Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

network formation. It appears that the developing vascular system has adopted several molecules that guide neural network formation and axonal growth cone migration, which resembles vascular tip cell migration. These molecules include the netrins, semaphorins, slits, and ephrins and their receptors (Carmeliet and Tessier-Lavigne, 2005; Dorrell and Friedlander, 2006). Specifically, Sema3E-PlexinD1 and Netrin-1-UNC5B interactions have been characterized to provide repulsive signals to the endothelial tip cells during mouse vascular development.

Ephrin ligands and Eph receptors have been the most extensively studied guidance molecules in regard to the formation of vascular and neural networks. The Eph receptor family is the largest group of receptor tyrosine kinases (RTK) known to date, currently made up of 15 receptors that engage nine ephrin ligands. Ephrin ligands contain an extracellular and transmembrane domain. However, ephrin ligands of the A class lack a cytoplasmic tail, while ephrin ligands of the B class contain a cytoplasmic domain, which can initiate an intracellular signaling cascade on interaction with an Eph receptor. Several of the ephrin ligands and ephrin receptors have been implicated in adult angiogenesis, while both ephrin-B2 (EFNB2) and ephrin receptor-B4 (EPhB4) have emerged as key regulators of vascular development. Ephrin-B2 ligand and EphB4 receptor identify arterial and venous ECs, respectively, helping define molecular boundaries between arteries and veins, arresting EC migration at the arterial-venous interface. Both Efnb2 and Ephb4 are essential for vascular development, and targeted disruptions of these genes results in embryonic lethality due to defects in arteries and veins and failure to properly remodel vascular networks. EphrinB2 and EPhB4 have recently been localized to the developing retinal vasculature in the mouse retina, and examination of the superficial vascular network allows one to follow the establishment of arteries, veins, and capillaries. Uemura et al (2006) characterized Efnb2 and Ephb4 expression in the retinal vessels by detecting LacZ expression in retinas from mutant mice heterozygous for each gene. At P2, most of the vascular sprouts around the optic nerve express Ephb4, indicating venous origin. However, between P3 and P4, Efnb2 is expressed in the arteries and associated capillaries, while Ephb4 expression becomes restricted to veins. Other investigators have also detected Ephb4 expression in retinal veins as well as in some of the capillaries by in situ hybridization, indicating that the retinal capillaries may have both arterial and venous origins (SaintGeniez et al., 2003). Retinal mRNA expression of Efnb2 has been shown to be constitutively expressed in the developing mouse retina, while expression of Ephb4 was modestly reduced between P12 and P17 (Zamora et al., 2005).

Of interest, oxygen levels can regulate Ephb2 expression in retinal ECs in the developing retina (Claxton and Fruttiger, 2005). Mice raised in 10% oxygen from birth

exhibit reduced capillary-free zones around retinal arteries on P6, and concurrently, Efnb2 expression was absent from the arteries. Increasing the oxygen exposure to 60% at birth resulted in widening of the capillary-free zone around arteries but did not alter the expression of Efnb2. Mice exposed to hypoxic conditions develop a retinal vascular network that exhibits an abnormal double superficial capillary network and unusual spacing between arteries and veins. Despite the lack of Efnb2 expression in response to hypoxia, the arterial ECs do not express vein-specific markers, indicating the arteries do not convert to veins, and oxygen levels do not directly determine cell fate. However, it appears that arterial ECs require a threshold of oxygen to express Efnb2, and its absence results in an abnormal spatial organization of the developing retinal vasculature, perhaps from inability to develop proper boundaries between arteries and veins.

