Keirnan L. Willett

Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis

The mouse model of oxygen-induced retinopathy (OIR) has been widely used in studies related to retinopathy of prematurity, proliferative diabetic retinopathy and in studies evaluating the efficacy of antiangiogenic compounds. In this model, 7-d-old (P7) mouse pups with nursing mothers are subjected to hyperoxia (75% oxygen) for 5 d, which inhibits retinal vessel growth and causes significant vessel loss. On P12, mice are returned to room air and the hypoxic avascular retina triggers both normal vessel regrowth and retinal neovascularization (NV), which is maximal at P17. Neovascularization spontaneously regresses between P17 and P25. Although the OIR model has been the cornerstone of studies investigating proliferative retinopathies, there is currently no harmonized protocol to assess aspects of angiogenesis and treatment outcome. In this protocol we describe standards for mouse size, sample size, retinal preparation, quantification of vascular loss, vascular regrowth, NV and neovascular regression.

The Mouse Retina as an Angiogenesis Model

The mouse retina has been used extensively over the past decades to study both physiologic and pathologic angiogenesis. Over time, various mouse retina models have evolved into well-characterized and robust tools for in vivo angiogenesis research. This article is a review of the angiogenic development of the mouse retina and a discussion of some of the most widely used vascular disease models. From the multitude of studies performed in the mouse retina, a selection of representative works is discussed in more detail regarding their role in advancing the understanding of both the ocular and general mechanisms of angiogenesis.

5-Lipoxygenase Metabolite 4-HDHA Is a Mediator of the Antiangiogenic Effect of ω-3 Polyunsaturated Fatty Acids

In mice, ω-3 fatty acids in the diet are converted by 5-lipoxygenase to a metabolite that inhibits destructive vessel growth similar to diabetic retinopathy. LOX and Vessels In a dreaded morning ritual, parents once force-fed cod liver oil—a source of vitamin D—to their children to prevent rickets. Today, fish oil has a new star: ω-3 polyunsaturated fatty acids (PUFAs). These molecules—eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)—can protect against the abnormal growth of blood vessels, a prime contributor to diabetes-related blindness and retinopathy associated with premature birth. Sapieha et al. have now enriched our understanding of the mechanism behind this protection by showing that the production of a DHA metabolite, 4-hydroxy-docosahexaenoic acid (4-HDHA), by the enzyme 5-lipoxygenase (5-LOX) is one of the main metabolic pathways required for the antiangiogenic effect. 4-HDHA acts via peroxisome proliferator–activated receptor γ (PPARγ) to directly inhibit the sprouting and proliferation of endothelial cells—those that line the inner surfaces of blood vessels. Mice that are exposed to high amounts of oxygen (75%) from day 7 to 12 after birth and then returned to ambient air develop abnormal revascularization of the retina. Using this system as an approximation of human retinal revascularization, the authors found that feeding mice a diet high in ω-3 PUFAs, but not ω-6 PUFAs, could prevent this event. By genetically removing, one by one, each of the four main enzymes that convert these ω-3 PUFAs to active metabolites, the authors found that protection against revascularization required 5-LOX, a non–heme iron dioxygenase that generates leukotrienes, lipoxins, and the recently identified ω-3 PUFA–derived resolvins and protectins. After identifying the critical 5-LOX as the one present in white blood cells, Sapieha et al. found that large amounts of DHA metabolites were generated under stress conditions such as those that produce retinopathy in premature newborns. One metabolite, 4-HDHA, was detected in the serum and white blood cells of mice and humans and in retinas of mice with oxygen-induced retinopathy. 4-HDHA is known to act through the PPARγ receptor, and pharmacological inhibition of PPARγ interfered with the ability of the ω-3 diet to alleviate oxygen-induced retinopathy in the mice. These results elucidate an important pathway through which ω-3 oils protect against retinopathy and perhaps exert some of their other beneficial effects: oxidation of ω-3 PUFAs by 5-LOX and subsequent inhibition of angiogenesis via PPARγ activation. One implication of this work is good news for those who take aspirin and ibuprofen: The cyclooxygenase inhibition by these agents will not interfere with the healthy effects of your daily dose of fish oil. Lipid signaling is dysregulated in many diseases with vascular pathology, including cancer, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. We have previously demonstrated that diets enriched in ω-3 polyunsaturated fatty acids (PUFAs) effectively reduce pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy, in part through metabolic products that suppress microglial-derived tumor necrosis factor–α. To better understand the protective effects of ω-3 PUFAs, we examined the relative importance of major lipid metabolic pathways and their products in contributing to this effect. ω-3 PUFA diets were fed to four lines of mice deficient in each key lipid-processing enzyme (cyclooxygenase 1 or 2, or lipoxygenase 5 or 12/15), retinopathy was induced by oxygen exposure; only loss of 5-lipoxygenase (5-LOX) abrogated the protection against retinopathy of dietary ω-3 PUFAs. This protective effect was due to 5-LOX oxidation of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxy-docosahexaenoic acid (4-HDHA). 4-HDHA directly inhibited endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator–activated receptor γ (PPARγ), independent of 4-HDHA’s anti-inflammatory effects. Our study suggests that ω-3 PUFAs may be profitably used as an alternative or supplement to current anti–vascular endothelial growth factor (VEGF) treatment for proliferative retinopathy and points to the therapeutic potential of ω-3 PUFAs and metabolites in other diseases of vasoproliferation. It also suggests that cyclooxygenase inhibitors such as aspirin and ibuprofen (but not lipoxygenase inhibitors such as zileuton) might be used without losing the beneficial effect of dietary ω-3 PUFA.

