Susan E. Yanni

The role of PGE2 receptor EP4 in pathologic ocular angiogenesis.

PURPOSE PGE(2) binds to PGE(2) receptors (EP(1-4)). The purpose of the present study was to investigate the role of the EP(4) receptor in angiogenic cell behaviors of retinal Müller cells and retinal microvascular endothelial cells (RMECs) and to assess the efficacy of an EP(4) antagonist in rat models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (LCNV). METHODS Müller cells derived from COX-2-null mice were treated with increasing concentrations of the EP(4) agonist PGE(1)-OH, and wild-type Müller cells were treated with increasing concentrations of the EP(4) antagonist L-161982; VEGF production was assessed. Human RMECs (HRMECs) were treated with increasing concentrations of L-161982, and cell proliferation and tube formation were assessed. Rats subjected to OIR or LCNV were administered L-161982, and the neovascular area was measured. RESULTS COX-2-null mouse Müller cells treated with increasing concentrations of PGE(1)-OH demonstrated a significant increase in VEGF production (P < or = 0.0165). Wild-type mouse Müller cells treated with increasing concentrations of L-161982 demonstrated a significant decrease in VEGF production (P < or = 0.0291). HRMECs treated with increasing concentrations of L-161982 demonstrated a significant reduction in VEGF-induced cell proliferation (P < or = 0.0033) and tube formation (P < 0.0344). L-161982 treatment significantly reduced pathologic neovascularization in OIR (P < 0.0069) and LCNV (P < or = 0.0329). CONCLUSIONS Preliminary investigation has demonstrated that EP(4) activation or inhibition influences the behaviors of two retinal cell types known to play roles in pathologic ocular angiogenesis. These findings suggest that the EP(4) receptor may be a valuable therapeutic target in neovascular eye disease.

The development of the rat model of retinopathy of prematurity

Retinopathy of prematurity (ROP) is a potentially blinding disease affecting premature infants. ROP is characterized by pathological ocular angiogenesis or retinal neovascularization (NV). Models of ROP have yielded much of what is currently known about physiological and pathological blood vessel growth in the retina. The rat provides a particularly attractive and cost effective model of ROP. The rat model of ROP consistently produces a robust pattern of NV, similar to that seen in humans. This model has been used to study gross aspects of angiogenesis. More recently, it has been used to identify and therapeutically target specific genes and molecular mechanisms involved in the angiogenic cascade. As angiogenesis occurs as a complication of many diseases, knowledge gained from these studies has the potential to impact nonocular angiogenic conditions. This article provides historical perspective on the development and use of the rat model of ROP. Key findings generated through the use of this model are also summarized.

Foveal avascular zone and foveal pit formation after preterm birth

Background Vascularisation of the macula takes place between 24 and 27 weeks post-conception. Preterm birth may affect the formation of the foveal avascular zone (FAZ) and foveal depression, and displacement of inner retinal layers away from the incipient fovea. Objective To examine whether vascular abnormalities accompany an inner retinal abnormality, and whether they are coincident. Methods High-density spectral domain optical coherence tomography volume scans were obtained from 24 preterm children and 34 full-term controls (5–16 years). Matlab programs were used to quantify total retinal thickness, thickness of individual retinal layers and metrics of foveal morphology. Summed voxel projections for the ganglion cell layer–inner nuclear layer were used to identify the FAZ. Results Preterm children had significantly smaller FAZ diameters than controls (p<0.0001). The foveal pits of preterm children were significantly shallower and less steep (p<0.0001) and total retinal thickness at the fovea was significantly increased (p<0.0001) compared to controls. The ganglion cell layer–inner plexiform layer and outer nuclear layer were significantly (p≤0.0001) thicker in preterm children than in controls. Conclusions Preterm birth results in abnormal foveal vascularisation, a failure of the inner retinal neurons to migrate away from the fovea, and an elevated outer nuclear layer ratio. The spatial coincidence of inner retinal and vascular abnormalities in preterm children supports the hypothesis that aspects of foveal development are interdependent.

Genetic deletion of COX-2 diminishes VEGF production in mouse retinal Müller cells

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 microM PGE(2), or PGE(2) + 5 microM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE(2) was significantly increased by hypoxia (p < 0.02). Exogenous PGE(2) significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE(2), signaling through the EP(2) and/or EP(4) receptor and PKA, mediates the VEGF response of Müller cells.