Pericytes are mural cells that colocalize with retinal vessels. These cells are distributed evenly throughout the developing vascular network but always lag slightly behind the leading edge of the vessel sprout (Fruttiger, 2002). Pericytes, which express PDGFR-β, proliferate and migrate in response to the PDGF-B, which is expressed by the EC tip cells. The major role of pericytes is to provide stability to the nascent vessel. Pericytes encircle the vessel with long cytoplasmic extensions, making focal contacts with ECs via junctional complexes located at sites where the shared basement membrane is absent (Armulik et al., 2005). The critical role of PDGF-B in EC-pericyte interactions was confirmed in endothelium-specific Pdgf-b knockout mice. In this strain, pericyte recruitment is altered, resulting in a diabetic-like retinopathy (Enge et al., 2002). Transforming growth factorbeta1 (TGF-β1, Tgfb1) expressed by EC is also important for pericyte differentiation and function. There are numerous studies of gene deletion of TGF-β ligands, TGF-β receptors, binding proteins, and other downstream signaling molecules, resulting in cardiovascular defects and vascular malformations (Armulik et al., 2005). Recent coculture studies have shown that mesenchymal cells can express VEGF-A as they differentiate into pericytes in response to TGF-β, while in vivo studies have confirmed that capillary-associated pericytes express VEGF-A in the developing mouse retinal vasculature (Darland et al., 2003). These findings suggest that astrocytes and pericytes are sources for VEGF-A and both cell types are important for vessel stabilization.

Pericyte-derived angiopoietin-1 (Angpt1) also contributes to vessel stabilization by activating the TEK receptor on vascular ECs, mediating maturation and quiescence of the retinal microvessels (Jain, 2003). Angpt1 null mice die in midgestation and exhibit microvascular defects of altered pericyte coverage. In the absence of pericytes, exogenous Angpt1 helps restore vessel integrity in the developing mouse retina, reducing edema and hemorrhage (Armulik et al., 2005).

Figure 23.2 Schematic summary of cytokine interactions between endothelial cells and resident cells during postnatal retinal vascular development.

Figure 23.2 is a schematic summary of cytokine interactions between ECs and resident cells during retinal vasculature development, which are subject to alteration by high oxygen exposure.

Bone marrow–derived hematopoietic stem cells (HSCs) can differentiate into nonhematopoietic (Lin−) lineages, function as endothelial progenitor cells (EPCs), and participate in retinal neovascularization (Grant et al., 2002). In the neonatal mouse retina, EPCs can target retinal astrocytes and incorporate into the developing vasculature (Otani et al., 2002). Alternatively, when a subset of Lin− HSC cells (CD44hi) are intravitreally injected into eyes of neonatal mice, the CD44hi cells differentiate into microglia and promote revascularization of the central retina, as well as reduce neovascular tuft formation in the mouse model of OIR. These studies suggest there is a subpopulation of Lin− HSCs that participates in normal vascular development and pathological retinal neovascularization. The use of cellbased therapies certainly needs further investigation, but they hold promise for future approaches to the treatment of ischemic retinopathies and retinal degenerative diseases.

Historical perspective: Mouse model of retinopathy of prematurity

The association between high levels of supplemental oxygen and ROP was characterized by several investigators in the early 1950s (Ashton, 1968; Madan and Penn, 2003). These studies recognized the obliteration of developing retinal vessels from hyperoxia and subsequent pathological retinal neovascularization on returning to room air. Concurrently with these clinical observations, several groups were also investigating the effect of hyperoxia on the mouse retina (for a review, see Ashton, 1968; Madan and Penn, 2003). These

studies entailed using 100% oxygen for variable lengths of time, with the exposure initiated at birth or shortly after birth. The studies demonstrated obliteration of vascular development in newborn mice from hyperoxia exposure. Furthermore, when these mice recovered in room air, preretinal neovascularization was also observed around the optic disc. When older mice were exposed to hyperoxia, neovascularization was observed in the peripheral retina. In addition to the retinal changes observed, marked dilation and proliferation of hyaloid vessels were noted.

The early studies of oxygen toxicity to developing mouse retina were reviewed and repeated by Ashton in the late 1960s to directly compare the mouse studies with the kitten and rat models of oxygen-induced retinopathy (Ashton, 1968). Ashton confirmed that if the mouse retina is exposed to hyperoxia before 5 days of age, a severe hyaloidopathy is the major pathology observed, with a mild retinal neovascularization confined to the disc region. However, when a P5 mouse was exposed to 80%–90% oxygen for 4 days, a starshaped pattern of vaso-obliteration was observed, but in contrast to earlier studies, Ashton did not observe abnormal retinal neovascularization when the animals were returned to room air (Ashton, 1968). Gole et al. in 1990 suggested that the mouse is a suitable model of OIR, based on their ultrastructural findings of vitreous vessels, and suggested that histological cross sections could be used to grade preretinal neovascularization in future studies. Hence, the early studies using the mouse as a model of OIR were inconsistent and lacked reproducible quantification, and the animals developed a severe hyaloidopathy.

