0.3% Triton X-100 in PBS, 1 h) and incubated with isolectin GS-IB₄-Alexa594 (1:300, Thermo Fisher Scientific) at 4°C overnight. Retinas were washed in PBS, mounted on glass slides and analyzed by fluorescence microscopy (Axioplan/ApoTome; Zeiss). Blood vessels were reconstructed in three dimensions using Imaris software (Versions 7.7.2/8.3.0, Bitplane AG, Zurich, Switzerland). For better recognition and distinction of the vascular plexi, the z-value of the z-stacks was increased five times.

Retinas were isolated through a slit in the cornea, frozen in liquid nitrogen and stored at -80°C. RNA was extracted using an RNA isolation kit (RNeasy; Qiagen, Hilden, Germany) including a DNAse treatment. 1 μg of RNA, oligo (dT) and M-MLV reverse transcriptase (Promega, Fitchburg, WI, USA) were used to prepare cDNA. To analyze gene expression by real-time PCR, 10 ng of cDNA template was amplified using a PCR polymerase ready mix (LightCycler 480 SYBR Green I Master, Roche Diagnostics, Rotkreuz, Switzerland), specific primer pairs (Table 1) and a thermocycler (LightCycler 480, Roche Diagnostics). Expression levels were normalized to β-actin (Actb) and relative expression was calculated using the comparative threshold cycle method (∆∆C_T) of the LightCycler480 software (Roche Diagnostics). At least 3 mice per strain and time point were used.

Proteins were isolated by homogenizing the retinas in ice-cold Tris-HCl (100 mM, pH 7.5) using ultrasound at 4°C. Protein concentrations were determined spectrophotometrically by using Bradford reagent (Bio-Rad, Hercules, CA, USA). SDS-PAGE and Western blotting were performed as described [41] and proteins detected by rabbit anti-HIF1A (1:2000, NB100-479, Novus Biologicals, Littleton, CO, USA) and mouse anti-β-actin (1:10’000, A5441, Sigma-Aldrich) antibodies. HRP-conjugated secondary antibodies were applied for 1 h at RT and signals were visualized using Western lightning chemiluminescence reagent (PerkinElmer, Waltham, MA, USA) and X-ray films.

Mice were dark-adapted overnight and pupils were dilated under dim red light with 1% cyclogyl (Alcon Switzerland SA, Rotkreuz, Switzerland) and 5% neosynephrin-POS (Ursapharm Schweiz GmbH, Roggwil, Switzerland) 30 minutes prior to recording. Mice were anesthetized with a subcutaneous injection of ketamine (85 mg/kg, Pfizer PFE Switzerland GmbH, Zurich, Switzerland) and xylazine (Rompun 2%, 4mg/kg, Bayer, Leverkusen, Germany). To keep the cornea moist, a drop of mydriaticum dispersa (Omnivision AG, Neuhausen, Switzerland) was applied to each eye. Low background illumination for 5 min was used for light adaptation. Electroretinograms were recorded simultaneously from both eyes with an LKC UTAS Bigshot unit (LKC Technologies, Inc. Gaithersburg, MD, USA) as described [32]. Flashes of 8 different light intensities ranging from -10 to 25 dB (0.25–790.5694 cd*s/m²) were applied under photopic conditions. 10 responses were averaged per light intensity. Traces from n≥4 mice were averaged for each light intensity.

Fundus photographs were taken with a Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA). The pupils were dilated and the mice were anesthetized as described above. Methocel 2% (Omnivision) was applied to lubricate the eyes. Fluorescein images were captured 1-5 minutes after intraperitoneal injection of 20 μL of 2% fluorescein solution (Akorn, Lake Forest, IL, USA).

Two-way ANOVA with Šídák’s multiple comparison test was performed using GraphPad Prism (version 7.02, GraphPad Software, San Diego, CA, USA) for statistical analysis of gene expression levels and ERG traces. All data are shown as means ± SD.

