C. M. Aderman

Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.

Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of ω-3- and ω-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy. We show that increasing ω-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive ω-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of ω-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-α. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of ω-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in ω-3-PUFA, and premature infants lack the important transfer from the mother to the infant of ω-3-PUFA that normally occurs in the third trimester of pregnancy. Supplementing ω-3-PUFA intake may be of benefit in preventing retinopathy.

A mechanosensitive transcriptional mechanism that controls angiogenesis

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

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.

Erythropoietin deficiency decreases vascular stability in mice

Erythropoietin (Epo), a hormone known to stimulate bone marrow erythrocyte production, is widely used to treat anemia in patients at risk for vascular disease. However, the effects of Epo on angiogenesis are not well defined. We studied the role of Epo in a mouse model of retinopathy characterized by oxygen-induced vascular loss followed by hypoxia-induced pathological neovascularization. Without treatment, local retinal Epo levels were suppressed during the vessel loss phase. Administration of exogenous Epo prevented both vessel dropout and subsequent hypoxia-induced neovascularization. Early use of Epo also protected against hypoxia-induced retinal neuron apoptosis. In contrast, retinal Epo mRNA levels were highly elevated during the retinopathy neovascular phase. Exogenous late Epo treatment did not protect the retina, but rather enhanced pathological neovascularization. Epos early protective effect occurred through both systemic retinal recruitment of proangiogenic bone marrow-derived progenitor cells and activation of prosurvival NF-kappaB via Epo receptor activation on retinal vessels and neurons. Thus early retinal Epo suppression contributed to retinal vascular instability, and elevated Epo levels during the proliferation stage contributed to neovascularization and disease. Understanding the role of Epo in angiogenesis is critical to timing its intervention in patients with retinopathy or other diseases in which pathological angiogenesis plays a significant role.

IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth.

Vessel loss precipitates many diseases. In particular, vessel loss resulting in hypoxia induces retinal neovascularization in diabetic retinopathy and in retinopathy of prematurity (ROP), major causes of blindness. Here we define insulin-like growth factor binding protein-3 (IGFBP3) as a new modulator of vascular survival and regrowth in oxygen-induced retinopathy. In IGFBP3-deficient mice, there was a dose-dependent increase in oxygen-induced retinal vessel loss. Subsequent to oxygen-induced retinal vessel loss, Igfbp3−/− mice had a 31% decrease in retinal vessel regrowth versus controls after returning to room air. No difference in serum insulin-like growth factor 1 (IGF1) levels was observed among groups. Wild-type mice treated with exogenous IGFBP3 had a significant increase in vessel regrowth. This correlated with a 30% increase in endothelial progenitor cells in the retina at postnatal day 15, indicating that IGFBP3 could be serving as a progenitor cell chemoattractant. In a prospective clinical study, we measured IGFBP3 (and IGF1) plasma levels weekly and examined retinas in all premature infants born at gestational ages <32 weeks at high risk for ROP. The mean level of IGFBP3 at 30–35 weeks postmenstrual age (PMA) for infants with proliferative ROP (ROP stages 3>, n = 13) was 802 μg/liter, and for infants with no ROP (ROP stage 0, n = 38) the mean level was 974 μg/liter (P < 0.03). These results suggest that IGFBP3, acting independently of IGF1, helps to prevent oxygen-induced vessel loss and to promote vascular regrowth after vascular destruction in vivo in a dose-dependent manner, resulting in less retinal neovascularization.

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.