Contemporary perspective: Mouse model of oxygen-induced retinopathy

Characterization of Model Smith and colleagues in the early 1990s developed and characterized a consistent and reproducible model of OIR in neonatal mice (Smith et al., 1994). This model continues to be used today to investigate the pathogenesis and molecular mechanisms of the proliferative retinopathies (ROP, diabetic retinopathy). To develop the model, Smith et al. examined the effect of initiating the hyperoxia exposure from P0 (birth) to P7, while varying the oxygen concentrations between 50% and 95%. Eventually, P7 mice (C57BL/6J) were chosen in order to balance the oxygen-induced injury of the developing retinal vasculature with the regression of the hyaloid vasculature. This aided in avoiding the hyaloidopathy that was observed by earlier investigators. In addition, a 75% oxygen exposure protocol was chosen because it yielded a reproducible pathological preretinal neovascularization without the maternal or pup mortality that was associated with higher oxygen exposures. Neonatal mice (P7) were exposed to 75% oxygen for 5 days and then returned to room air on P12.

Mouse eyes were evaluated daily to assess the neovascular response from P12 until P44. Exposure of adult mice (P24) to 75% oxygen for 5 days did not result in vaso-obliteration or retinal neovascularization, confirming that developing vessels, but not mature retinal vessels, were sensitive to oxygen toxicity. A recent study also determined that mice exhibit a mature vascular response to hyperoxia by P15, by failing to exhibit a vaso-obliteration response (Gu et al., 2002).

Two different techniques were used to assess and quantify the vascular response. The first method used high- molecular-weight (HMW) fluorescein-labeled dextrans that were perfused through the left ventricle just prior to sacrifice, allowing visualization of the entire retinal vasculature with fluorescence microscopy. This technique had the advantage over the previously used India ink or low-molecular-weight fluorescein-labeled dextran perfusion in that the HMW dextrans filled the entire vasculature without leaking. As suggested by Gole et al., the second method used to evaluate retinal neovascularization was to examine histological cross sections of the retinas. Two to four sections on each side of the optic nerve, 30–90 μm apart, were evaluated from each sectioned eye. Preretinal vascular cell nuclei that extended into the vitreous beyond the inner limiting membrane (ILM) were counted from each cross section to quantify neovascularization. Cross sections that included the optic nerve were excluded to avoid counting regressing hyaloid vessels. GFAP was also immunolocalized in the retinal sections to examine whether astrocytes and Müller cells contributed to retinal response in OIR.

After 24 hours of hyperoxia, the central retina exhibited a nonreversible central vasoconstriction. After 5 days of hyperoxia exposure (P12), the central retina displayed a severe hypoperfusion with apparent vaso-obliteration of both the superficial network capillaries and deep vascular network (see figure 23.1D). Subsequent studies did confirm obliteration of the central superficial vascular capillaries at P12; however, in contrast to the superficial network, the entire deep vascular network failed to develop both centrally and peripherally in response to hyperoxia (Banin et al., 2006; Davies et al., 2003). This observation is consistent with the fact that the superficial network is almost complete at P7, but the deep network starts to develop between P7 and P8 (Dorrell et al., 2002; Fruttiger, 2002). Hence, normal retinal vascular development was altered by hyperoxia, with vaso-obliteration of superficial capillaries in the central retina and inhibition of the deep network capillaries in both the central and peripheral retina. After 5 days in hyperoxia and 2 days in room air (P14), the retinas continued to display a lack of central retinal capillaries; however, the arteries and veins now had a dilated and tortuous appearance.