Suppression of Retinal Neovascularization by Erythropoietin siRNA in a Mouse Model of Proliferative Retinopathy

PURPOSE Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy. METHODS The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17. RESULTS Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%. CONCLUSIONS Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

Wnt signaling mediates pathological vascular growth in proliferative retinopathy.

Background— Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. Methods and Results— In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor–related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5−/− vessels. Blocking claudin5 significantly suppresses Wnt pathway–driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. Conclusions— These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

Quantification and localization of the IGF/insulin system expression in retinal blood vessels and neurons during oxygen-induced retinopathy in mice.

PURPOSE Retinopathy is a result of pathologic angiogenesis influenced by insulinlike growth factor (IGF)-1. The authors examined the local expression of the IGF/insulin family. METHODS In retinas with and without oxygen-induced retinopathy, the authors assessed with real-time RT-PCR mRNA expression of the IGF-1 receptor (IGF-1R), insulin receptor (IR), IGF-1, IGF-2, insulin (Ins2), and IGF-binding protein 1 (IGFBP1) to IGFBP6 in total retina from postnatal day (P) 7 to P33 to examine changes over time with the induction of retinopathy and at P17 on laser-captured retinal components to quantitatively localize mRNA expression in the ganglion cell layer, the outer nuclear layer, the inner nuclear layer, normal blood vessels, and neovascular tufts. RESULTS IGF-1R and IR are expressed predominantly in photoreceptors and in vessels, with scant expression in the rest of the neural retina. IGF-1R expression is more than 100-fold greater than IR. The major local growth factor (expressed in photoreceptors and in blood vessels) is IGF-2 (approximately 1000-fold greater than IGF-1). IGF-1 (approximately 600 copies/10(6) cyclophilin) is expressed throughout the retina. IGFBP2, IGFBP4, and IGFBP5 expression is unchanged with increasing retinal development and with the induction of retinopathy. In contrast, IGFBP3 expression increased more than 5-fold with hypoxia, found in neovascular tufts. CONCLUSIONS IGF-1R, IR, and the ligand IGF-2 are expressed almost exclusively in photoreceptors and blood vessels. IGFBP3 and IGFBP5 expression increases in neovascular tufts compared with normal vessels. IGF-1 is expressed throughout the retina at much lower levels. These results suggest cross-talk between vessels and photoreceptors in the development of retinopathy and retinal vasculature.

Short communication: PPAR gamma mediates a direct antiangiogenic effect of omega 3-PUFAs in proliferative retinopathy.

Rationale Omega3 long-chain polyunsaturated fatty acids (&ohgr;3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing &ohgr;3-PUFA–dependent attenuation of angiogenesis. Objective This study aims to identify a major mechanism by which &ohgr;3-PUFAs attenuate retinal neovascularization. Methods and Results Administering &ohgr;3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)&ggr; almost completely abrogates this effect. Inhibition of PPAR&ggr; also reverses the &ohgr;3-PUFA–induced reduction of retinal tumor necrosis factor-&agr;, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor. Conclusions These results identify a direct, PPAR&ggr;-mediated effect of &ohgr;3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

Short Communication: PPARγ Mediates a Direct Antiangiogenic Effect of ω3-PUFAs in Proliferative RetinopathyNovelty and Significance

Rationale Omega3 long-chain polyunsaturated fatty acids (&ohgr;3-PUFAs) are powerful modulators of angiogenesis. However, little is known about the mechanisms governing &ohgr;3-PUFA–dependent attenuation of angiogenesis. Objective This study aims to identify a major mechanism by which &ohgr;3-PUFAs attenuate retinal neovascularization. Methods and Results Administering &ohgr;3-PUFAs exclusively during the neovascular stage of the mouse model of oxygen-induced retinopathy induces a direct neovascularization reduction of more than 40% without altering vasoobliteration or the regrowth of normal vessels. Cotreatment with an inhibitor of peroxisome proliferator-activated receptor (PPAR)&ggr; almost completely abrogates this effect. Inhibition of PPAR&ggr; also reverses the &ohgr;3-PUFA–induced reduction of retinal tumor necrosis factor-&agr;, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial selectin, and angiopoietin 2 but not vascular endothelial growth factor. Conclusions These results identify a direct, PPAR&ggr;-mediated effect of &ohgr;3-PUFAs on retinal neovascularization formation and retinal angiogenic activation that is independent of vascular endothelial growth factor.

Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5−/−) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5−/− mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5−/− and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5−/− retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5−/− retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5−/− retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

Proliferative Retinopathy Is Associated with Impaired Increase in BDNF and RANTES Expression Levels after Preterm Birth

Background: Extremely preterm delivery is, amongst other complications, associated with retinopathy of prematurity (ROP). Untreated, ROP can progress to visual impairment and blindness due to an overgrowth of new vessels in the retina and vitreous cavity. Objective: The aim of this study was to identify cytokine markers within the first weeks of life that could be used to predict the risk for development of ROP later in life. Methods: Serum levels of 27 different cytokines in infants born at gestational weeks 23–30 were analyzed using a multiplex immunoassay method and compared between infants who did not develop ROP and infants who later developed proliferative ROP. In addition, mRNA levels of brain-derived neurotrophic factor (BDNF) in retinas from mice exposed to hyperoxia were analyzed using quantitative real-time PCR. Results: At birth, serum levels of IL-5 were higher in infants with no ROP compared to infants with proliferative ROP. 10–14 days after birth, serum levels of BDNF and RANTES were lower in infants who later developed proliferative ROP compared to infants who did not develop ROP. Furthermore, mRNA expression levels of BDNF in retinas from mice exposed to hyperoxia were significantly lower at postnatal day 15 compared to retinas from mice in room air. Conclusions: These results indicate that BDNF and RANTES may be important factors in the selective vulnerability of ROP development in preterm infants.