Photoreceptors in the all-cone R91W;Nrl ^-/- mouse are predominantly blue-light sensitive S-cones [32, 34]. Therefore, we used BP-Cre transgenic mice, which express functional Cre recombinase under the transcriptional control of the blue cone opsin promoter (BP, [36]) to delete floxed sequences from cones in the all-cone mice. BPCre;R91W;Nrl ^-/- ;ZsGreen reporter mice verified that Cre expression was uniform over the entire retina and restricted almost exclusively to the majority of cones in the ONL (Fig. 1a). Green fluorescence of the activated reporter protein was only occasionally detected in few cells of the inner retina. We validated Vhl excision in the BPCre;R91W;Nrl ^-/- ;Vhl ^f/f (=cone ^ΔVhl ) mouse line by analyzing genomic DNA isolated from retinal tissues of 4-week-old cone ^ΔVhl mice. The detection of a PCR product corresponding to the excised fragment suggested a successful Cre-mediated deletion of Vhl in retinas of cone ^ΔVhl but not of ctrl mice (=R91W;Nrl ^-/- ;Vhl ^f/f , Fig. 1b). Genomic deletion of Vhl led to the accumulation of HIF1A protein (Fig. 1c) and to increased transcript levels of known hypoxic target genes such as adrenomedullin (Adm), egl-9 family hypoxia-inducible factor 1 (Egln1, also known as Phd2), glucose transporter 1 (Glut1) and BCL2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) in normoxic cone ^ΔVhl mice (Fig. 1d). This suggested that cells, presumably cones, activated a hypoxia-like response in retinas of cone ^ΔVhl mice.

We observed an age-dependent decline in HIF1A protein levels and hypoxia target gene expression in cone ^ΔVhl mice (Fig. 1c, d). This prompted us to analyze retinal function and morphology. B-wave amplitudes and photopic ERGs were not significantly different between cone ^ΔVhl mice and ctrl mice at 6 weeks of age (Fig. 2a). However, the amplitudes were strongly reduced in 12-week-old cone ^ΔVhl mice as compared to age-matched ctrl mice (Fig. 2b). This indicated an age-dependent loss of function potentially due to retinal degeneration. Indeed, we observed severe, progressive thinning of the outer nuclear layer (ONL) in cone ^ΔVhl mice with most cones lost at 26 weeks of age (Fig. 3a, b). Additionally, partial loss of the retinal pigment epithelium (RPE) and strong perturbations in the inner nuclear layer (INL) were detected in older cone ^ΔVhl mice (Fig. 3a, 12we cone ^ΔVhl ). In contrast, the ctrl mice showed only a slow age-related ONL thinning, as reported for the parental R91W;Nrl ^-/- mouse line before [32].

To gain insight into cellular mechanisms leading to cone degeneration, we analyzed expression of specific genes during aging in cone ^ΔVhl and ctrl mice (Fig. 3c). Caspase 1 (Casp1), a gene involved in retinal degeneration, was upregulated in cone ^ΔVhl mice at 8 weeks and peaked at 12 weeks of age. Expression of the stress signaling gene glial fibrillary acidic protein (Gfap) was upregulated in cone ^ΔVhl mice already at 4 weeks and remained elevated throughout the observation period. Interestingly, we observed increased expression of tissue inhibitor of metalloproteinase 3 (Timp3) during the aging process, a gene associated with age-related macular degeneration [43, 44]. Similarly, expression of vascular endothelial growth factor (Vegf), a hypoxic response gene that is involved in neovascularization in wet AMD, was strongly increased in cone ^ΔVhl mice at 4 and 6 weeks of age.

Increased transcription of Timp3 is potentially associated with a higher risk to develop neovascular AMD [43] and hypoxia-regulated genes such as Vegf contribute to retinal and choroidal neovascularization (reviewed in [45]). Therefore, we analyzed the vascular network of cone ^ΔVhl mice. All three vascular plexi were detected at 4 weeks of age (Fig. 4a, b, c). However, we observed vessels extending from the deep plexus into the normally avascular ONL in central retinas of cone ^ΔVhl mice (Fig. 4a, arrowheads). Vessels reached the RPE but did not cross Bruch’s membrane (Fig. 4d, e), as we never observed retinal-choroidal anastomoses. This suggests that abnormal vessels originated from the retinal vasculature and not from the choroid. Interestingly, abnormal vessel growth was characteristic for the central but not for the peripheral retina (Fig. 4b). To evaluate the integrity of retinal vessels we performed fluorescein angiography. Signs of leakage were observed in cone ^ΔVhl mice (Fig. 4f, g). To confirm these findings we stained retinal cross-sections for albumin (ALB), a marker for blood extravasation. Detailed analysis revealed strong ALB immunoreactivity in the ONL, RPE and inner plexiform layer (IPL) in cone ^ΔVhl mice, as opposed to the signal detected in ctrl mice that was confined to retinal vessels. The choroid with its extensive vasculature was also strongly positive for ALB in both types of mice (Fig. 4h, i). Microglia/macrophages detected by IBA1 staining were found within the photoreceptor and subretinal layer in cone ^ΔVhl but not ctrl mice (Fig. 4j, k).