At P17 and P21, preretinal neovascular tufts were appreciated at the transition zone between the nonperfused central retina and the perfused peripheral retina in both the

fluorescein-dextran-perfused retinal flat mounts and the retinal cross sections (see figure 23.1F). The neovascular tuft response occurred in 100% of the mice and was similar in both male and female mice (Smith et al., 1994). The retinal neovascularization that is observed in ROP also occurred at the transition zone between the vascular and avascular retina. However, in contrast to mice, in humans the avascular zone is located at the periphery of the retina and often exhibits a well-defined mesenchymal ridge. The neovascular tufts in the mice were noted to gradually regress after P21 and were absent after approximately P24–P26. In the initial studies by Smith et al., intense GFAP immunostaining was exhibited by astrocytes along the superficial layer of vessels, as well as in Müller cell processes beneath the areas of preretinal neovascularization. GFAP expression is increased in a variety of retinal injuries, and it appears to be a marker for intraretinal injury in this model. Despite the overall increased GFAP expression in the retina, the neovascular tufts were negative for GFAP, indicating that despite their activation, astrocytes and Müller cells are not physically associated with the tufts.

The extent of retinal neovascularization in the mouse model of OIR is also strain dependent because of differential expression of angiogenic factors (Chan et al., 2005). The mouse strain used to characterize the OIR model, C57BL/6J, is relatively resistant to angiogenic stimuli when compared with the 129/SvImJ strain (Chan et al., 2005; Rohan et al., 2000). Therefore it is essential to understand the genetic background of mice when evaluating the angiogenesis response in the OIR model. Investigators must ensure the use of proper genetic controls when comparing the effects of gene deletion (commonly made in 129 mice) or gene overexpression. The effect of genetic diversity on angiogenesis has been recently characterized and reviewed (Rogers and D’Amato, 2006).

Two recent studies have attempted to explain the selective vulnerability of central retinal vessels in mice to hyperoxia as opposed to the peripheral retina observed in humans (Claxton and Fruttiger, 2003; Shih et al., 2003a). One hypothesis is that the hyperoxia results in a larger capillaryfree zone around arteries emanating from the optic nerve head (including the regressing hyaloid) with an associated downregulation of astrocyte-derived VEGF-A. A reduction in VEGF-A would result in additional vascular regression in the central retina. The second hypothesis is that the pericytes that are located in the central neonatal mouse retina fail to express TGF-β, with a secondary failure to induce expression of FLT1 on their associated EC. Protection of capillaries from oxygen-induced injury is mediated by VEGF-A binding to FLT1 (Shih et al., 2003b). Of interest, the pericytes at the leading edge of the superficial network express TGF-β at P5, which correlates with the area of the mouse superficial network that is resistant to hyperoxia. In addition, TGF-β-

expressing pericytes are localized to all vessels in P15 retinas (resistant to oxygen-induced injury, as noted earlier). Hence, these studies suggest that the sensitivity of the neonatal vessels to hyperoxia-induced injury is related to reduced VEGF-A ligand and FLT1 expression in the central immature retina. The human hyaloid vasculature exhibits more regression prior to birth, even in the 25-week premature infant; therefore, excessive hyperoxia may not be present in the central retina compared with the periphery. Human studies examining the pericyte expression of TGF-β in the immature retina are lacking. Figure 23.3 is timeline of retinal vessel formation in normal development and in OIR in the mouse.

The mouse model of OIR continues to be further characterized, and additional methods of quantification have been developed. Higgins et al. (1999) developed a retinopathy scoring system using features from scoring systems used in kittens and humans. This scoring system grades blood vessel growth, blood vessel tufts, extraretinal neovascularization, central vasoconstriction, retinal hemorrhage, and blood vessel tortuosity as evaluated with HMW fluorescein-labeled dextran-perfused retinal flat mounts. This quantitative scoring system was validated by the quantification of preretinal neovascular nuclei as described by Smith et al. This scoring system has the advantage of evaluating several features of retinopathy that might be differentially altered by genetic manipulation or other therapeutic interventions but requires very consistent fluorescein perfusion and welltrained scorers. Mice with OIR were recently evaluated with three-dimensional in vivo imaging to assess the retinal vasculature in a living animal (Ritter et al., 2005). Imaging at P18 confirmed the presence of pathological neovascularization, as well as vascular leakage, a feature of the model not well appreciated when using HMW-fluorescein dextran perfusions, since they are designed not to leak out of the vasculature. This technique of imaging can provide new insights into retinal neovascularization that are not available when fixed tissues are used. However, improvements in optics and microscopy are likely required before investigators can routinely employ this methodology.

