Ocular Neovascularization: a Valuable Model System
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Oncogene (2003) 22, 6537–6548 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Ocular neovascularization: a valuable model system Peter Anthony Campochiaro*,1 and Sean Francis Hackett1 1The Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Maumenee 719, 600 N. Wolfe Street, Baltimore, MD 21287-9277, USA There is no unique formula for angiogenesis. Instead there related proteins, and/or their induced expression, which is a large group of potential participating proteins that may alter the angiogenesis cascade and may even alter interact in complex ways. Depending upon the surround- the effects of particular proteins. Some proteins promote ing cell types and the relative expression levels of angiogenesis in one setting and suppress it in others. angiogenesis-related proteins,the ‘angiogenesis cascade’ Proteins depend upon other proteins for their actions can vary. Therefore,it is valuable to study and compare and lack of expression of a binding partner in a the role of proteins in several well-characterized vascular particular locale may have a profound effect on the beds. The eye provides a useful model system,because it action of a protein. contains several vascular beds sandwiched between In order to define the potential actions of a protein avascular tissue. This allows for unequivocal identification and its interactions with other proteins in angiogenesis, and quantitation of new vessels. Retina-specific promoters it is useful to study its effects in several well-character- combined with inducible promoter systems provide a ized vascular beds and in different types of angiogenesis. means to regulate the expression of proteins of interest. The eye provides a valuable model system for such As a relatively isolated compartment,the eye also investigations, because it contains several vascular beds provides advantages for gene transfer. By gaining insight separated by avascular tissue. The vascular beds can be regarding the molecular signals involved in various types visualized in vivo and the presence of neovascularization of ocular angiogenesis,general concepts can emerge that can be unequivocally identified and quantified because may apply to other settings,including tumor angiogenesis. of the surrounding avascular tissue. Also, retina-specific One concept that has emerged is that despite participation promoters combined with inducible promoter systems of multiple stimulatory factors for ocular neovasculariza- provide a useful way to control expression of proteins of tion,VEGF plays an essential role and interruption of interest. By observing the different effects of proteins in VEGF signaling is an important therapeutic strategy. different ocular vascular beds, at different stages of Another concept is that while most studies have focused on development, and in different disease models, a more prevention of ocular neovascularization,regression of new complete picture of the protein’s actions and interac- vessels is desirable and is achievable with at least three tions with other proteins can emerge. agents,combretastatin A-4 phosphate,pigment epithe- lium-derived factor,and angiopoietin-2. Finally,endosta- tin and angiostatin,which have been sources of Vascular beds within the eye controversy because of inconsistent results in tumor In the eye, there are three vascular beds that have been models,have been shown to have good efficacy when intensively studied, the hyaloidal, retinal, and choroidal delivered by gene transfer in models of ocular neovascu- circulations. The hyaloidal vasculature develops in the larization. These results provide leads for new ocular embryonic eye and extends from the optic nerve through treatments and perspective for evaluation of studies of the vitreous to surround the developing lens. Near the neovascularization in extraocular tissues. end of development, when other circulatory systems in Oncogene (2003) 22, 6537–6548. doi:10.1038/sj.onc.1206773 the eye are completed, the hyaloidal vessels regress. This provides an opportunity to study molecular signals Keywords: VEGF; angiogenesis; angiopoietins; involved in vascular regression. retinopathies; age-related macular degeneration The retinal circulation, unlike the hyaloidal circula- tion, can be studied in adult animals as well as during its development. In rodents, retinal vascular development Introduction occurs postnatally, facilitating the study of develop- mental or physiologic angiogenesis. On the day of birth, Numerous gene products participate in angiogenesis, postnatal day 0 (P0), there are no retinal vessels and flat but that participation may vary somewhat in different mounts of the retina after perfusion with fluorescein- settings. Vascular