Three days after RPE-specific Vegf
inactivation, we observed complete ablation of the choriocapillaris (Figure A). Since almost the entire blood supply to the outer two-thirds of the retina (including the photoreceptors) is provided by the choriocapillaris, we predicted that its loss would induce dramatic secondary effects on the retinal neurons. In fact, following Vegfa
inactivation in RPE cells, we observed profound retinal dysfunction, especially in cone photoreceptors. Within 3 days after induction, rapid and progressive dysfunction of cone photoreceptors was detected using electroretinography (ERG) (Figure B) compared with multiple controls, including Vegfaf/f
(no Cre), Vegfaf/f
;VMD2-Cre with doxycycline, and Vegfaf/f
;VMD2-Cre without doxycycline (Supplemental Figure 1; supplemental material available online with this article; doi:
). Using immunohistochemistry to selectively stain for the cone photopigment opsin, we observed a nearly complete absence of cone photoreceptor outer segments (Figure C). Cone dysfunction was observed by at least 7 months after Vegfa
deletion in RPE (Supplemental Figure 2). These data demonstrate that RPE-derived VEGF is required for maintenance of the choriocapillaris and for cone photoreceptors, the most metabolically demanding cells in the central nervous system (14
). Photoreceptors also express VEGF receptors (9
), and paracrine-derived VEGF may have a direct role in their maintenance. In humans, loss of cones results in legal blindness, since fine (or “reading”) visual acuity is maintained by cones located in the central retina, or macula.
Inducible Vegfa deletion in adult RPE cells promotes rapid choriocapillaris degeneration and vision loss.
In contrast, rod photoreceptor function (responsible for achromatic and night vision) is insensitive to Vegfa
inactivation at the same time points when we observe cone degeneration. Immunohistochemical analyses revealed that the layer of rod photoreceptor outer segments was as thick as that of controls (Figure A). Since the murine retina is composed largely of rods and the thickness of the entire retina does not change as late as 7 months after Cre recombinase induction (Figure B), this suggests that there is no significant rod photoreceptor atrophy. Scotopic (dark adapted) ERGs demonstrate that rod photoreceptors are fully functional, since there was no change in rod photoreceptor light responsiveness in recordings before and after induction in Vegfa
mutant mice (Figure C). The preservation of rod photoreceptors for several months after gene deletion suggests that these cells are supported by a minimal vasculature not dependent on RPE-derived Vegfa
for its survival. Indocyanine green angiography demonstrated 2 blood supply sources for photoreceptors in Vegfa
mutants; intraretinal and outer, major choroidal vessels (Figure D). Utilizing electron microscopy, we determined that the choriocapillaris remained atrophic through 7 months and choroidal vessels were observed only sporadically (Figure E). These findings are consistent with results from a previous study that examined the effects of global deletion of the soluble VEGF isoforms (VEGF188/188
Cone but not rod photoreceptor dysfunction in RPE-specific Vegfa mutants.
Atrophy and thinning of the choriocapillaris is a known age-related event in humans (18
). To compensate for choriocapillaris thinning, the RPE is known to synthesize proangiogenic factors that maintain the choriocapillaris (19
). Our findings suggest that VEGF may act both directly and indirectly to regulate this phenomenon. Using gene-profiling assays, we demonstrated that the loss of Vegfa
affects the expression of multiple angiogenesis-related genes (most are downregulated) in isolated RPE/choroid preparations (Figure F and Supplemental Table 1). Vegfa
is tightly regulated by HIFs during development and pathologically in disease states (21
). To determine the role for HIFs in maintenance of the choriocapillaris, we genetically ablated Hif1a
(encoding HIF-1α protein), Epas1
(HIF-2α), and Hif1a/Epas1
in adult RPE. No obvious morphological, functional, or transcriptional differences, including Vegfa
mRNA, were observed in naive-state adult HIF mutants (Supplemental Figures 3 and 4, and Supplemental Table 1). Several pathways, such as NF-κB/JunB and PGC1α/ERRα are known to induce Vegfa
gene expression independently of HIFs (23
). These factors, or others, rather may maintain VEGF levels at sufficient levels in healthy retinas. Using laser photocoagulation to induce choroidal neovascularization (CNV), we determined that the extent of CNV was significantly reduced in Vegfa
mutants (and partially reduced in Hif1a
mutants; Figure , A and B). The upregulation of angiogenic genes was observed in wild-type C57BL/6 mice, but normal expression of these genes was observed in HIF mutants and expression was attenuated in Vegfa
mutants compared with naive controls (Figure C and Supplemental Table 2). These data strongly suggest that HIFs become activated in pathological states to promote VEGF-mediated neovascularization, but are not required to maintain physiological vasculature.
Attenuated angiogenic gene regulation in pathological states of Vegfa conditional mutants.
VEGF-mediated neovascularization is associated with vision loss in diseases such as AMD (10
). The majority of patients receiving currently approved anti-VEGF drugs to treat AMD experience no adverse events. However, long-term safety studies of the effects of VEGF antagonism in the eye have not yet been completed, and there are isolated clinical reports showing progressive RPE/photoreceptor atrophy in patients on anti-VEGF therapies (25
). Due to the differences in rod and cone photoreceptor distribution in humans and mice, it is difficult to speculate on what the effect of near-complete VEGF antagonism may be in human subjects. However, if a certain threshold of VEGF could be neutralized, the fovea would be very susceptible and the loss of macular function could result in the devastating loss of central vision. These observations, coupled with reported renal, gastrointestinal (GI), and mucosal complications in patients on systemic VEGF inhibition, contribute to the concern that VEGF antagonism may not be completely effective or safe (26
). The data in this study reinforce this concept and suggest that HIF-targeted or combination therapies targeting multiple angiogenic pathways may be safer and more effective than increased VEGF suppression therapy. Newer VEGF antagonists that bind VEGF with higher affinities and less rapid clearing from the eye are coming into use to treat a variety of neovascular diseases (28
). Our results suggest that “off-target” effects of potent, persistent VEGF antagonism in the eye may exacerbate degeneration of the choriocapillaris, RPE, and photoreceptors due to the diminished trophic effect of RPE-produced VEGF.