Banin et al. have developed a new, complementary methodology of quantification for the mouse model of OIR that can provide a detailed time course of the vaso-obliteration and retinal neovascularization (Banin et al., 2006). Isolectin Griffonia simplicifolia (isolectin GS)-stained retinal flat mounts

were analyzed using confocal microscopy and image analysis software. To measure the area of vaso-obliteration in the retina, the border of the avascular retina was traced using the “freehand” selection tool from Photoshop. To measure neovascularization, the vascular tufts were traced using the “magic wand” tool. The total areas were measured in pixels and then converted to square micrometers. Dextranperfused retinas were also stained with the isolectin GS, with the vascular tufts being more clearly visualized with isolectin GS and therefore used for quantification. In contrast, both the dextran-perfused and isolectin GS–stained retinas delineated the avascular regions in a comparable fashion. This neovascular tuft quantification method correlated well with results obtained by counting preretinal neovascular tufts in tissue sections. The data analysis revealed good correlation between both eyes from each animal with regard to the extent of vaso-obliteration and vascular tuft formation. This allows one eye to be used for intraocular treatment while the other eye is used as a control, with a predictable and consistent response. The data analysis also revealed that vasoobliteration significantly recovers by P16 and is nearly resolved on P21, while the neovascularization peaks on P17, with a gradual regression until P22. Figure 23.4 shows a retinal flat mount from a P17 oxygen-injured animal with preretinal neovascularization. The sample has been stained with isolectin GS and digitally imaged using a conventional fluorescence microscope in the author’s laboratory.

Studies of VEGF, Growth Factors, and Cytokines The mouse model of OIR has played an important role in defining the contribution of VEGF-A to the pathogenesis of pathological retinal neovascularization and its link to the human diseases of ROP and diabetic retinopathy (Lutty et al., 2006a; Saint-Geniez and D’Amore, 2004). Smith and colleagues initially investigated the expression and regulation of VEGF-A in the mouse retina using the model (Pierce et al., 1995, 1996). During the hyperoxia phase (P7–P12), VEGF-A expression was downregulated within 6 hours and remained reduced after 5 days (P12). After animals were returned to room air on P12, VEGF-A expression was significantly increased compared with that seen in room air controls within 12 hours and remained elevated through P17, the peak of retinal neovascularization. VEGF-A expression was localized to Müller cell processes and their end-feet along the inner retina. Intravitreal injection of

Figure 23.4 Visualization of vessels in P17 retinal flat-mount preparations stained with GS-lectin from a mouse model of OIR. A, Low-power (magnification ×25) image of the retina shows areas of central vaso-obliteration, as well as neovascular tufts. B, Higher magnification (×200) shows neovascular tuft formation at the transition zone between vascular and central avascular retina. C, At even greater magnification (×400), multiple neovascular tufts are evident. See color plate 13.

recombinant VEGF-A on P7 reduced the extent of vasoobliteration typically observed in the model.

These early findings shed light on the interactions between VEGF-A and the retinal vasculature: (1) VEGF-A is downregulated in response to hyperoxia, (2) reduced VEGF-A expression is associated with vaso-obliteration, and (3) overproduction of VEGF-A in avascular (relative hypoxic) regions of the retina results in abnormal retinal neovascularization. Subsequent studies have experimentally determined that the avascular areas of the mouse retina are indeed hypoxic, and that a transcription factor that stimulates Vegfa expression, hypoxia inducible factor-1α (HIF-1α), is increased in the mouse model of OIR, with a temporal and spatial correla-

tion with VEGF-A expression (P12.5–P17) (Ozaki et al., 1999; West et al., 2005). The central role that VEGF-A plays in proliferative retinopathies was further confirmed by the suppression of retinal neovascularization with intravitreal injection (P12 and P14) of a soluble VEGF receptor (ca. 42% reduction) and antisense oligodeoxynucleotides targeting Vegfa (ca. 50% reduction) (Aiello et al., 1995; Robinson et al., 1996). The fact the specific inhibitors did not completely block the pathological neovascularization suggests that additional angiogenic factors are involved or the inhibitors did not penetrate into the intraretinal locations of VEGF-A.