S-opsin expression starts shortly before birth in mice [46, 47] and we observed Cre-activity in BPCre;R91W;Nrl ^-/- ;ZsGreen reporter mice as early as at postnatal day (PND) 1 (data not shown). The primary plexus in mice develops along a central-to-peripheral gradient and reaches the periphery around PND8-10 [48]. Vessels sprout from the primary plexus into the retina and turn laterally when they reach the outer and inner boundaries of the INL to first form the deep plexus and subsequently the intermediate plexus [48]. To determine the onset of neovascularization in cone ^ΔVhl mice we stained retinal flat mounts with isolectin at PND7 and PND11. Using 3D-reconstruction of blood vessels, no difference in early vessel formation was detected between cone ^ΔVhl and ctrl mice at PND7 (Fig. 5a). However, at PND11, before formation of the intermediate plexus, vessels growing from the deep plexus into the ONL were observed in cone ^ΔVhl mice (Fig. 5b).

Formation of the deep plexus is preceded by Vegf expression in the INL [49]. We analyzed gene expression levels in cone ^ΔVhl , ctrl and 129S6 (wt, rod-dominant retina) mice to test for different regulation of genes involved in angiogenesis and vessel guidance during and after the process of vessel formation (PND7, 14 and 28, Fig. 5c). Vegf mRNA was not elevated at PND7, slightly increased by 1.3-fold at PND14 and significantly increased at PND28 (2-fold) in cone ^ΔVhl mice compared to controls. Fibroblast growth factor 2 (Fgf2), a potent angiogenic factor that has been shown to be involved in retinal stress [50‐52], was upregulated at PND28, at a time when developmental vascularization is completed. Recently, protective effects of semaphorin 3F (Sema3f) against subretinal neovascularization have been demonstrated [53]. We thus analyzed Sema3f gene expression levels to test for potential differential expression of this anti-angiogenic factor in cone ^ΔVhl mice. However, expression of Sema3f in all-cone mice did not differ between ctrl and cone ^ΔVhl mice (Fig. 5c). On the other hand, a pro-angiogenic factor, platelet derived growth factor, B polypeptide (Pdgfb), was upregulated in cone ^ΔVhl compared to ctrl mice at PND28. Pdgfb expression by endothelial cells is essential for pericyte recruitment and is increased under hypoxia [54‐57]. Surprisingly, expression levels of von Willebrand factor (vWf), a marker for endothelial cells, as well as of Pdgfb receptor (Pdgfrb), which is expressed by pericytes/mural cells [58‐61], were not increased in cone ^ΔVhl mice as compared to controls. The reason for this unexpected pattern of expression is not clear and requires further investigation.

We hypothesized that stabilized HIF1A, and not other Vhl targets, might promote retinal degeneration and neovascularization in cone ^ΔVhl mice. Therefore, we additionally deleted Hif1a in cone photoreceptors (BPCre;R91W;Nrl ^-/- ;Vhl ^f/f ;Hif1a ^f/f =cone ^ΔVhlHif1a ). After we confirmed the presence of deletion alleles for both Hif1a and Vhl in genomic DNA of cone ^ΔVhlHif1a retinas (not shown) we determined retinal function. At 12 weeks of age, photopic ERG traces and b-wave amplitudes were similar in cone ^ΔVhlHif1a and ctrl (R91W;Nrl ^-/- ; Vhl ^f/f ;Hif1a ^f/f ) mice (Fig. 6a, b) indicating no functional loss upon combined Hif1a and Vhl inactivation. Similarly, retinal morphology (Fig. 6c) and ONL thickness (Fig. 6d) of cone ^ΔVhlHif1a mice was comparable to ctrl mice, whereas the thickness of the ONL in cone ^ΔVhl mice was prominently reduced (same data as in Fig. 3b) at 12 weeks of age.

Combined deletion of Hif1a and Vhl also prevented pathological neovascularization into the ONL but had no effect on the developmental formation of the three vascular plexi (Fig. 6e). No induction of the expression of hypoxic target genes such as Adm, Egln1, Vegf and Timp3 was detected in cone ^ΔVhlHif1a mice, confirming that HIF1 was the responsible HIF isoform for regulating their expression in cones (Fig. 6f). Altogether, these data demonstrate that combined deletion of Hif1a and Vhl (cone ^ΔVhlHif1a mice) rescues the retinal phenotype observed in cone ^ΔVhl mice and identify HIF1 as the causative factor for retinal degeneration and pathological vessel growth in cone ^ΔVhl mice.