One of the long-standing observations in ophthalmology is that there is a high correlation between retinal nonperfusion and neovascularization— when there is substantial dropout of the retina vessels, retinal neovascularization occurs.
We now understand that hypoxia-inducible factor-1 (HIF- 1) is the main protagonist in this process. HIF-1 is a transcription factor that under normoxic conditions is hydroxylated, binds to the Von Hippel-Lindau (VHL) protein and is rapidly degraded. In the setting of hypoxia, however, it is stabilized and translocates to the nucleus where it binds to the hypoxia response element, which increases transcription of a whole host of genes.
VASCULAR ENDOTHELIAL GROWTH FACTOR
One of the most important hypoxia-regulated genes is
vascular endothelial growth factor (VEGF). Mouse retinal
vessels do not develop until after birth. Right after birth,
the retinal vessels begin to develop at the optic nerve and
grow out to the periphery of the retina, a process driven
in part by VEGF. If infant mice are put into a hyperoxic
environment, HIF-1 is destabilized and degraded and the
expression of VEGF is significantly reduced (Figure 1). The
development of retinal vessels ceases and the newly developed
vessels, which are dependent upon VEGF for survival,
regress. After 5 days in a high-oxygen environment,
there are large areas of nonperfused retina and return of
the mice to room air results in retinal ischemia. This
results in stabilization of HIF-1, increased production of
VEGF, and retinal neovascularization.
In choroidal neovascularization (CNV), the blood vessels grow under the retina and so it is not clearly related to hypoxia (Figure 2). To determine if VEGF plays a role in that we tried to mimic that disease process by creating transgenic mice using the rhodopsin promoter (rho) to express VEGF in photoreceptors (Figure 3).1 The rho/VEGF transgene begins production of VEGF at postnatal day (P) 7. By P10 the endothelial cells begin to migrate into the photoreceptor layer and at P21, well-developed blood vessels that extend from the deep capillary bed into the subretinal space are visible (Figure 4). The hyperfluoresence represents significant neovascularization with feeder vessels from the deep capillary bed extending into the subretinal space forming clusters of new vessels partially surrounded by retinal pigment epithelial cells (Figure 5). After longer periods of time, these blood vessels form an extensive complex of vessels in the subretinal space. Fluorescein angiography shows hyperfluorescent spots beneath the retina that increase in size over time (Figure 6), similar to what is seen in human patients with neovascular age-related macular degeneration (AMD). Because the new vessels extend from retinal vessels into the subretinal space, this is actually a model for retinal angiomatous proliferation (RAP). In order to get new vessels that grow from the choroid into the subretinal space, it is necessary to perturb Bruch's membrane. One way to achieve this is to rupture Bruch's membrane with laser photocoagulation, which results in CNV (Figure 7).
VEGF TRAP
VEGF trap is a recombinant protein consisting of VEGFbinding
domains from VEGF receptors 1 and 2, which binds
VEGF-A and placental growth factor, both hypoxia-regulated
gene products. Administration of VEGF trap strongly
suppresses laser-induced CNV2 indicating that VEGF and
possibly placental growth factor are critical stimuli in this
model.
PDGF-B
Another hypoxia-regulated gene is platelet-derived growth
factor B (PDGF-B). While VEGF is a survival factor for newly
developed endothelial cells, PDGF-B is a survival factor for
newly developed pericytes. The pericytes provide survival
factors other than VEGF to endothelial cells. Elimination of
pericytes makes endothelial cells more dependent upon
VEGF for survival. We made transgenic mice with inducible
expression of PDGF-B; their phenotype is similar to that of
mice with inducible expression of VEGF in the retina; both
develop severe neovascularization and retinal detachment.3
Compared to agents that target only VEGF, kinase inhibitors
that block both VEGF and PDGF receptors are more effective
inhibitors of CNV. PKC412 is a nonselective VEGF and
PDGF inhibitor that strongly suppresses CNV (Figure 8).4
Pazopanib is another kinase inhibitor that blocks both PDGF
and VEGF receptors. Unlike specific VEGF antagonists, which
only suppress CNV, pazopanib causes regression of established
CNV (Figure 9).5
SDF-1
SDF-1 and its receptor, CXCR4, are hypoxia-regulated
genes. CXCR4 is located on monocytes and macrophages
and is involved in macrophage recruitment from the bone
marrow and blood stream into tissues. In ischemic retina,
both SDF-1 and CXCR4 are increased.6 Even in the absence
of ischemia, elevated levels of VEGF cause increases in SDF-1
and CXCR4. Under normal conditions, there is very little
SDF-1 in the retina, while in hypoxic retina, it is produced by
glial cells identified by staining for glial fibrillary acidic protein.
The production of SDF-1 by glial cells promotes influx and retention of macrophages into the retina and because
the glial cells wrap around blood vessels, the macrophages
also surround blood vessels (Figure 10). Thus, the production
of SDF-1 by ischemic retina allows macrophages to
home in on retinal vessels and participate in stimulation of
neovascularization.
VEGF, PDGF-B, and SDF-1 are three soluble signals that participate in ocular neovascularization. Each is transcriptionally regulated by HIF-1. The receptors for these agents are also upregulated by HIF-1 as is angiopoietin-2, which helps to regulate signaling derived from extracellular matrix. Thus, a reasonable strategy might be to target HIF-1 itself because it regulates all of the soluble signals.
The laboratory of Gregg L. Semenza, MD, PhD, has determined that several drugs approved by the US Food and Drug Administration for other actions also act as inhibitors of HIF-1.7 One of these is digoxin, an agent used for cardiac failure or arrhymias. In ischemic retina, digoxin is a strong suppressor of HIF-1 (Figure 11)8 and a large number of hypoxia-regulated genes, including VEGF, SDF-1, placental growth factor, PDGF-B, and angiopoietin 2. Digoxin also reduces influx of macrophages into ischemia retina and prevents retinal neovascularization.
Thus, retinal hypoxia is a major driving force for retinal neovascularization. Does it play a role in excessive vascular permeability and macular edema? To investigate the role of retinal hypoxia in macular edema, my colleagues and I performed a study where we gave patients with diabetic macular edema (DME) continuous supplemental oxygen by nasal cannula.9 Figure 12A is a baseline OCT from one patient, showing thickening throughout the central area of the retina. After 3 months of supplemental oxygen (Figure 12B), there is a marked decrease in thickening. Nine eyes of patients DME experienced a reduction in edema while on supplemental oxygen (Figure 13) and when the oxygen was discontinued, the edema returned in most patients, although some patients in whom macula thickening had normalized had persistent benefit for at least 3 months.
Which of the hypoxia-regulated gene products is most important in macular edema? Not surprisingly, it is VEGF. Injecting a pellet that releases VEGF into the vitreous cavity of monkeys (Figure 14A) results in tremendous leakage from the retinal vessels (Figure 14B).10 VEGF antagonists provide great benefit in macular edema due to DME or retinal vein occlusions.
SUMMARY
Hypoxia-regulated genes play an important role in ocular
neovascularization and macular edema. VEGF is particularly
important and most efforts thus far have been directed at
suppressing VEGF. Over the next several years agents that
target other hypoxia-regulated gene products will be tested
in combination with VEGF antagonists to see if additional
benefit can be obtained.
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