Several additional studies have used the mouse model of OIR to demonstrate a major role for VEGF-A in ischemic retinopathies, with potential translation of VEGF-A inhibition to human disease (Al-Shabrawey et al., 2005; Deng et al., 2005; Shen et al., 2006). For example, reduction of VEGF-A expression and retinal neovascularization has been achieved after treatment with virus-mediated expression of small VEGF peptides, inhibition of NAD(P)H oxidase, or siRNA targeting of Flt-1. Insulin-like growth factor-I (IGF-I) also modulates VEGF-A function during angiogenesis and is an additional target for inhibition of retinal neovascularization (Smith et al., 1999). Mice treated with an IGF-I receptor antagonist exhibited a reduction in neovascularization by 53% compared to control. The IGF-I receptor antagonist did not alter levels of VEGF-A or VEGF receptors but rather modulated VEGF-A-mediated intracellular signaling pathways. IGF-I has also been linked to ROP in infants (Hellstrom et al., 2003).

As previously discussed, the angiopoietin/Tek system is critical for normal retinal vasculature development, playing a role in the EC–mural cell interactions. In postnatal angiogenesis, Angpt2 is also specifically upregulated in microvascular ECs by hypoxia in vitro and in the mouse model of OIR in vivo, and localizes to neovascular tufts in P17 mice. The coexpression of Angpt2 and VEGF-A in response to hypoxia is required for retinal neovascularization to occur (Hackett et al., 2000). In the mouse model of OIR, retinal neovascularization was suppressed by 23% by the intraocular injection of a soluble Tek (sTek-Fc). However, when the sTek-Fc was combined with a soluble Flt1 (sFlt-1-Fc), neovascularization was inhibited by 50% compared to control. In the absence of VEGF-A, Angpt2 will induce vascular regression of nascent vessels but have no effect on more mature vessels (Campochiaro, 2006). This finding implies that Angpt2 has the potential to be a therapeutic antiangiogenic agent if used in combination with a specific VEGF-A antagonist.

Briefly, two cytokines that have been implicated in human proliferative retinopathy, erythropoietin and interleukin-8, are also increased during the phase of retinal neovascularization in the mouse model of OIR (Powers et al., 2005; Watanabe et al., 2005). Expression of retinal erythropoietin

mRNA is increased dramatically on P17 compared to control, and intraocular injection of a soluble erythropoietin receptor reduces the neovascularization when evaluated on P19. The chemokine KC, a mouse homologue of inter- leukin-8, is markedly increased on P17 and P21 compared to control, immunolocalizes to the Müller cell processes, and is induced by interleukin-1.

As outlined in this section, multiple angiogenic factors contribute to neovascularization in the immature retina. For clinical use, inhibitors of retinal neovascularization will likely need to be administered in combined fashion, and they should affect only pathological vessels, not the normal pattern of retinal vascular development.

Endothelial Cell Apoptosis and Oxygen-Induced Retinopathy It is now recognized that there is a critical link between angiogenesis and apoptosis (Folkman, 2003). After ECs are incorporated into new vessels, they continue to depend on angiogenic factors (survival factors) to block apoptotic pathways while simultaneously stimulating intracellular survival pathways. Alon et al. (1995) correlated downregulation of VEGF-A by hyperoxia with the apoptosis of retinal ECs in a rat model of OIR, implicating VEGF-A as an EC survival factor. Thus, during the phase of hyperoxia exposure, growth factor withdrawal results in EC apoptosis and contributes to the subsequent vaso-obliteration. Survival factor withdrawal has since been characterized to activate the mitochondrial pathway of apoptosis, although specific intracellular apoptotic pathways need to be delineated during vaso-obliteration in the mouse model of OIR.