Development of Refractive Error in Individual Children With Regressed Retinopathy of Prematurity

PURPOSE We investigated longitudinally the refraction development in children with regressed retinopathy of prematurity (ROP), including those with and those without a history of peripheral retinal laser photocoagulation. METHODS Longitudinal (0-7 years) cycloplegic refraction data were collected prospectively for two groups of preterm children: severe ROP group included those with regressed ROP following bilateral panretinal laser photocoagulation (n = 37; median gestational age [GA] = 25.2; range, 22.7-27.9 weeks) and mild/no ROP group included those with spontaneously regressed ROP or no ROP (n = 27; median GA = 27.1; range, 23.1-32.0 weeks). Analyses were based on spherical equivalent (SEQ), anisometropia, astigmatism, and age (corrected for gestation). RESULTS The prevalence, magnitude, and rate of myopic progression all were significantly higher in the severe ROP group than in the mild/no ROP group. Longitudinal SEQ in the severe ROP group were best fit with a bilinear model. Before 1.3 years old, the rate of myopic shift was -4.7 diopters (D)/y; after 1.3 years, the rate slowed to -0.15 D/y. Longitudinal SEQ in the mild/no ROP group was best fit with a linear model, with a rate of -0.004 D/y. Anisometropia in the severe ROP group increased approximately three times faster than in the mild/no ROP group. In the severe ROP group, with-the-rule astigmatism increased significantly with age. CONCLUSIONS The severe ROP group progressed rapidly toward myopia, particularly during the first 1.3 years; anisometropia and astigmatism also increased with age. The mild/no ROP group showed little change in refraction. Infants treated with laser photocoagulation for severe ROP should be monitored with periodic cycloplegic refractions and provided with early optical correction.

The effects of nepafenac and amfenac on retinal angiogenesis

PURPOSE Nepafenac is a potent NSAID that rapidly penetrates the eye following topical ocular administration. In the eye, nepafenac is converted to amfenac, which has unique time-dependent inhibitory properties for COX-1 and COX-2. The purpose of the present study was to investigate the capacity of amfenac to inhibit discrete aspects of the angiogenic cascade in vitro, and to test the efficacy of amfenac and nepafenac in vivo, using the rat OIR model. METHODS Müller cells were treated with amfenac, celecoxib (COX-2), or SC-560 (COX-1), and hypoxia-induced VEGF and PGE(2) assessed. Endothelial cells were treated with amfenac, celecoxib, or SC-560, and VEGF-induced proliferation and tube formation assessed. Rat pups were subjected to OIR, received intravitreal injections of amfenac, celecoxib, or SC-560, and neovascularization (NV), prostanoid production, and VEGF assessed. Other OIR-exposed pups were treated with topical nepafenac, ketorolac, or diclofenac, and inhibition of NV assessed. RESULTS Amfenac treatment failed to inhibit hypoxia-induced VEGF production. Amfenac treatment significantly inhibited VEGF-induced tube formation and proliferation by EC. Amfenac treatment significantly reduced retinal prostanoid production and NV in OIR. Nepafenac treatment significantly reduced retinal NV in OIR; ketorolac and diclofenac had no effect. CONCLUSIONS Nepafenac and amfenac inhibit OIR more effectively than the commercially available topical and injectable NSAIDs used in this study. Our data suggests there are COX-dependent and COX-independent mechanisms by which amfenac inhibits OIR. Because it is bioavailable to the posterior segment following topical delivery, nepafenac appears to be a promising advancement in the development of therapies for neovascular eye diseases.

Retinal angiogenesis suppression through small molecule activation of p53.

Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3-mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.

RODENT MODELS OF OXYGEN-INDUCED RETINOPATHY

Retinopathy of prematurity (ROP), a condition affecting premature infants, is characterized by pathological ocular angiogenesis, or retinal neovasculariztion (NV). Much of what is known about the development of the retinal vasculature and the progression of ROP has been acquired through the use of animal models of oxygen-induced retinopathy (OIR), which approximate ROP. Animal models of OIR have provided a wealth of information regarding the cellular and molecular pathogenesis of ROP. This information has contributed to a better understanding of other, non-ocular, neovascular conditions. The aim of this chapter is to explore the significance of the two most prevalent animal models of OIR, the mouse and the rat.