Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

measurement. In other words, an image of the eye obtained during room air breathing alone cannot be used to measure retinal oxygenation, but the images acquired before and during carbogen breathing can be compared to measure the change in pO2. The agreement between the MRI and oxygen electrode data support this interpretation.71

Carbogen is a gas mixture of carbon dioxide (5% CO2) and oxygen (95% O2) that has been used clinically, instead of 100% oxygen, to minimize the vasoconstrictive effects of pure O2 on retinal blood flow. We and others have confirmed that, in the rat, carbogen increases retinal oxygenation relative to O2 breathing.71,73 When this retinal oxygenation response (ROR or pO2) is measured in the posterior vitreous (within 200 μm from the retina), we refer to it as a measure of inner retinal oxygenation.71 The functional MRI pO2 measurement is particularly advantageous because (1) it is noninvasive, (2) it is applicable to a wide range of species including mice, rats, and humans,71,74-77

(3) it simultaneously measures the retinal oxygenation response from superior to inferior ora serrata, and (4) it is not affected by media opacities, such as cataract.68 There are currently no other techniques that can noninvasively measure the panretinal oxygenation response in rats, mice, and humans.

6.3Results

To determine if pO2 is sensitive to retinal NV, we measured the spatial and temporal pO2 patterns associated with abnormal retinal vessel growth in the 50/10 newborn rat NV model described above. The small size of the newborn rodent eye and presence of hyaloidal circulation has made the measurement of retinal oxygenation and hemodynamic parameters in newborn rodent models difficult with other methods.78,79 In this model, a subnormal pO2 was measured over avascular retina and, somewhat surprisingly, over vascular retina before (P14), during (P20), and after the appearance of retinal NV on P34.7,69 Subnormal pO2 was clearly associated with NV histopathology in all cases.

As discussed earlier, constantly applied supplemental oxygen treatment (SOT), unlike variable magnitude SOT, significantly reduces the risk of developing experimental retinal NV.5-7,10 We therefore investigated the consequences of constantly applied SOT on pO2 as well as its association with NV incidence and severity. In the newborn rat 50/10 model, retinal

pO2 was measured during the appearance of NV (P20) in rats recovered in 28% supplemental oxygen instead of room air between P14 and P20.7 We found that, after 28% SOT, the expected decrease in NV incidence and severity occurred (P <0.05), but there was an unexpected decrease (P < 0.05) in panretinal pO2. In addition, on further recovery in room air (at P26),

animals with a history of supplemental oxygen had a higher incidence of NV compared with room air recovered pups (53% vs. 17%, respectively) even though NV severity was 1 clockhour in both groups. In other words, worse treatment outcome at P26 was associated with subnormal pO2 at P20. These results reveal that a higher incidence of NV is a potential consequence of SOT and that retinal pO2 is potentially a surrogate marker of early treatment response in retinopathy.

The data from the above experimental proliferative retinopathy studies highlight the value of functional MRI measures of pO2 as a powerful tool to monitor retinopathy. These basic research results have motivated the development of similar approaches for use clinically. In an initial study, a practical approach for eliminating blinking-related artifacts was developed that, for the first time, enabled measurement of pO2 in control subjects and patients with diabetes.77,80 In over 50 patients studied to date, it appears that the procedure is well tolerated, with few reports of eyestrain or overall fatigue from subjects.

7.SUMMARY AND CONCLUSIONS

For over 50 years, retinal hypoxia has been considered to be a major causative factor in the development of retinal neovascularization (NV), a condition associated with blindness and vision loss in a variety of retinopathies. Review of the existing literature and results of new experiments from our laboratory strongly suggest that the oxygen-based pathophysiology stimulating retinal NV is more complicated than previously thought. Static hypoxia found at the border of vascular and avascular retina appears to be necessary but not sufficient to cause NV. We have now identified a new factor strongly associated with retinal NV: a dynamic mismatch between oxygen supply and demand that can be evaluated using MRI and a hyperoxic provocation and that is found over both vascular and avascular retina. Interestingly, in the field of tumor biology, the tight focus on static hypoxia as the sole causative agent of abnormal angiogenesis has now also been widened by evidence that other factors (similar to those discussed in this chapter, such as temporal oxygen instability and inflammatory cells) are also strongly associated with angiogenesis.55 In practical terms, these new insights into the intersection of static hypoxia, oxygen supply dysfunction, and inflammation are expected to lead to improved therapeutic strategies for preventing vision loss and blindness from retinal NV.

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Chapter 9

HYPOXIA INDUCIBLE FACTOR-1

AND VEGF INDUCTION

Ashima Madan, MD

Stanford University School of Medicine, Stanford, California

1.RETINAL VASCULARIZATION

Retinal vascularization occurs by a process of vasculogenesis, de novo formation of capillaries from endothelial cells that have differentiated from spindle cell precursors,1,2 and angiogenesis, formation of blood vessels from existing blood vessels.3 Vasculogenesis begins at approximately 16 weeks’ gestation in the posterior region around the optic disc. With advancing gestation, blood vessels spread across the surface of the retina in the superficial and deep plexus following the central peripheral gradient of retinal ganglion maturation toward the peripheral retina.4

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen.5-7 Physiological hypoxia created by the increased metabolic demands of the fetal retina at the onset of neuronal activity is the major stimulus for secretion of VEGF from strategically

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located populations of neuroglia in utero. The hypothesis that new capillaries grow toward tissues that produce and secrete VEGF is supported by studies that show increased expression of VEGF in tissues with active angiogenesis and increased expression of VEGF receptors on target endothelial cells of blood vessels in the vicinity.8-12 As the vessels grow and become patent, the hypoxic stimulus is relieved when vessel formation is matched to oxygen demand.

The retina is vascularized by a process of vasculogenesis, de novo formation of blood vessels from mesodermal angioblasts, which begins around 16 weeks of gestation as well as by angiogenesis, formation of blood vessels from pre-existing vessels, around 25 weeks’ gestation. VEGF is important for both vasculogenesis and angiogenesis. However, unlike angiogenesis, vasculogenesis in the human retina, although dependent on VEGF, is independent of metabolic demand and hypoxia-induced VEGF expression.

2.ROLE OF VEGF AND HIF-1 IN RETINAL NEOVASCULARIZATION

Although VEGF is but one factor in the angiogenic response, it is a key activator of vascular endothelial cells and plays an essential role in angiogenesis.13,14 Numerous ocular cell types, including retinal pigment epithelial cells, retinal endothelial cells, retinal pericytes, and Müller cells, secrete VEGF in response to reduced oxygen tension.15-17

Several lines of evidence indicate that VEGF plays a central role in both the natural development of blood vessels in the retina and the development of abnormal retinal vascularization in various disease states. The temporal and spatial increase in VEGF mRNA and protein expression in the retina of various animal models of retinopathy is associated with the onset of ischemia-induced neovascularization.18-22 In the hyperoxia-induced ischemic mouse model of retinopathy, VEGF levels are decreased in the initial phase of hyperoxic injury and subsequently increased in the hypoxic phase of retinopathy.23,24 In situ hybridization studies in this model have localized the site of production of VEGF to the inner nuclear layer of the retina.21 Inhibition of VEGF by injection of VEGF receptor chimeric proteins, antiVEGF antibodies, or antisense oligonucleotides in animal models of retinopathy has been shown to decrease neovascularization.25-27

In a separate series of experiments using immunohistochemistry and in situ hybridization in the mouse model of retinopathy, hypoxia induced factor-1-alpha (HIF-1α) was increased in the hypoxic inner retina but not in the normoxic outer retina. The peak increase in HIF-1α occurred at 2 hours