In addition to growth factor withdrawal, proliferating ECs upregulate the death receptor Fas on their cell surface, making immature vessels vulnerable to regression via FasLinduced apoptosis (Stuart et al., 2003). In a variety of mouse models, the Fas/FasL pathway has been shown to play an important role in retinal vessel regression during normal vascular development and neovascular tuft regression (Barreiro et al., 2003; Davies et al., 2003; Ishida et al., 2003b). Two independent groups utilized FasL-defective mice (Gld) in the mouse model of OIR and observed a significant increase (ca. 50%) in preretinal neovascularization on P17 (Barreiro et al., 2003; Davies et al., 2003). The gld mice also exhibited a significantly reduced number of apoptotic EC (ca. 50%) in the neovascular tufts as compared to control mice at P17. These findings suggest that Fas/FasLmediated apoptosis helps to regulate the extent of pathological neovascularization observed in the mouse model of OIR. However, other apoptotic mediators also likely contribute to the delicate balance between angiogenic and antiangiogenic factors, because even in the Gld mice the tufts eventually regressed by P26. Another observation from these studies was that normal vascular development (in room air controls) and vaso-obliteration were not altered in the Gld mice on P8

or P12 compared to C57BL/6 controls. Additional studies are needed to further define the apoptosis pathways that regulate development of the retinal vasculature and their role in hyperoxia-induced EC death.

Mice deficient in Bcl-2 (an intracellular apoptosis inhibitor) exhibited significantly (70%) reduced neovascularization compared to controls during OIR despite the characteristic increase in VEGF-A expression on P15 (Wang et al., 2005). Bcl-2 inhibits apoptosis in ECs and is normally upregulated by VEGF-A to stabilize new vessel formation. Wang et al. (2005) found that VEGF-A is unable to counterbalance the endogenous pro-apoptotic factors, owing to lack of functional Bcl-2; thus, the balance is tipped toward vascular regression and not vascular proliferation. Endostatin, a cleavage product of collagen XVIII and endogenous antiangiogenic factor, inhibits angiogenesis by inducing EC apoptosis through the downregulation of Bcl-2 and VEGF-A (Dhanabal et al., 1999; Zhang et al., 2006). Retinal neovascularization on P17 is nearly completely inhibited by exogenous intraocular injection of endostatin on P12 in the mouse model of OIR. The inhibition of NV was also associated with a 3.5-fold reduction in retinal VEGF-A expression, downregulating an antiapoptotic factor (VEGF-A), again tipping the balance toward vascular regression. Thrombospondin 1 (Thbs1) is an endogenous inhibitor of angiogenesis and induces EC apoptosis through the upregulation of FasL (Quesada et al., 2005). Overexpression of ocular thrombospondin-1 in transgenic mice results in significant inhibition of retinal neovascularization on P17 in the mouse model of OIR. These studies highlight the potential novel approaches in developing antiangiogenic therapies, through the modulation of apoptosis pathways with specific agonists or antagonists, or by enhancing endogenous antiangiogenic factors. During the phase of vaso-obliteration, prevention of EC apoptosis should prove an invaluable approach to treating ischemic retinopathies. For example, thrombospondin- 1-deficient mice (decreased apoptosis) are less sensitive to hyperoxia-mediated vessel obliteration (Wang et al., 2003). If vaso-obliteration can be minimized, there should be less hypoxia during the room air recovery phase, less VEGF-A expression, and ultimately less retinal neovascularization.

Role of Microglia and Leukocytes in the Mouse Model of OIR Microglia (myeloid-derived) and leukocytes have the potential to play many different roles in the process of angiogenesis and vascular regression, depending on the temporal and spatial biological context. For example, macrophages can produce many angiogenic growth factors, but they are also capable of expressing several antiangiogenic/ pro-apoptotic factors. Infiltrating leukocytes (lymphocytes) have been observed to contribute to vaso-obliteration via a FasL-dependent pathway in P7 mice that had been exposed to 80% oxygen for 2 days. However, this study did not

evaluate the impact of leukocytes during the period of neovascularization (Ishida et al., 2003b).