beds differ with regard to surrounding labeled dextran show hyaloidal vessels, which have not cell types, constitutive expression of angiogenesis- yet regressed, but no retinal vessels (Figure 1a). The central retinal artery enters the eye through the optic *Correspondence: PA Campochiaro; E-mail: [email protected] nerve and retinal vessels develop from the optic nerve to Ocular neovascularization PA Campochiaro and SF Hackett 6538 Figure 1 Retinal vascular development. Mice were perfused with fluorescein-labeled dextran on the day of birth, postnatal day (P) 0 (a, b), P4 (c), P7( d), P10 (e), or P18 (f), and retinal flat mounts were examined by fluorescence microscopy. The large vessels seen in flat mounts from P0 mice are hyaloidal vessels; retinal vessels are just beginning to develop at the border of the optic nerve (b, arrowheads). By P4, the margin of the developing blood vessels on the surface of the retina is located halfway between the optic nerve and the peripheral edge of the retina (c, arrowheads). Large hyaloidal vessels are still seen overlying the avascular peripheral retina. At P7, superficial vessels cover about 80% of the retina (d, arrowheads). The hyaloidal vessels have started to regress. At P10, the superficial vessels have reached the peripheral edge of the retina and the deep capillary bed has begun to develop posteriorly (e). By P18, retinal vascular development and remodeling is completed (f) the periphery of the retina. At P4, superficial retinal make up the outer avascular half of the retina. It vessels extend from the optic nerve halfway to the receives its oxygen and nutrients from the choroidal peripheral edge of the retina (Figure 1b). At P7, vessels circulation, a high-flow system supplied by multiple long cover the entire surface of the retina (Figure 1c) and and short posterior ciliary arteries, which all feed into an sprouts from superficial vessels begin to grow into the extensive network of fenestrated capillaries, the chor- retina to form the intermediate and deep capillary beds. iocapillaris. The choriocapillaris allows plasma to pool By P18, all the three vascular beds of the retina have beneath the retinal pigmented epithelium (RPE), which been formed and remodeled, resulting in an adult retinal has tight junctions and specialized transport systems, circulation, which is relatively stable thereafter and it constitutes the outer blood retinal barrier. (Figure 1d). Retinal vascular endothelial cells have tight junctions and modified vesicular transport, and are the Developmental retinal neovascularization sites of the inner blood–retinal barrier. This is in part due to their interactions with pericytes and retinal glia Retinal astrocytes enter the retina from the optic nerve (Janzer and Raff, 1987). (Watanabe and Raff, 1988). As they migrate away from The retinal circulation supplies the inner half of the the optic nerve into avascular retina, they become retina (Figure 2a and b). The outer nuclear layer, which hypoxic and express increased levels of vascular is made up of the cell bodies of the photoreceptors, and endothelial growth factor A (VEGF) (Stone et al., the inner and outer segments of the photoreceptors, 1995). VEGF stimulates the growth of blood vessels Oncogene Ocular neovascularization PA Campochiaro and SF Hackett 6539 Figure 2 Mice with oxygen-induced ischemic retinopathy develop severe retinal neovascularization. (a, b) Paraffin-embedded ocular sections stained with Griffonia simplicifolia lectin (GSA), which selectively stains vascular cells, from a normal postnatal day (P) 17 mouse shows normal superficial, intermediate, and deep capillaries. There are no vessels extending above the internal limiting membrane (ILM). Hematoxylin counterstain shows retinal neurons. (c, d) Sections from P17mice with ischemic retinopathy stained with GSA show prominent neovascularization that has broken through the ILM and is on the surface of the retina (arrows). Dropout of the normal retinal blood vessels throughout the posterior retina is seen in the low magnification view in (c). This is also illustrated by the black (nonperfused) area in the retinal flat mount from a fluorescein dextran-perfused P17mouse with ischemic retinopathy ( e) from the optic nerve to the periphery of the retina and as provide an alternative source of survival factors. the blood vessels become functional; they alleviate Platelet-derived growth factor-B (PDGF-B) secreted by hypoxia, resulting in decreased expression of VEGF. endothelial cells is critical for the recruitment of The relative hypoxia of the deeper layers of the retina pericytes, and PDGF-deficient mice lack pericyte results in a VEGF gradient that favors sprouting from envelopment of retinal vessels (Lindahl et al., 1997). the superficial vessels, resulting