Two recent studies explored the contribution of endogenous retinal microglia to the development of the retinal vasculature and the retinal response to oxygen-induced injury (Checchin et al., 2006; Ritter et al., 2006). The depletion of microglia during vascular development resulted in reduced vascular density, while mice exposed to 75% oxygen from P7 to P12 exhibited a marked loss of microglia in the central retina, correlating with the regions of vasoobliteration. Mice strains that are more resistant to oxygeninduced vaso-obliteration and that have a quicker vascular recovery also have a greater number of microglial cells in the central retina. These studies imply that microglia promote the survival of nascent vessels in the immature retina.

Macrophages/microglia also colocalize to the neovascular tufts in the OIR model, as determined by the mouse macrophage marker F4/80 (Banin et al., 2006; Davies et al., 2006). The contribution of retinal microglial cells cannot be excluded because F4/80 specifically labels both macrophages and microglia. The monocyte/macrophage chemokine, monocyte chemoattractant protein-1 (MCP-1, CCL2), is also significantly elevated on P14–P17, indicating that many of the F4/80-positive cells observed in the neovascular tufts are likely blood-borne macrophages recruited to the retina (Davies et al., 2006). Retinal sections also revealed that microglial cells exhibit an activated morphology and are localized to the inner retina/neovascular tufts by P17. Quantification of F4/80-positive cells from P12–P21 indicated the monocytes/macrophages infiltrate into the retina after P14, peaking on P21 (a fivefold increase), with continued localization to the neovascular tufts. In a rat model of OIR, depletion of monocytes led to a suppression of retinal neovascularization, suggesting an angiogenic role for infiltrating monocytes/macrophages, while blocking lymphocytes increased the neovascularization (Ishida et al., 2003a). In the mouse model of OIR, the number of macrophages peaks at a time when the neovascular tufts are beginning to regress (after P17). Activated macrophages are quite capable of inducing EC apoptosis, a finding that may suggest that macrophages could contribute to vascular tuft regression in the mouse model of OIR (Lobov et al., 2005). This concept does not exclude an angiogenic function during an earlier phase of disease injury (Checchin et al., 2006; Ritter et al., 2006). Studies in MCP-1-deficient mice demonstrated reduced infiltration of F4/80 cells into the retina, reduced apoptosis in the neovascular tufts, and increased retinal neovascularization on P21 and P24 compared to controls (Davies and Powers, 2005).

Miscellaneous Molecules in the Mouse Model of

OIR More than 250 publications referenced the mouse model of OIR within the past decade. The details of many

of these studies could not be outlined here. A plethora of molecules have been evaluated to determine whether they contribute to the pathogenesis of pathological retinal neovascularization. A short list of the types of molecules examined in these studies includes TNF, integrins, matrix metalloproteinases and their inhibitors, nitric oxide and nitric oxide synthase, cyclooxygenases, angiotensin-con- verting enzyme, and several transcription factors. Many of these contributions have been recently reviewed (Das and McGuire, 2003; Lutty et al., 2006a; Saint-Geniez and D’Amore, 2004).

Summary

Basic scientific studies are critical if we are to advance our understanding of normal retinal vascular development and the response of developing retinal vessels to oxygen-induced injury. As noted by Patz, “The mouse model of OIR has become the gold standard for research on retinal angiogenesis” (cited in Lutty et al., 2006a). Studies in mice have created a fundamental understanding of blood vessel guidance, the role of VEGF-A isoforms in both normal and pathological retinal neovascularization, the critical interactions of retinal ECs and mural cells, and the role of apoptosis in balancing angiogenic and antiangiogenic factors. The results of these studies have also translated to human clinical trials, such as the application of a specific VEGF inhibitor to the VEGF165 isoform, in order to selectively treat pathological retinal neovascularization without altering normal vessels (Lutty et al., 2006a). Future studies of angiogenesis in the mouse retina should lead to new targets for selective therapies in human proliferative retinopathies.

acknowledgments I thank Michael Davies for his dedicated assistance, and David Zamora for critically reading the manuscript. Work was supported by grant no. EY011548 from the National Eye Institute, Fight For Sight, the Collins Medical Trust, and Research to Prevent Blindness.

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